Correction: 12 July 2012

www.sciencemag.org/cgi/content/full/336/6089/1711/DC1

Supplementary Material for

Uniform ripening Encodes a Golden 2-like Transcription Factor Regulating Tomato Fruit Chloroplast Development

Ann L.T. Powell,* Cuong V. Nguyen, Theresa Hill, KaLai Lam Cheng, Rosa Figueroa-Balderas, Hakan Aktas, Hamid Ashrafi, Clara Pons, Rafael Fernández-Muñoz, Ariel Vicente, Javier Lopez-Baltazar, Cornelius S. Barry, Yongsheng Liu, Roger Chetelat,

Antonio Granell, Allen Van Deynze, James J. Giovannoni,* Alan B. Bennett

*To whom correspondence should be sent. E-mail: alpowell@ucdavis.edu (A.L.T.P.); jjg33@cornell.edu

(J.J.G.)

Published 29 June 2012, Science 336, 1711 (2012) DOI: 10.1126/science.1222218

This PDF file includes:

Materials and Methods

Figs. S1 to S8

Tables S1 to S3, S7, and S8

References (32–58) Other Supplementary Material for this manuscript includes the following: (available at www.sciencemag.org/cgi/content/full/336/6089/1711/DC1)

Tables S4 to S6 as a separate Excel file Correction: On page 2, the accession number of Ailsa craig was corrected to “LA2838A” on the third line of Materials and Methods.

2

Materials and Methods MATERIALS AND METHODS Plant materials, fruit staging and harvesting. Tomato plants (Solanum sps.) were

grown in fields and greenhouses in Davis, CA and Ithaca, NY, USA and Malaga, Spain.

The S. lycopersicum varieties ‘Ailsa Craig’ (LA2838A), the monogenic u mutant of

‘Ailsa Craig’ called ‘Craigella’ (LA3247) (36), ‘Castlemart’, ‘Fireball’, ‘E6203’ and

‘M82’ (LA3475) were germinated from stock seed collections (Tomato Genetics

Resource Center, UC Davis), transplanted and grown in two gallon pots in greenhouses

or in furrow irrigated fields. Seed for S. lycopersicum var. cerasiforme (PI114490) was

provided by A. Van Deynze and SolCAP and grown in greenhouses in Davis, CA, USA.

Seed for lines, N93 (u/u) and 73X (U/U), T91 (U/U) were provided by Hanoi University

of Agriculture, Viet Nam and grown in Ithaca, NY, USA. S. pennellii (LA0716), IL10-1

(LA4087), IL10-1-1 (LA4088) and IL10-2 (LA4089) lines from the S. lycopersicum

(‘M82’) x S. pennellii IL population were grown in greenhouses in Davis and Ithaca. The

BC2S1 and RIL S. lycopersicum (‘Moneymaker’) x S. pimpinellifolium (‘TO-937’)

population and additional IL10-1, IL10-1-1, IL10-2, and LA0716 plants were grown in

plastic greenhouses in Malaga, Spain. The ‘Cuatomate’ landrace was provided by J.

Lopez-Baltazar, Oaxaca, Mexico, and grown in greenhouses in Davis. Transgenic tomato

(S. lycopersicum cv. ‘T63’) lines expressing the transcription factors AtGLK1

(At2g20570) or AtGLK2 (At5g44190) regulated by the CaMV35S (p35S), the

Arabidopsis lipid transfer protein (pLTP) (37), the tomato rubisco small subunit 3b

(pRbcS) (38, 39) or the tomato phytoene desaturase (pPDS) (38, 40) promoters were

provided by Mendel Biotech. Inc., Hayward, CA, USA and Seminis Vegetable Seeds-

3

Monsanto, Woodland, CA, USA. The AtGLK expressing lines were obtained by crossing

two types of parental transgenic tomato lines. In the cross, one parent line was

transformed using Agrobacterium tumefaciens with a TDNA construct containing either

the AtGLK1 or the AtGLK2 coding sequence fused to the E. coli LexA operator binding

site (LexA:AtGLK1, LexA:AtGLK2) and the CaMV35S regulated sulfonamide selectable

marker (dihydropteroate synthase, SULII). The second parental lines used in the crosses

were transformed with the LexA-Gal4 activation domain coding sequence linked to the

CaMV35S, LTP, RbcS or PDS promoters (p35S:LexA-Gal4, pLTP:LexA-Gal4,

pRbcS:LexA-Gal4, pPDS:LexA-Gal4) and the CaMV35S regulated GFP and the

CaMV35S regulated kanamycin (NPTII) as a selectable marker (41). The LexA:GLK1 and

LexA:GLK2 parental lines were each crossed with each of the four promoter containing

parental lines, p35S:LexA-Gal4, pLTP:LexA-Gal4, pRbcS:LexA-Gal4, pPDS:LexA-Gal4

and progeny contained a promoter, the LexA-Gal4, an AtGLK, a CaMV35::GFP and both

selectable markers. Therefore, AtGLK1 and AtGLK2 were each expressed with four

different promoters in four lines. In the progeny, the two transgenic constructs in each

line were selected by their resistance to kanamycin and sulfonamide. The identity of the

transgenic constructs in each line was confirmed by PCR amplification of genomic DNA

using primers for the selectable markers and primers for each promoter with a LexA

reverse primer and gene specific primers for each transcription factor and the selectable

marker genes (Table S7). The transgenic lines were screened for plant and fruit

phenotypes by comparisons to ‘T63’ lines expressing only the CaMV35S promoter

construct (TControl) with no AtGLK sequences and non-transformed ‘T63’. The

genotype of the ‘T63’ cultivar is u/u and no plant or fruit phenotype differences were

4

observed between the TControl and the nontransformed ‘T63’ plants. The initial

observations of the green fruit phenotype were made in the heterozygous progeny of the

cross; the phenotypes were consistent in the homozygous progeny through five

generations in greenhouse and field trials in Davis, CA (2004-2012). The full-length

sequence of SlGLK2 (Genbank accession numbers JX163897/JQ316459, Table S8)

cDNA was cloned downstream of the CaMV35S promoter into a pBI121 derived vector

pBTEX (42) digested with SmaI/SalI. The fidelity of the construct was confirmed by

DNA sequencing and transgenic ‘Ailsa Craig’ (U/U) and ‘M82’ (u/u) tomato plants were

generated by Agrobacterium tumefaciens (strain LBA4404) mediated transformation

using previously described methods (43). Ten transgenic plants were selected by

kanamycin resistance, crossed with themselves and homozygous progeny identified. Five

transformed U/U lines and more than 5 transformed u/u lines showed the overexpression

fruit phenotype. Four other transformed U/U lines showed the co-suppression phenotype.

Tomato fruit were tagged 3-4 days post anthesis (dpa) when they were 0.5 cm

diameter. Mature green and red ripe fruit were harvested 32 and 46 dpa, respectively,

using the tags for reference. Immature green fruit was collected at 10-25 dpa. To examine

the effects of light, 3 dpa fruit attached to the plants were enclosed in a three layer (red,

black and white) paper bag (kindly provided by Yasutaka Kubo, Okayama University,

Okayama, Japan, through Fujii-Seitai Co., Okayama, Japan) that was crimped closed with

a twist-tie. The fruit remained enclosed in the light-blocking bags (>99% of the incident

light was blocked) on the plants until they were harvested for analysis. Fruit which

developed in the absence of light were harvested and compared to fruit of the same age

that developed in typical greenhouse or field light conditions.

5

Genomic DNA was prepared from leaf tissue from for PCR amplification using

Phire Plant Direct PCR Kit (Finnzymes, Thermo Fisher Scientific, Inc.) reagents. To

amplify the GLK and promoter sequences for genotyping, the primers shown in Table S7

were used.

Sequence alignments were generated using Geneious Pro v 5.3 (44).

Mapping SlGLK2 on tomato chromosome 10. The S. lycopersicum cv. ‘M82’ x S.

pennellii acc. ‘LA0716’ introgression line (IL) population (45) and an F2 population of

1100 individuals derived from a backcross of S. pennellii IL 10-1 (U/U) to its recurrent

parent ‘M82’ (u/u) were used for low and higher resolution mapping, respectively. Initial

analysis positioned U between markers TG303 (SL2.40ch10:1773625) and CT234

(SL2.40ch10:2641027); additional CAPS markers more precisely mapped U to between

markers B (SL2.40ch10:2275056) and C (Sl2.40ch10:2358687) (Figure 2A). A BC2S1

population of 40 individuals and a recombinant inbred line (RIL) population of 110

individuals from S. lycopersicum cv. ‘Moneymaker’ x S. pimpinellifolium acc. ‘TO-937’

were also phenotyped for fruit with dark shoulders (Figure 2A) and they were genotyped

with markers saturating the end of chromosome 10. Linkage analysis revealed that U was

located between solcap_snp_sl_29163 (SL2.40ch10:2113226) and solcap_snp_sl_17859

(SL2.40ch10:2335463) markers. Linkage analysis using the overlapping minimal regions

flanking the U locus from the three populations narrowed U to the region of chromosome

10 between markers B (SL2.40ch10:2275056) and solcap_snp_sl_17859

(SL2.40ch10:2335463) (Figure 2A). In this 60,507 bp segment, eight gene models,

including SlGLK2, are predicted (Table S1).

6

Identification of GLK-like sequences in pepper and potato. The pepper (C.

annuum) transcriptome was assembled using mRNA prepared from vegetative and fruit

tissues of three pepper varieties, ‘CM334,’ ‘Maor’ and ‘Early Jalapeño’. The assemblies

from the three varieties were combined using CAP3 to generate a common assembly for

pepper. The total pepper assembly and the individual variety assemblies were used to

perform basic local alignments (BLAST) searches with the Arabidopsis and tomato GLK

sequences to identify CaGLK1 (JF807944) and CaGLK2 (JF807945). To identify potato

GLK sequences, homology of the Arabidopsis, tomato and pepper sequences to the

diploid potato, S. phurjea, genome and transcript sequences

(http://potatogenomics.plantbiology.msu.edu/index.html) identified CaGLK1 (JF807946) and CaGLK2

(JF807948).

RNA extraction. Total RNA was prepared using Qiagen RNeasy Plant kits (Qiagen)

from cotyledons, sepals, petals, young leaves and fully developed leaves and using

standard protocols (46) from immature green fruit (12-15 dpa), mature green fruit (32

dpa) and ripe fruit (46 dpa). At least three biologically replicated samples of RNA were

prepared from each genotype, tissue and ripening stage from more than four plants.

Extraction of RNA from fruit was done using a modified version of a CTAB based RNA

extraction protocol (47). Outer pericarp and epidermis were excised with a sterile scalpel

and frozen and ground with liquid nitrogen to a fine powder. Two grams of this tissue

were transferred in a 50 mL tube to which 10 mL of the RNA extraction buffer (CTAB

2% v/v, PVP 2% v/v, 100mM Tris pH 8, 2M NaCl, 25mM EDTA, 0.5 g/L spermidine

10mM β-mercaptoethanol, DEPC water) was added. The sample was incubated 5 min at

65 °C and extracted twice with 1 volume of chloroform-isoamyl alchohol 24:1 (CIA)

7

mixed by vortexing. The tubes were centrifuged at 3,500 rpm for 45 min at 4 °C. The

supernatant was placed in a new 15 mL tube. 1/10 volume of 1M KOAc was and the

samples were vortexed and centrifuged at 3,500 rpm for 30 min. The supernatant was

recovered, 25% volume of 10 M LiCl was added followed by mixing gently by inversion.

The tubes were kept overnight at -20 °C. The samples were centrifuged at 4,000 rpm for

45 min, the supernatant was discarded and an RNA clean up protocol was done with the

RNA Plant Mini Kit (Qiagen). The RNA pellet was resuspended in 35 µL of nuclease-

free H2O. The RNA concentration and purity were measured using NanoDrop 2000c

Spectrophotometer (Thermo Scientific, Inc.). The RNA integrity was checked by agarose

gel electrophoresis.

RNA gel-blot analysis. Total RNA was extracted from frozen tissues and gel blot

analyses were done as described (48) using 25 µg total RNA per lane. An SlGLK2-

specific probe was generated by PCR using primers GLK2E1F and GLK2E2R (Table

S7) to amplify a 402 bp fragment from a pGEM-T Easy vector that contained the SlGLK2

full length cDNA.

Semi-quantitative and quantitative real time reverse transcription-PCR (RT-PCR).

cDNA was synthesized using M-MLV Reverse Transcriptase (Promega) from total RNA

extracted as described above using 1 µL of 100 mM Oligo dT (Applied BioSystems), 3

µg of RNA and nuclease-free H2O up to 12 µL. This mixture was heated at 70°C for 10

min and cooled on ice. To this, 5 µL of 5X first strand buffer, 1 µL of 10 mM dNTP´s

and 1 µL of RNA inhibitor (all from Applied BioSystems); to reverse transcribe the RNA

to cDNA, 1 µL of 200 u/ µL M-MLV Reverse transcriptase (Promega) was added. The

tube was heated 2 min at 42 °C, 50 min at 37 °C and kept at 4 °C until use. Semi-

8

quantitative RT-PCR was done simultaneously with the cDNA product and gene specific

and actin control primers (see Table S7). Three separate biologically replicated samples

were amplified and analyzed by agarose gel electrophoresis and ethidium bromide

staining; gel images are an example of the results.

Quantitative RT-PCR (qRT-PCR) was done to quantitate the amount of AtGLK and

SlGLK expression in 4-5 biological replicated preparations of RNA of each genotype or

tissue from green (10-25 dpa) fruit. qRT-PCR was performed on the StepOnePlus

(Applied Biosystems) using SYBR GREEN. The reaction volume contained 2 µL of

template, 0.3 µL of forward primer (10 mM), 0.3 µL of reverse primer (10 mM), 7.5 µL

of Fast SYBER GREEN Master Mix (Applied Biosystems) and 4.9 µL of sterile

molecular biology-grade water (total 15 µL). All qRT-PCR reactions were performed

with the following cycling conditions: 95°C for 10 min, followed by 40 cycles of 95 °C

for 3 s and 60 °C for 30 s. A melting curve for every target analyzed was included using

the following conditions: 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s. Tomato

actin primers (LeACT, The Institute for Genomic Research no. TC116117) were used as

an internal control and processed in parallel with reactions with gene specific primers

(Table S7).

The 2-ΔΔCT method (49) was used to determine the relative mRNA abundance and

compared to tomato actin and the sample with the least expression. Comparisons of gene

expression between different genes were justified because all primers had similar

efficiencies and were > 93%. All templates and primer concentrations were optimized for

the reactions initially using conventional RT-PCR.

9

Microarray analysis. RNA was extracted as three biological replicates of immature

green (15 dpa) pericarp tissue taken from three or more fruit as described above. Samples

of total RNA were checked for integrity and quality using an Agilent Bioanalyser

(Agilent Technologies). The three biologically replicated RNA samples were labeled and

hybridized to the 34,000 gene EUTOM3 exon array (http://www.eu-

sol.net/science/bioinformatics/data-and-databases/all-databases) according the

manufacturer's instructions (Affymetrix) at Unitat Central d’Investigació (Universitat de

Valencia, Spain). Briefly, 300 ng of total RNA from each sample were labeled using the

Ambion WT expression array kit (Ambion Inc.). The end labeling and hybridization were

performed according to the GeneChip whole transcript (WT) sense target labeling assay

manual (Ambion Inc.). Hybridization was performed using an Affymetrix Hybridization

Oven 640 (Affymetrix) for 17 hr. at 45oC. Following hybridization, the chips were

washed and stained with a phycoerythrin-strepavidin conjugate using the GeneChip®

Fluidics Station (Affymetrix) with the FS450-0001 protocol. The chips were scanned

using the Affymetrix® GeneChip® Scanner 30007G and the Affymetrix® GeneChip®

Command Console software (Affymetrix) was used to generate non-scaled RNA signal

intensity files (.cel). Raw data are MIAME compliant and are deposited at the

Arrayexpress site (http://www.ebi.ac.uk/arrayexpress/ with experiment accession number

E-MEXP-3652)

Data was pre-processed and analyzed using Partek Genomic Suite software v6.6 (Partek

Inc.) with the probes matching only once with the iTAG annotation v2.3. The

configuration consisted of a pre-background adjustment for GC content, Robust Multi-

array Analysis (50) for background correction, quantile normalization and probe set

10

summarization using median polishing. All signals were log2 transformed. Library files

were eutom3gene_v2_ucprobes.cdf and the annotation file version was eutom3-

annotation-per-scaffold-modif.txt which represents 30,000 genes of tomato genome.

To identify statistically significant differentially expressed genes between WT (Tcontrol)

and AtGLK expressing lines, probe set information is summarized into information for

the genes. A 3-way ANOVA mixed model (51) was used to analyze the effects of type

(transgenic or WT), sample genotype (WT, AtGLK1- and AtGLK2-expressor) and

replicate and the interaction between type and sample genotype. The ANOVA model

was:

Yijkl = μ + typei+replicatej+sample genotype(type)ik+ εijkl

Where Yijkl represents the lth observation on the ith type jth replicate kth sample genotype

μ is the common effect for the whole experiment. εijkl represents the random error present

in the lth observation on the ith type jth replicate kth sample genotype .The errors εijkl are

assumed to be normally and independently distributed with mean 0 and standard

deviation δ for all measurements.

To determine specific group differences in case of significant main effects (or

interaction), the ANOVA analysis was followed by Fisher’s LSD post hoc contrast to

generate p-values and fold changes for comparisons between type and sample type. Gene

lists of pair-wise contrasts were divided into up- and down-regulated genes (compared

with wildtype (WT) TControl). Genes whose expression changed as a consequence of

AtGLK expression were defined independently for each AtGLK expressing line using a

11

mean fold change ≥2 (relative to WT TControl samples) and a P value of ≤0.05. A total

of 3216 genes (≈ 10% of the EUTOM3 probes on the microarrays) were significantly up

or down regulated in AtGLK1 and/or AtGLK2 overexpressing tomato lines (Table S4).

Venn diagrams (Figure S6A) were used to identify sets of common and specifically

regulated genes. Genes differentially expressed (either up or down-regulated) in both

transgenic lines were defined as regulated by GLKs and genes up or down regulated only

in the material expressing either AtGLK1 or AtGLK2 were defined as specifically

regulated by GLK1 or GLK2, respectively. These classes of genes were used for

subsequent analyses. Two dimensional hierarchical agglomerative clustering using

Euclidean distance and average linkage were performed. The differentially expressed

genes identified genes were grouped into clusters to calculate Gene Ontology (GO)

enrichment scores for molecular function categories by applying Fisher Exact tests using

a local, customized version of the 'catscore.pl' Perl script (52) was used. Only GO terms

with a p<0.05, and three or more regulated genes for the GO-term were defined as over-

represented. Complete functional enrichment results are provided in Tables S5 and S6.

The EUTOM3 microarrays were designed and annotated by Stephane Romabauts (VIB

Department of Plant Systems Biology, Ghent University Technologiepark 927, 9052

Ghent, Belgium). The MIAME–compliant microarray data are available at

http://ted.bti.cornell.edu and at http://www.ebi.ac.uk/arrayexpress/ with the accession

number E-MEXP-3652.

Chlorophyll. Chlorophyll was measured in the youngest fully expanded apical leaves

in a truss and in immature green (15 dpa) fruit. Tissues from the outer fruit pericarp and

epidermis (~50 mg each) and leaf (~7 mg) from well irrigated plants were extracted into

12

1 ml N,N dimethylformamide (DMF) overnight at 4 oC. The amounts of chlorophyll a

and chlorophyll b, were determined spectrophotometrically using published equations

(53). Total chlorophyll was calculated as chlorophyll a + chlorophyll b. Results were

expressed as µg chlorophyll per mg of tissue and the results agreed with chlorophyll

determinations made with material extracted in 80% acetone. A minimum of five

biologically replicated samples was used for each genotype and tissue.

Starch measurements. For starch quantitation, two grams of outer fruit pericarp

were ground in 10 mL ethanol. The samples were centrifuged and the pellet was re-

extracted two more times with 10 mL ethanol. After centrifugation, the pellet was dried at

50 oC and resuspended in 5 mL of 50 mM NaAc buffer (pH 5.0). 100 µL containing 10

units of amylase and 3 units of amylo-glucosidase were added and samples were

incubated at 30 oC with stirring overnight. The samples were centrifuged and adjusted to

6 mL with water. The content of reducing sugars was determined using a modification of

the Somogyi-Nelson method and measured with a spectrophotometer at 520 nm (54).

Transmission electron microscopy. Pericarp fragments were excised from fruit at

the mature green stage and from fully expanded leaves. Fragments were fixed in

Karnovsky’s fixative using vacuum-microwave combination as described by Russin and

Trivett (55) and washed in 0.1 M sodium phosphate buffer, pH 7.2, microwaved under

vacuum at 450 W for 40 seconds, post-fixed for 2 hours in 1 % (w/v) osmium tetroxide

buffered in 0.1 M sodium phosphate buffer and microwaved a second time at 450 W for

40 seconds. After incubation in 0.1% (w/v) tannic acid in water for 30 minutes on ice and

in 2 % (w/v) aqueous uranyl acetate for 1 hour, samples were dehydrated in acetone and

embedded in Epon/Araldite resin. Ultrathin sections were examined with a Philips

13

CM120 Biotwin Lens transmission electron microscope (FEI Company). Images in

Figure 4A were taken at 11,000 magnification and 0.5 µm scale bars are shown.

Measurement of soluble solids. Soluble solid contents (oBRIX) were of total fruit

juice from freshly harvested red ripe fruit were measured with a digital refractometer

(PR100, Atago Co., Ltd.).

Sugar analysis. For simple sugar analysis, 5 to 7 g of total mature green and red fruit

tissue was extracted with 20 mL 95% (v/v) ethanol. The samples were centrifuged and

the pellets re-extracted with 10 mL 95% (v/v) ethanol. The supernatants were pooled and

adjusted with 95% (v/v) ethanol to a final volume of 45 mL. From these pooled

supernatants, 200 µL samples were dried and resuspended in 1 mL water. Forty

microliters of the resuspended sample were diluted to 10 mL with water and 200 µL were

injected in the HPLC for sugar analysis. Sugar profiles were analyzed using a DX-500

HPLC system (Dionex Corp.) equipped with an analytical Carbopac PA1 column and an

ED-40 electrochemical detector for pulsed amperimetric detection (PAD). A linear

sodium carbonate gradient at a flow rate of 0.6 mL min-1 was used. Glucose and fructose

were identified and quantified by using authentic standards. Results were expressed in

grams of sugar per 100 g of fresh fruit.

Carotenoid analysis. Carotenoid compounds were extracted and measured by

HPLC from three independent biological samples of red ripe (42 dpa) fruit epidermis and

pericarp as described previously (56). For spectrophotometric measurements, 0.25 g of

pericarp and epidermis from red ripe fruit (42 dpa) were ground in liquid N2, and

extracted with 8 ml hexane:ethanol:acetone (2:1:1) with shaking at room temperature

overnight. 1 ml water was added to each sample which was mixed by vortexing and the

14

solvent layers separated. The absorbance of the organic phase at 503 nm was used to

calculate the amount of lycopene (57, 58).

Statistical analysis. Experiments were performed according to a factorial design.

Data were analyzed by ANOVA, if the number of experimental replicates was equal, or

by General Linear Model (GLM) if the number of replicates was unequal followed by

post hoc testing using Tukey’s Honestly Significant Difference (HSD) or Bonferonni

Multiple Comparison Test (MCT) with JMP 9.0 (SAS, Cary, NC). Genetic linkage

analysis was performed by using JoinMap 4.0 software (Van Ooijen, J. W., Kyazma

B.V., Wageningen, the Netherlands, 2006).

15

Fig. S1.

Figure S1. Total soluble solids as measured by oBRIX of juice from red ripe (42 days post anthesis, dpa) fruit that had dark green shoulders (‘Ailsa Craig’ U/U) or were uniformly light green (‘Craigella’ u/u) prior to ripening. n=100 fruit of each genotype. Significant statistical differences determined by means of ANOVA and Tukey’s HSD at p<0.05 are indicated by different letters.

16

Fig. S2 A

17

B

18

C

19

D BLOSUM50 BLOSUM62 PAM250

Name Length Name Length Identity Similarity Identity Similarity Identity Similarity

AtGPRI1/GLK1 420.0 AtGLK2 386.0 46.5 60.0 46.1 58.6 44.5 66.4

CaGLK1 447.0 AtGLK2 386.0 47.3 59.3 46.4 57.9 44.9 64.9

CaGLK1 447.0 AtGPRI1/GLK1 420.0 49.8 65.1 49.1 63.1 47.5 71.6

CaGLK1 447.0 CaGLK2 313.0 47.1 55.7 45.7 55.0 44.5 57.9

CaGLK1 447.0 SpGLK2 317.0 46.2 58.2 46.0 56.8 46.3 60.4

CaGLK1 447.0 SpGLK1 415.0 80.5 85.7 80.5 85.2 80.3 87.5

CaGLK1 447.0 SlGLK1 464.0 84.3 89.2 84.3 88.4 84.1 90.9

CaGLK1 447.0 SlGLK2 310.0 45.1 55.7 44.6 54.1 44.1 58.4

CaGLK2 313.0 AtGLK2 386.0 43.1 57.3 41.9 54.4 40.4 60.9

CaGLK2 313.0 AtGPRI1/GLK1 420.0 41.3 56.4 41.1 53.3 38.6 59.0

CaGLK2 313.0 SpGLK2 317.0 75.1 86.1 75.1 84.9 74.5 89.9

CaGLK2 313.0 SpGLK1 415.0 48.2 57.6 48.2 57.1 45.8 60.2

CaGLK2 313.0 SlGLK1 464.0 46.4 55.8 45.1 54.3 43.6 56.9

SpGLK2 317.0 AtGLK2 386.0 43.1 57.8 42.3 54.7 40.3 61.9

SpGLK2 317.0 AtGPRI1/GLK1 420.0 41.9 55.7 41.2 53.3 38.1 57.6

SpGLK2 317.0 SpGLK1 415.0 45.3 58.3 44.7 56.6 44.0 60.5

SpGLK1 415.0 AtGLK2 386.0 46.1 60.5 45.7 58.6 43.9 66.0

SpGLK1 415.0 AtGPRI1/GLK1 420.0 51.1 68.1 50.9 65.5 47.1 70.7

SlGLK1 464.0 AtGLK2 386.0 45.8 57.5 44.5 54.1 42.9 62.3

SlGLK1 464.0 AtGPRI1/GLK1 420.0 49.7 64.7 48.3 60.1 46.6 69.2

SlGLK1 464.0 SpGLK2 317.0 44.1 55.8 44.1 53.9 43.4 57.8

SlGLK1 464.0 SpGLK1 415.0 85.6 87.5 85.6 87.3 85.6 87.9

SlGLK2 310.0 AtGLK2 386.0 42.2 56.5 40.9 52.8 40.7 60.1

SlGLK2 310.0 AtGPRI1/GLK1 420.0 40.1 53.6 39.4 51.4 38.9 56.7

SlGLK2 310.0 CaGLK2 313.0 74.7 85.0 74.7 83.7 74.7 90.1

SlGLK2 310.0 SpGLK2 317.0 87.1 92.1 87.1 91.8 87.1 95.6

SlGLK2 310.0 SpGLK1 415.0 44.7 54.9 44.3 54.0 42.9 58.3

SlGLK2 310.0 SlGLK1 464.0 43.4 53.2 43.4 51.3 41.9 55.2 Figure S2. Amino acid alignments showing the sequences of the predicted GLK1 and GLK2 proteins from Arabidopsis (AtGLK1, AtGLK2), domesticated tomato (S. lycopersicum, (A) cv. Ailsa Craig (B) cv.’Craigella’) (SlGLK1, SlGLK2), diploid potato (S. phureja) (SpGLK1, SpGLK2) and pepper (Capsicum annuum) (CaGLK1, CaGLK2) cDNA sequences. To identify potato GLK genes, homology searches of the potato transcriptome assembly and genomes using Arabidopsis GLK1 and GLK2 were used to identify two GLK-like genes in potato (SpGLK1 and SpGLK2). The exon structures are based on comparisons with Arabidopsis, tomato and potato genomic sequences. Sequence alignment was generated by using Geneious Pro v 5.3. (C) A phylogenetic tree

20

of GLKs generated using a Bayesian inference of phylogeny. In addition to the Arabidopsis, tomato, potato and pepper GLKs, BLAST searches against protein and EST databases at NCBI and EMBL were used to identify multiple GLK sequences from Monocot, Rosid Dicot and Asterid Dicot groups. Predicted amino acid sequences were aligned with T-Coffee using the PSI-Coffee method followed by an additional alignment evaluation using Core (http://tcoffee.crg.cat/). Sequences were trimmed to remove regions that showed inconsistent alignment (0-5 reliability score out of 10). Trimmed sequences were used to construct the tree using MrBayes 3.2.1 (http://mrbayes.sourceforge.net/index.php) with mixed amino acid and co-varion models run for 300,000 iterations at 2 runs by 1 chain per run. The branch lengths indicate the evolutionary distances, and numbers indicate percent probabilities for each node. Abbreviations and accession numbers used are found in Table S8. (D) Pairwise sequence identity and similarity between tomato, potato, pepper and Arabidopsis GLKs was calculated using MatGAT 2.02 (http://bitincka.com/ledion/matgat/) run with BLOSUM50, BLOSUM62 and PAM250 alignment matrices.

21

Fig. S3

Figure S3. Maturing fruit from Tcontrol (A) and transgenic plants expressing p35S::AtGLK1 (B,) or p35S::AtGLK2 (C). From left to right fruit are green (4, 6, 12, 18, 25, 32 dpa) turning (35 dpa) and fully red ripe (42 dpa).

22

Fig. S4 A

B

Figure S4. Chlorophyll contents of green fruit as a function of exposure to light during maturation. A. Phenotypes of ‘Ailsa Craig’ U/U and ‘Craigella’ u/u mature green (32 dpa) fruit after maturation in the absence (Dark) and presence (Light) of light. B. Chlorophyll contents of pericarp from the pedicel (shoulders) or style (stylar) region of mature green fruit. Significant statistical differences determined by means of GLM and Tukey’s HSD at p<0.05 are indicated by different letters.

23

Fig. S5

Figure S5. Expression and phenotypes of fruit expressing p35S::SlGLK2. A. SlGLK2 expression as determined by hybridization of SlGLK2 specific probes to gel blots of RNA from ‘Ailsa Craig’ U/U or ‘M82’ u/u transformed with p35S::SlGLK2. B. Fruit phenotypes from representative plants of lines overexpressing (OE) or with co-suppressed expression (CS) of SlGLK2. Five transformed U/U lines and more than 5 transformed u/u lines showed the overexpression fruit phenotype. Four other transformed U/U lines showed the co-suppression phenotype.

24

Fig. S6

Figure S6. Summary of transcript abundance analysis by hybridization to EUTOM3 microarrays. A. Comparison of 3216 genes differentially expressed in AtGLK expressing lines relative to Tcontrol lines. B. Cellular components Gene Ontology (GO) terms of significantly (p<0.05, fold change >2) down-regulated genes in IM green fruit identified in EUTOM3 microarray hybridizations. The total number of genes with known GO terms is shown below bars. C. Hierarchical average linkage clustering of 3216 genes differentially expressed in AtGLK expressing lines relative to Tcontrol. Red and blue correspond to up- and down-regulation, respectively. D. 672 genes differentially expressed in both AtGLK1 and AtGLK2 expressing lines. E. Genes differentially expressed only in the AtGLK1 expressing lines. F. Genes differentially expressed only in the AtGLK2 expressing lines.

25

Fig. S7

Figure S7. Exposure of fruit to light determines soluble solids in ripe fruit. A. Ripe fruit (42 dpa) fruit phenotype of u/u ‘T63’ fruit that developed in normal light (Light) and in light blocking bags (Dark). B. Total soluble solids of juice from red ripe (42 dpa) fruit. Significant statistical differences determined by means of GLM and Tukey’s HSD at p<0.05 are indicated by different letters.

26

Fig. S8

A

B Phytoene Phytofluene Lutein γ-Carotene β-Carotene cis-Lycopene trans-Lycopene Total Carotenoids

Average ± SE Average ± SE Average ± SE Average ± SE Average ± SE Average ± SE Average ± SE Average ± SE

Tcontrol 4.23 ± 0.10 a 2.83 ± 0.44 a 0.95 ± 0.20 a 2.44 ± 0.09 a 4.35 ± 0.33 a 2.25± 0.21 a 121.58 ± 0.39 a 138.18 ± 0.81 a

p35S::AtGLK1 6.45 ± 0.87 a 4.41 ± 0.40 b 1.56 ± 0.14 a 3.42 ± 0.10 a 6.47 ± 0.51 a 3.58 ± 0.42 a 252.29 ± 17.28 b 278.51 ± 17.68 b

p35S::AtGLK2 4.61 ± 0.35 a 2.68 ± 0.20 a 1.80 ± 0.19 a 3.18 ± 0.14 a 6.72 ± 0.35 a 4.04 ± 0.73 a 166.28 ± 14.51 a 189.43 ± 16.18 a Figure S8. Carotenoid compounds in ripe fruit. A. Lycopene content of red fruit pericarp and epidermis measured as the absorbance at 503 nm of ethanol/hexane/acetone extracts of pulverized pericarp tissue. B. Carotenoid compound contents of red fruit measured by HPLC. Statistical significance determined by means of GLM and Bonferonni MCT at p<0.05 are indicated by different letters.

27

Table S1. Table S1. Eight predicted genes in the 60,507 bp region of S. lycopersicum chromosome 10 (ITAG2.4 Release: genomic annotations, http://solgenomics.net) between SL2.40ch10:2275056 and SL2.40ch10:2335463. SlGLK2 (yellow highlight) is within this region, specifically between Sl2.40chr10:2291209 and Sl240chr10:2295578. Start Stop Gene ID Identifier 2276303 2278610 Solyc10g008140 Unidentified, length=2308

2281545 2281874 Solyc10g008150 Glutaredoxin (AHRD V1 ***- B9MYC1_POPTR); B contains Interpro domain(s) IPR011905 Glutaredoxin-like%2C plant II

2293088 2295945 Solyc10g008160 Transcription factor (Fragment) Slglk2/u (AHRD V1 *--- D6MK15_9ASPA)

2300710 2304760 Solyc10g008170 26S proteasome regulatory subunit (AHRD V1 ***- C6HL17_AJECH)

2305283 2311789 Solyc10g008180

26S proteasome regulatory subunit (AHRD V1 ***- A8J3A4_CHLRE); B contains Interpro domain(s) IPR016643 26S proteasome regulatory complex%2C non-ATPase subcomplex%2C Rpn1 subunit

2311949 2314392 Solyc10g008190

OB-fold nucleic acid binding domain containing protein (AHRD V1 ***- B6SHT0_MAIZE); B contains Interpro domain(s) IPR012340 Nucleic acid-binding%2C OB-fold

2315211 2319689 Solyc10g008200

Tyrosine aminotransferase (AHRD V1 **** D3K4J1_PAPSO); B contains Interpro domain(s) IPR005958 Tyrosine%2Fnicotianamine aminotransferase

2325581 2332352 Solyc10g008210 Os07g0507200 protein (Fragment) homolog (AHRD V1 *-*- C7J530_ORYSJ); B contains Interpro domain(s) IPR009675 Targeting for Xklp2

28

Table S2. Table S2. Relative expression of SlGLK1 and SlGLK2 as determined by qRT-PCR of cDNA prepared from young fully expanded leaves and from the pedicellar shoulder (Pedicel) and stylar (Style) portions of ‘Ailsa Craig’ U/U or ‘Craigella’ u/u green fruit (25 dpa) that developed in normal light conditions. qRT-PCR reactions were done on 4 - 5 biological replicated samples. Values were normalized to the expression of actin and expression of SlGLK1 and SlGLK2 is relative to the expression of SlGLK1 in the pedicellar portion of u/u. Standard errors are indicated.

Relative Expression SlGLK1 SlGLK2 Leaves Pedicel Style Leaves Pedicel Style U/U 353.4 ± 22.7 1.0 ± 0.8 -1.8 ± 1.5 394.1 ± 72.6 329.7 ± 112.5 41.1 ± 8.1 u/u 222.0 ± 37.1 -1.4 ± 0.4 -1.9 ± 0.2 10.6 ± 22.5 86.3 ± 44.9 16.3 ± 4.0

29

Table S3 Table S3. Relative expression of SlGLK1 and SlGLK2 as determined by qRT-PCR of cDNA prepared from the pedicellar shoulder (Pedicel) or the blossom stylar (style) portions of green ‘Ailsa Craig’ U/U or ‘Craigella’ u/u fruit (25 dpa) that developed in normal light conditions (Light) or in light blocking bags (Dark). qRT-PCR reactions were done on 4-5 biologically replicated samples. Values were normalized to the expression of actin and expression of SlGLK1 and SlGLK2 is relative to the expression of SlGLK1 in the pedicellar portion of u/u fruit that developed in the light. Standard errors are indicated.

Relative Expression SlGLK1 SlGLK2 Pedicel Style Pedicel Style U/U (Light) 1.0 ± 0.8 -1.8 ± 1.5 329.7 ± 112.5 41.1 ± 8.1 U/U (Dark) 1.0 ± 0.7 -1.9 ± 1.3 127.1 ± 54.3 10.4 ± 21.0 u/u (Light) -1.4 ± 0.4 -1.85 ± 0.2 86.3 ± 44.9 16.3 ± 4.0 u/u (Dark) -6.8 ± 1.1 -4.0 ± 1.1 6.3 ± 21.7 13.5 ± 0.6

30

Table S4 – attached separately Table S4 (ST.4). Genes (3215 genes) identified as differentially expressed in p35S::AtGLK expressing lines versus wild-type (WT, TControl). Functional annotations are based on ITAG2.3 and ANOVA 3 way-LSD statistics. The sample types are WT (Tcontrol), GLK1 (expressing p35S::AtGLK1) and GLK2 (expressing p35S::AtGLK2). The annotations in this file are: Gene identity (ID) corresponding to the mRNA ID (Tomato whole genome sequence (WGS) cDNA ITAG 2.3) and Cluster based on Venn diagrams. Gene Functional Annotations:GO terms, Tomato WGS cDNA ITAG 2.3 hit name (exons); ITAG description; nearest 3-prime marker; nearest 5-prime marker; pseudo-molecule and position; tomato cDNA TAIR10 best hit; Arabidopsis gene symbol; Arabidopsis gene description; Arabidopsis component GO term and general cellular component. Matches with previous work with GLKs, photomorphogenesis regulators: Genes reported in Waters et al. (32), Savitch et al. (33), Rohrmann et al. (34) and Enfissi et al. (35), Kolotilin et al. (2007). Statistics: probe id, p-value(type), p-value(replicate), p-value(sample type(type)), p-value(WT * WT vs. transgenic * GLK1), Fold-Change(WT * WT vs. transgenic * GLK1), Fold-Change (WT * WT vs. transgenic * GLK1) (Description), p-value (WT * WT vs. transgenic * GLK2), Fold-Change (WT * WT vs. transgenic * GLK2), Fold-Change (WT * WT vs. transgenic * GLK2) (Description), F (type), SS (type), F (sample type(type)), SS (sample type(type)), F (replicate), SS (replicate), SS (Error), F (Error). Red cells indicate p<0.05.

31

Table S5- attached separately

Table S5 (ST.5). Results of GO term enrichment analysis of AtGLK expressing lines vs WT (Tcontrol). For enrichment all Venn diagram sectors are considered.

32

Table S6- attached separately

Table S6 (ST-6). GO term description for functional categories with p-value <0.1

33

Table S7

Table S7. Primers used for PCR and qRT-PCR.

34

Sequences Forward Primer Reverse Primer

SULII CGGACAGTTTCTCCGATGGA GGATAGAACGCAGCGTCTGG

NPTII GGCCGCTTGGGTGGAGAG GGTAGCCGGATCAAGCGTATG

At5g44190 (AtGLK2) AGCGGAAGAGATGAGGAACA CTAAGGCAGGAGCTGTCCAC

At5g44190 (AtGLK2) ATGTTAACTGTTTCTCCGGCTCCAG TCAAGGAAGAGGAGGAACATTAGAAACTCC

At2g20570 (AtGLK1) AGGTGGATTGGACACCAGAG CTGGCGGTGCTCTAAATCTC

AtGLK1 – qRT-PCR Efficiency 97.29%

ATTTAGAGCACCGCCAGTTG ACGCTCTCTTTTGACGGATG

AtGLK2 – qRT-PCR Efficiency 93.73%

AGCAACCACTCTATCCACAG TAACGTCCCCAATAGCTG

LexA activator GCCTTCAGATGTTCTTCAGC

35s promoter GAACTCGCCGTAAAGACTGG

LTP promoter ATGCAAAGAAGGACGTAGGC TGTGGTGTGAATGCGATAGA

PDS promoter TAACTGCCAAACCACCACAA

Rbc3b promoter TCCAATGGTTATGGTTGCTCT

SlGLK1 (Solyc07g053630) ATGGAAAGTTTCGCGATAGGAGGA CTATGCACAAGTTGGTGGTATTTTA

SlGLK2 (Solyc10g008160) ATTTTCTCTCTTTTGATGTCACC CYTTGATAATGTGGATGCCAAAA

SlGLK1 – qRT-PCR (3), Efficiency 95.53%

CCGTAAGCAGTGGTGATGAGTCTG AACCCGAACCTACATCCGAAGC

SlGLK2 – qRT-PCR Efficiency 94.18%

CCTTACATGTTTGGGGGCATCCAC GGGGTGCAAATCAGAGGC

SlGLK2(GLK2E1F/GLK2E2R) ATGCTTGCTCTATCTTCATCATTGA TTGAAGATGACTAGCAATGTTATGTCT

SlActin – qRT-PCR CCTCAGCACATTCCAGCAG CCACCAAACTTCTCCATCCC

35

Table S8 Table S8. GenBank accession numbers.

Protein Name Full name GenBank Accession number

AaGLK Artemisia annua UniGene Aan.2135

AtGLK2 Arabidopsis thaliana GOLDEN2-like 2 protein NP_199232.1

AtGLK1 Arabidopsis thaliana GPRI1/GLK1 NP_565476.1

BdGLKA Brachypodium distachyon probable transcription factor GLK1-like XP_003563963.1

BdGLKB Brachypodium distachyon probable transcription factor GLK2-like XP_003565554.1

BnGLKB Brassica napus cDNA 5', mRNA sequence GR447064.1 & EE428427.1 *

BrGLKB Brassica rapa subsp. pekinensis FY423077.1 & EX098749.1 *

CaGLK1 Capsicum annuum golden 2-like 1 transcription factor GLK1 JF807944

CaGLK2 Capsicum annuum golden 2-like 2 transcription factor GLK2 JF807945

GmGLKA Glycine max PREDICTED: transcription activator GLK1-like XP_003543323.1

GmGLKB Glycine max PREDICTED: transcription activator GLK1-like XP_003540379.1

GmGLKC Glycine max uncharacterized protein LOC100799248 NP_001241943.1

GmGLKD Glycine max PREDICTED: transcription activator GLK1-like XP_003539944.1

HaGLK Helianthus annuus cDNA clone UniGene Han.12384

HvGLKA Hordeum vulgare subsp. vulgare predicted protein BAJ98698.1

HvGLKB Hordeum vulgare subsp. vulgare predicted protein BAJ84790.1

MtGLK Medicago truncatula Two-component response regulator-like APRR2 XP_003607509.1

OsGLK1 OsG2-like, Oryza sativa Japonica Group golden2-like BAD62070.1

OsGLK2 Oryza sativa Japonica Group OsGLK2 BAD81484.1

PtGLK Populus trichocarpa predicted protein XP_002310413.1

RcGLK Ricinus communis DNA binding protein, putative XP_002517855.1

SbGLKA Sorghum bicolor hypothetical protein SORBIDRAFT_10g008400 XP_002436765.1

SbGLKB Sorghum bicolor hypothetical protein SORBIDRAFT_03g000400 XP_002454868.1

SlGLK1 Solanum lycopersicum GLK1 JF807943/JQ316460

SlGLK2 Solanum lycopersicum GLK2 (U) JX163897/JQ316459

Slglk2 Solanum lycopersicum glk2 (u) JF807947

SpGLK1 Solanum phureja GLK1 JF807946

SpGLK2 Solanum phureja GLK2 JF807948

TaGLKA Triticum aestivum golden 2-like protein ABL10089.1

TaGLKB Triticum aestivum MYB-related protein AEV91189.1

ThGLKA Thellungiella halophila mRNA, complete cds AK352526.1 *

ThGLKB Thellungiella halophila unnamed protein product BAJ33719.1

36

VvGLK Vitis vinifera PREDICTED: transcription activator GLK1-like XP_002275230.1

ZmGLK1 Zea mays G2-like1 NP_001105018.1

ZmG2 Zea mays putative transcription factor Golden2 AAK50391.1 * EMBL accessions

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