comparative genetic and metabolic characterization between
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Comparative genetic and metabolic characterizationbetween two table grape varieties with contrasted color
berry skin : red Globe and Chimenti GlobeClaudia Santibanez
To cite this version:Claudia Santibanez. Comparative genetic and metabolic characterization between two table grapevarieties with contrasted color berry skin : red Globe and Chimenti Globe. Agricultural sciences.Université de Bordeaux; Pontificia universidad católica de Chile (Santiago de Chile), 2017. English.�NNT : 2017BORD0918�. �tel-01809532�
0
THÈSE EN COTUTELLE PRÉSENTÉE
POUR OBTENIR LE GRADE DE
DOCTEUR DE
L’UNIVERSITÉ DE BORDEAUX
ET DE
LA PONTIFICIA UNIVERSIDAD CATÓLICA DE CHILE
ÉCOLE DOCTORALE DE SCIENCES DE LA VIE ET LA SANTÉ
SPECIALITÉ : BIOLOGIE VÉGÉTALE
ET DU
DOCTORAT EN SCIENCES BIOLOGIQUES
SPECIALITÉ : GENETIQUE MOLECULAIRE ET MICROBIOLOGIE
Par Claudia SANTIBAÑEZ ORELLANA
COMPARATIVE GENETIC AND METABOLIC CHARACTERIZATION BETWEEN
TWO TABLE GRAPE VARIETIES WITH CONTRASTED COLOR BERRY SKIN:
RED GLOBE AND CHIMENTI GLOBE.
Sous la direction de Eric GOMÈS
et de Patricio ARCE JOHNSON
Soutenue le 18 de décembre 2017
Membres du jury : M. GUTIERREZ, Rodrigo, Professeur à La Pontificia Universidad de Chile Président
M. HANFORD, Michael, Professeur à L’Universidad de Chile Rapporteur
M. INOSTROZA, Claudio, Chargé de Recherche à L’ Universidad Catolica de Temuco Rapporteur
M. GOMÈS, Eric, Professeur de l’Université de Bordeaux Examinateur
M. ARCE, Patricio, Professeur à La Pontificia Universidad de Chile Examinateur
M. DELROT, Serge, Professeur de l’Université de Bordeaux Examinateur
Mme MEISEL, Lee, Professeur à L’ Universidad de Chile Invité
1
Comparative genetic and metabolic characterization between two table grape
varieties with contrasted color berry skin: Red Globe and Chimenti Globe
Abstract Grape berry development is a dynamic process exhibiting a double sigmoid curve separated by
a lag phase, characterized by coordinated biosynthesis of primary and secondary metabolites.
At the end of lag phase, a phenomenon called veraison occurs, where the fruit begins to take
color and maturation process is initiated. Anthocyanins are the responsible of berry skin
coloration and its regulation has been widely studied. However, few studies have focused on
metabolic and genetic characterization using varieties with contrasted color berry skin. Using
RNA-seq technology and metabolic analysis, we performed a new comparative characterization
of two table grapes, Chimenti Globe (CG) and Red Globe (RG), with contrasted color berry
skin: CG has a bright light red color and RG has a dark purple color. The originality of this
model is that CG was generated from a spontaneous event in field from a branch in a vine of
RG plant. Thus, the genetic background responsible for the color change is practically the same.
Berry metabolic content analysis showed the importance of veraison and ripening stages, in both
varieties in the study. Particularly, striking difference in metabolites concentration of
phenylpropanoid pathway: shikimate, UDP-glucose, phenylalanine and trehalose-6-phosphate,
among others. Also, differences between the varieties were due to changes in metabolites related
to the biosynthesis of sucrose and anthocyanin. CG only contained dihydroxylated
anthocyanins, peonidin and cyanidin, and not the trihydroxylated ones, malvidin, delphinidin
and petunidin, which was consistent with the observed color phenotype. From transcriptomic
analysis, we generated a heat map with 109 genes differentially expressed in CG in comparison
to RG, being many of these related to the flavonoid pathway. In addition, 11 gene copies of
flavonoid 3’5’-hydroxylase, a key enzyme for biosynthesis of trihydroxylated anthocyanins,
were not induced in CG. From this analysis, we selected a candidate gene to contribute in the
study of the anthocyanin biosynthetic pathway: Cytochrome b5 (Cytb5) encoding a key electron
donor protein not characterized in grapevine. Cytb5 overexpression in V. berlandieri x V.
Rupestris 110R embryos strongly suggested its participation in the pathway, since transgenic
embryos exhibited reddish color and in addition, an accelerated development compared to
control. With these results, we were able to provide new insights in anthocyanins regulation in
grapevines responsible for berry skin coloration, opening new fields in the search for molecular
regulators of the flavonoid pathway.
Keywords : Chimenti Globe, Red Globe, RNA-seq, skin, anthocyanin, cytb5.
2
Analyse génétique et métabolique comparée de deux variétés de raisins de
table présentant des pigmentations contrastées au niveau des pellicules des
baies : Red Globe et Chimenti Globe.
Résumé Le développement de la baie de raisin est un processus dynamique présentant une courbe de
croissance sigmoïde avec deux phases de croissance séparée par une phase de latence. Il se
caractérise par une biosynthèse coordonnée de métabolites primaires et secondaires. À la fin de
la phase de latence, un phénomène appelé véraison se produit, au cours duquel le fruit
commence à prendre de la couleur et le processus de maturation est initié. Les anthocyanes sont
responsables de la coloration des baies et la régulation de leur biosynthèse été largement étudiée.
Cependant, peu d'études ont porté sur la caractérisation métabolique et génétique de variétés de
baies de couleurs fortement contrastées, dans un même fond génétique. En combinant la
technologie RNAseq et des analyses métabolomiques, nous avons effectué une caractérisation
comparée de deux variétés de raisin de table : Chimenti Globe (CG, rose pâle) et Red Globe
(RG, rouge foncé). L'originalité de ce modèle est que CG a été généré à partir d'un événement
de mutation spontanée de RG, dans un vignoble de production, permettant ainsi l’étude de la
régulation de la biosynthèse des anthocyanes dans un même fond génétique.
L'analyse du contenu métabolique des baies a démontré l'importance des stades de
développement, de la véraison à la maturation, dans les deux variétés de raisin. En particulier,
des différences marquées dans les concentrations de certains métabolites de la voie des
phénylpropanoïdes (shikimate, UDP-glucose, phénylalanine) et du tréhalose-6-phosphate ont
été mises en évidence en post-véraison. De plus, les différences entre les variétés étaient dues à
des changements dans les métabolites liés à la biosynthèse du saccharose et de l'anthocyanine.
Les baies de CG ne contenaient que des anthocyanines dihydroxylées (péonidine et cyanidine),
et aucune quantité détectable d’anthocyanes trihydroxylées (malvidine, delphinidine et
pétunidine), qui sont abondamment présentes dans les pellicules des baies de RG. Ceci explique
le phénotype de couleur rose pâle des baies de CG. Une analyse transcriptomique globale par
RNAseq montre que 109 gènes sont exprimés de façon différentielle chez CG, en comparaison
avec RG, y compris de nombreux gènes liés au métabolisme des flavonoïdes. Notamment, 11
gènes codant pour des 3'5'-hydroxylases flavonoïde, une enzyme-clé pour la biosynthèse des
anthocyanes trihydroxylées, sont réprimés dans CG. A partir de cette analyse, un gène candidat
pour la régulation de la voie de biosynthèse des anthocyanes a été sélectionné : le gène Cytb5,
qui code pour un cytochrome b5, non caractérisé à ce jour chez la Vigne. La surexpression de
Cytb5 par transgénèse dans des vignes hybrides V. berlandieri x V. rupestris cv. 110R suggère
fortement un rôle clé pour ce gène dans la régulation de la biosynthèse des anthocyanes chez la
vigne : les embryons obtenus présentaient une forte coloration rouge, indiquant la présence
d’anthocyanes en quantités importantes dans les tissus végétatifs. De plus les embryons
transgéniques ont un développement accéléré par rapport aux embryons sauvages.
Ces travaux ont permis de mieux comprendre la régulation de l’accumulation des anthocyanes
responsables de la coloration des baies de raisin, et plus largement, la régulation du métabolisme
des flavonoïdes.
Mots clés : Chimenti Globe, Red Globe, RNA-seq, pellicule, anthocyane, cytb5.
3
Caracterización genética y metabólica comparativa entre dos tipos de uva
de mesa con color de piel contrastante: Red Globe y Chimenti Globe
Resumen El desarrollo de la uva es un proceso dinámico caracterizado por una curva de crecimiento doble
sigmoidea, separada por una fase lag, en donde ocurre una biosíntesis coordinada de metabolitos
primarios y secundarios. Al final de la fase lag, ocurre un fenómeno llamado pinta
correspondiente al comienzo de la coloración de la baya e iniciándose también el proceso de
maduración. Las antocianinas son las responsables de la coloración de la piel de las bayas y su
regulación ha sido ampliamente estudiada. Sin embargo, pocos estudios se han enfocado en la
caracterización genética y metabólica, utilizando variedades contrastantes de color de piel.
Utilizando análisis metabólico y la tecnología de RNA-seq, se realizó una nueva caracterización
comparativa de dos uvas de mesa, Chimenti Globe (CG) y Red Globe (RG), que poseen un color
de piel de la baya contrastante: CG tiene un color rojizo claro y RG posee un color morado. La
originalidad de este modelo es que CG fue generada en un evento espontáneo de campo desde
una rama de una planta RG. Por lo tanto, el background genético responsable del cambio de
color es el mismo.
El análisis del contenido metabólico de las pieles de las bayas reveló la importancia de las etapas
de desarrollo, pinta y maduración, en ambas variedades en estudio. En particular, la diferencia
en la concentración de metabolitos de la ruta fenilpropanoide, tales como shikimato y
fenilalanina y otras moléculas como UDP-glucosa y trehalosa-6-fosfato, entre otros. Asimismo,
las diferencias entre las variedades estuvieron dadas por cambios relacionados con la biosíntesis
de sacarosa y antocianinas. CG solo contenía antocianinas dihidroxiladas, cianidina y peonidina,
y no las del tipo trihidroxiladas, malvidina, delfinidina y petunidina, lo cual fue consistente con
el fenotipo del color de piel observado. A partir del análisis transcriptómico, generamos un
heatmap con 109 genes expresado diferencialmente en CG en comparación con RG, siendo
muchos de estos asociados a la ruta biosíntesis de flavonoides. Además, observamos que 11
copias del gen flavonoide 3'5'-hidroxilasa, que codifica una enzima clave para la biosíntesis de
antocianinas trihidroxiladas, no estaban inducidas en CG. A partir de este análisis, se seleccionó
un gen candidato para contribuir en el estudio de la ruta de biosíntesis de antocianinas:
citocromo b5 (Cytb5) que codifica una proteína clave donadora de electrones no caracterizada
en vides. La sobreexpresión de Cytb5 en embriones V. berlandieri x V. rupestris cv. 110R sugirió
fuertemente la participación en la ruta, ya que los embriones transgénicos exhibieron un color
rojizo e incluso, un desarrollo acelerado en comparación con el control. Con estos resultados,
hemos sido capaces de proporcionar información sobre la regulación de las antocianinas en
vides responsables de la coloración de la piel de las bayas, abriendo nuevos terrenos en la
búsqueda de reguladores moleculares de la vía flavonoide.
Palabras clave: Chimenti Globe, Red Globe, RNA-seq, piel, antocianinas, cytb5.
4
Unité de recherche dans Université de Bordeaux UMR 1287 Ecophysiologie et Génomique Fonctionnelle de la Vigne
Institut des Sciences de la Vigne et du Vin
210 Chemin de Leysotte
33140 Villenave d’Ornon, France
Research Unit in Pontificia Universidad Católica de Chile Laboratorio de Biología Molecular y Biotecnología Vegetal
Facultad de Ciencias Biológicas
Departamento de Genética Molecular y Microbiología
Avenida Libertador Bernardo Bernardo O’Higgins 340
8331150 Región Metropolitana Santiago de Chile
5
"Happiness can be found, even in the darkest of times,
if one only remembers to turn on the light."
Albus Dumbledore, Harry Potter and the Prisoner of Azkaban.
6
This work was supported by:
Scholarship of Comisión Nacional de Investigación (CONICYT) Nº 21120432.
Operational Expenses Scolarship of CONICYT year 2014 and 2015, Nº 21120432.
Millennium Nucleus of Plant Systems and Synthetic Biology NC130030.
Chimenti S.A.
7
ACKNOWLEDGEMENT
This thesis has been performed under co-supervision between the Pontificia Universidad
Católica de Chile and the University of Bordeaux, and thus, most of my activities were carried
out at the Laboratorio de Biología Molecular y Biotecnología Vegetal (Facultad de Ciencias
Biológicas, Departamento de Genética Molecular y Microbiología from PUC) and at the
Ecophysiologie et Génomique Fonctionnelle de la Vigne (EGFV), Institut des Sciences de la
Vigne et du Vin (ISVV) (INRA and Université de Bordeaux).
My thesis supervisors, Dr. Patricio Arce and Dr. Eric Gomes, for their help in the supervision
of this thesis. My most sincere thanks for all the time that this thesis was carried out.
Listy Martínez, Valeria Borjas, Don Julio, Toño, Ghislaine Hilbert and Christel Renaud for their
technical assistance and interest in the experimental part of my thesis.
The researchers of the institutions I have been working in: Dr. Zhanwu Dai, Dr. John Lunn and
Dr. José Tomás Matus for their contribution in key thesis experiments and analysis of them.
Dr. Michael Hanford (Raporteur), Dr. Claudio Inostroza (Raporteur), Serge Delrot
(Examinateur), Lee Meissel and Rodrigo Gutiérrez for accepting the hard work of being the jury
of this private and public doctoral defense.
Dr. Sarah Frusciante, Dr. María José Clemente, Dr. Roberta Triolo, Dr. Noé Cococh and Dr.
Hugo Campbell, for their role in my adaptation in my stays at the ISVV.
Dr. Rodrigo Loyola, Dr. Alejandra Serrano, Brenda Riquelme, Francisca Parada, Dr. Carmen
Espinoza, Dr. Grace Armijo, Dr. Mario Agurto, Aníbal Arce, Carlos Meyer and Tomás Moyano,
for all your comments and corrections to my thesis that helped to shape what is finally. Special
thanks to Consuelo Medina for accompanying me during my stay in Bordeaux, France and for
being a key person in the end of this thesis.
Dr. Daniela Muñoz, Evelyn Poblete, Rowena Cortés, Camila Almendra, Yessica Arcos,
Elizabeth Torres, Susan Hitschfeld, Daniela Herrera and Amparo Rodríguez, for being the best
friends and for being by my side in this long process.
Paola Cárdenas and Marisol Carrasco for being with me in the difficult moments and know how
to get a smile on me. My Harry Potter’s friends!
And most importantly, my family, Joaquín, Mary, Alejandra, Álvaro, Nicolás, Adolfo,
Francisca, Victoria, Lucía, Emilia, Joaquín junior, Alejandro, Rebeca, for allowing me to fulfill
my dreams and every single lab mate who have been there for me during this challenging period.
8
TABLE OF CONTENTS
CHAPTER I 9
GENERAL INTRODUCTION 9
1. Vitis vinifera 10
2. Metabolic changes during grapevine development 11
2.1 Phase I 11
2.2 Phase II 12
2.3 Phase III 13
3. Anthocyanins, responsible of grape berry coloration 13
4. Anthocyanins biosynthesis 16
5. Transportation of anthocyanins to the vacuole 21
6. Regulation of anthocyanin biosynthesis 22
6.1 Transcriptional regulation 22
6.2 Environmental factors that can affect anthocyanin accumulation 24
6.3 Hormonal factors that can affect anthocyanin accumulation 25
7. New advances in comparative studies between contrasted berry skin varieties
27
REFERENCES FROM CHAPTER I 31
CHAPTER II 47
PROBLEM STATEMENT: Hypothesis and Objectives 47
CHAPTER III 49
TRANSCRIPTOMIC AND METABOLIC ANALYSIS REVEALS NEW GENETIC
DIFFERENCES BETWEEN TWO TABLE GRAPES WITH CONTRASTED COLOR
BERRY SKIN
CHAPTER IV 125
OBJETIVE 3: Evaluate the function of at least one gene of interest, selected form the
above objetives, related with the branching point F3’-H and F3’5’-H of the anthocyanin
biosynthesis pathway
INTRODUCTION 126
MATERIAL AND METHODS 128
RESULTS 132
DISCUSSION AND CONCLUSION 138
CHAPTER V 141
GENERAL DISCUSSION 141
CHAPTER VI 156
CONCLUSION 156
REFERENCES FROM CHAPTER IV and V 159
9
CHAPTER I
GENERAL INTRODUCTION
10
In the present chapter the literature and accumulated knowledge of grapevine development,
anthocyanins biosynthesis and regulation of anthocyanin pathway are reviewed.
1. Vitis vinifera
Grapevine (Vitis vinifera) is an economically important perennial fruit crop whose main
destinations are fresh fruits as table grape, raisins and wine making (Coombe, 1992; Coombe &
Mccarthy, 2000). Vitis vinifera L. plants have been propagated asexually for a long time, as
cuttings grafted into rootstocks. As grapevine berries develop, they display modifications in
composition, texture, size, flavour, color and in pathogen susceptibility. The grape berry is
composed of seeds and 3 major types of tissue called exocarp, corresponding to skin; mesocarp
corresponding to flesh; and endocarp which correspond to surrounding seed tissue, the latter
being very difficult to differentiate from the mesocarp (FIGURE 1) (J. Kennedy, 2002). Skin
cells are smaller than flesh cells, but are much thicker, allowing less water loss. The outermost
layer of cells of the exocarp corresponding to epidermis is covered by cuticle on their external
faces, a wax that acts as the primary protective barrier against environmental stress (Hardie,
O’Brien, & Jaudzems, 1996; Pensec et al., 2014).
Grape berry is water and nutrient supplied through the pedicel by a vascular system composed
of phloem elements and xylem. The phloem is the vasculature involved in photosynthate
(hexoses) transport from the canopy to the vine. The phloem has a reduced function in early
berry development but, becomes the primary source of ingress after veraison. The xylem is the
vasculature responsible for transporting nutrients, minerals, water and growth regulators from
the root system to the rest of the plant. Unlike the phloem, the xylem does have a fundamental
11
role in the first stages of berry developmental, but less after veraison (Greenspan, Shackel, &
Matthews, 1994).
FIGURE 1: Grape berry structure. A simple diagram to describe the main tissues of the ripe
grapevine berry. Diagram from (Conde et al., 2007).
2. Metabolic changes during grapevine development
2.1 Phase I
Grape berry development consists of two successive sigmoidal growth periods separated by a
lag phase (FIGURE 2) (Coombe, 1992; Coombe & Mccarthy, 2000). Phase I correspond to the
first growth period and is initiated with fruit set. Also called herbaceous growth, it lasts 60 days,
depending on the variety and environmental conditions. It is characterized by important by an
early cellular divisions phase, followed later on by cellular expansion, which results in an
accumulation of water in the vacuoles as solutes accumulate. During this phase, seed embryos
12
are also produced. In metabolic terms, phase I is characterized by the accumulation of organic
acids, mostly tartaric and malic acid, stored in the vacuoles. Tartaric acid accumulates mainly
at the beginning of development and malic acid towards to the end of the herbaceous phase,
with a maximum concentration reached just before veraison (Conde et al., 2007; Downey,
Dokoozlian, & Krstic, 2006). These compounds provide acidity to the wine and therefore are
critical to its quality. Other important metabolites that accumulate in Phase I are
hydroxycinnamic acids, tannins, methoxypyrazines, citric acid, succinic acid, all strongly
involved in the organoleptic qualities of wine (J. A. Kennedy, Hayasaka, Waters, & Jones, 2001;
Serrano et al., 2017). Conversely, the concentration of sugars remains low throughout this phase
and chlorophyll is then the major pigment of berries. To support itself, the grape berry produces
a few sugars via photosynthesis in situ and massively imports carbon from the leaves (Conde et
al., 2007).
2.2 Phase II
Also called lag phase, Phase II is marked by the slowing down of cell growth (FIGURE 2). It
lasts between 8 and 12 days and during this phase, the seeds change color after desiccation of
their integument (Ristic & Iland, 2005) and also the increase in berry volume is exclusively due
to cell growth (Ebadi, Sedgley, May, & Coombe, 1996). At the end of this phase the
phenomenon called veraison occurs, in which the grape berry begins to gain color, thus third
phase of growth is initiated.
13
2.3 Phase III
This last phase in berry development, starts with veraison, which corresponds to the onset of
ripening and as its name says, it is known by the name of ripening stage. Grape berries switch
to a status where they are larger, softer, sweeter, less acidic, and strongly flavored and colored
(Conde et al., 2007; Coombe & Mccarthy, 2000) (FIGURE 2). Organic acids concentration
decreases and sucrose produced from photosynthesis is imported into the grape berry during
ripening stage as well as water, doubling its size (Dai et al., 2013). Once transported into the
berries, the sucrose is hydrolyzed into glucose and fructose. Beyond sugar accumulation, it
begins to synthesize secondary metabolites, contributing to the organoleptic qualities of wine.
For colored cultivars, skin tissue changes from green to purple, due to the accumulation of
anthocyanins. Near the end of the third phase of berry development, other secondary metabolites
are also accumulated, such as flavors and their precursors as well as flavonols. Ripening stage
ends around 120 days after flowering (Costantini, Battilana, Lamaj, Fanizza, & Grando, 2008;
J. Kennedy, 2002; Kuhn et al., 2014) (FIGURE 2).
3. Anthocyanins, responsible of grape berry coloration
Anthocyanins are the main pigments in flowers and fruits and are responsible for the red color
of pigmented grape berries. Their localization is in the berry skin, although in some cultivars
they are found in flesh, i.e. in the so-called “teinturier” varieties (Castillo-Muñoz, Fernández-
González, Gómez-Alonso, García-Romero, & Hermosín-Gutiérrez, 2009; Guan et al., 2012).
Principal anthocyanin functions are attraction of the animals that allow seed dispersion (Jaakola,
14
2013), protection of berry against solar exposure and UV-B radiation, reduction of oxidative
stress (Adams, 2006; Dixon, Xie, & Sharma, 2005), defense against many different pathogens,
among others (Chalker-scott, 1999).
The basic structure of anthocyanins is the flavylium cation, that consists of the typical flavonoid
carbon skeleton of C6-C3-C6, but has two double bonds on C ring, thus getting a positive charge
(FIGURE 3A) (He et al., 2010). The aglycone part of anthocyanin is called anthocyanidin and
the structure of the different anthocyanidins varies according to the position and the numbers of
hydroxyl and methyl groups on the B ring (FIGURE 3B and 3C) (Jimenez-garcia et al., 2012;
Tanaka, Sasaki, & Ohmiya, 2008).
The common anthocyanidins found in grapes are cyanidin, delphinidin, peonidin, petunidin and
malvidin, being malvidin the predominant anthocyanidin in most pigmented berry grape (Holton
& Cornish, 1995; Mazza, 1995). Blueness is increased with free hydroxyl groups, whereas
redness intensifies with the raising of the methylation of the hydroxyl groups. Thus, malvidin is
the reddest individual anthocyanidin. Sugars are bound to the anthocyanidins through a
glycosidic bond at the C3 and C5 positions, monoglycosylated anthocyanins in C3 being the
most common found in Vitis vinifera (Yamazaki et al., 2015). Methylation takes place after
glycosylation, after the anthocyanins are already formed. Both process contribute to the
solubility and stabilization of anthocyanidins by protecting them from oxidation. In Vitis
vinifera anthocyanins of 3-O-monoglucoside, acetylated and p-coumaroyl forms have been
identified (Acevedo et al., 2012; Haibo Wang, Race, & Shrikhande, 2003).
15
FIGURE 2: Graphical representation of grape berry development. The diagram shows
berry size and coloration during development and major developmental events. Also, it shows
an inflow rate of phloem and xylem into the berry and accumulation periods of different
compounds. Illustration from (J. Kennedy, 2002)
16
FIGURE 3: Anthocyanin structure. (A) Basic structure of an anthocyanidin showing the
numbering of the different carbon groups. The common modification positions are R1, R2, R3,
R4, R5 and R6. (B) Hydroxylated anthocyanins in which hydroxyl group is show in purple color.
(C) Methoxylated anthocyanins in which methoxyle group is show in green color. Modified
from (Y. Zhang, Butelli, & Martin, 2014).
4. Anthocyanins biosynthesis
Anthocyanins belong to the flavonoid class of compounds and are synthesized along the general
phenylpropanoid pathway by the activity of a cytosolic multi-enzymatic complex, associated to
the cytoplasmic face of the endoplasmic reticulum (Boss, Davies, & Robinson, 1996). Once
produced anthocynins are later on transported and stored in the skin cell vacuoles (Winkel-
Shirley, 2001). Most of the genes encoding enzymes of the anthocyanin biosynthesis pathway
have been well identified in grapevine (FIGURE 4).
Phenylpropanoid biosynthesis begins with the amino acid phenylalanine, which is a product of
the shikimate pathway (Maeda & Dudareva, 2012). The shikimate pathway begins with
erythrose-4-phosphate and phosphoenol pyruvate and is responsible for producing the other
aromatic amino acids: tyrosine and tryptophan. The first enzyme responsible for the phenolic
biosynthesis is phenylalanine ammonia-lyase (PAL), which converts phenylalanine into
17
cinnamic acid. PAL is encoded by a multigene family in the grapevine (Sparvoli, Martin,
Scienza, Gavazzi, & Tonelli, 1994) and is transcriptionally regulated in response to biotic and
abiotic factors (Boss et al., 1996). Cinnamic acid suffers a series of transformations resulting in
the synthesis of precursors of several simple phenolics, such as phenolic acids, lignin precursors,
etc. The incorporation of 3 molecules of malonyl-CoA, produced via the acetate pathway, with
the 4-coumaroyl-CoA starts the phenylpropanoid pathway. These last compounds are condensed
by chalcone synthase (CHS), the first committed enzyme of flavonoid biosynthesis. There are
at least in grapevines three genes encoding CHS, CHS1, CHS2 and CHS3, each one may act in
three different pathways to produce different secondary metabolites (Castillo-Muñoz et al.,
2009; Goto-Yamamoto, Wan, Masaki, & Kobayashi, 2002; Huiling Wang et al., 2016).
Then, CHS and chalcone isomerase (CHI) are the enzymes involved in a two-step condensation,
producing a colourless flavanone named naringenin. In Vitis vinifera, there is a single gene
encoding a putative CHI, that is expressed strongly at the onset of veraison (Goes et al., 2005).
Later, naringenin is oxidized by flavanone 3-hydroxylase (F3-H) giving rise the formation of
dihydrokaempferol (colourless dihydroflavonol). This compound is also the substrate for
flavonoid 3’-hydroxylase (F3'-H) and flavonoid 3’5’-hydroxylase (F3'5'-H), which allows
hydroxylation to dihydroquercetin and dihydromyricetin, respectively (FIGURE 4). In
grapevines, there are two genes encoding F3-H, two coding F3’-H and sixteen copies of the
gene coding for F3’5’-H, of which only eight encode full-length proteins (Falginella et al.,
2010). It has been showed that these genes are differentially expressed in the different tissues of
the berry, also according to the stage of development. The high copy number of these genes
would increase the complexity and diversity among pigmented varieties and F3′-H and F3’5’-
H expression directly affects the anthocyanin composition (Ali et al., 2011; Jochen Bogs, Ebadi,
18
McDavid, & Robinson, 2006; Falginella et al., 2010). A recent study showed that in Cabernet
Sauvignon and Shiraz, F3’5’H is as a key molecular player driving the phenylpropanoid
metabolic flux towards bluish anthocyanins production (Degu et al., 2014). These two genes,
F3′-H and F3’5’-H are members of P450 cytochrome family (de Vetten et al., 1999) and in
several studies it had been showed another member of P450 cytochrome family, cytochrome b5
(Cytb5) has previously been involved in F3’5’-H activity modulation in Petunia flowers, by
catalyzing the transfer of electrons to their prosthetic heme group (de Vetten et al., 1999). In
2006, (Jochen Bogs et al., 2006) demonstrated that a putative Cytb5 from grapevine cv Shiraz
modulated both F3’5’-H and F3’-H activities, even though the exact mechanism remains to be
deciphered.
Continuing with the following steps of the pathway, dihydroquercetin and dihydromyricetin are
converted to anthocyanidins (coloured but unstable pigments) by two reactions catalyzed by
dihydroflavonol reductase (DFR) and anthocyanidin synthase (ANS or also called LDOX). The
DFR converts dihydroquercetin and dihydromyricetin into leucocyanidin and leucodelphinidin
(colourless), respectively; and LDOX catalyzes the oxidation of leucocyanidin into cyanidin
(red-magenta anthocyanidin) and leucodelphinidin into delphinidin (purple-blue anthocyanidin)
(Abrahams et al., 2003; Sparvoli et al., 1994). In grapevines, DFR is coded by a single gene (He
et al., 2010; Winkel-Shirley, 2001), which it first expresses in the grape berry development
during phase I, decreased at veraison and it increases again during the phase III (Jochen Bogs
et al., 2005). Anthocyanidins formed by the reaction catalyzed by LDOX are immediately
modified by glycosylation, methylation and acylation, strongly necessary for their stabilization
as vacuolar anthocyanins (He et al., 2010; Springob, Nakajima, Yamazaki, & Saito, 2003). For
19
example, it had been reported that red grapes mutant lacking gene expression for the initial
glycosylation of anthocyanidins, did not generate anthocyanins (Boss et al., 1996).
Anthocyanidins are glycosylated by the action of UDP-glucose:flavonoid 3-O-
glucosyltransferase (UFGT), which catalyze the O-glycosylation in C3 position (Vitis vinifera
sp), using UDP-glucose as sugar donor (Ford, Boss, & Hoj, 1998). Other sugar donors can be
also being used occasionally by UFGT, such as UDP-xylose, UDP-galactose, UDP-arabinose,
UDP-rhamnose and UDP-glucuronic acid (Springob et al., 2003). In general, UFGT gene
expression is only detected in berry skin after veraison (Boss et al., 1996) and it is expressed
only in red cultivars (Kobayashi, Goto-Yamamoto, & Hirochika, 2004). In most non-V. vinifera
grape species the O-glycosylation can also occur at both of the C3 and C5 positions to form
anthocyanidin-3,5-O-diglucoside (Lamikanra, 1989).
With the participation of S-adenosyl-L-methionine (SAM), a O-methyltransferase (OMT)
catalyzes the methylation of the hydroxyl groups at the C3’ positions or both the C3’ and C5’
positions on the B rings of the anthocyanins (FIGURE 3A) (Ibrahim, Bruneau, & Bantignies,
1998). Acylation is also one of the most common modifications of anthocyanins catalized by an
anthocyanin acyltransferase (ACT) which produce 3-O-acetyl-, 3-O- coumaroyl-, and 3-O-
caffeoyl-monoglucosides by attaching acyl groups to the C6’’ position of the Glucose moiety
(Mazza, 1995; C.-L. Zhao et al., 2017). A recent study showed that Vv3ACT has novel activity
found in grapevine because it has a broad anthocyanin substrate specificity and is able to use
both aliphatic and aromatic acyl donors (Rinaldo et al., 2015).
20
FIGURE 4: Flavonoid biosynthesis pathway in grapevine. Enzymes that constitue the
pathway are shown, as well as the main intermediates and the transcription factors that regulate
the pathway (in light blue color). Blue boxes contain delphinidin-type anthocyanins and red
boxes cyaniding-type anthocyanins. CHS, chalcone synthase; CHI, chalcone isomerase; F3H,
flavanone 3-hydroxylase; F3’H, flavonoid 3’-hydroxylase; F3’5’H, flavonoid 3’5’-hydroxylase;
DFR, dihydroflavonol 4-reductase; LDOX, leucoanthocyanin dioxygenase; UFGT,
UDPglucose:flavonoid 3-O-glucosyltransferase; OMT, O-methyltransferase; FLS, flavonol
synthase; GST glutathione S-transferase; AM1/AM3, anthocyanin multidrug and toxic
extrusion transporters; ABCC1, ATP-binding cassette transporter. Adapted from (Kuhn et al.,
2014).
21
5. Transportation of anthocyanins to the vacuole
Anthocyanins are synthesized in the cytosol and stored into the vacuole (Chen, Wen, Kong, &
Pan, 2006). Three distinct, but potentially nonexclusive mechanisms for flavonoid transport in
plant cells have been proposed: vesicle trafficking, membrane transporter, and GST mediated
transport (J. Zhao, Pang, & Dixon, 2010). Recent evidence suggests that the three mechanisms
collaborate in vacuolar sequestration of flavonoids or flavonoid secretion through the plasma
membrane to the apoplast (J. Zhao et al., 2010).
Vesicular transport is mostly supported by microscopy observations (Gomez et al., 2009, 2011;
H. Zhang, Wang, Deroles, Bennett, & Davies, 2006). Concerning the membrane transporter
mechanism, molecular and genetic evidences support the involvement of both ATP Binding
Cassette (ABC) proteins and Multidrug and Toxic Extrusion (MATE) in the transport of
anthocyanins (Gomez et al., 2009; Goodman, Casati, & Walbot, 2004; J. Zhao et al., 2010).
MATE transporters include a large family of membrane transporters. The first gene identified
was Transparent Testa 12 (TT12) involved in flavonoid biosynthesis in Arabidopsis thaliana
seed testa (Debeaujon, Peeters, Léon-kloosterziel, & Koornneef, 2001) and was able to transport
cyanidin 3-O-glucoside, when expressed in heterologous form in yeast (Marinova et al., 2007).
The grapevine VvGST genes are also involved in anthocyanin transport (Conn, Curtis, Bézier,
Franco, & Zhang, 2008), and in VvGST-knockdown hairy roots, anthocyanins accumulated only
in small vesicles but did not make it to the vacuole. Also, hairy roots with knockdown of AM1
and AM3 had no anthocyanin-filled small vesicles (Gomez et al., 2011). This suggests that AM1
and AM3 proteins sequestrate anthocyanins from the cytoplasm to the small vesicles before
22
VvGST-aided transport to the central vacuole occurs via vesicle trafficking (Gomez et al.,
2011).
Genetic studies have shown that multidrug resistance-associated protein (MRP)/C-type of ABC
(ABCC) transporters are also involved in anthocyanin accumulation with the assumption that
they transport flavonoid conjugates with glutathione (GSH) (Banasiak, Biała, Staszków,
Swarcewicz, & Ewa, 2017; J. Zhao et al., 2010). In grapevine, it was shown that free GSH is
strictly required for transport of malvidin 3-O-glucoside into yeast vacuoles with ABCC1 (Goes
et al., 2005). Therefore, grapevine ABCC1 is a GSH-dependent anthocyanin transporter, rather
than an anthocyanin–GSH transporter, but the actual mechanism remains unclear.
6. Regulation of anthocyanin biosynthesis
6.1 Transcriptional regulation
Anthocyanin biosynthesis pathway is under the control of several different families of
regulatory genes. These correspond to transcription factor type of R2R3-MYB proteins, bHLH
(basic helix–loop-helix) proteins and WDR (tryptophan–aspartic acid repeat) proteins, which
also play crucial roles in the regulation of other flavonoid products, such as proanthocyanidins
and flavonols (He et al., 2010; Tian, Pang, & Dixon, 2008). FIGURE 4 from (Kuhn et al., 2014)
in addition to the structural genes of the pathway, also shows the transcription factors
characterized so far. These transcription factors could interact as ternary complexes MYB-
bHLH-WDR to regulate the expression of genes encoding enzymes involved in the final steps
of flavonoid biosynthetic pathway (Imène Hichri et al., 2011).
23
In grapevine, 108 members of R2R3-MYB gene subfamily have been classified and described
(José Tomás Matus, Aquea, & Arce-Johnson, 2008). MYB transcription factors are positive
regulators of the flavonoid pathway such as MYBPA1 for tannin production (J Bogs, Jaffe,
Takos, Walker, & Robinson, 2007); MYBF1 for flavonol production (Czemmel et al., 2009);
MYB5a and MYB5b in anthocyanin and proanthocyanidin production (Cavallini et al., 2017;
L. Deluc et al., 2008); and MYBA1 and MYBA2 for direct control of anthocyanin biosynthesis
(Kobayashi et al., 2004). These two genes are located in tandem in a single locus on
chromosome 2, which is called the “berry color locus” (Kobayashi et al., 2004; Walker, Lee, &
Robinson, 2006). MYBA1 transcriptionally regulates UFGT, which glycosylates
anthocyanidins, stabilizing them and therefore responsible for the anthocyanins accumulation
in grape berry skins of pigmented varieties (Ford et al., 1998). White skinned grape contains a
mutated allele of MYBA1 due to the presence of the retrotransposon Gret1 in its promoter
region, resulting in a white berry allele (Kobayashi et al., 2004). Pigmented cultivars possess at
least one allele of the MybA1 locus that does not contain Gret1 and is thus called a red berry
allele (This, Lacombe, Cadle-Davidson, & Owens, 2007). Also, a 33bp insertion was found in
the second intron of MYBA1, generating low gene expression levels in grape pink berry skin
varieties (Shimazaki, Fujita, Kobayashi, & Suzuki, 2011) and a deletion of a large region of
chromosome 2 including MYBA1 and MYBA2 genes produced the bud sports of Cabernet
Sauvignon called “Malian” with red-grey berry skin (Walker et al., 2006).
The others transcription factors WDR and bHLH have been characterized to a lesser extend in
grapevine. WDR1 promotes anthocyanin accumulation when ectopically expressed in
Arabidopsis thaliana (J. T. Matus et al., 2010). MYC1 (a bHLH transcription factor) is involved
in the anthocyanin and tannin biosynthesis regulation and trans-activates gene promoter of CHI,
24
of ANR (anthocyanidin reductase which catalyze the synthesis of flavan-3-ols such as catechin
and epicatechin) and of UFGT. MYC1 also interacts with different MYB partners to active
transcription in yeast (Imne Hichri et al., 2010)
Concerning negative regulators of anthocyanin biosynthesis, only a single MYB transcription
factor, MYB4, was identified and it could repress UFGT gene expression (José Tomás Matus et
al., 2009)
6.2 Environmental factors that can affect anthocyanin accumulation
In recent years, our knowledge about control of environment in berry ripening has greatly
increased. Several studies have shown that light exposure can increase anthocyanins
concentration, especially in skin cells, and that shading can have the opposite effect (Azuma &
Yakushiji, 2012).
In Cabernet Sauvignon it was shown that light exclusion or leaf shading significantly decreases
berry anthocyanin content and expression of genes associated directly with anthocyanin
biosynthesis such as MYBA1, MYBA2 and UFGT (Koyama & Goto-yamamoto, 2008; José
Tomás Matus et al., 2009). A recent study showed that several MYB transcription factors related
to flavonoid biosynthesis in grapevine respond differently to light and temperature conditions
(Greer & Weston, 2015; Jeong, Goto-yamamoto, Kobayashi, & Esaka, 2004). It was also
observed that gene expression of MYBA1-2, MYBA1-3 and MYBA2 was different in grapevine
berry skin in darkness or light at low (15ºC) or high (35ºC) temperature conditions, whereas
25
other studies concluded MYB5a and MYB5b did not respond to these environment stimuli
(Azuma & Yakushiji, 2012).
Water deficit (Deis, Cavagnaro, Bottini, Wuilloud, & Silva, 2011; L. G. Deluc et al., 2009) can
also affect anthocyanins content and other polyphenols like stilbenes and sugars, increasing their
concentrations, affecting final berry weight and up-regulating LDOX, UFGT and MYBA1
(Castellarin et al., 2007).
Also, light quality can affect anthocyanins biosynthesis, especially UV light. UV-B significantly
affects grape berry ripening, increasing anthocyanin and flavonols content and decreasing berry
weight (Berli et al., 2010; Berli, Fanzone, & Piccoli, 2011). Another environmental factor can
affect anthocyanin content is the infection by pathogens such as viruses (Singh, Singh, Swinny,
& Cameron, 2008; Vega, Gutierrez, Peña-Neira, Cramer, & Arce-Johnson, 2011).
Some of these environmental signals are also involved in active hormone contents regulation,
affecting berry ripening. For example, ABA levels increase in response to water deficit and low
temperatures exposure (L. G. Deluc et al., 2009; Yamane, Jeong, Goto-yamamoto, Koshita, &
Kobayashi, 2006).
6.3 Hormonal factors that can affect anthocyanin accumulation
Several studies have suggest abscissic acid (ABA) as one of the key signal triggering grape
berry ripening, due to the studies that show a significantly increase in ABA berry content at the
beginning of phase III (Lacampagne, Gagné, & Gény, 2010; Sun et al., 2010). In Cabernet
Sauvignon, a rapid increase in ABA content coincides with the rise in total anthocyanin content,
however, anthocyanins keep increasing during ripening stage, while ABA levels start to
26
decrease at the same time, suggesting that ABA only is necessary to trigger anthocyanin
biosynthesis (Wheeler, Loveys, Ford, & Davies, 2009). In addition, exogenous application of
ABA also increases anthocyanin content, berry weight and decrease acidity (Berli et al., 2010;
Gambetta, Matthews, Shaghasi, McElrone, & Castellarin, 2010; Wheeler et al., 2009). Increased
sugar concentrations in grape berry is associates with an increase in ABA content, but ABA
treatment does not affect sugar content (Peppi & Fidelibus, 2008). Regarding gene regulation
of ABA, it had been showed that expression of PAL, CHS, F3-H, GST, VvMYBA1, among others,
is increased in Cabernet Sauvignon skin treated with ABA at veraison stage (Koyama &
Sadamatsu, 2010). Another interesting fact is that ABA treatments repress putative auxin
response genes and at the same time increase the expression of genes related to ethylene
biosynthesis, suggesting ABA effects are mediated in conjunction with other hormones.
Auxin level, which is high in the green phase of rapid berry growth, strongly decreases levels
during veraison and ripening stages (Böttcher, Keyzers, Boss, & Davies, 2010). It had been
reported that grape berries treated with naphthalene acetic acid (NAA) 1 week before veraison
shows a delay in anthocyanins and sugar accumulation, parallel to changes in transcripts
involved in up-regulation of genes related to ethylene biosynthesis and down-regulation of
genes related to ABA biosynthesis (Ziliotto et al., 2012).
When berries are treated at veraison stage with an ethylene-releasing compound (2-CEPA), an
increase occurs in anthocyanin content (El-Kereamy et al., 2003), and on the other hand, when
an inhibitor of ethylene receptors is used (1-MCP) produce an decreased in anthocyanin content
accompanied by an increase in acidity in grape berries (Chervin et al., 2004). 1-MPC use also
suggest a relationship of ethylene with ABA, because its use produces a decrease in ABA
content in grape berry (Sun et al., 2010)
27
It had been showed that cytokinins increase berry weight but decrease sugar and anthocyanin
content (Peppi & Fidelibus, 2008) Finally, jasmonic acid increases anthocyanin production in
grape cell suspensions (Belhadj et al., 2008).
7. New advances in comparative studies between contrasted berry skin
varieties
In the last decade, several studies demonstrated that transcriptomic remodeling drives the
transition from the vegetative to the ripening stage, a process which is highly coordinated at the
transcriptional level (Fasoli et al., 2012). There has been advances in deep-sequencing
technologies that, integrated with the availability of grapevine genomic and transcriptomic data,
allow to carry out RNA-transcript expression analysis on a global scale at key points during
grapevine development (Serrano et al., 2017).
Many transcriptomic studies focused on berry development on a specific variety. A study in
Cabernet Sauvignon using Affymetrix technique, had reported that approximately 60% of the
detected transcripts correspond to differentially expressed genes between at least two out of the
seven stages of berry development, demonstrating the dynamic nature of the developmental
process in grapevine. These genes were related with many development processes in which 21
Unigenes encoding biosynthetic enzymes of the general phenylpropanoid and flavonoid
pathways were found to exhibit differential mRNA expression patterns across berry
development (L. G. Deluc et al., 2007). Studies in other pigmented varieties such as Shiraz also
showed that gene clusters for ripe berry contain genes related with phenylpropanoid metabolism
and secondary metabolism (Sweetman, Wong, Ford, & Drew, 2012). Another similar study but,
28
using a white-skinned berry variety called Garganega, realized its metabolomic and
transcriptomic characterization. It was shown that Garganega was highly plastic in different
environments and indeed more plastic than ripening Corvina berries, predominantly concerning
phenolic compounds accumulation (Dal Santo et al., 2016).
In relation with comparative studies between varieties, there are several examples that compare
varieties with the same berry skin color. Using berry skin of Norton variety, an important North
American wine grape, compared with Cabernet Sauvignon, Ali et al. 2011, showed an up-
regulation of flavonoid metabolism. Higher transcript levels in F3’-H, F3’5’-H, LDOX, UFGT,
among others, were reported in Norton variety compared to Cabernet Sauvignon (Ali et al.,
2011). Another study in the table grape ‘Kyoho’ variety versus its bud mutant ‘Fengzao’ indicate
that the mutant reached ripening stage 30 days earlier than ‘Kyoho’ (Guo et al., 2016). Another
comparative transcriptomic study concluded that differentially expressed genes may suggest
that gene activities related to ROS and pathogenesis are involved in contributing to the
phenotype observed, due to flavonoid metabolism differences (Guo et al., 2016). In addition,
light-red-skinned ‘Ruby Okuyama’ and more intense and uniform rosy-skinned ‘Benitaka’
correspond to bud sports of white-skinned ‘Italia’ (Azuma et al., 2009). ‘Ruby Okuyama’ was
caused by the recovery of VvmybA1 expression and ‘Benitaka’ due to presence of a novel
VvmybA1BEN allele that restored VvmybA1 transcripts (Azuma et al., 2009). Much less
information exists about studies that take into account the full range of berry skin color shades
that is present in V. vinifera cultivars. That is why we were interested in contributing to fill this
gap by considering more types of color berry skin, in near isogenic backgrounds.
29
We have two grapes that present contrasted differences in berry skin. One of them is Red Globe
(RG), a table grape whose berries are characterized by be seeded, of a large size, thick and
consistent skin of dark purple color. And the second one corresponds to Chimenti Globe (CG)
that has similar characteristics but differs in color skin that is bright light red. It originates from
a spontaneous mutation event that occurred in the vineyard from a branch of a vine of RG plant
in Talagante, Metropolitan Region of Chile, and was subsequent propagated through clonal
multiplication (FIGURE 5). As a first approximation, we asked if the color change was due to
a change in the key regulator known in grapevines, MYBA1, which it has been associated as
responsible for the majority color of berry skin found in grapes. We think that something similar
to what was observed in the work of Walker et al., 2006 with the variety Cabernet Sauvignon
and his bud sport was happening. We performed an analysis gene expression of the white and
red allele of MYBA1 by RT-PCR. We found surprisingly that both varieties, CG and RG, are
heterozygous for color berry locus since they express both alleles at the same time (FIGURE
6). With this observation, we discard MYBA1 like a possible explanation and not directly related
with the color change observed in this new variety. We are wondering about the relationship in
specific berry color and the type of anthocyanins that could have, which led us directly to think
of some change or modification in the so-called branching point in anthocyanin biosynthesis,
shaped by the enzymes F3’5’-H and F3’-H. Anyway, we do not have the certainty to which level
of anthocyanin biosynthesis was affected. For that reason, we consider CG and RG varieties an
interesting study model due to the genetic background responsible for the color change is the
almost identical (i.e. near isogenic), with differences affecting the color of the berry, therefore
allowing us to study the process of accumulation of anthocyanins using two near-isogenic
varieties with contrasting berry skin color.
30
FIGURE 5: Chimenti Globe and Red Globe table grapes. In the picture is show varieties
selected for this thesis as model of study. CG corresponds to the one on the left of the picture
and RG on the right. This picture was taken in ripening stage on season 2013. Both varieties are
located in Talagante, Metropolitan Region of Chile
FIGURE 6: Analysis of MybA1 gene structure in CG and RG. (A) PCR analysis of white
allele in CG, RG varieties and control Merlot (Me), Cabernet Sauvignon (CS), Chardonnay (Ch)
and Sauvignon Blanc (SB). (B) PCR analysis of red allele in CG and RG varieties and control
described above. (C) Integrity control of actin gene. Numbers on the left indicate the positions
of the molecular size marker 1 Kb Plus DNA Ladder and on the right the exact size of white and
red allele.
31
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47
CHAPTER II
PROBLEM STATMENT: Hypothesis and Objectives
48
According to all the previously mentioned in the General Introduction, I propose the following
hypothesis of my thesis:
Color differences in berry skin between Chimenti Globe and Red Globe are due to
metabolic and transcriptomic changes related with the branching point F3’-H and F3’5’-
H of the anthocyanin biosynthesis pathway
The main objective of this thesis was:
To determine transcriptomic and metabolic changes associated with the branching point F3’-H
and F3’5’-H of the anthocyanin biosynthesis pathway, responsible for the color differences in
berry skin between Chimenti Globe (CG) and Red Globe (RG).
The general objective was divided into the following partial objectives:
1. Characterize metabolic changes related to differences in anthocyanin accumulation
between CG and RG.
2. Analyze transcriptomic changes involved in differences in anthocyanin accumulation
between CG and RG.
3. Evaluate the function of at least one gene of interest, selected from the above objectives,
related with the branching point F3’-H and F3’5’-H of the anthocyanin biosynthesis
pathway.
The objectives 1 and 2 have been developed in the Chapter 3 and objective 3 in Chapter 4.
49
CHAPTER III
TRANSCRIPTOMIC AND METABOLIC ANALYSIS REVEALS NEW
GENETIC DIFFERENCES BETWEEN TWO TABLE GRAPES WITH CONTRASTED
COLOR BERRY SKIN
This article will be submitted to Frontiers in Plant Science
50
TITLE:
TRANSCRIPTOMIC AND METABOLIC ANALYSIS REVEAL NEW
GENETIC DIFFERENCES BETWEEN TWO TABLE GRAPES WITH CONTRASTED
COLOR BERRY SKIN
Claudia Santibáñez1,2, Carlos Meyer1, Litsy Martínez1, Tomás Moyano3, John Lunn4, Regina
Feil4, Zhanwu Dai2, Ghislaine Hilbert2, Christel Renaud2, Serge Delrot2, Patricio Arce1* and
Eric Gomès2*
1 Laboratorio de Biología Molecular y Biotecnología Vegetal, Departamento de Genética
Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica
de Chile, Santiago 8331150, Chile.
2 Université de Bordeaux, ISVV, INRA, UMR 1287 Ecophysiology and Functional Genomic of
Grapevine, Villenave d’Ornon, France.
3 FONDAP Center for Genome Regulation, Millennium Nucleus Center for Plant Systems and
Synthetic Biology, Departamento de Genética Molecular y Microbiologia, Facultad de Ciencias
Biológicas, Ponticia Universidad Católica de Chile, Santiago 8331150, Chile
4 Max Plant Institute of Molecular Plant Physiology, Wissenschaftspark Golm, Am Mühlenberg
1, 14476 Potsdam (OT) Golm, Germany.
*Correspondence: [email protected] , [email protected]
51
ABSTRACT
Anthocyanins are pigments responsible of grape berry skin coloration and its biosynthesis
regulation has been widely studied. However, few studies focus on metabolic and genetic
characterization of such metabolism using varieties of the same genetic background with
contrasting berry skins color. Using metabolic profiling and RNA-seq technology, we
performed a comparative characterization of two table grapes, Chimenti Globe (CG) and Red
Globe (RG), with contrasting color berry skin: CG has a bright light red color and RG has a
purple color. The originality of this model is that CG was generated from a spontaneous
mutation event in field from a branch in a vine of RG plant, allowing us to study anthocyanin
metabolism regulation in near isogenic backgrounds. Berry metabolic content analysis showed
the importance of developmental stages, veraison and ripening, in both varieties of the study.
Striking differences in metabolite concentrations of the phenylpropanoid pathway, such as
shikimate, phenylalanine and other molecules such as UDP-glucose and trehalose-6-phosphate,
among others, the latter being a signalling molecule with major implications in plant growth,
development, and metabolism regulation. Also, in contrast to RG, CG only contained
dihydroxylated anthocyanins, peonidin and cyanidin, and not the trihydroxylated ones,
malvidin, delphinidin and petunidin, which was consistent with the observed color phenotype.
From transcriptomic analysis using Illumina Hiseq200, we generated a heat map with 109 genes
differentially expressed in CG in comparison to RG, many of these being related to the flavonoid
pathway. In addition, 11 gene copies of flavonoid 3’5’-hydroxylase, a key enzyme for the
biosynthesis of trihydroxylated anthocyanins, were repressed in CG. These results, provide new
52
insights in anthocyanins biosynthesis regulation in grape, offering new clues in the search for
molecular regulators of the flavonoid pathway.
Keywords: RNA-seq, skin, anthocyanin, grape, Red Globe, Chimenti Globe.
53
INTRODUCTION
Grapevine (Vitis vinifera L.) is the most important non-climacteric fruit crop worldwide, used
for fresh consumption as table grapes, dried into raisins and processed into wine and liquors.
Berry development is a process displaying a double sigmoid curve with two growth phases
separated by a lag phase. In the first phase a fruit growth is observed, during which organic
acids, tannins, hydroxycinnamates and several phenolic compound precursors are synthesized
and the number of fruit cells is defined (Adams, 2006). Then, there is a lag phase where there is
no significant growth of the fruit. At the end of this phase a phenomenon called veraison take
place, where the fruit initiates its ripening and begins to take color (COOMBE, 1995). In the
third phase, a second growth of the berry occurs, organic acids levels decrease (mostly due to
malate breakdown), sugars and secondary metabolites (aroma, phenolics) are synthetized and
accumulated, to eventually end up with a fruit exhibiting desirable quality traits (Conde et al.,
2007).
Anthocyanins are secondary metabolites and one of the main pigments present in red grape berry
skin (Souquet, Cheynier, Brossaud, & Moutounet, 1996), which are associated to organoleptic
properties in red wine such as color, bitterness, astringency, and are also antioxidants with
beneficial effects on human health (Dixon, Xie, & Sharma, 2005). The biosynthesis of
anthocyanins occurs via the flavonoid pathway at the cytosolic surface of the endoplasmic
reticulum, in grape cells (P. K. Boss, Davies, & Robinson, 1996). Most of the structural genes
encoding the enzymes of the anthocyanin biosynthesis pathway have been identified and
characterized (Kuhn et al., 2014). The regulation of these genes is achieved by ternary
54
complexes, formed by transcription factors from the R2R3-MYB (L. Deluc et al., 2006, 2008;
Kobayashi, Goto-Yamamoto, & Hirochika, 2004; This, Lacombe, Cadle-Davidson, & Owens,
2007) and bHLH (basic helix–loop–helix) families and WDR (tryptophan–aspartic acid repeat)
proteins (Hichri et al., 2010; Matus et al., 2010).
In the last decade, several studies demonstrated that transcriptomic remodeling drives the
transition from the vegetative to the ripening stage, a process which is highly coordinated at the
transcriptional level (Fasoli et al., 2012). There has been advances in deep-sequencing
technologies that, integrated with the availability of grapevine genomic and transcriptomic data,
allow to carry out RNA-transcript expression analysis on a global scale at key points during
grapevine development (Serrano et al., 2017). However, most of these studies focus on
characterization of one single cultivar such as Cabernet Sauvignon (L. G. Deluc et al., 2007);
comparison between red cultivars from contrasted genotypes (Guo et al., 2016; Sweetman,
Wong, Ford, & Drew, 2012); or between red and white cultivars (Yakushiji et al., 2006). They
do not, however, take into account the full range of berry color shades that is present in V.
vinifera cultivars, nor do they consider the comparison or closely related, near-isogenic
cultivars.
Here, we performed a metabolic and transcriptomic characterization of berry development and
ripening using as a model two closely related varieties with different berry color skin. The first
one corresponds to Red Globe (RG) variety, which is a table grape whose berries are of a large
size, thick and consistent skin of dark purple color. The second one, Chimenti Globe (CG), has
similar characteristics but differs in color skin that is bright light red. It originates from a
55
spontaneous mutation event in field from a branch in a vine of RG plant in Talagante,
Metropolitan Region of Chile that was propagated through clonal multiplication (FIGURE 1C).
The interest of this model is the genetic background responsible for the color change is the
almost identical, with differences affecting the color of the berry therefore allowing us to study
the process of accumulation of anthocyanins using two near-isogenic varieties with contrasting
berry skin color.
56
MATERIALS AND METHODS
Plant material and experimental design
Plant material used in this study corresponds to six plants of commercial table grape cv. ‘Red
Globe’ (RG) and six plants of cv. ‘Chimenti Globe’ (CG), located in Camino Loreto, Parcela
8-9, Talagante, Región Metropolitana, Santiago of Chile. All plants were in the same plot, one
plant next to the other (FIGURE 1). Sampling were performed during seasons 2013 and 2014.
Veraison stage was classically determined as the time at which clusters were 30–50% colored,
and ripening stage when berry sugar content reached 22-23°Brix (22-23% w/w soluble solids).
A random cluster for each sampling stage representing biological unit was selected for each
plant in both cultivars, stage and season. Berries were immediately peeled and frozen in liquid
nitrogen, and stored at –80°C until required for further analysis.
Metabolites extraction and analysis
For metabolite analysis, berry skin from 2013 and 2014 season were used and each sample was
analyzed separately, except for anthocyanin analysis where were used only five samples for
each variety.
Anthocyanins were extracted from 300 mg freeze-dried ground powder from skin of RG and
CG using 1 ml methanol containing 0.1% HCl (v/v). Extracts were filtered across 0.45 μm
polypropylene syringe filter (Pall Gelman Corp., Ann Harbor, MI, USA) for HPLC analysis.
Each individual anthocyanin was analyzed as described in (Dai et al., 2013) using HPLC. For
anthocyanin quantification was integrated peak area at 520 nm and using Malvidin 3-glucoside
as standard (Extrasynthese, Lyon, France).
57
Primary metabolites such as Tre6P, glycolytic and TCA cycle intermediates were extracted from
25 mg of frozen powder with chloroform/methanol solution, and measured by high-performance
anion-exchange liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) as
described by (John E. Lunn et al., 2006). Amino-acids were extracted and analyzed by high
performance liquid chromatography as described by (Martínez-Lüscher et al., 2014).
For sugars and organic acids, an aliquot of 150-200 mg of skin fine powder was extracted
sequentially with ethanol 80% and 50%, dried in a SpeedVac and re-dissolved in 1 ml of sterile
ultrapure water. Hexose content (glucose and fructose) were measured enzymatically with an
automated micro-plate reader (Elx800UV, Biotek Instruments Inc., Winooski, VT, USA)
according to (Gomez, Bancel, Rubio, & Vercambre, 2007). Tartaric acid content was measured
by using the colorimetric method based on ammonium vanadate reactions (Pereira et al., 2006).
Malic acid content was determined using an enzyme-coupled spectrophotometric method that
measures the change in absorbance at 340 nm from the reduction of NAD+ to NADH (Pereira
et al., 2006).
RNA extraction
Total RNA were isolated from berry skins according to the procedure of (Reid, Olsson,
Schlosser, Peng, & Lund, 2006), using a CTAB-spermidine extraction buffer. This material was
used for transcriptome sequencing by RNA-seq technology and validation of data by
quantitative real-time RT-PCR (qRT-PCR). For RNA sequencing, only berry skin from 2013
season was used and to carry out this analysis was necessary to reduce all material into 3 pools
per cultivar (CG and RG) and stage (veraison and ripening), reaching a total number of 12
58
samples for this study. Each pool was made by mixing 2 individual RNA extractions (1 µg each
one) and then mixing them to obtain a concentration of 2 µg per pool (TABLE S1).
Total RNA were then sent to Macrogen (Macrogen Inc. Seoul, Korea) according to their delivery
protocol consisting in ethanol precipitation condition. Briefly, it was added 0.1 volume of 3 M
Sodium Acetate pH 7-8 to total RNA solution, and mixed gently; then it was added 2 volumes
of 100% ethanol, leaving the samples reading to be sent.
RNA Sequencing and Reads Mapping
mRNA were isolated from 10 μg of total RNA, fragmented, converted to cDNA, and amplified
by PCR to Illumina ® TruSeqTM RNA Sample Preparation Kit (Illumina, Inc., USA). Pair-end
100bp sequence reads were generated using the Illumina Genome Analyzer II (Illumina) and
Illumina HiSeq 2000 (Illumina) at Macrogen Inc. (https://dna.macrogen.com/) according to the
manufacturer’s recommendations. Trimmomatic (Bolger, Lohse, & Usadel, 2014) was used to
clean the reads prior to mapping and to remove the Illumina adapter sequences as well as the
low-quality sequences from the ends of the reads. All the distinct clean reads were aligned to
the Grape Genome Database hosted at CRIBI V2 (http://genomes.cribi.unipd.it/grape/) (Vitulo
et al., 2014). Uniquely mapped reads were counted by HITSAT2 software (Kim, Langmead, &
Salzberg, 2015) and featureCounts in the Rsubread package (Liao, Smyth, & Shi, 2013).
Screening of DEGs
Analyses for discovering differentially expressed genes (DEGs) were performed with DEseq2
using false discovery rate (FDR) with a threshold of 0.05 and absolute value log2 ratio ≥ 1 (Love,
Huber, & Anders, 2014). MultiExperiment Viewer (MeV) was used for gene clustering analysis
59
that was performed by the k-means method with Pearson’s correlation distance. MeV also was
used for graphical representation of DEGs and it was performed a heatmap using Fold Change
≥ 1 (Howe, Sinha, Schlauch, & Quackenbush, 2011).
PANTHER™ Version 12.0 program (Mi et al., 2017) with default parameters was used to
perform the GO functional enrichment analysis with the Bonferroni correction for multiple
testing (P ≤ 0.05). Corrected P-values < 0.05 were used to obtain significant GO enrichment.
Validation of RNA-seq data by quantitative real-time RT-PCR (qRT-PCR)
Two ug of total RNA were treated with TURBO DNA-freeTM DNase (Ambion®) and were
reverse transcribed with random primers using SuperScript II RT (InvitrogenTM Co., Carlsbad,
CA, USA), according to the manufacturer’s instructions. Relative transcript quantification of
DEGs was performed by real-time RT-PCR (qRT-PCR) using the BRILLIANT II SYBR®
GREEN QPCR Master Mix and the Mx3000P qPCR system (Stratagene, Agilent Technologies
Inc., Santa Clara, CA, USA) according to the manufacturer’s protocol. Expression levels of all
evaluated genes were calculated from six biological replicates, relative to Vv60SRP
housekeeping control gene. We used the 2-∆∆Ct method for the statistical analysis (Schefe,
Lehmann, Buschmann, Unger, & Funke-Kaiser, 2006).
Pimers used in qRT-PCR for DEGs correspond to F3’5’H multicopies (IDs:
VIT_206s0009g02805, VIT_206s0009g02810, VIT_206s0009g02840, VIT_206s0009g02860,
VIT_206s0009g02873, VIT_206s0009g02880, VIT_206s0009g02920, VIT_206s0009g02970
and VIT_206s0009g03010): F5’-TGGATAAAGTGATTGGAAGGA-3’ and R5’-
GGATGGGTGCTTCCGGAAGCTT-3’; CYTB5 (ID: VIT_218s0001g09400): F5’-
GGCTCCCCAAAAAGTTTTCA-3’ and R5’-TGCCATGAATGACGAACCAG-3’;
UFGT2230 (ID: VIT_216s0039g02230): F5’-GCTTTGATCAAATTCTCTCACA-3’ and R5’-
60
ACACTAAATCCACCAGGGTTTT-3’; CCOAMT (IDs: VIT_201s0010g03490 and
VIT_201s0010g03510): F5’-TTGATCCAGATAAAGAAGCGTA-3’ and R5’-
CGGCAATGAGATCATTTAGAAC-3’; ANP-like (IDs: VIT_216s0050g00900 and
VIT_216s0050g00910): F5’-ATTCGGGTCTCCAATGAGCT-3’ and R5’-
GTGAGAAACAGCCTCTTGCA-3’; GST13 (ID: VIT_207s0104g01800): F5’-
AATTCCCATGTCCACATGCACC-3’ and R5’-GGGATTCTTGGCAAGAAAAGGA -3’;
F3H (ID: VIT_204s0023g03370): F5’-AGGCAACCGTGTATCCTCTG-3’ and R5’-
TTCTCCACGTCTTGCAACTG-3’; CHS (ID: VIT_205s0136g00260): F5’-
CCCATGTTCGAATTGGTTTC-3’ and R5’- TGTGCAATCCAGAAAATGGA-3’; MYB24
(ID: VIT_214s0066g01090): F5’-ACCCTGCAATTCTCAGGATG-3’ and R5’-
CAGGCCTCAGGTAGTTCAGC-3’; COMT-like (IDs: VIT_215s0048g02480.1,
VIT_215s0048g02490.1 and VIT_215s0048g02490.2): F5’-
TACCCTGGCAAGGACCCCAGAT-3’ and R5’-AGGTGCTCAAAACCCTTGTAGG-3’;
VvABCTRANSPORTER (ID: VIT_214s0060g00720): F5’-AATGTGAAGAGCGAGGTGCT-
3’ and R5’-CGACTGCCTTTCGTTCTTTC-3’; VvSRP60 (ID: VIT_05s0077g02060): F5’-
ATCTACCTCAAGCTCCTAGTC-3’ and 5’-CAATCTTGTCCTCCTTTCCT-3’. Also were
analyzed 6 gene copies of F3’5’H: VIT_206s0009g02810, F5’-
AGCATGGTGGTGGCCTCAACTC-3’ and R5’-TAGCTTTCTCAGTAGCTTCCAC-3’;
VIT_206s0009g02830, F5’- GACCGGTTATTAACAAAGATGA-3’ and R5’-
TGTCTGTTCCAGGAGGAGTGCT-3’; VIT_206s0009g02860, F5’-
CACTAAGCCATGGCCATAGATA-3’ and R5’- GCAACATGAGGCATGTTACCTA-3’;
VIT_206s0009g02880, F5’-AGTCAAATGAGTTCAAGGACATGG-3’ and R5’-
AGGAGCACACGGCGTCAGCCCA-3’; VIT_206s0009g02920, F5’-
61
TTTGGATCCTTCAAATATTC-3’ and R5’-GAAAAGATGGAGATGGCTCT-3’;
VIT_206s0009g03010, F5’-TAACGGTATAAGATGTATTAGC-3’and R5’-
GAAAAATAGAAACTATAACCAA-3’.
Statistical data analysis
Data were analyzed with multivariate analysis methods using the R statistics environment (R
Core Team, 2010). In order to evaluate alterations in metabolite levels during stages of
grapevine development, principal component analysis (PCA) was performed on mean-centered
and scaled data using the ade4 package in R (Dray & Dufour, 2007). Differences between
development stages and seasons were analyzed by a two-way ANOVA followed with a Tukey
multiple comparison post-hoc test at P < 0.05, for example, in the case of PCA analysis,
component 1. For differences between varieties were analyzed using unpaired T-test, for
example in the case of PCA analysis, component 2 and qRT-PCR analysis. GraphPad Prism
version 6.00 for Windows (GraphPad Software, La Jolla California USA, www.graphpad.com)
was used for graphics representation and analysis. For RNA-seq analysis, Trimmomatic (Bolger
et al., 2014), HISAT2 (Kim et al., 2015) and Rsubread package (Liao et al., 2013) were used
with default parameters. DEGs analysis were performed with DEseq2 using FDR with a
threshold of 0.05 and absolute value log2 ratio ≥ 1 (Love et al., 2014). (MeV) was used for
cluster analysis by the k-means method with Pearson’s correlation distance and graphical
representation of DEGs with a heatmap using FC ≥ 1 (Howe et al., 2011). PANTHER™ Version
12.0 program (Mi et al., 2017) with default parameters was used to perform the GO analysis
with the Bonferroni correction for multiple testing (P ≤ 0.05). We used corrected P-values <
0.05 to obtain a significant enrichment of the genes.
62
RESULTS
Metabolic characterization of Chimenti Globe and Red Globe
Primary metabolite profiling of RG and CG
Principal component analysis (PCA) was conducted to obtain a comprehensive view of
distribution of metabolites during veraison and ripening stages, between both cultivars on study,
CG and RG, in two seasons evaluated, 2013 and 2014. The first two principal components (PC1
and PC2) explained about 64% of the total variance and allowed to discriminate developmental
stages between CG and RG (FIGURE 2A). PC1 (43,75%) separated developmental stages,
veraison and ripening in both seasons evaluated. This discrimination was mainly determined by
the concentrations of metabolites related with phenylpropanoid metabolism such as shikimate,
UDP-glucose and phenylalanine. In addition, it was observed that the molecular regulator
trehalose-6-phosphate and TCA cycle intermediates such as citrate, isocitrate and other several
amino-acids also contributed to separated veraison from ripening (FIGURE 2B and FIGURE
S1). PC2 (20,26%) resolved cultivar type, CG and RG, even in 2013 and 2014 years, but this
discrimination only was observed at the ripening stage. These results were explained in changes
observed in metabolism related with biosynthesis of sucrose: glyceraldehyde 3-phosphate,
fructose 6-phosphate, glucose 6-phosphate, fructose 1,6-biophosphate, glucose 1,6-
biophosphate, glycerol 3-phosphate, fructose, glucose and phosphoenolpyruvate; and also, by
anthocyanins compounds (FIGURE 2B and FIGURE S2).
63
Anthocyanin contents of RG and CG berry skins
We analyzed total anthocyanin levels present in berries skins from CG and RG cultivars during
same developmental stages and seasons studied before. During veraison stage, CG showed
almost null concentration of any types of anthocyanins, and in ripening we observed significant
differences in total anthocyanin concentration between CG and RG. RG contains about 7 times
more of total anthocyanins than CG, in samples from both seasons (FIGURE 3A and 3C,
respectively). For example, in ripening stage 2013, RG had 14.39 mg.g-1 DW and CG 1.93
mg.g-1 DW of total of anthocyanin content (TABLE 1). Detailed analysis of individual
anthocyanins revealed that CG mainly contains only two types of anthocyanins: peonidin (in the
form of peonidin 3-glucoside and peonidin 3-(p-coumaryl)-glucoside) and cyanidin (in the form
of cyanidin 3-glucoside and cyanidin 3-(p-coumaryl)-glucoside) which confers the most reddish
tonality to grapevines berries (FIGURE 3B and 3D). The others 3 types of anthocyanins were
present in minute amounts (or even no detectable at all) at both developmental stages and for
both seasons analyzed. These corresponded to malvidin (in the form of malvidin 3-glucoside),
petunidin (in the form of petunidin 3-glucoside) and delphinidin (in the form of delphinidin 3-
glucoside and delphinidin 3-(acetyl)-glucoside), which are bluish anthocyanins (TABLE 1).
Conversely, in RG we observed all 5 types of anthocyanins mentioned above, in higher
concentrations than in the CG cultivar, both at veraison and ripening stages in 2013 and 2014
samples (FIGURE 3B and 3D, respectively).
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Transcriptomic characterization between Chimenti Globe and Red Globe
RNA-Seq Data mapping and alignment to Grapevine Genome Database
Based on the model study used in this work, we proposed that the gene expression program
responsible for changes in berry skin color for CG could have been differentially altered
compared to RG. To investigate this hypothesis, we performed a genome-wide transcriptome
comparison using Illumina Hiseq2000, and NGS technology. Twelve RNA samples were
analyzed corresponding to pools of biological replicates of CG and RG, from veraison and
ripening stages during 2013 season (TABLE S1). From these samples, we obtained an average
of ~4.8 – 8.1 million total sequencing reads pairs, for each sample (TABLE S3). These total
reads were cleaned from adapters and removed from low-quality bases from the ends of the
reads using Trimmomatic (Bolger et al., 2014). and were aligned to Grapevine Reference
Genome hosted at CRIBI V2 (http://genomes.cribi.unipd.it/grape/) (Vitulo et al., 2014) using
HISAT2 (Kim et al., 2015) and Rsubread package (Liao et al., 2013) (TABLE S3).
Approximately ~3.3 – 4.9 million reads aligned 1 time onto the genome, ~0.5 – 0.8 million reads
aligned 0 times and ~0.7 – 1.1 million reads aligned more than 1 time (72%, 11-20% and 15-
17% of the total reads after quality analysis, respectively) (TABLE S2). The reads that aligned
1 time to genome were used for following analysis.
Chimenti Globe exhibited altered flavonoid metabolism compared to Red Globe
To further explore the expression profiles of genes between CG and RG cultivars, all clean reads
were aligned to the grape genome database V2 (Vitulo et al., 2014) and then a comparative
transcriptomic analysis was performed to screen for DEGs using the DEseq2 program (Love et
65
al., 2014). As shown in FIGURE 4, a heat map was generated where a total of 109 genes were
differentially expressed in CG in comparison to RG, during veraison and ripening stages, in the
2013 samples. At veraison a total of 64 genes were down-regulated and 45 genes were up-
regulated in CG cultivar; and at ripening stage, 66 genes were down-regulated and 43 genes
were up-regulated in CG cultivar, compared to RG.
Many of the 109 DEGs were related to flavonoid metabolism and were categorized into six
clusters: cluster A (1 gene), cluster B (23 genes) and cluster C (31 genes) that were not induced
in CG at both developmental stages. Flavonoid metabolism genes such as flavonoid 3,5-
hydroxylases (VIT_206s0009g02805-2810-2830-2840-2846-2860-2873-2880-2970-2920-
3010) flavanone 3-hydroxylase (VIT_204s0023g03370) glutathione transferases
(VIT_204s0079g00690-710), chalcone synthase (VIT_205s0136g00260), cytochrome b5
(VIT_218s0001g09400), caffeoyl-CoA O-methyltransferase (VIT_201s0010g03490-3510) and
UDP-glucose:flavonoid 3-O-glucosyltransferase (VIT_216s0039g02230) fall in that category
(TABLE 2). Cluster E (34 genes) encompass genes that were induced at both developmental
stages in CG such as cytochrome p450 (VIT_201s0137g00540), catechol-O-methyltransferases
(VIT_215s0048g02480-2490), MYB24 (VIT_214s0066g01090) and ABC TRANSPORTER
(VIT_214s0060g00720) (TABLE 3). Finally, Cluster D (11 genes) and cluster F (9 genes)
combined genes that were induced and not induced at veraison and ripening, such as for example
stilbene synthases (VIT_210s0042g00840-880-920) (TABLE 4).
66
GO Analysis of DEGs
We performed a Gene Ontology (GO) enrichment analysis for enlight functional categories
represented in the DEGs. In order to be able to use the available PHANTER Classification
System tool (Mi et al., 2017), we had to convert the gene ID references of 109 DEGs to the V1
version of the grapevine reference genome (http://genomes.cribi.unipd.it/DATA/V1/), which
caused 7 genes to be removed for the GO analysis for they were absent from this older
annotation of the grape genome. Based on these 102 DEGs, 46 DEGs were found to be enriched
for the term “molecular function” (FIGURE 5A): the DEGs were enriched in the categories
“binding” (GO:0005488, 7 genes), “signal transducer activity” (GO:0004871, 2 genes),
“catalytic activity” (GO:0003824, 30 genes) and “transporter activity” (GO:0005215, 7 genes).
A total of 59 genes were enriched into the term “biological process” (FIGURE 5B). The DEGs
were enriched in the categories “response to stimulus” (GO:0050896, 7 genes), “developmental
process” (GO:0032502, 1 gene), “cellular process” (GO:0009987, 17 genes), “transporter
activity” (GO:0005215, 7 genes), “metabolic process” (GO:0008152, 26 genes), “biological
regulation” (GO:0065007, 4 genes), “cellular component organization or biogenesis”
(GO:0071840, gene) and “localization” (GO:0051179, 3 genes). Finally, 29 genes were enriched
into the term “cellular component” (FIGURE 5C): the DEGs were enriched in the categories
“cell junction” (GO:0030054, 1 gene), “membrane” (GO:0016020, gene), “macromolecular
complex” (GO:0032991, gene, “cell part” (GO:0044464, 15 genes) and “organelle”
(GO:0043226, 11 genes).
67
Confirmation of the RNA-Seq Results
To validate the RNA-seq results, 12 of DEGs were selected and qRT-PCR analysis was
performed (FIGURE 6). From clusters of not induced genes (clusters A, B and C) 9 genes were
selected: F3’5’-H (Flavonoid 3’5’-hydroxylase) whose expression signal corresponds to 9
copies of this gene, CYTB5 (Cytochrome b5), UFGT (UDP-glucose:flavonoid 3-O-
glucosyltransferase), CCOAOMT (caffeoyl-CoA O-methyltransferase), ANP-like (anthocyanin
permease like), GST13 (glutathione S-transferase 13), F3H (Flavanone 3-hydroxylase), CHS
(Chalcone synthase). Seven of these genes (F3’5’-H, CYTB5, UFGT, CCOAOMT, GST13, F3H
and CHS) showed significant differences between CG and RG at both veraison and ripening
stages, fully supporting the RNA-seq results; ANP-like gene only showed differences during
ripening stage.
To gain more insight in gene expression 9 copies of F3’5’-H we designed specific primers for
6 of them: 2810, 2830, 2860, 2880, 2920 and 3010. In general, we observed significant
differences in each gene copies evaluated, in which there was not induction in CG compared to
RG, in veraison and ripening stages (FIGURE S4); also, this observation was correlated with
specific reads extracted from RNA-seq results for each gene copy of F3’5-H (TABLE S5).
From cluster of induced genes (cluster E) 3 genes were selected: MYB24, ABC transporter,
COMT-like (Catechol-O-methyltransferase) (FIGURE 6). It was observed that MYB24 was
significantly induced in veraison and ripening stages in CG and ABC transporter and COMT-
like genes were induced in CG only during veraison. Finally, the STS gene (Stilbene synthase)
was selected from combined clusters (cluster D and F). It exhibits a higher expression at ripening
and a lower one at veraison in CG compared to RG, but not with significant differences.
68
Eleven copies of F3’5’-H genes that were closely located in the same physical region of
chromosome 6 were repressed in Chimenti Globe
We observed that within the list of genes belonging to the repression cluster there were 11 copies
of the gene F3’5’-H repressed in CG: VIT_206s0009g02805, VIT_206s0009g02810,
VIT_206s0009g02840, VIT_206s0009g02860, VIT_206s0009g02873, VIT_206s0009g02880,
VIT_206s0009g02920, VIT_206s0009g02970 and VIT_206s0009g03010. This prompted us to
search for their physical location on the grape genome assembly, and this led to the conclusion
that all these genes were located closely in the same region of chromosome 6 (FIGURE 7). This
corresponded to a segment of the chromosome that span from approximately 15,660 to 16,060
MB.
69
DISCUSSION
Metabolic changes during veraison and ripening stages in CG and RG
Berry ripening is an active process that involves regulation of global gene expression (Fasoli et
al., 2012) and changes in the accumulation of primary and secondary metabolites (P. K. Boss et
al., 1996; Conde et al., 2007). Berry organoleptic quality depends mainly on sugars, organic
acids and secondary metabolites such as tannins, volatile compounds, flavonols, aroma
precursors and anthocyanins, that accumulate during fruit development. Anthocyanins are the
responsible for the coloration process of berry skin. Many studies have focused on this process
and the vast majority centered on varieties with same skin color (Degu et al., 2014; Fraige,
González-Fernández, Carrilho, & Jorrín-Novo, 2015; Guo et al., 2016; Wu et al., 2014). Fewer
have focused on comparatives studies using varieties with red color skin and white color skin
(no presence of anthocyanins) (Ghan et al., 2015; Massonnet et al., 2017; Negri, Prinsi, Failla,
Scienza, & Espen, 2015; Niu, Wu, Yang, & Li, 2013) and even less includes pink-colored
skinned berries (Cardoso, Lau, Dias, Fevereiro, & Maniatis, 2012; Shimazaki, Fujita,
Kobayashi, & Suzuki, 2011; Walker, Lee, & Robinson, 2006).
To understand in depth the molecular changes related to coloration of grape berry skin, we used
RG and CG varieties in this study, two varieties of the near isogenic genetic background, which
differ in berry color skin from purple to bright light red. First, we performed a comparative
metabolic analysis of skin at two developmental stages, veraison and ripening, using LC-
MS/MS, HPLC, colorimetric and enzymatic assays for various types of metabolites. We chose
these developmental stages for their relevance and importance during berry development and
70
determination of crucial characteristics in the grape fruit: veraison marks the beginning of
ripening period and includes the start of coloration of berry skin, and the ripe stage represents
the culmination of berry developmental process (He et al., 2010; Kuhn et al., 2014). We
observed with PCA analysis that developmental stages were the primary discriminant
parameters in such comparative study, despite the use of different varieties, when PC1 was
analyzed (FIGURE 2A). This observation is concordant with previous published studies (Guo
et al., 2016; Massonnet et al., 2017; Wong, Lopez Gutierrez, Dimopoulos, Gambetta, &
Castellarin, 2016; Wu et al., 2014) in which, despite the use of different varieties, the importance
of developmental stages appeared to overrule variety origin. Another interesting result was that
T6P, the main influential metabolite in PC1 that explained 43,75% of data variability and
separated veraison from ripening in both CG and RG. T6P is a sugar phosphate considered as a
metabolic signaling molecule in plants; which regulates developmental growth (John Edward
Lunn, Delorge, Figueroa, Van Dijck, & Stitt, 2014) and was demonstrated to be involved in
sugar signaling in Arabidopsis thaliana (John E. Lunn et al., 2006). In Vitis vinifera there is no
clear evidence of control of T6P during berry development, although several transcriptomic
studies have shown that orthologue genes encoding T6P synthase, T6P synthase/phosphatase
and SnRK1 (Sucrose-non-fermentative Related Kinase 1, another sugar signaling pathway
component) are tightly regulated during berry development (L. G. Deluc et al., 2007; Gambetta,
Matthews, Shaghasi, McElrone, & Castellarin, 2010). Our data showed an increase in T6P
concentration in both CG and RG berry skin, from veraison to ripe stage (FIGURE S1). This
constitute a new finding in grape berry development, because previous studies analyzing T6P
in grapevine berries used total berries (Dai et al., 2013), and did not separate skin and flesh,
which probably lead to dilution of T6P signal from the skin. Due to the fact that sugar stimulates
71
anthocyanin biosynthesis in grape berry and grape cell culture (Dai et al., 2013; Larronde et al.,
1998), it would be interesting to study the expression of genes related to sugar accumulation
and metabolism in our RNA-seq data, to check whether there are differences between our
contrasted color skin varieties CG and RG.
Another important metabolite distinguishing veraison and ripe stages in PC1 (FIGURE 2A and
FIGURE S1) was shikimate, an intermediary in shimikate pathway that mediates the carbon
flow from carbohydrate metabolism to phenylpropanoid and aromatic compound biosynthesis
(Maeda & Dudareva, 2012; Zhang et al., 2012). This strongly suggest the importance of this
metabolite in species such as vitis vinifera, in which berry ripening involved phenylalanine
consumption (one of the main destination of chorismate, the final product of shikimate pathway)
for biosynthesis of flavonoid compounds such as anthocyanins.
According to PC2 which discriminated varieties, CG from RG, we observed many metabolites
directly related with early products of sucrose biosynthesis such as glyceraldehyde 3-phosphate,
fructose 1,6-biphosphate, fructose 6-phosphate and glucose 6-phosphate; and other metabolites
less related to this process (FIGURE S2). Also, another important metabolite that discriminated
varieties were anthocyanins (FIGURE 3A and 3C) where we observed that only traces of
malvidin, petunidin and delphinidin anthocyanins were detected in CG (FIGURE 3B and 3C),
indicating a disruption in the tri-hydroxylated branch of the anthocyanin biosynthesis pathway,
linked to flavonoid 3’5’-hydroxylase enzyme (F3’5’H) activity, an observation latter confirmed
by RNA-seq results (discussed below). Accordingly, an increase in F3’5’H activity have been
previously reported to explain higher concentration of tri-hydroxylated anthocyanins in
72
Cabernet Sauvignon and Shiraz, pointing F3’5’H plays as a key molecular player driving the
phenylpropanoid metabolic flux towards bluish anthocyanins production (Degu et al., 2014).
If we take both results together, we can link them because there is a relationship between sugars
and anthocyanin accumulation described in previous studies. For example, studies in Shiraz
grape berries showed that soluble solids (measured as Brixº and represent glucose and fructose
in grape berry) began to increase 8 weeks post flowering and continue to increase, and
anthocyanins are then synthetized 10 weeks post flowering (Paul K. Boss, Davies, & Robinson,
1996). Also, in Vitis vinifera cell cultures was found that sucrose modulated anthocyanins
content, and this was observed also with fructose and glucose (Larronde et al., 1998). In our
study the anthocyanin biosynthesis pathway was affected in last steps in CG, and maybe this
observation can be linked to modifications in biosynthesis of sucrose in CG or otherwise,
changes in anthocyanins in CG modified intermediates of sucrose. That is an interesting point
of discussion.
Transcriptomic changes during veraison and ripening in CG an RG varieties
RNA-seq analysis had been often used for searching and characterizing new gene expression
patterns in pathway related to grape berry development (Degu et al., 2014; Fortes et al., 2011;
Ghan et al., 2015, 2017; Guo et al., 2016; Li et al., 2014; Lijavetzky et al., 2012; Massonnet et
al., 2017; Sweetman et al., 2012; Wong et al., 2016; Wu et al., 2014; Zenoni et al., 2010).
However, few studies focused on the global gene expression in contrasted color berry skin from
near isogenic backgrounds, and even less considered pink varieties. In our study, besides
metabolic studies, skin transcriptome was analyzed at two developmental stages using Illumina
Hiseq2000 in two near isogenic varieties, RG and CG. According to DEGs analysis, we reported
73
that several genes related to phenylpropanoid pathway, which grouped in three main clusters of
repression, induction or a combination of both, were differentially expressed in CG compared
to RG (TABLE 2, 3 and 4, respectively). Among them, we found that 11 copies of F3’5’-H,
encoding a key enzyme in anthocyanin biosynthesis pathway were repressed in CG. This
enzyme corresponds branching point of the flavonoid pathway, where dihydrokaempferol is the
substrate shared in common (Kuhn et al., 2014). Dihydrokaempferol is converted via F3’5’-H
to dihydromyricetin, producing the glucoside, acetyl, coumaroyl and caffeoyl forms of
delphinidin, petunidin and malvidin, that correspond to the delphinidin-type (tri-hydroxylated)
anthocyanins of bluish color. Dihydrokaempferol is also used by F3’-H to produce
dihydroquercetin and gives rise to the biosynthesis of glucoside, acetyl and coumaroyl forms of
cyanidin and peonidin, that correspond to cyanidin-type (di-hydroxylated) anthocyanins or
reddish color (Degu et al., 2014). Indeed, our results unambiguously showed that CG berry skins
did not contain delphindin-type anthocyanins (FIGURE 3B and 3D) and therefore, is consistent
with the repression of 11 gene copies of F3’5’-H in CG. Additionally, another cunning
observation was the adjacent location of each of these copies in a single cluster within
chromosome 6 (FIGURE 7). It is known that there are many copies of F3’5’-H in Vitis vinifera,
and it was shown that F3’5’-H in grapevine are highly redundant and predominately expressed
in berry skins (Castellarin et al., 2006; Falginella et al., 2010). However, to the best of our
knowledge, there was no evidence of the repression of such high number of gene copies at the
same time in a single variety. This raises the question of how the not induction of the F3’5’-H
genes occur in CG. At least two mechanisms can be foreseen, among others. Firstly, since all
these genes are physically close together in a single region of chromosome 6, an epigenetic can
be postulated. Although left uninvestigated until recently, the field of epigenetic regulation in
74
grapevine has gain considerable attention in the last years; and epigenetic regulation were shown
to be potentially part of the so-called “terroir” effect in Shiraz grapes from Australia (Xie et al.,
2017). Secondly, a still to be discovered master regulator (i.e a transcription factor) might be
defective in the CG. For example, an identical transcription factor biding motif might be present
in the promoter regions of all 11 gene copies, which would be in agreement with the hypothesis
of a master regulator for these F3’5’-H genes. Further work is necessary to decide between these
two hypotheses.
In parallel, several genes putatively involved in anthocyanin transport to the vacuoles were
differentially expressed in CG compared to RG at both veraison and ripening stages, such as
GST13 and ABC transporter (FIGURE 6). GST13 (VIT_207s0104g01800) expression was
down-regulated at both developmental stages in CG. Previous studies in model plants showed
that members of the multigenic GST family have been described to be necessary for anthocyanin
accumulation such as AN9 in petunia (Alfenito et al., 1998), TT19 in Arabidopsis (Kitamura,
Shikazono, & Tanaka, 2004) and BZ2 in maize (Goodman, Casati, & Walbot, 2004). A recent
study in vitis vinifera performed molecular dynamics and docking analysis together with gene
analysis expression for GST1 and GST4 in different organs during berry development and also.
The ability of GST1 and GST4 to rescue the phenotype of the Arabidopsis tt19-1 mutant was
also tested. The results showed that at least GST4 behaved like Arabidopsis TT19 in the
transport of anthocyanins and proanthocyanidins, revealing a novel function for his gene in
flavonoid transport (Pérez-Díaz, Madrid-Espinoza, Salinas-Cornejo, González-Villanueva, &
Ruiz-Lara, 2016). In our RNA-seq data, we noticed that an ABC (ATP-binding cassette)
transporter (VIT_214s0060g00720) was induced only at veraison in CG. ABC transporters have
75
also been involved in vacuolar sequestration of flavonoids in maize (Goodman et al., 2004) and
in the transport of malvidin 3-O-glucoside into vacuoles, in a strictly GSH-dependent fashion
in grapevine (Zarrouk et al., 2013). In addition, ANP-like genes differentially expressed in our
RNA-seq study (VIT_216s0050g00900 and VIT_216s0050g00910) encodes anthocyanin
vacuolar transporters that were repressed in CG, and have been suggested to participate in
anthocyanin vacuolar sequestration in grapes (Mathews et al., 2003; Wu et al., 2014). These
findings reported in our study related to GST13 and ANP-like, repressed in CG and on the other
hand ABC transporter induced in CG, might suggest a dynamic process in transport and
sequestration of anthocyanin into vacuoles occurring in this variety context. Anyways, due to
these genes have not been characterized in grapevines, they become in new possible candidates
to study for better understanding anthocyanin transport.
In addition, several genes related to anthocyanin O-methylation, such as CCoAOMT and
COMT-like, were also found to be differentially expressed between RG and CG in our study.
CcoAOMT is a multifunctional O-methyltransferase that might contribute to anthocyanin
methylation activity in grapevine, but this was only observed under drought stress conditions
(Giordano et al., 2016). Our data showed that CcoAOMT was repressed in veraison and ripening
stage in CG, suggesting that maybe abiotic stress regulation in CG was modified for some
unknown reason. Stress in grapevine had been generally associated with biosynthesis of tri-
hydroxylated anthocyanins (delphinidin-type), which would make sense according to the
phenotype observed in CG. Finally, COMT-like is an OMT using catechol as substrate and
characterized in S. lycopersicum (Mageroy et al., 2012) but not in grapevine so far. It was found
to be induced in CG during veraison and at ripening stage. Further studies will be necessary to
understand the function of this enzyme in CG.
76
Another key gene found to be down-regulated in CG was CHS (FIGURE 6), that encodes the
first enzyme of the flavonoid pathway (Petrussa et al., 2013; Wang et al., 2016) and this would
be coherent with the observed metabolic profile of CG, which contains less total anthocyanins
than RG. Also, STS gene, encoding another early enzyme in flavonoid metabolism and involved
in resveratrol biosynthesis (Dubrovina & Kiselev, 2017; Kiselev, Aleynova, Grigorchuk, &
Dubrovina, 2016) was induced in CG at veraison and ripening stages (FIGURE 6). This
possibly reveals a possible compensation point in flavonoid metabolism: CHS utilizes the same
substrates as STS but, is responsible for flavonoid-type compound formation. Thus, STS would
act as an escape valve to absorb the excess of substrate flux generated in the upper part of the
flavonoid metabolism because of the decrease in CHS activity. Taken together, this suggests a
multilevel alteration of the flavonoid pathway, and not only of its tri-hydroxylated branch in CG
variety.
Most studies investigating the mechanisms of changes in berry skin color explain them through
the regulatory effect of MybA transcription factors, whose genes are closely clustered in a single
locus on chromosome 2, referred to as the “berry color locus” (Kobayashi et al., 2004; Walker
et al., 2006). One of these is the transcription factor MybA1, a major determinant of grape berry
anthocyanin biosynthesis pathway (Kobayashi et al., 2004; Lijavetzky et al., 2006). MybA1
transcriptionally regulates UFGT gene, which glycosylates anthocyanidins, stabilizing them and
therefore responsible for the anthocyanins accumulation in grape berry skins (Ford, Boss, &
Hoj, 1998). White skinned grape varieties that do not produce anthocyanins, contain a mutated
allele of MYBA1 in which the presence of the retrotransposon Gret1 in promoter region
interrupts the normal transcription process, resulting in a white berry allele (Kobayashi et al.,
2004). Pigmented cultivars possess at least one allele of the MybA1 locus that does not contain
77
Gret1 and is called red berry allele (This et al., 2007). In the case of pink skin berry varieties of
oriental origin, a different situation was reported: a 33bp insertion was found in the second
intron of MybA1, generating low expression levels of MybA1 in grape from these cultivars
(Shimazaki et al., 2011). Another different example is a bud sports of Cabernet Sauvignon called
“Malian” with red-grey berry skin, associated to the deletion of a large region of chromosome
2 that includes both MybA1 and MybA2 genes (MybA2 is located adjacent to MybA1 in the color
berry locus) (Walker et al., 2006). We reported that UFGT gene (VIT_216s0039g02230) is
repressed in CG at veraison and ripening stages (FIGURE 6), which could suggest an alteration
in MybA1 expression, according to literature describe above. MybA1, however, was not deemed
significantly affected in DEGs analysis (FIGURE 4). Nevertheless, we decided to check for the
presence of white and red allele in CG and RG, using the same set of primer used in (Shimazaki
et al., 2011). We observed the presence of white and red allele of MybA1 in all CG and RG
samples tested (FIGURE S5A and S5B, respectively), demonstrating that both varieties are
heterozygous for this gene and that our results cannot be explained by structural changes in
MybA1 DNA sequence.
Another MYB gene, MYB24 (VIT_214s0066g01090) was found to be induced in CG (TABLE
3, FIGURE 6). Recently, it was reported that MYB24 was associated with terpene biosynthesis
in berries in response to drought (Savoi et al., 2016); also was highly co-expressed with flower
and fruit specific terpene synthase (TPS) (Wong et al., 2016); and induced by solar UV radiation
in Tempranillo berry skins, in conjunction with terpenoid metabolism induction (Carbonell-
Bejerano et al., 2014). Therefore, it would seem that this transcription factor would be in charge
of leading the responses towards the activation of biosynthesis of terpenes. So, how could this
be related to berry color? With these antecedents we hypothesized that this transcription factor
78
could be linked by activating the terpene biosynthesis pathway in CG, which would therefore
affect the flow towards anthocyanin biosynthesis, or vice versa, a change or block in the
anthocyanin biosynthesis pathway, takes the flow to the terpenes, which would be mediated via
MYB24. How to know what is happening in CG? Future studies using this transcription factor
to evaluate berry color in CG would be necessary.
Another gene of particular interest reported in our study was Cytb5, which was repressed in CG
at veraison and ripening stages (FIGURE 6). Cytb5 has previously been involved in F3’5’-H
activity modulation in Petunia flowers, catalyzing the transfer of electrons to their prosthetic
heme group (de Vetten et al., 1999). In 2006, (Bogs, Ebadi, McDavid, & Robinson, 2006)
demonstrated that a putative Cytb5 from grapevine cv Shiraz) modulated both F3’5’-H and F3’-
H activites even though the exact mechanism remains to be deciphered. This is of interest
because this suggests a possible link between the drastic down-regulation of 11 gene copies of
F3’5’H and the repression of the Cytb5 gene observed in CG. Additional work will be required
to test that hypothesis.
79
CONCLUSION
This comparative study revealed important differences at the metabolic and transcriptomic level
between two contrasted color berry skin, but nevertheless near isogenic, table grapes. Using
RNA-seq we showed that flavonoid metabolism was affected at different levels in CG compared
to RG at both veraison and ripening stage. While the nature of the mutation responsible for color
changes in berry skin of CG from its progenitor RG remains unknown, the comparative RNA
profiling of the berry development between CG and RG showed the deregulation of genes
directly related to anthocyanin biosynthesis: CHS, STS, CYTB5, UFGT, MYB24, F3’5’-H, F3’-
H, CCoAOMT, COMT, GST13, ABC transporter and ANP-like. A prominent result was the
drastic down-regulation of 11 gene copies of F3’5’H in CG positioned in a single cluster in
chromosome 6, which was totally consistent with the anthocyanin content observed in CG, with
the absence of delphinidin-type (tri-hydroxylated) anthocyanins. This left in evidence an
arrangement occurring in this metabolic process in CG compared to RG and open new types of
regulation undescribed in anthocyanin biosynthesis in grapevines.
This study provides new insights in the genetic and metabolic background that may be
responsible of color change in CG compared to RG, that constitute a relevant biological material
to study in depth the mechanisms underlying berry skin color intensity.
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AUTHOR CONTRIBUTIONS
The manuscript was written by Claudia Santibáñez. Tomás Moyano and Carlos Meyer made the
bioinformatics data analysis and datamining. John Lunn and Regina Feil carried out metabolic
analysis by LC-MS/MS. Ghislaine Hilbert and Christel Renaud helped in hexose, anthocyanin
and organic acids experiments. Litsy Martínez took care of biological material. Claudia
Santibáñez performed vegetal material sampling, RNA extraction, qRT-PCR and data analysis
of all study. Eric Gomès, Patricio and Serge Delrot contribute in supervision, design of
experiments and revision of the manuscript.
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ACKNOWLEDGMENTS
We thank to CONICYT scholarship doctorate 2012 Nº 21120432, Operational Expenses
Scolarship of CONICYT Nº 21120432, Millennium Nucleus of Plant Systems and Synthetic
Biology NC130030 and CHIMENTI S.A.
82
FIGURE 1: Field photographs of cultivars RG and CG in the different stages during 2013 season sampling. (A) RG cluster during
veraison stage. (B) CG cluster during veraison stage. (C) RG and CG clusters during ripening stage. (D and E) RG and CG plants used
in this study. RG correspond to left side of photograph and CG to right side, during veraison and ripening stage, respectively.
83
FIGURE 2: Principal component analysis of metabolites present in berry skin of RG and CG. (A) Discriminations of cultivars RG
and CG, veraison and ripening stages for the 2013 and 2014 seasons. (B) Loading plots of metabolites analyzed for the first two
components (PC1 and PC2).
84
0%
20%
40%
60%
80%
100%
VeraisonRipening
VeraisonRipening
% A
nth
ocyan
ins c
on
ten
tm
g g
-1 s
kin
DW
Axis Title
Malvidin
Peonidin
Petunidin
Cyanidin
Delphinidin
Chimenti Red Globe
0%
20%
40%
60%
80%
100%
VeraisonRipening
VeraisonRipening
% A
nth
ocyan
ins c
on
ten
tm
g g
-1 s
kin
DW
Axis Title
Chimenti Globe Red Globe
A B
C D
Verai
son
Rip
enin
g
0
5
10
15
Stages
To
tal a
nth
oc
ya
nin
s
mg
g-1
sk
in D
W
Chimenti Globe
Red Globe
****
**
Stages
To
tal a
nth
oc
ya
nin
s
mg
g-1
sk
in D
W
Verai
son
Rip
enin
g
0
5
10
15Chimenti Globe
Red Globe
***
**
85
FIGURE 3: Content of anthocyanins in Chimenti globe and Red Globe berry skin, at
veraison and ripening stages (mg g-1 DW). (A) and (C) Total anthocyanins content in 2013
and 2014 season, respectively. The results are the mean of five biological replicates, error
bars represent standard error of mean. Asterisks indicate the result of unpaired T-test. **, P
< 0.0081; ***, P = 0.0001; ****, P < 0.0001. (B) and (D) Percentages of main anthocyanins
in 2013 and 2014 season, respectively. Malvidin corresponds to malvidin 3-glucoside
(purple); Peonidin to peonidin 3-glucoside and peonidin 3-(p-coumaryl)-glucoside
(magenta); Petunidin to petunidin 3-glucoside (indigo); Cyanidin to cyanidin 3-glucoside and
cyanidin 3-(p-coumaryl)-glucoside (red); and Delphinidin to delphinidin 3-glucoside and
delphinidin 3-(acetyl)-glucoside (blue). DW, Dry Weight.
86
TABLE 1: Average content of different types of anthocyanins in Chimenti Globe and Red Globe berry skin (mg g-1 DW).
CHIMENTI GLOBE RED GLOBE
Veraison Ripening Veraison Ripening
2013 2014 2013 2014 2013 2014 2013 2014
Delphinidin 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.01 ± 0.00a 0.07 ± 0.02e 0.09 ± 0.09b 0.52 ± 0.08e 0.53 ± 0.03c
Petunidin 0.00 ± 0.00a 0.00 ± 0.00a 0.01 ± 0.01a 0.01 ± 0.00a 0.06 ± 0.02e 0.09 ± 0.09a 0.60 ± 0.11e 0.48 ± 0.23c
Malvidin 0.00 ± 0.00a 0.00 ± 0.00a 0.01 ± 0.01a 0.03 ± 0.01a 0.21 ± 0.06e 0.32 ± 0.37a 2.07 ± 0.57e 1.38 ± 0.64c
Cianidin 0.11 ± 0.03a 0.04 ± 0.02a 0.69 ± 0.49a 0.65 ± 0.14a 0.39 ± 0.19c 0.65 ± 0.26d 2.17 ± 0.24d 2.43 ± 0.65d
Peonidin 0.16 ± 0.04a 0.06 ± 0.02a 1.21 ± 0.40a 1.13 ± 0.32a 0.75 ± 0.33c 1.41 ± 0.86c 9.04 ± 1.22e 7.90 ± 2.08e
Total 0.27 ± 0.05a 0.11 ± 0.04a 1.93 ± 0.85a 1.84 ± 0.44a 1.49 ± 0.57c 2.57 ±1.57c 14.39 ± 1.21e 12.71 ± 3.48d
The letters represent the result of an unpaired T-test between Chimenti Globe and Red Globe in each developmental stage during 2013
and 2014 season: a represent no significant differences; b represent *, P = 0.0497; c represent **, P < 0.0093; d represent ***, P < 0.0009; e represent ****, P < 0.0001.
87
88
FIGURE 4: Hierarchical clustering heat map and cluster tree from 109 DEGs in Chimenti
Globe and Red Globe. Each row represents a gene and each column represents a developmental
stage, veraison (left panel) and ripening (right panel). The color key indicates logFC values ≥ 1,
ranging from bright yellow for most up-regulated to bright blue for the most down-regulated
genes, considering a FDR of 0.05. Genes that showed a similar developmental profile were
clustered together. Six clusters were generated.
89
TABLE 2: The DEGs ID of repression cluster A, B and C during veraison and ripening stages
Nº Cluster Genoscope ID Log2FCV Log2FCR Blast NCBI
1 A VIT_204s0079g00710 -4,66 -5.44 glutathione s-transferase
2 B VIT_206s0009g02830 -2.04 -5.20 flavonoid 3', 5'- hydroxylase
3 B VIT_206s0009g02840 -1.27 -4.88 flavonoid 3', 5'- hydroxylase
4 B VIT_207s0104g01650 -1.64 -4.30 potassium transporter 5
5 B VIT_206s0009g02810 -1.73 -4.42 flavonoid 3', 5'- hydroxylase
6 B VIT_206s0009g02920 -2.04 -4.23 flavonoid 3', 5'- hydroxylase
7 B VIT_206s0009g02970 -2.15 -4.38 flavonoid 3', 5'- hydroxylase
8 B VIT_206s0009g02805 -2.41 -3.72 flavonoid 3', 5'- hydroxylase
9 B VIT_206s0009g02880 -2.22 -3.50 flavonoid 3', 5'- hydroxylase
10 B VIT_207s0104g01800 -1.99 -3.53 glutathione s-transferase f13
11 B VIT_206s0009g02846 -1.54 -3.98 flavonoid 3', 5'- hydroxylase
12 B VIT_206s0009g02873 -1.70 -3.64 flavonoid 3', 5'- hydroxylase
13 B VIT_200s0256g00120 -1.22 -3.30 protein
14 B VIT_214s0036g00330 -1.02 -2.68 disease resistance protein at4g27190-like
15 B VIT_205s0077g01730 -1.03 -2.67 uncharacterized protein loc100243551
16 B VIT_214s0036g00100 -1.05 -2.39 disease resistance protein at4g27190-like
17 B VIT_207s0031g01040 -1.11 -2.34 l-ascorbate oxidase
18 B VIT_207s0197g00230 -1.15 -2.35 probable disease resistance protein at5g66900-like
19 B VIT_216s0050g00900 -1.25 -2.46 multidrug resistance
20 B VIT_206s0009g02860 -1.75 -2.36 flavonoid 3', 5'- hydroxylase
21 B VIT_219s0027g01070 -1.75 -2.48 disease resistance protein rga4-like
22 B VIT_203s0088g00260 -1.93 -2.45 serine carboxypeptidase-like 18-like
23 B VIT_216s0050g00910 -2.08 -2.67 multidrug resistance
90
24 B VIT_204s0023g03370 -1.99 -2.90 flavanone 3- hydroxylase
25 C VIT_216s0022g00670 -1.30 -1.04 vacuolar invertase
26 C VIT_200s0282g00040 -1.05 -1.01 protein spinster homolog 1-like
27 C VIT_203s0091g00410 -1.03 -1.07 uncharacterized protein
28 C VIT_214s0030g01160 -1.06 -1.16 disease resistance protein at4g27190-like
29 C VIT_215s0046g01560 -1.10 -1.15 bcl-2-associated athanogene 6
30 C VIT_207s0197g00140 -1.12 -1.28 probable disease resistance protein at5g66900-like
31 C VIT_208s0040g01140 -1.14 -1.27 serine carboxypeptidase-like 45-like
32 C VIT_205s0077g01720 -1.08 -1.29 uncharacterized protein loc100256873
33 C VIT_216s0013g01500 -1.08 -1.35 leucine-rich repeat receptor protein kinase exs-like
34 C VIT_204s0044g00170 -1.03 -1.34 dna mismatch repair protein mlh3
35 C VIT_210s0003g02000 -1.06 -1.42 protein
36 C VIT_208s0007g03560 -1.23 -1.63 solute carrier family 35 member f1-like
37 C VIT_200s0160g00100 -1.11 -1.61 tmv resistance protein n-like
38 C VIT_203s0017g00870 -1.13 -1.65 protein
39 C VIT_210s0003g01995 -1.07 -1.71 atp binding
40 C VIT_216s0100g00670 -1.10 -1.84 homeobox-leucine zipper protein anthocyaninless 2-like
41 C VIT_206s0061g01230 -1.11 -1.87 glucomannan 4-beta-mannosyltransferase 2
42 C VIT_214s0036g00310 -1.05 -2.17 disease resistance protein at4g27190-like
43 C VIT_217s0000g07375 -1.02 -2.06 enhanced disease susceptibility 1
44 C VIT_208s0007g03570 -1.13 -2.04 solute carrier family 35 member f1-like
45 C VIT_205s0136g00260 -1.54 -2.19 chalcone synthase
46 C VIT_200s0245g00030 -1.49 -1.86 uncharacterized protein loc100852932
47 C VIT_206s0009g03010 -1.94 -1.78 flavonoid 3', 5'- hydroxylase
48 C VIT_214s0066g00900 -1.77 -1.41 cysteine-rich receptor-like protein kinase 42
91
49 C VIT_205s0049g00480 -2.00 -1.38 at1g04360
50 C VIT_216s0039g02220 -2.18 -1.45 exportin-1-like isoform 1
51 C VIT_201s0010g03510 -2.20 -1.41 caffeoyl- o-methyltransferase
52 C VIT_201s0010g03490 -2.36 -1.65 caffeoyl- o-methyltransferase
53 C VIT_216s0039g02230 -3.15 -1.35 udp-glucose:flavonoid 3-o-glucosyltransferase
54 C VIT_218s0001g09400 -3.82 -1.99 cytochrome b5
55 C VIT_204s0079g00690 -3.96 -2.53 glutathione s-transferase
92
TABLE 3: The DEGs ID of induction cluster E during veraison and ripening stages
Nº Cluster Genoscope ID Log2FCV Log2FCR Blast NCBI
67 E VIT_202s0087g00380 2.49 1.32 hypothetical protein
68 E VIT_209s0002g03460 1.73 1.26 clavaminate synthase-like protein
69 E VIT_204s0044g01230 1.56 1.38 hypothetical protein
70 E VIT_213s0067g02690 1.07 1.39 dimethylguanosine trna methyltransferase
71 E VIT_206s0004g03910 1.11 1.38 hypothetical protein
72 E VIT_202s0234g00110 1.13 1.41 oleosin low molecular weight isoform
73 E VIT_209s0002g03450 1.13 1.33 clavaminate synthase-like protein
74 E VIT_201s0137g00540 1.11 1.29 cytochrome p450
75 E VIT_203s0038g01380 1.01 1.23 calcium sensor
76 E VIT_208s0007g01470 1.06 1.21 copper transporter
77 E VIT_216s0098g00890 1.08 1.18 harpin-induced family protein
78 E VIT_219s0090g01350 1.03 1.16 hypothetical protein
79 E VIT_212s0028g03620 1.12 1.15 uncharacterized protein
80 E VIT_219s0014g04790 1.16 1.15 solute carrier family 22 member 15-like
81 E VIT_202s0025g02560 1.17 1.15 o-succinylhomoserine sulfhydrylase
82 E VIT_210s0003g03270 1.29 1.43 potassium channel
83 E VIT_205s0077g00635 1.32 1.40 hypothetical protein
84 E VIT_200s0684g00030 1.25 1.52 senescence-associated protein
85 E VIT_214s0060g00720 1.29 1.56 abc transporter family protein
86 E VIT_208s0058g00930 1.21 1.70 alanine--glyoxylate aminotransferase 2 homolog mitochondrial
87 E VIT_207s0005g00870 1.34 1.74 hypothetical protein
88 E VIT_215s0048g02490 1.45 1.76 catechol o-methyltransferase
89 E VIT_214s0066g01090 1.53 1.69 myb transcription factor 24
93
90 E VIT_214s0108g00450 1.05 1.93 hypothetical protein
91 E VIT_219s0014g05080 1.10 1.90 oxygen-evolving enhancer protein 3-1
92 E VIT_205s0102g00790 1.20 1.88 hypothetical protein
93 E VIT_201s0137g00680 1.21 1.99 hypothetical protein
94 E VIT_207s0129g00510 1.40 2.12 uncharacterized protein
95 E VIT_205s0020g04110 1.12 2.25 early light-inducible protein
96 E VIT_216s0050g01820 1.35 2.48 hypothetical protein
97 E VIT_201s0011g05440 1.92 2.24 hypothetical protein
98 E VIT_202s0025g01710 1.89 2.51 glutaredoxin family protein
99 E VIT_215s0048g02480 1.74 2.88 catechol o-methyltransferase
100 E VIT_218s0001g04780 1.64 3.76 sesquiterpene synthase 10 (TPS10)
94
TABLE 4: The DEGs ID of combined induction/repression clusters D and F during veraison and ripening stages
Nº Cluster Genoscope ID Log2FCV Log2FCR Blast NCBI
56 D VIT_215s0048g02420 1.03 -3.27 hypothetical protein
57 D VIT_210s0042g00880 1.31 -3.05 stilbene synthase
58 D VIT_216s0022g00650 1.22 -2.40 hypothetical protein
59 D VIT_210s0042g00840 1.28 -2.16 stilbene synthase
60 D VIT_209s0002g03560 1.05 -2.07 atp-binding cassette
61 D VIT_200s0483g00020 1.12 -2.03 suppressor-like protein
62 D VIT_218s0041g02020 1.11 -1.82 12-oxophytodienoate reductase
63 D VIT_202s0025g04860 1.31 -1.59 hypothetical protein
64 D VIT_210s0042g00920 1.30 -1.59 stilbene synthase
65 D VIT_201s0011g05890 1.42 -1.52 hypothetical protein
66 D VIT_201s0182g00130 1.54 -1.78 phosphate transporter pho1 homolog 3-like
101 F VIT_212s0028g01130 -1.00 1.86 hypothetical protein
102 F VIT_217s0000g09800 -1.02 1.62 hypothetical protein
103 F VIT_200s0301g00070 -1.32 1.59 hypothetical protein
104 F VIT_212s0142g00160 -1.23 1.51 hypothetical protein
105 F VIT_215s0046g00080 -1.17 1.39 hypothetical protein
106 F VIT_200s0400g00030 -1.41 1.25 leucine-rich repeat receptor protein kinase exs-like
107 F VIT_205s0020g02210 -1.46 1.12 histidine phosphotransfer protein
108 F VIT_203s0038g01310 -1.28 1.10 saur family protein
109 F VIT_204s0023g03230 -1.03 1.03 saur family protein
95
FIGURE 5: Gene ontology classification of DEGs. GO annotations for differentially
expressed genes during veraison and ripening stages in CG and RG varieties. (A) Molecular
Function, (B) Biological Process and (C) Cellular Component.
96
97
FIGURE 6: Real-time quantitative PCR (qRT-PCR) validation of 12 selected DEGs
identified by RNA-seq in berry skin of CG and RG. Samples were collected at veraison
and ripening stages in 2013, and genes were selected from repression, induction and
combined repression/induction clusters. The results are the mean of six biological replicates,
error bars represent standard error of mean. Asterisks indicate the result of unpaired T-test.
*, P < 0.0465; **, P < 0.0053; ***, P < 0.0009; ****, P < 0.0001. FW, Fresh Weight.
98
FIGURE 7: Localization of 11 gene copies of flavonoid 3’5’-hydroxylases on
chromosome 6 in Vitis vinifera. Diagram representation showing the position on Ch6 of
F3’5’-H genes. Ch6 has a size of 21.5MB and F3’5’-H gene copies from results are located
in an area 0.5MB
99
SUPPLEMENTARY MATERIAL
100
FIGURE S1: Metabolites whose percentage of contribution are higher in PC1 from
PCA (nmol g-1 FW), at veraison and ripening stages. (A, C, E, G, I) Metabolites
corresponding to the season 2013, and (B, D, F, H, J) corresponding to the season 2014. The
results are the mean of six biological replicates, error bars represent standard deviations.
Asterisks indicate the result of two-way ANOVA, followed by Tukey’s test. ****, P <
0.0001; ***, P < 0.0005; **, P < 0.0052; *, P < 0,0282. FW, Fresh Weight.
101
102
103
FIGURE S2: Metabolites whose percentage of contribution are higher in PC2 from
PCA (nmol g-1 FW). (A, C, E, G, I, K, M, O, Q) Metabolites corresponding to the season
2013; (B, D, F, H, J, L, N, P, R) metabolites corresponding to the season 2014. The results
are the mean of six biological replicates, error bars represent standard error of mean.
Asterisks indicate the result of unpaired T-test. *, P < 0.0443; **, P < 0.0085; ***, P <
0.0003.; ****. FW, Fresh Weight.
104
FIGURE S3: Analysis of MybA1 gene structure in CG and RG. (A) PCR analysis of
white allele in CG, RG varieties and control Merlot (Me), Cabernet Sauvignon (CS),
Chardonnay (Ch) and Sauvignon Blanc (SB). (B) PCR analysis of red allele in CG and RG
varieties and control described above. (C) Integrity control of actin gene. Numbers on the
left indicate the positions of the molecular size marker from KBplus and on the right the
exact size of white and red allele.
105
106
FIGURE S4: Real-time quantitative PCR (qRT-PCR) validation of 6 gene copies of
flavonoid 3’-5’ hydroxylases identified by RNA-seq in berry skin of CG and RG.
Samples were collected at veraison and ripening stages in 2013. The gene copies of F3’5-H
corresponded to 2810, 2830, 2860, 2880, 2920 and 3010. The results are the mean of six
biological replicates, error bars represent standard error of mean. Asterisks indicate the result
of unpaired T-test. **, P < 0.0085; ***, P < 0.0003; ****, P < 0.0001. FW, Fresh Weight.
107
TABLE S1: Pool designation of CG and RG plants for RNA-seq analysis.
SKIN SAMPLE ID POOL NAME
Veraison Chimenti Globe 1
POOL-1A Veraison Chimenti Globe 2
Veraison Chimenti Globe 3
POOL-1B Veraison Chimenti Globe 4
Veraison Chimenti Globe 5
POOL-1C Veraison Chimenti Globe 6
Veraison Red Globe 1
POOL-2A Veraison Red Globe 2
Veraison Red Globe 3
POOL-2B Veraison Red Globe 4
Veraison Red Globe 5
POOL-2C Veraison Red Globe 6
Ripening Chimenti Globe 1
POOL-3A Ripening Chimenti Globe 2
Ripening Chimenti Globe 3
POOL-3B Ripening Chimenti Globe 4
Ripening Chimenti Globe 5
POOL-3C Ripening Chimenti Globe 6
Ripening Red Globe 1
POOL-4A Ripening Red Globe 2
Ripening Red Globe 3
POOL-4B Ripening Red Globe 4
Ripening Red Globe 5
POOL-4C Ripening Red Globe 6
108
TABLE S2: Alignment of RNA-seq reads to the Grapevine Reference Genome V2
using HISAT2
Sample
ID
Total reads
paired
Reads aligned
0 times
Reads aligned
1 time
Reads aligned
>1 time
Pool-1A 4575605
(100.00%)
508068
(11.1%)
3301327
(72.15%)
766210
(16.75%)
Pool-1B 4871122
(100.00%)
766210
(11.6%)
3508905
(72.03%)
797273
(16.37%)
Pool-1C 5350777
(100.00%)
642477
(12.01%)
3808111
(71.07%)
900189
(16.82%)
Pool-2A 6169343
(100.00%)
670998
(10.88%)
4482441
(72.66%)
1015904
(16.47%)
Pool-2B 6180772
(100.00%)
654089
(10.58%)
4493751
(72.71%)
1032932
(16.71%)
Pool-2C 5010252
(100.00%)
868444
(17.33%)
3387979
(67.62%)
753829
(15.05%)
Pool-3A 7690225
(100.00%)
1588674
(20.66%)
4964146
(64.55%)
1137405
(14.79%)
Pool-3B 5624673
(100.00%)
883057
(15.70%)
3820294
(67.92%)
921322
(16.38%)
Pool-3C 5360934
(100.00%)
715581
(13.35%)
3748929
(69.93%)
896424
(16.72%)
Pool-4A 6895816
(100.00%)
791137
(11.47%)
4928442
(71.47%)
1176237
(17.06%)
Pool-4B 7147979
(100.00%)
767762
(10.74%)
5160674
(72.20%)
1219543
(17.06%)
Pool-4C 6229341
(100.00%)
729131
(11.7%)
4436080
(71.21%)
1064130
(17.08%)
109
TABLE S3: Cleaning raw reads using Trimmomatic
Sample
ID
Input total
reads pairs
Both
Surviving
Forward Only
Surviving
Reverse Only
Surviving
Dropped
Pool-1A 4852418
(100.00%)
4575605
(94.30%)
178246
(3.67%)
60885
(1.25%)
37682
(0.78%)
Pool-1B 5173857
(100.00%)
4871122
(94.15%)
201139
(3.89%)
60598
(1.17%)
40998
(0.79%)
Pool-1C 5681319
(100.00%)
5350777
(94.18%)
213111
(3.75%)
72260
(1.27%)
45171
(0.80%)
Pool-2A 6553961
(100.00%)
6169343
(94.13%)
246950
(3.77%)
85694
(1.31%)
51974
(0.79%)
Pool-2B 6570611
(100.00%)
6180772
(94.07%)
258552
(3.93%)
79087
(1.20%)
52200
(0.79%)
Pool-2C 5311185
(100.00%)
5010252
(94.33%)
197744
(3.72%)
64427
(1.21%)
38762
(0.73%)
Pool-3A 8183865
(100.00%)
7690225
(93.97%)
309652
(3.78%)
111186
(1.36%)
72802
(0.89%)
Pool-3B 5982329
(100.00%)
5624673
(94.02%)
232378
(3.88%)
76608
(1.28%)
48670
(0.81%)
Pool-3C 5694271
(100.00%)
5360934
(94.15%)
213180
(3.74%)
74459
(1.31%)
45698
(0.80%)
Pool-4A 7331098
(100.00%)
6895816
(94.06%)
286456
(3.91%)
90148
(1.23%)
58678
(0.80%)
Pool-4B 7604337
(100.00%)
7147979
(94.00%)
298053
(3.92%)
95992
(1.26%)
62313
(0.82%)
Pool-4C 6606565
(100.00%)
6229341
(94.29%)
241663
(3.66%)
84617
(1.28%)
50944
(0.77%)
110
TABLE S4: Gene mapping analysis from aligned reads to Grapevine Reference Genome V2
Status Pool-1A Pool-1B Pool-1C Pool-2A Pool-2B Pool-2C Pool-3A Pool-3B Pool-3C Pool-4A Pool-4B Pool-4C
Assigned 4166463 4429083 4850826 5613766 5621724 4087521 6382908 4990397 4860962 6323211 6573172 5691548
Unassigned_Ambiguity 0 0 0 0 0 0 0 0 0 0 0 0
Unassigned_MultiMapping 310015 321664 355914 403352 433391 299855 316418 363941 369011 545779 528626 499145
Unassigned_NoFeatures 129072 141636 140843 185174 179281 379406 408854 121440 92241 131729 139122 125051
Unassigned_Unmapped 154182 170911 214395 205819 204309 420061 770711 369383 260927 230418 225009 218959
Unassigned_MappingQuality 0 0 0 0 0 0 0 0 0 0 0 0
Unassigned_FragmentLength 0 0 0 0 0 0 0 0 0 0 0 0
Unassigned_Chimera 0 0 0 0 0 0 0 0 0 0 0 0
Unassigned_Secondary 0 0 0 0 0 0 0 0 0 0 0 0
Unassigned_Nonjunction 0 0 0 0 0 0 0 0 0 0 0 0
Unassigned_Duplicate 0 0 0 0 0 0 0 0 0 0 0 0
111
TABLE S5: Reads from RNA-seq results for each 11 gene copies of F3’5-H.
Flavonoid 3’5’ hydroxylase
CG
Veraison
RG
Veraison
CG
Ripening
RG
Ripening
Gene 2805 2 50 0 38
Gene 2810 15 91 0 65
Gene 2830 32 214 1 150
Gene 2840 18 186 1 135
Gene 2846 11 59 0 38
Gene 2860 9 68 15 153
Gene 2873 4 38 2 70
Gene 2880 2 50 1 38
Gene 2920 20 154 2 104
Gene 2970 12 106 1 81
Gene 3010 30 182 61 354
112
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CHAPTER IV
OBJECTIVE 3: Evaluate the function of at least one gen of interest, selected
from the above objectives, related with the branching point F3’-H and F3’5’-
H of the anthocyanin biosynthesis pathway.
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INTRODUCTION
In plant species including grapevine, the biosynthesis of cyanidin and delphinidin type
anthocyanins is determined by the activity of two enzymes, flavonoid 3’- and 3’,5’-
hydroxylases (F3’-H and F3’,5’-H) that add either a single hydroxyl group at the 3’ position or
two hydroxyl groups at the 3’ and 5’ positions to dihydrokaempferol and/or dihydroquercetin,
respectively. Once converted into 3’-dihydroquercetin (cyaniding type) or 3’,5’-
dihydromyricetin (delphinidin type), these intermediates flow through common downstream
enzymes to form di-substituted and tri-substituted anthocyanins and also other polyphenols
(flavonols, leucoanthocyanins, catechins, proanthocyanins, tannins) at different developmental
stages (He et al., 2010; Petroni & Tonelli, 2011; Tanaka, 2006). F3’-H and F3’,5’-H genes code
for CYP75 cytochrome P450 hydroxylases that have been well characterized in the ornamental
plants such as Petunia hybrid (Brugliera et al., 1994), Antirrhinum majus (Martin, Prescott,
Mackay, Bartlett, & Vrijlandt, 1991) among others. In ornamental species, F3’H and F3’,5’H
are found as low/medium copy number genes and have been genetically manipulated to produce
commercial new flower pigmentation which naturally do not produce certain types of
anthocyanins. In grapevine, gene copy number of F3’5’-H has increased through recurrent
cycles of duplication, give rise to a single copy gene on chromosome 8 and the others multiplies
copy gene in tandem on chromosome 6 (Falginella et al., 2010).
Cytochrome b5 proteins are components of electron transport systems found in various species
such as DIF-F in petunia flowers, which has been implicated as an alternative electron donor for
F3’5’-H (Hf1) that contributes to efficient accumulation of delphinidin-based anthocyanins (de
Vetten et al., 1999). Ectopic expression of these two genes of petunia, F3’5’H and cytochrome
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b5, in a carnation cultivar that lack F3’5’H activity, resulted in efficient production of
delphinidin-based anthocyanins and subsequent petal color change (Tanaka & Aida, 2009). In
grapevine, it was observed in Syrah cultivar, that expression of the genes encoding these two
enzymes is increase in veraison stage related to the beginning of anthocyanin accumulation in
berry skin and were only identified in pigmented cultivars (Bogs, Ebadi, McDavid, & Robinson,
2006). Taking into account all these observations has been proposed to cytochrome b5 such a
possible a putative modulator of F3’5’-H and may regulate anthocyanin accumulation.
However, its relationship in the anthocyanins accumulation in grapevines has not been
demonstrated. Due to the fact that in RNA-seq results from Objetive 2 we identified 11 gene
copies of F3’5’-H repressed in CG compared to RG, and at the same time we observed Cytb5
repressed, we focus our interest in this gene to demonstrate its relationship anthocyanin
accumulation in grapevines We isolated Cytb5 coding sequence from CG and RG and we
generated a construction to later transform V. berlandieri x V. Rupestris 110R embryos that
overexpressed it. Our results showed that transgenic embryos overexpressing Cytb5 exhibited a
rapid growth and also colored areas where they were placed into light condition, not observed
in transgenic control line. The knowledge of their specific regulation could be important for
future attempts to engineer flavonoid composition in fruit.
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MATERIAL AND METHODS
Binary vector constructions
From the same RNA extraction samples and subsequent cDNA synthesis from Objective 2 used
for qRT-PCR analysis (Santibanez et al., in preparation), were now used for generating binary
vector constructions containing CYTB5 gene. We amplified the coding sequence (CDS) of
CYTB5 (444bp) by PCR from CG and RG during veraison and ripening stages of season 2013.
Pimers used correspond to CYTB5 (ID: VIT_218s0001g09400): F5’-
CACCATGGCTCCCCAAAAAGTTT-3’ and R5’-TTAGGTGGTGAGGTGGGCAGCT-3’.
Also, was used primers of a housekeeping gene actin that amplified 666pb: F5’-
ACCAGAATCCAGCACAATC-3’ and R5’-ATGGCCGATACTGAAGATAT-3’.
PCR reactions were carried out using a T100™ Thermal Cycler (Bio-Rad Laboratories, Inc.) in
a 25 µl reaction mixture (1 µl template cDNA, 2.5 µl PCR Buffer 10X, 1 µl MgCl2 50mM, 0.3
µl DMSO, 0.5 µl each primer 10mM, 0.5 µl dNTPs 10mM, 0.13 µl Taq DNA polymerase
(Invitrogen)). Thermocycling conditions were as follows: one cycle at 95ºC for 7 min; 35 cycles
at 95ªC for 30 secs, 60ºC for 30 secs and 72ºC for 30 secs followed by one cycle at 72ºC for 7
min. Then, were cloned into pENTR™ Directional / SD/D-TOPO® (Invitrogen) using
chemically competent E. coli. Later, positive colonies transformed were used for plasmid
extraction using GeneJET Plasmid Miniprep Kit (Promega) according to manufacturer's
instructions, and then were sequenced. CYTB5-containing vector were recombined into
pK7FWG2 (Karimi, Inzé, & Depicker, 2002) using GatewayTM LR ClonaseTM II Enzyme
Mix (Invitrogen), using chemically competent E. coli and subsequent positive colonies
transformed were used for plasmid extraction using GeneJET Plasmid Miniprep Kit (Promega)
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according to manufacturer's instructions. Two expression vector were generating,
P35S::VvCYTB5::T35S and control of empty pK7FWG2. Each expression vector was
introduced in A. tumefaciens strain GV3101 using electroporation.
Preparation of Agrobacterium culture
An overnight suspension culture of Agrobacterium already transformed with each binary vector
was prepared by inoculating 25 ml of LB (Luria-Bertani) liquid medium supplemented with 50
mg/l kanamycin and 100 mg/l rifampicin in a 125 ml flask with single colony-derived bacteria.
The culture was maintained at 28ºC on an orbital shaker (185 rpm) in dark. When the optical
density at 600 nm (OD600) reached a value of 0.8–1.0, bacterial suspension was transferred into
a sterile 50 ml centrifuge tube and spun down at 6000 rpm for 10 min at room temperature. The
supernatant was removed and 100 ml of liquid G1SCA medium (2.1 g of NITSCH and NITSCH
medium, 0.1 g of myo-inositol, 60 g of sucrose, 2.5 g of activated carbon, 5 g of Gelrite, 1 ml
of Gamborg 1000X vitamins, 1 mL of NOA (stock: 2 mg/ml), 1.66 ml of AIA (stock: 1.66
mg/ml), 0.2 ml of BAP (stock: 2 mg/ml), adjusted to a pH of 5.8 prior to add agar and activated
carbon), but without antibiotics and activated carbon was added to resuspend the cells by gentle
swirling agitation in a flask.
Inoculation of V. berlandieri x V. Rupestris 110R embryos with Agrobacterium tumefaciens
and subsequent maintenance in culture media
From flask containing 100 ml bacterial suspension, 10 ml was added in a sterile 15 ml centrifuge
tube for each binary vector construction, together with 0.5 g embryogenic tissue V. berlandieri
x V. Rupestris 110R and was maintained at 28ºC on an orbital shaker (185 rpm) for 10 min.
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Bacterial suspension was withdrawn using a transfer pipette and any excess moisture was
removed by blotting with sterilized Whatman 3MM filter paper. The transformed embyros were
transferred in to GISCA medium with acetosyringone 200 mM and also with Whatman 3MM
filter paper and were cultivated for two days at 28ºC in dark. Subsequently, these embryos were
transferred into a flask containing G1SCA liquid medium with Timentin (300 mg/ml), to
eliminate the overgrowth of A. tumefaciens, during 3 days at 24ºC in dark. Again, embryos were
withdrawn using a transfer pipette and any excess moisture was removed by blotting with
sterilized Whatman 3MM filter paper. Dried embyros were transferred into GISCA medium
containing Timentin (300 mg/ml) and Kanamycin (50 mg/ml) to prevent the bacteria
overgrowth and transgenic embryo selection, respectively, for 1 month at 24ºC in dark. After
this month, embryos in torpedo stage were transferred into DE medium (2.21 g of MS basal
medium with vitamins, 0.05 g of micronutrients MS, 1 ml of vitamins MS 1000X, 0.1 g of myo-
inositol, 30 g of sucrose, 2.5 g of activated carbon and 5 g of Gelrite, adjusted to a pH of 5.8
prior to add agar and activated carbon) containing Timentin (300 mg/ml) and Kanamycin (50
mg/ml). After 2 months elapsed, the embryos were transferred during approximately 3 months
in GE medium (similar to DE medium but with additional 583μl of IAA hormone (stock: 3
mg/ml) and 350μl of GA3 hormone (stock: 1 mg/ml). When the germinated embryos will be
seedlings, it will be transferred to MS/2 medium (2.21 g of MS basal medium with vitamins,
0.1 g of myo-inositol, 30 g of sucrose, 2.5 g of activated carbon, 8.5 g of agar-agar and 3 ml
PPM).
Photographic record of phenotype of transgenic and control embryos
131
Each month of embryo subculture a photographic record was made using Camera Powershot
G16 Canon.
132
RESULTS
Generating constructions overexpressing CYTB5
For CYTB5 cloning in a binary destination vector, first was done PCR amplification from
cDNAs form CG and RG during veraison and ripening stages (FIGURE 7). From all samples
was obtained fragments of 444pb, which were cloned into pENTR™ Directional / SD/D-
TOPO®, and each reaction was used for chemically competent E. coli transformation and
subsequent plasmid extraction. A new fragment of 444pb were amplified by PCR from this
extraction (FIGURE 8) confirming that were cloned. CYTB5 cloned in TOPO-SD were
analyzed by sequencing and we observed that CG and RG presented the same sequence
described in database (data no show), suggesting that repression in gene expression of CYTB5
observed in RNA-seq and qRT-PCR results from objective 2, were not due to point mutations
in the CYTB5 coding sequence. Understanding the importance of demonstrate the relationship
of CYTB5 in the anthocyanin biosynthesis pathway in grapevine, an RG sequence was selected
for the following experiments. Then, a recombination reaction was done using GatewayTM LR
ClonaseTM II Enzyme Mix for obtain final expression vector pK7FWG2 (Karimi et al., 2002).
Recombination was checked with, a new CTYB5 PCR amplification was done (FIGURE 9A)
confirming a correct reaction. The next step was transform A. tumefaciens with final vector
P35S::VvCYTB5::T35S and control of empty pK7FWG2 using electroporation method. PCR
amplification of CYTB5 was performed in bacterial suspension for check positive colonies
(FIGURE 9B) and then it was done the plasmid extraction. Finally, a co-culture between A.
tumefaciens and V. berlandieri x V. Rupestris 110R embryos was performed.
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FIGURE 7: CYTB5 amplification from cDNA using of CG and RG berries skin during
veraison and ripening stages. PCR reaction was done using specific forward primer containing
the sequence 5’CACC. Housekeeping corresponded to auxin gene and molecular marker
corresponded to 1 Kb Plus DNA Ladder.
Transgenic embryos overexpressing CTYB5
Transformed embryos were transferred into G1SCA medium for one month in dark at 24ºC,
containing kanamycin 50 mg/ml and timentin 300 mg/ml for transgenic embryos selection and
avoid overgrowth of A. tumefaciens used for transformation. It was observed a rapid grown in
transgenic embryos overexpressing CTYB5 compared to transgenic control condition (FIGURA
10A and 10B, respectively). Embryos that were in torpedo state were transferred into DE
medium for generate differentiation in them, and also DE medium contained antibiotic selection
for transgenic embryos and overgrowth of bacteria.
FIGURE 8: CYTB5 amplification from TOPO-SD of CG and RG berries skin during
veraison and ripening stages. PCR reaction was done using specific forward primer containing
the sequence 5’CACC. Molecular marker corresponded to 1 Kb Plus DNA Ladder.
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After two more months in this medium (during each month the culture medium was renewed)
fulfilling 3 months in total of cultivation, another rapid growth was observed in transgenic
embryos compared to control (FIGURA 10C and 10D, respectively), where embryos
overexpressing CTYB5 were more differentiated. Also, in these embryos was observed
developed structures such the cotyledons. These differentiated embryos therefore, were
transferred into GE medium, similar to DE medium but containing growth hormones, auxin and
gibberellic acid, and were placed in light at 24ºC for generate germination in them. After one
month in GE medium, fulfilling 4 months in total of cultivation, transgenic embryos
overexpressing CYTB5 exhibited pigmented areas of red color indicated in yellow arrows and
also cotyledons more developed compared with control embryos that still remained in dark in
DE medium in that month (FIGURE 10E, 10F and 10G, respectively).
Finally, after another month in GE medium, fulfilling 5 months in total of cultivation, these
transgenic embryos overexpressing CYTB5 exhibited the same red color demonstrated that was
stable for 2 months, and also covering more areas along the embryo (FIGURE 10H). In
addition, it was observed that cotyledons started to produce chlorophyll due to green color
observed in them. The transgenic control embryos were passed to light conditions only in this
fifth month, showing a much slower growth (FIGURE 10I).
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FIGURE 9: CYTB5 amplification of TOPO-SD recombination with binary destination
vector pK7FWG2 and final vector construction. (A) PCR amplification from recombination
reaction in E. coli. (B) PCR amplification in final expression vector in A. tu
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FIGURE 10: Phenotype observed in transformed embryos during 5 months in media
culture. (A) and (B) Transgenic embryos overexpressing CYTB5 gene and transgenic control
embryos transformed with empty pK7FWG2, respectively. Correspond to the first month in
GISCA medium in dark at 24ºC. (C) and (D) Transgenic embryos overexpressing CYTB5 gene
and transgenic control embryos transformed with empty pK7FWG2, respectively. Correspond
to third month in DE medium in dark at 24ºC. (E) and (F) Transgenic embryos overexpressing
CYTB5 gene placed in light for one month in GE medium. Yellow arrows indicate embryos
areas generating reddish coloration. (G) transgenic control embryos transformed with empty
pK7FWG2 in dark condition in DE medium. (H) Transgenic embryos overexpressing CYTB5
gene placed in light for two months in GE medium. Yellow arrows indicate embryos areas
generating reddish coloration. (I) Transgenic control embryos transformed with empty
pK7FWG2 in dark in DE medium. All embryos transformed correspond to V. berlandieri x V.
Rupestris 110R.
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DISCUSSION AND CONCLUSION
F3’-H and F3’5’-H genes have been intensively studied in flowers of various plant species (e.g.
carnation, petunia, or rose) due to the anthocyanin hydroxylation importance in determining
flower color shades. In petunia, it had been demonstrated that Cytb5 is required for full activity
of F3’5’-H, in order to produce the anthocyanins of the delphinidin type (de Vetten et al., 1999).
In grapevines, only it had been demonstrated that Cytb5 from Syrah variety, was expressing
after veraison stage at the same time with F3’5’-H and both genes were presented in red berry
skin varieties (Bogs et al., 2006). However, there is no evidence to suggest the participation of
this gene in the accumulation of anthocyanins in grapevines. Knowing that grapevine
embryogenic cultures have been for a long time as the targets of choice for inserting genes of
interest, since single cells on their surface can be prompted to develop into complete plants; and
in addition, that gene insertion into such totipotent cells results in grape plants that stably express
the required characteristic (Dhekney, Li, Grant, & Gray, 2016), we were interested in correlate
the expression of this gene with anthocyanins accumulation. The aim of this work was to isolate
Cytb5 CDS from RG and observed its participation in this process in grapevines. Here we
provide evidence suggestin that Cytb5 from RG (ID: VIT_218s0001g09400) can produced
coloration in transgenic embryos that normally do not produce it (FIGURE 10E, 10F and 10H).
Reporter genes have long proved useful for distinguishing transformed from untransformed cell
tissues or embryos and optimizing transformation protocols through expression quantification
(Matthews, Saunders, Gebhardt, Lin, & Koehler, 1995). Typical examples of reporter genes
correspond to the uidA gene (GUS) as well as fluorescent proteins (GFP or RFP) (Vidal et al.,
2010). In addition, expression of MYBA1 in somatic embryos in Thompson seedless variety
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allowed to observed visible anthocyanin accumulation and it was demonstrated the potential of
this gene as a reporter gene for transient expression assays (Li, Dhekney, & Gray, 2011). A
similar result was observed in our experiment, suggesting that Cytb5 could be used such a new
reporter gene, due to the capacity to generate color in V. berlandieri x V. Rupestris 110R
embryos. However, we need more experiments to evaluate the gene stability during plant
development, if it produces normal plants and also considering that the use of these genes that
produced coloration, is restricted to experiments for which this phenomenon does not interfere
with the expression of investigated genes or pathways.
Overexpression experiments are very helpful approaches in learning about a gene of unknown
function. In our case, we fusion Cytb5 coding sequence with the strong constitutive promoter
cauliflower mosaic virus 35S (CaMV35S), a common promoter used for plant transformation
(Hull et al., 2017). However, the main disadvantage of this approach is that the gene product is
synthesized in excessive amounts, and this can be in tissues where it is not usually present (Jelly,
Valat, Walter, & Maillot, 2014). That is a possible explanation for our case with Cytb5, since
normally grapevine embryos do not produce pigments during their development to plants, and
this gene altered that process, a similar effect produced by Myba1.
It was observed that transgenic embryos overexpressing Cytb5 exhibited a rapid growth
compared with transgenic control (FIGURE 10H compare to 10I for example). F3’-H and
F3’5’-H are cytochrome P450 proteins whose activities are dependent on associated proteins,
such as cytochrome b5, catalyzing the transfer of electrons to their prosthetic heme group
(Vergeres and Waskell, 1995). It could be possible that the overexpression of it in grapevine
embryos would be improving the transfer of electrons in both cytochromes P450, as well as in
other reactions in which they could participate that still remains unknown. However, we need
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more evidence for understand this process and wait for complete developed plants to compare
to control.
In conclusion, with these previous results in evaluation of gene expression of Cytb5, we may
suggest that for a first time this cytochrome from grapevine was involved in its participation in
anthocyanin accumulation and also may contribute in accelerated growth metabolism, but it is
only part of a speculation and therefore, several tests are needed to prove this.
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CHAPTER V
GENERAL DISCUSSION
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In this work grapevine ripening were studied at anthocyanin accumulation level related to
flavonoid biosynthesis, according to understand changes in transcripts and also metabolites in a
contrasted model of grapevine varieties, CG and RG. In first instance, study model could not be
explained in the traditional way related to changes in color berry skin, which is through genetic
changes in the MYBA1 transcription factor. We showed that anthocyanin biosynthesis was
altered at different level of structural genes codifying enzymes of this pathway according with
RNA-seq results, with particular interest in down-regulation of 11 multicopies of F3’5’-H in
CG variety during veraison and ripening stages of berry development. F3’-H together with
F3’5’-H are part of the branching point in flavonoid biosynthesis where both use as a substrate
dihydrokaempferol to produce 3’-dihydroquercetin (cyaniding type anthocyanin) or 3’,5’-
dihydromyricetin (delphinidin type anthocyanin), becoming a key step in the pathway to
produce the type of anthocyanins in the grapevines.
Metabolic changes between contrasted CG and RG varieties
The spontaneous generation of this new CG variety in the field, from a branch of the RG variety,
led us to wonder ourselves what was happening at metabolic level and then relate this to the
transcriptomic changes to be perform later. First, we performed a comparative metabolic
analysis of skin at two developmental stages, veraison and ripening, between CG and RG in two
seasons, 2013 and 2014. We evaluated primary metabolites, phosphorylated intermediates,
organic acids, trehalose 6-phosphate, hexoses and anthocyanins. The selection of veraison and
ripening stages was done due their relevance in the establishment of beginning and end of
anthocyanin accumulation in grape (Kennedy, 2002). We observed with PCA analysis that
developmental stages were the primary discriminant parameters in the comparative study when
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PC1 was analyzed (Chapter 3). This observation is concordant with previous published studies
(Guo et al., 2016; Massonnet et al., 2017; Wong, Lopez Gutierrez, Dimopoulos, Gambetta, &
Castellarin, 2016; Wu et al., 2014) in which, despite the use of different varieties, the importance
of developmental stages appeared to overrule variety origin. The metabolites that explained el
43.75% of data variance due to veraison and ripening stages, some of importance appeared in
this process as T6P, a sugar phosphate considered as a metabolic signaling molecule in plants;
which regulates developmental growth (Lunn, Delorge, Figueroa, Van Dijck, & Stitt, 2014). In
vitis vinifera there is no clear evidence of control of T6P during berry development, although
several transcriptomic studies have shown that orthologue genes encoding T6P synthase, T6P
synthase/phosphatase and SnRK1 (Sucrose-non-fermentative Related Kinase 1, another sugar
signaling pathway component) are tightly regulated during berry development (Deluc et al.,
2007; Gambetta, Matthews, Shaghasi, McElrone, & Castellarin, 2010). Anyway, find T6P in
our results, may suggest its participation in grape berry development, focused on skin, an
approach that had not been observed (Dai et al., 2013).
Another metabolites involved in explained developmental stages between CG and RG were
shikimate and phenylalanine (Chapter 3) which have relation with general phenylpropanoid
pathway where is part flavonoid biosynthesis (Petrussa et al., 2013). Shikimate correspond to
an intermediary in shimikate pathway that mediates the carbon flow from carbohydrate
metabolism to phenylpropanoid and aromatic compound biosynthesis (Maeda & Dudareva,
2012; Zhang et al., 2012). On the other hand, phenylpropanoid biosynthesis begins with the
amino acid phenylalanine, which is a product of the shikimate pathway (Maeda & Dudareva,
2012), therefore, these two metabolites are directly linked (Tohge, Watanabe, Hoefgen, &
Fernie, 2013). In addition, phenylalanine content was higher in CG compared to RG during
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veraison and ripening stages, maybe involved in alteration that caused color change in our study
model.
According to anthocyanin content, it was observed extreme differences between CG and RG
associated to total content and type of anthocyanins accumulated (Chapter 3). We observed that
CG contained less content of total anthocyanin compared to RG and also did not contain
delphinid-type anthocyanins, catalyzed by one of the two enzymes belong to branching point in
anthocyanin biosynthesis pathway, F3’5’-H (He et al., 2010). Taken together, these observations
suggest that according to our results, the anthocyanin biosynthesis pathway is affected.
Accordingly, an increase in F3’5’H activity have been previously reported to explain higher
concentration of tri-hydroxylated anthocyanins in Cabernet Sauvignon and Shiraz, pointing
F3’5’H plays as a key molecular player driving the phenylpropanoid metabolic flux towards
bluish anthocyanins production (Degu et al., 2014), but these will be discussed latter in RNA-
seq results.
In addition, there is another important point to analyze, that could also explain the difference in
anthocyanins between varieties. This refers to results in the PCA analysis, specifically related
to Component 2 that explained the differences between CG and RG. The main metabolites
involved were related to the sucrose biosynthesis pathway. It is known from literature that
increasing concentrations of glucose and fructose increase the accumulation of anthocyanins in
grapevine cell cultures (Larronde et al., 1998). Therefore, according to our results, we realize
that the lower number of intermediaries in CG towards the sucrose biosynthesis, could have an
implication at the final level of anthocyanins in CG. Or it could even be a consequence of the
change or blockage that is occurring in the pathway of anthocyanin biosynthesis in CG through
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F3’5’-H. At least the question of what was happening is raised, and more studies would be
needed, for example, with applications with sugars in the CG variety and observe the results.
Transcriptomic changes between contrasted CG and RG varieties
Color mutations in grape berry skin are relatively frequent events and can be easily seen in the
vineyard. Most studies investigating the mechanisms of changes in berry skin color explain them
through the regulatory effect of MybA transcription factors, whose genes are closely clustered
in a single locus on chromosome 2, referred to as the “berry color locus” (Kobayashi, Goto-
Yamamoto, & Hirochika, 2004; Walker, Lee, & Robinson, 2006). One of these is the
transcription factor MybA1, a major determinant of grape berry anthocyanin biosynthesis
pathway (Kobayashi et al., 2004; Lijavetzky et al., 2006). MybA1 transcriptionally regulates
UFGT gene, which glycosylates anthocyanidins, stabilizing them and therefore responsible for
the anthocyanins accumulation in grape berry skins (Ford, Boss, & Hoj, 1998). White skinned
grape varieties that do not produce anthocyanins, contain a mutated allele of MYBA1 in which
the presence of the retrotransposon Gret1 in promoter region interrupts the normal transcription
process, resulting in a white berry allele (Kobayashi et al., 2004). Pigmented cultivars possess
at least one allele of the MybA1 locus that does not contain Gret1 and is called red berry allele
(This, Lacombe, Cadle-Davidson, & Owens, 2007). In the case of pink skin berry varieties of
oriental origin, a different situation was reported: a 33bp insertion was found in the second
intron of MybA1, generating low expression levels of MybA1 in grape from these cultivars
(Shimazaki, Fujita, Kobayashi, & Suzuki, 2011). Another different example is a bud sports of
Cabernet Sauvignon called “Malian” with red-grey berry skin, associated to the deletion of a
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large region of chromosome 2 that includes both MybA1 and MybA2 genes (MybA2 is located
adjacent to MybA1 in the color berry locus) (Walker et al., 2006). In addition, light-red-skinned
‘Ruby Okuyama’ and more intense and uniform rosy-skinned ‘Benitaka’ correspond to bud
sports of white-skinned ‘Italia’ (Azuma et al., 2009). ‘Ruby Okuyama’ was caused by the
recovery of VvmybA1 expression and ‘Benitaka’ due to presence of a novel VvmybA1BEN allele
that restored VvmybA1 transcripts (Azuma et al., 2009). In our results, we reported that UFGT
gene (VIT_216s0039g02230) was repressed in CG at veraison and ripening stages, which could
suggest an alteration in MybA1 expression, according to literature describe above presented.
MybA1, however, was not deemed significantly affected in DEGs analysis. Nevertheless, we
decided to check for the presence of white and red allele in CG and RG, using the same set of
primer used in (Shimazaki et al., 2011), observing that both varieties are heterozygous for this
gene and that our results cannot be explained by structural changes in MybA1 DNA sequence.
This observation therefore led us to ask ourselves what was going on in CG variety and to be
able to answer this question we decided to carry out an analysis of global expression to in some
way “take a picture” of the genes that are being expressed in CG to be then compared with RG
and thus be able to arrive at a possible explanation of the change of coloration in berry skin.
We performed a skin transcriptome analysis using the same samples for metabolic analysis, at
the same two developmental stages using Illumina Hiseq2000 in these two near isogenic
varieties, RG and CG, in 2013 season. According to DEGs analysis, we reported that several
genes related to flavonoid pathway, which grouped in three main clusters of repression,
induction or a combination of both, were differentially expressed in CG compared to RG
(Chapter 3). The most interesting observation was that we found that 11 copies of F3’5’-H,
encoding a key enzyme in anthocyanin biosynthesis pathway were repressed in CG. This
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enzyme corresponds branching point of the flavonoid pathway, where dihydrokaempferol is the
substrate shared in common (Tanaka, 2006). Dihydrokaempferol is converted via F3’5’-H to
dihydromyricetin, producing the glucoside, acetyl, coumaroyl and caffeoyl forms of
delphinidin, petunidin and malvidin, that correspond to the delphinidin-type (tri-hydroxylated)
anthocyanins of bluish color. Dihydrokaempferol is also used by F3’-H to produce
dihydroquercetin and gives rise to the biosynthesis of glucoside, acetyl and coumaroyl forms of
cyanidin and peonidin, that correspond to cyanidin-type (di-hydroxylated) anthocyanins or
reddish color (Degu et al., 2014). Indeed, this result allowed us to connect directly previously
metabolic results related to anthocyanin content, where CG berry skins did not contain
delphindin-type anthocyanins and therefore, is consistent with the repression of 11 gene copies
of F3’5’-H. In addition, another important observation was the adjacent location of each of these
copies in a single cluster within chromosome 6. It is known that there are many copies of F3’5’-
H in Vitis vinifera, and it was shown that F3’5’-H in grapevine are highly redundant and
predominately expressed in berry skins (Castellarin et al., 2006; Falginella et al., 2010).
However, to the best of our knowledge, there was no evidence of the repression of such high
number of gene copies at the same time in a single variety. This raises the question of how the
repression of the F3’5’-H genes occur in CG. At least two mechanisms can be predicted, among
others. Firstly, since all these genes are physically close together in a single region of
chromosome 6, an epigenetic can be postulated. Although left uninvestigated until recently, the
field of epigenetic regulation in grapevine has gain considerable attention in the last years; and
epigenetic regulation were shown to be potentially part of the so-called “terroir” effect in Shiraz
grapes from Australia (Xie et al., 2017). Secondly, a still to be discovered master regulator (i.e
a transcription factor) might be defective in the CG. For example, an identical transcription
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factor binding motif might be present in the promoter regions of all 11 gene copies, which would
be in agreement with the hypothesis of a master regulator for these F3’5’-H genes. One possible
finding that could validate this theory is that all the hydroxylases under study are integrated into
the cytoplasmic membrane (data not showed, using UniProt) (Bateman et al., 2017), which
makes sense because anthocyanin are synthesized in the cytosol of the plant cell, and then they
are finally translocated in the vacuole for storage. And therefore, the regulation of all could be
given at the level of a master regulator since all hydroxylases would be fulfilling the same
function, in the same place in the cell. However, further work is necessary to decide between
these two hypotheses.
In parallel, several genes putatively involved in anthocyanin transport to the vacuoles were
differentially expressed in CG compared to RG at both veraison and ripening stages, such as
GST13 and ABC transporter proteins, which expression was down-regulated at both
developmental stages in CG. Several studies in Arabidopsis had been reported the ability of
GST1 and GST4 to rescue the phenotype of the Arabidopsis tt19-1 mutant, resulting in a
restoration of transport of anthocyanins and proanthocyanidins at least with GST4 (Pérez-Díaz,
Madrid-Espinoza, Salinas-Cornejo, González-Villanueva, & Ruiz-Lara, 2016). ABC
transporters have also been involved in vacuolar sequestration of flavonoids in maize
(Goodman, Casati, & Walbot, 2004) and in the transport of malvidin 3-O-glucoside into
vacuoles, in a strictly GSH-dependent fashion in grapevine (Zarrouk et al., 2013). These
findings reported in our study might suggest a dynamic process in transport and sequestration
of anthocyanin into vacuoles occurring in this variety context. Also, it would be interesting to
related if this alteration in anthocyanin transportation was caused by previous variation in
branching point related to F3’5’-H downregulated in CG variety. Anyways, due to these genes
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have not been characterized in grapevines, they become in new possible candidates to study for
better understanding anthocyanin transport.
Another key genes found to be down-regulated and up-regulated in CG were CHS that encodes
the first enzyme of the flavonoid pathway (Petrussa et al., 2013; Wang et al., 2016) and STS,
the early enzyme in flavonoid metabolism and involved in resveratrol biosynthesis (Dubrovina
& Kiselev, 2017; Kiselev, Aleynova, Grigorchuk, & Dubrovina, 2016).
This possibly reveals a possible compensation point in flavonoid metabolism: CHS utilizes the
same substrates as STS but, is responsible for flavonoid-type compound formation. Thus, STS
would act as an escape valve to absorb the excess of substrate flux generated in the upper part
of the flavonoid metabolism because of the decrease in CHS activity. Taken together, this
suggests a multilevel alteration of the flavonoid pathway, and not only of its tri-hydroxylated
branch in CG variety.
The only transcription factor found in our DEGs results was MYB24 (VIT_214s0066g01090)
which was induced in CG. Recently, it was reported that MYB24 was induced by solar UV
radiation in Tempranillo berry skins, in conjunction with terpenoid metabolism induction
(Carbonell-Bejerano et al., 2014), and also was associated with terpene biosynthesis in berries
in response to drought (Savoi et al., 2016) and highly co-expressed with flower and fruit specific
terpene synthase (TPS) (Wong et al., 2016). So maybe MYB24 in CG activated terpene
biosynthesis pathway, theory that could be valid due to the induction of the sesquiterpene
synthase 10 (TPS10) in CG, observed in DEGS results (Chapter 3). In addition, in personal
communication with Tomás Matus, a recognized specialist in the MYB type transcription
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factors in grapevine, told us about his work not yet published with MYB24. In a novel grape
mutant that had half red and half white berries, MYB24 was induced in half white part, and also
MYB24 can activate a terpene synthase, passing terpenes to fulfill a more photoprotective
function (such as anthocyanins) than aromatic function. Therefore, we think that what could be
happening in CG, is a change in the flow of the types of secondary metabolites synthesized,
where MYB24 would be taking this flow towards the synthesis of terpenes in CG and not
towards anthocyanins. Consequently, the terpenes would be supplying the photoprotective
function in CG, making this variety successful despite having a more attenuated color compared
to RG.
Finally, a gene that had not been previously functionally characterized in grapevines, and only
had been extrapolated its function in the anthocyanin biosynthesis according to observations in
ornamental plants, corresponds to Cytb5, another gene repressed in CG during veraison and
ripening stage (Chapter 3). Cytb5 has previously been involved in F3’5’-H activity modulation
in Petunia flowers, catalyzing the transfer of electrons to their prosthetic heme group (de Vetten
et al., 1999). In 2006, Bogs et al., demonstrated that a putative Cytb5 from grapevine cv Shiraz
modulated both F3’5’-H and F3’-H activities even though the exact mechanism remains to be
deciphered. Due to its participation in modulate F3’5’-H activity, we suspect that can exist a
possible link between the drastic down-regulation of 11 gene copies of F3’5’H and the
repression of the Cytb5 observed in CG. To demonstrate the relationship between Cybt5 in
anthocyanin accumulation we decided to overexpressed in V. berlandieri x V. Rupestris 110R
embryos, due to the fact that grapevine embryogenic cultures are very useful in study a particular
gene of interest (Dhekney et al., 2016). We observed that transgenic embryos overexpressing
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Cytb5 exhibited coloration since they were placed into light conditions and in addition, was
observed a rapid growth of them compared to transgenic embryo control (FIGURE 10). We
proposed that Cytb5 could be used as reporter gene as MYBA1, but it need a longer follow-up
of the plants to really postulate it as a new candidate in genetic engineering in grapevines.
As summary, in FIGURE 11, the main events at the genetic level in CG are presented in a
simplified overview. In general, there is a decrease in the induction of several genes in the
anthocyanin biosynthesis pathway and also, activation of genes that drive the carbon flux to
other branches of the phenylpropanoid pathway, such as the induction of the COMT, goes
towards the biosynthesis of lignin; TPS10, which goes towards the biosynthesis of
sesquiterpenes; STS, with combined induction and repression dependent on the development
stage; and MYB24, previously characterized in induction works towards the biosynthesis of
terpenes . Therefore, we believe that in CG what could be happening is a switch in the carbon
flux from the anthocyanins to the biosynthesis of other secondary metabolites, thus supplying
in some way the photoprotective function of these compounds, since by containing less amount
of anthocyanins, and at the same time only contains dihydroxylated anthocyanins, could be a
variety without evolutionary success, but it not the case. And this change in the flow could be
led by MYB24, however, we know that it could also be an opposite effect, in which the
activation of these other secondary metabolites could have repressed the biosynthesis of
trihydroxylated anthocyanins, generating the phenotype observed in CG. Both theories could be
correct, and to test them, several experiments are needed to prove which one is appropriate to
the phenotype in CG.
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FIGURE 11: General scheme proposed for Chimenti Globe variety. Simplified overview of
biosynthesis of flavonols, lignins, stilbenes, anthocyanins that belong to phenylpropanoid
pathway and terpenes, and also show the dynamics that would be occurring in CG at molecular
level. According to DEGS results, many genes were not induced (cyan color circles) and induced
(yellow color circles) in CG during veraison and ripening stages. In the case of STS, this gene
was not induced in veraison and induced during ripening stage. The final result was that
trihydroxylated anthocyanins such as delphinidin 3-glucoside, petunidin 3-glucoside and
malvidin 3-glucoside were not detected in CG, pathway branch leading by F3'5'H enzyme.
Therefore, the phenotype observed in CG would be given only by dihydroxylated anthocyanins,
cyanidin 3-glucoside and peonidin 3-glucoside leading by F3’H enzyme (showed in the scheme
enclosed with a pink rectangle). Terpene pathway is an additional route showed here, because
in CG were induced MYB24 and TPS10, related to activating terpene biosynthesis.
Abbreviations: PAL, Phenylalanine Ammonia Lyase; C4H, Cinnamate 4-Hydroxylase; 4CL, 4-
Coumaryol CoA Ligase; CHS, Chalcone Synthase; CHI, Chalcone Isomerase; F3H, Flavanone
3-Hydroxylase; F3’H, Flavonoid 3’-Hydroxylase; F3’5’H, Flavonoid 3’5’-Hydroxylase;
CYTB5, Cytochrome b5; FLS, Flavonol Synthase; DFR, Dihydroflavonol-4-reductase; LDOX,
Leucoanthocyanidin dioxygenase; UFGT, UDP glucose:flavonoid-3-O-glucosyltransferase;
OMT, O-Methyltransferase; GST13, Glutathione S-Transferase 13, ABC TRANSPORTER,
ATP-binding cassette transporter; TPS10, Sesquiterpene Synthase 10; DMAPP, Dimethylallyl
Pyrophosphate; IPP, Isopentenyl Pyrophosphate; COMT, Catechol-O-Methyltransferase; STS,
Stilbene Sythase.
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Impact of the study model in agriculture
In general, berry color is a fundamental attribute for the quality of wine and in particular in table
grapes, for the quality of the fruit to be consumed. In table grapes, most of the commercial
varieties that now exist are white or black, only a few correspond to pink varieties, as is the case
of the Chimenti Globe characterized in this thesis.
The red berry trait is a very desired attribute in the fruit industry. It is common that the grape
berry is harvested before reaching maturity in order to have this desired characteristic, despite
not having all the organoleptic properties, suitable for consumption. From this point of view, it
is of great relevance to contribute with the pertinent scientific information to explain the
generation of color in grapevines and to suggest to genetic improvement programs the type of
genes that should be repressed or induced, if it want to obtain varieties with these characteristics.
In this sense a rapid procedure, without the need to wait for successive fructifications in the field
(from 3 to 4 years approximately) until the color stabilizes, could be to perform an RT-PCR for
the expression of F3'-H and F3'5 'H and based on its expression predict the type of anthocyanins
that will accumulate and the color that the grapes would have. Possibly the incorporation of this
variety found in the field, Chimenti Globe, in genetic improvement programs allows to transfer
these characteristics to new varieties of table grapes to achieve the desired colors. This would
be a very relevant attribute for genetic improvement programs in the world and especially for
breeding programs in Chile, given the potential of Chile as a main exporter of table grapes.
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In addition, the finding that the type and concentration of anthocyanins in the grape berry could
be modulated through the differential expression of F3'-H and F3'5'-H, led us to believe that it
could be extrapolated to other fruits characterized by accumulating anthocyanins as is the case
of cherry (Prunus avium), raspberry (Rubus idaeus), strawberry (Fragaria x ananassa),
blackberry (Rubus spp.) and blueberry (Vaccinium corymbosum) and even native Chilean
berries such as Maqui (Aristotelia chilensis), calafate (Berberis microphylla) or murta (Ugni
molinae). And this would allow to modify these existing varieties to generate new colors, being
a great scientific contribution towards the biotechnological development of new commercial
fruit varieties
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CHAPTER VI
CONCLUSION
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This comparative study revealed important differences at the metabolic and transcriptomic level
between two contrasted color berry skin, but nevertheless near isogenic, table grapes. It was
possible to link the phenotype observed to an alteration in branching point of flavonoid pathway
that involved two cytochrome P450, F3’-H and F3’5’-H. Using RNA-seq we showed that
flavonoid metabolism was affected at different levels in CG compared to RG at both veraison
and ripening stage. While the nature of the mutation responsible for color changes in berry skin
of CG from its progenitor RG remains unknown, the comparative RNA profiling of the berry
development between CG and RG showed the deregulation of genes directly related to
anthocyanin biosynthesis: CHS, STS, Cytb5, UFGT, MYB24, F3’5’-H, F3’-H, CCoAOMT,
COMT, GST13, ABC transporter and ANP-like. A prominent result was the drastic down-
regulation of 11 gene copies of F3’5’H in CG positioned in a single cluster in chromosome 6,
which was totally consistent with the anthocyanin content observed in CG, with the absence of
delphinidin-type (tri-hydroxylated) anthocyanins. This left in evidence an arrangement
occurring in this metabolic process in CG compared to RG and open new types of regulation
undescribed in anthocyanin biosynthesis in grapevines, independent to MYBA1 alterations
previously described in other changes of color berry skin in grapevines. In addition, with
preliminary results observed in coloration in grapevine embryos due to overexpression of Cytb5,
we may suggest that for a first time this cytochrome from grapevine could be involved in its
participation in anthocyanin accumulation and regulating F3’5’-H activity as previously
demonstrated in ornamental flowers.
This study provides new insights in the genetic and metabolic background that may be
responsible of color change in CG compared to RG, that constitute a relevant biological material
158
to study in depth the mechanisms underlying berry skin color intensity. In addition, provides
new possible candidates to study in depth, as CYTB5 and MYB24, directly related with
anthocyanin accumulation in berry skin, being able to use them in generate changes in color
berry skin of new varieties, or even extrapolate their function in other species.
159
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