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Title: Expression patterns of flower specific SEPALLATA-like MADS-box genes in crocus (Crocus sativus L.).

Article Type: Full Length Article

Section/Category:

Keywords: Crocus; monocots; MADS-box genes; SEPALLATA

Corresponding Author: Prof. Athanasios S Tsaftaris, Ph.D

Corresponding Author's Institution: CERTH

First Author: Athanasios S Tsaftaris

Order of Authors: Athanasios S Tsaftaris; Alexios N Polidoros; Konstantinos Pasentsis; Shiri Freilich;

Christos Ouzounis

Manuscript Region of Origin:

Abstract: MADS-box genes in plants encode transcription factors involved in numerous steps of plant

development including flowering, flower meristem formation and flower organ identity. In an effort to

understand flower formation in the monocot crop crocus (Crocus sativus L.) cultivated for saffron production,

we have cloned four MIKCc type II MADS-box genes of the SEPALLATA (SEP) subfamily (E-type) using a

modified RCA-RACE method, named famRCA-RACE. All proteins contain the typical for this subfamily SEP

I and SEP II motifs. Phylogenetic analysis indicated that the isolated genes belong to the SEP3 subclade

showing high similarity with Asparagus officinalis AOM1 and AoMADS2 SEP-like genes. Expression

analysis revealed that all genes are strongly expressed in flowers but not in leaves. Furthermore the genes

are expressed in all flower organs: outer whorl (whorl 1) tepals, inner whorl (whorl 2) tepals, stamens and

carpels. Expression of the CsatSEP-like genes in the outer tepals together with A-type (CsatAP1/FUL) and

B-type (CsatAP3 and CsatPI) genes fits nicely the modified ABCE model proposed to explain the homeotic

transformation of whorl1 sepals into tepals in Liliales and Asparagales. In contrast, expression of both B-

type (CsatAP3 and CsatPI) together with E-type (CsatSEP-like) genes in the fourth whorl of crocus carpel

without any phenotypic effects raises questions about the role of A-, B- and E-function genes in carpel

formation in this non-grass monocot.

CENTER FOR RESEARCH AND TECHNOLOGY HELLAS (CERTH)

INSTITUTE OF AGROBIOTECHNOLOGY (ΙΝΑ)

6th km. Charilaou-Thermis, 570 01 Thermi, P.O. Box 361

Thermi 1 February 2008

To: M. Delseny

Manuscript submissionTitle: Expression patterns of flower specific SEPALLATA-like MADS-box genes in crocus (Crocus sativus L.).

Dear Dr. Delseny,

We submit our article to be considered for publication in Plant Science because

we believe that it fits the aims and scope of the journal and provides novel and

original data on the structure and regulation of a class of crocus genes as well as

their role in flower development, which comprises a valuable commercial product

of this plant. This manuscript is a continuation of our work with the monocot

crocus crop aiming at not only understanding tepal formation but also to

characterize a number of mutants that could be helpful for saffron production.

Since we now have A-type, B-type, C-type and with this manuscript the E-type

MADS-box TFs of crocus, we present in this manuscript for the first time for such

a perennial monocot with corms, a domain analysis for this important TF family.

I hope that you will find our manuscript a complete and worthy contribution for

publication in PLANT SCIENCE.

We would like to suggest the following scientists who are experts in related fields as possible reviewers:

Dr. Brendan DAVIESCentre for Plant SciencesFaculty of Biological SciencesUniversity of LeedsLeeds LS2 9JTUKemail: b.h.davies@leeds.ac.uk

Cover Letter

Dr. Chang-Hsien YANGGraduate Institute of BiotechnologyNational Chung hsing UniversityTaichung, Taiwan. 40227Tel: 886-4-22853126email: chyang@dragon.nchu.edu.tw

Gerco C. AngenentPlant Research International, Business Unit Bioscience, 6700 AA Wageningen, The Netherlands e-mail gerco.angenent@wur.nl ; fax 31-317-423110.

Akira KannoGraduate School of Life Sciences, Tohoku University, Katahira 2-1-1, Aoba-ku,Sendai 980-8577, JapanE-mail: kanno@ige.tohoku.ac.jp

Thank you again for your kind consideration and help in publishing our results.

Best RegardsProf. A. TsaftarisAUTh and INA/CERTH

Expression patterns of flower specific SEPALLATA-like MADS-box genes in crocus (Crocus sativus L.).

Athanasios S. Tsaftaris1,2,*, Alexios N. Polidoros1, Konstantinos Pasentsis1, Shiri Freilich1

, Cristos Ouzounis1

1Institute of Agrobiotechnology, Center for Research and Technology Hellas, Thermi, Greece, GR-570 01

2Department of Genetics and Plant Breeding, Aristotle University of Thessaloniki, Thessaloniki, Greece, GR-541 24

*Corresponding Authoremail: tsaft@certh.gr

KEYWORDS: Crocus sativus L., monocots, MADS-box genes. SEPALLATA.

ManuscriptClick here to view linked references

Abstract

MADS-box genes in plants encode transcription factors involved in numerous

steps of plant development including flowering, flower meristem formation and

flower organ identity. In an effort to understand flower formation in the monocot crop

crocus (Crocus sativus L.) cultivated for saffron production, we have cloned four

MIKCc type II MADS-box genes of the SEPALLATA (SEP) subfamily (E-type)

using a modified RCA-RACE method, named famRCA-RACE. All proteins contain

the typical for this subfamily SEP I and SEP II motifs. Phylogenetic analysis indicated

that the isolated genes belong to the SEP3 subclade showing high similarity with

Asparagus officinalis AOM1 and AoMADS2 SEP-like genes. Expression analysis

revealed that all genes are strongly expressed in flowers but not in leaves.

Furthermore the genes are expressed in all flower organs: outer whorl (whorl 1)

tepals, inner whorl (whorl 2) tepals, stamens and carpels. Expression of the CsatSEP-

like genes in the outer tepals together with A-type (CsatAP1/FUL) and B-type

(CsatAP3 and CsatPI) genes fits nicely the modified ABCE model proposed to

explain the homeotic transformation of whorl1 sepals into tepals in Liliales and

Asparagales. In contrast, expression of both B-type (CsatAP3 and CsatPI) together

with E-type (CsatSEP-like) genes in the fourth whorl of crocus carpel without any

phenotypic effects raises questions about the role of A-, B- and E-function genes in

carpel formation in this non-grass monocot.

1. Introduction

Flowering plants represent one of the most successful and diverse groups of

organisms on the planet, with more than 250,000 extant species in the wild and

thousands more varieties generated by horticulturists through hybridization and other

breeding efforts. Most flowers contain just four distinct organ types namely from

outside to inside: sepals in whorl 1, petals in whorl 2, stamens in whorl 3, and carpels

in whorl 4. Depending on the species, this model can be adapted replacing for by

species-specific homologous organs in each whorl. But plant like orchids, roses,

asparagus, maize, crocus and many other have varied distinctive flowers. A common

departure from the typical flower for example, observed in several monocots

including crocus (Crocus sativus L.) is that there is no clear distinction between sepals

and petals, and the petaloid organs in whorls 1 and 2 are therefore referred to as tepals

[1]. Crocus is a monocot triploid species belonging to the Iridaceae family of

Asparagales, whose whorl 4 red stigmatic styles constitute saffron, a popular food

additive with delicate aroma and attractive color used also for couloring and medical

purposes. The flower of crocus is bisexual and it is sterile. The perianth consists of 6

petaloid tepals in whorls 1 and 2. Androecium consists of 3 distinct stamens in whorl

3 and the gynoecium consists of a single compound pistil with 3 stigmas in whorl 4.

Several phenotypic flower mutants have been described, such as flowers with larger

numbers of styles and stamens [2] and references therein), as well as a flower without

stamens and a double flower described in this study. Crocus blooms only once a year

during mid-November and is harvested by hand. After a wind separation of tepals the

red stigmas are separated from the yellow stamens by hand. Consequently, the

cultivation of this crop for its flowers and specifically its stigmas is very labor-

intensive leading to high costs [3]. Thus, understanding flower development in crocus

could not only help us understand tepal formation in this monocot but in addition

could reveal ways to increase yield and lower production costs.

The characteristics of the different floral organs during flower development are

determined by actions of floral organ identity genes. The first attempt to explain

flower development through a unifying model was made in the early 1990s based on

genetic experiments in Antirrhinum and Arabidopsis. The proposed model envisaged

the action of flower-specific genes with three distinct functions termed A, B and C,

hence the name of the original ABC model, where A-function genes alone could

determine sepals, A+B function genes determine petals, B+C function genes

determine stamens, and C-function genes alone determine carpels [4]. The ABC

model was broadly adopted because of its simplicity and its applicability to a wide

range of angiosperm species, both dicots and monocots, including economically

important grass species such as rice and maize [5, 6]. Parallel studies identified that

almost all genes responsible for the A, B, and C-function involved in flower

development belong in a large family of MADS-box transcription factors, which

specify flowering time, floral meristem identity and space-time regulation of flower

organ formation [7-10].

However, it was soon realized that flower development was more complex than

the ABC model predicted. Expression of ABC genes throughout a plant does not

transform leaves into floral organs. Thus the ABC functions, although necessary, are

not sufficient to superimpose floral organ identity on a leaf development program

[11]. An additional class of MADS-box genes recognized later, the E-type

SEPALLATA (SEP) genes of Arabidopsis, provide redundant control over the ABC

system. The three different SEP genes in Arabidopsis (SEP1, SEP2 and SEP3) that

were identified through their sequence similarity to AGAMOUS (AG) [12, 13] when

expressed together with the ABC genes are sufficient for specification of petals,

stamens and carpels, and their ectopic expression may turn leaves into floral organs

[14]. Expression of the SEP-like FBP2 in petunia and TM5 in tomato as well as of

many other SEP-like genes was found in petal, stamen, and carpel primordia [15-17].

Triple-null mutants sep1/2/3 produce indeterminate flower structures consisting only

of sepal-like organs and are strikingly similar to the phenotype produced by double

mutants of B and C class floral homeotic genes [18]. A recently recognized SEP4

gene of Arabidopsis is probably still expressed and enough to confer the sepal-like

structure in the triple mutants since in quadruple sepl/2/3/4 mutants leaf-like

structures replace flowers (Ditta et al. 2004). Furthermore, transgenic plants with

inhibited expression of FBP2 in petunia and TM5 in tomato by co-suppression or

antisense technology developed highly aberrant flowers with modified whorl 2, 3 and

4 organs [19, 15, 17].

SEP1/2/3 are still expressed in B and C loss-of-function mutants, and the initial

expression patterns of B and C class genes are not altered in the sep1/2/3 triple

mutant, indicating that SEP 1/2/3 do not act downstream of the B and C genes, and

that they are not required for the initial activation of the B and C class genes,

respectively [18]. Thus, SEP genes comprise a separate class of floral homeotic genes

that provide an additional function. The SEP proteins form higher-order complexes

together with class A, B, or C proteins, several of which are sufficient to transform

leaves into floral organs [14]. These findings led to the suggestion of a protein-based

combinatorial "floral quartet model" that could explain how the different floral organ

identity genes interact at the molecular level [7, 11]. This model predicts that different

floral homeotic proteins form quaternary DNA-binding transcription factor complexes

that may bind to two cis-regulatory elements termed CArG-boxes (consensus: 5'-

CC(A/T)6GG-3’). These factors which result from the expression of A, B, and C

function genes and in addition SEP genes regulate the expression of downstream

target genes responsible for the development of the identities of different floral

organs. More specifically, the revised "ABCE" model postulates that sepals are

specified by A protein activity alone, petals by A+B+E, stamens by B+C+E, and

carpels by C+E [20, 7, 11]. Thus, E activity and consequently SEP expression is

required for the formation of whorl 2, 3 and 4 organs.

Previous results from our group indicated that expression of the B-type paleo AP3-like

homologs in crocus is extended in all four whorls including whorl 4 and whorl 1 and may be

important for the homeotic transformation of whorl 1 sepals into tepals in this species [21].

We also demonstrated the presence of PI homologs, too in all four whorls of crocus flower

[22]. In the present study we have isolated and characterized four SEP-like sequences

from crocus and show that expression of SEP that should be required for whorl 1 tepal

formation under the predictions of the ABCE "quartet model" is also present in whorls 1 and

the combined action of AP3, PI, and SEP may be responsible for morphogenetic patterning

of tepals during flower development in crocus proving further support for the need of

ABCE activities for the formation of tepals. Furthermore the expression of ABE activities

in the reproductive whorls including whorl 4 of carpel together with the previously reported

expression of C-type CsatAG gene [23] raises question of their role in carpel formation in

crocus.

2. Materials and methods.

2.1. Plant material.

Crocus field growing plants were collected from Kozani, Greece. Tissues from

wild type crocus and two mutant plants, one lacking stamens and one with double

number of flower organs were collected. Sampling was during the late flowering

season in October. Tissues were separated and immediately frozen in liquid nitrogen

and stored at –80 0C until used.

2.2. RNA isolation and cDNA synthesis.

Total RNA from flowers and leaves of crocus field growing plants were

extracted using Trizol reagent (Invitrogen) following the manufacturers instructions.

First-strand cDNA synthesis was performed with 200u SuperScript II-RT (Invitrogen)

in a 20μl reaction volume containing a pool of 5 μg total RNA from flowers and

leaves (2.5μg each), 0.5μg NdT-Adaptor primer (5’-

TGACTAATGAATGTGGTAATGA(T)18-3’) 5’ phosphate (synthesized at VBC-

genomics, Austria), 0.5mM dNTPs, 1x first strand buffer (Invitrogen) and 10mM

DTT (Invitrogen) . The reactions were incubated 50min at 420C followed by 15 min at

70 0 C to inactivate the RT. After the addition of 2 units RNAseH (Invitrogen) the

reactions were incubated 20 min at 37 0 C. The reactions were purified using the

QIAquick PCR purification kit (Qiagen).

2.3. Circularization and rolling-cycle amplification of cDNA

Ten μl representing 1/3 of the purified cDNA were used in a circularization

reaction containing 100 units CircLigase (EPICENTRE), 1x reaction buffer and 50

μM ATP (all reagents from EPICENTRE). The reactions were performed for 1h at 600 C followed by inactivation of the enzyme 10 min at 80 0 C. The reactions were

purified using the QIAquick PCR purification kit (Qiagen). Rolling circle

amplification reactions were performed in 50 μl volume containing 1/6 (5μl) of the

circularized cDNA, 1mM dNTPs, 200μg/ml BSA (NEB), 1x phi29 DNA Polymerase

reaction buffer (NEB), 10u phi29 DNA Polymerase (NEB) and 1μM NSFInF primer (

5’-TTCATTAGTCAG-3’), with two PTO linkages on the 3’-end (custom synthesized

at VBC-genomics). The reactions were incubated for 1h at 300C followed by the

addition of 1μM NSFInvR (5’-TGTGGTAATGAT-3’) and further incubated for 1h at

300C. Finally heat inactivation was performed for 10min at 600C.

2.4. Direct cloning of CsatSEP genes.

A 1 μl aliquot of a 1/10 dilution of the rolling circle amplification reaction was

used for the PCR reactions. The reactions were performed in 50 μl volume containing

0.2mM dNTPs,1u DyNAzyme II DNA polymerase (Finnzymes), 0.4μM MADS-2F

degenerate primer (5’- GTKCTYTGYGAYGCYGAGGT -3’) designed from the

conserved amino acid sequence V L C D A E V and in sense orientation and either

0.4μM MADS5R degenerate primer (5’- AAYGTNACYTGNCGRTTDAT -3’)

designed from the conserved amino acid sequence I N R Q V T F and in antisense

orientation or 0.4μM MADS6R degenerate primer (5’-

CGRTTDATYTTRTTYTCDAT -3’) designed from the conserved amino acid

sequence I E N K I N R and in antisense orientation. The cycling conditions were: 3

min at 940C, 40 cycles of 45sec at 940C, 45sec at 500C, 1.5min at 720C and a final

extension step of 10min at 720C.

2.5. Cloning of MADS-box genes using famRCA-RACE.

In a second approach, 1μl of the RCA-RACE library from crocus prepared with

the InVUP primer as described [24], was used as template for inverse PCR using now

degenerate MADS-box primers under the same conditions as above. Primer

combination MADS-2F/MADSPromR degenerate primer (5’-

CTTVSAGWAGGTSACYTG-3’) corresponding to the conserved amino acid

sequence Q V T F S K of the MADS-box genes and designed in antisense orientation

was used under the same conditions as above. Several PCR products ranging from

900bp to 1300bp were excised from an agarose gel, ligated into pCR 2.1-TOPO

vector using the PCR II-TOPO TA cloning kit (Invitrogen) and transformed in DH5a

competent cells. Several individual clones were screened for the presence of an insert

and sequenced.

Based on the sequence information obtained from SEP-like sequences isolated,

gene specific primers were designed and used for PCR to complete small missing

parts (39-57 nucleotides between the two outward oriented primers) and obtain the

full length coding sequence of the CsatSEP3-like genes. Having as template 1/30 of

the single stranded cDNA used in the RCA-RACE experiment the reactions contained

also 0.2mM dNTPs and 1u DyNAzyme II DNA polymerase (Finnzymes) and 0.4μM

of the corresponding primers. The cycling conditions were: 2 min at 940C, 35 cycles

of 45s at 940C, 30s at 540C, 1.5min at 720C and a final extension step of 10min at

720C. The primers used were: Sep3-F (5’-AATAAATTGGGCTCTCAGAA-3’) and

Sep3-2R (5’-ATCGAAGGGCTGATAATTAACC-3’) for amplifying CsatSEP3a,

Contig3F (5’-TTCTAGAGAGAGAAATTAGGTAGT-3’) and Contig3R (5’-

ATGTTCTTTTGATCGATTTGGGAC-3’), for amplifying CsatSEP3b and

Contig5/6F (5’-GAAGGAGAGAAATCGTTGGTAATT-3’) and Contig5/6R (5’-

GCAAGTAACCAAGAGCAAATCACT-3’) for amplifying CsatSEP3c and d. A

single PCR product from each primer pair was cloned as above. Several individual

clones were screened for the presence of an insert and sequenced (Macrogen, Korea).

2.6. Phylogenetic analysis.

The deduced amino acid sequences of the isolated CsatSEP alleles were used in

BLAST searches in the GenBank and the best hits were for the SEP-like protein from another

monocot Asparagales species, the Asparagus officinalis AOM1 (AAQ83834; [25] with

86% identity over the entire sequence. CsatSEP phylogeny was reconstructed using

sequences from Oryza sativa (AAG35652, AAB64250, AAF21900, AF058697,

AAF04972 and AAB71434), Zea mays (AAB00078, AF112150 and AJ430695),

Arabidopsis thaliana (P29382, P29384, P29386, O22456 and Z16421), Asparagus

officinalis (AAQ83835 and AAQ83836), Vitis vinifera (AAM21343),

Chrysanthemum x morifolium (AAO22987), Lolium perenne (AAO45876),

Ranunculus bulbosus (AAP83408), Triticum aestivum (AAQ11687 and BAA33458),

Aristida longiseta (AAT07925), Avena sativa (AAT07926), Magnolia

praecocissima (BAB70739), Agapanthus praecox (BAC66964), Oncidium Gower

Ramsey [26], Antirrhinum majus (CAA64742), Gnetum gnemon (CAB44457), Pisum

sativum (AJ279089), Eucalyptus globulus (AF305076) and the three previously

isolated crocus AP1/FUL-like sequences CsatAP1/FULa (AY337928),

CsatAP1/FULb (AY337929), and CsatAP1/FULc (AY337930) [3, 27].

Alignment was performed by the ClustalW method [28]. Phylogenetic relationships

of the sequences were examined using the Neighbor-Joining Method with p-distance

correction [29]. Bootstrap values were derived from 1000 replicate runs. A phylogenetic tree

was constructed using the MEGA 2.1 software [30].

2.7 Domain distribution across MADS-box proteins from crocus

All known MADS-box proteins from Crocus sativus were retrieved from

genebank. The domain compositions of the proteins were determined according to the

PRODOM database [31]. The full sequence of each protein was subject to a BLAST

search [32] against the PRODOM database with default BlastP parameters. Only

domains with an E-value smaller than 1 X 10-4 or more than 60% conserved residues

were considered to be recognized, as described in [33]. Since in PRODOM domains

are conserved segments of sequence and they do not necessarily represent structural

domains, a sequence can be represented by more than a single PRODOM domain. In

case of overlapping domains, only the domain with the highest score was presented.

2.8. Expression analysis.

The expression analysis of the isolated CsatSEP3-like genes was performed

with RT-PCR. Total RNA from leaves, flowers, sepals, petals, stamens, carpels,

carpels of a mutant flower lacking stamens and a double flower were extracted using

the RNeasy plant mini kit (Qiagen).On-column digestion of DNA during RNA

purification was performed using the RNase-Free DNase Set (Qiagen). One μg of

total RNA from each sample were used in a reverse transcription reactions containing

200u SuperScript II-RT, 0.5 μg NdT-Adaptor primer, 0.5mM dNTPs, 1x first strand

buffer and 10mM DTT in a 20μl reaction volume. The reactions were incubated

50min at 420C followed by 15 min at 70 0 C to inactivate the RT. PCR was performed

having as template 1/20 of the samples cDNAs supplemented with 0.2mM dNTPs ,1u

DyNAzyme II DNA polymerase and 0.4μM of the corresponding primers. A control

PCR having as template 50ng genomic DNA from crocus was included for each

primer pair. Successful cDNA synthesis was monitored by amplifying a fragment of

the actin-beta gene as described [21]. The primers used were: Sep3-F/Sep3-2R for

CsatSEP3a (annealing at 500C), CsSEP3B-F (5’-

CTTGAAAGACAACTTGATTCGTCG-3’)/Contig3R for CsatSEP3b (annealing at

560C), CsSEP3C-F (5’-GAGCCTCCGCAAAAAGCTGGAAG-3’)/ CsSE3C/D-R (5’-

GCAAGTAACCAAGAGCAAATCAC-3’) for CsatSEP3c (annealing at 580C) and

CsCEP3D-F (5’-GAGCCTCCGCAAAAAGTCTCCTT-3’)/CsSE3C/D-R for

CsatSEP3d (annealing at 580C). The cycling conditions were: 2 min at 940C, 35

cycles of 30s at 940C, 45s at annealing temperature, 45s at 720C and a final extension

step of 10min at 720C. The PCR products were separated on 2% agarose gels where

amplification products of the expected size could be observed.

3. Results.

3.1. Isolation of CsatSEP3-like genes.

Thirty individual clones obtained from cloning of MADS-box genes using

degenerate MADS primers in the famRCA-RACE as well as SEP-like genes

specifically, with inserts ranging from 900bp to about 1300bp were sequenced. In a

first analysis 4 sequences were discarded because represented PCR and circularization

artefacts. BLAST similarity searches revealed that 26 remaining clones showed

homology to MADS-box genes from different plant species. These sequences were

grouped to seven distinct contigs using DNA STAR software. BLAST similarity

searches revealed 4 of the 7 contigs showed homology to other Sep-like MADS-box

genes from different plant species while differences were found among them. Two of

them were very similar differing only in a 15bp insertion/deletion.

To verify the above four sequences and complete small missing parts between

inverse primers (see Materials and Methods), gene specific primer designed were used

in RT-PCR as described in the materials and methods section. All three primer pairs

used produced a clear single RT-PCR product. Five to six individual clones for each

one of the 4 contigs were sequenced and analyzed. The sequences obtained together

with the sequences of the original generated RCA-RACE clones were reassembled to

four different sequences witch were termed CsatSEP3a, CsatSEP3b, CsatSEP3c and

CsatSEP3d (GenBank accession nos. EU424137, EU424138, EU424139, EU424140,

respectively). Part of the 3’ end of CsatSEP3c was identified in the recently published

partial collection of crocus stigma-specific 6.803 ESTs [34] under the cluster id.

cr.saCl001179. The rest crocus SEP-like sequences isolated in this study were not

represented in that partial collection.

3.2. Sequence alignment and C-terminal motifs

The alignment of CsatSEP3 deduced amino acid C-terminal sequences with

other SEP-like proteins using Clustalw is shown in Figure 1. The C-terminal motifs

are typical for members of the SEP lineage [35-37]. The crocus sequence has a motif

(YMPGWLQ) characteristic of SEP3-like MADS-box proteins that form a separate

branch in the SEP lineage. Similar motifs as shown in Fig.1 are present in Arabidopsis

thaliana SEP3, Antirrhinum majus DEFH72, and Lilium longiflorum LMADS3 and

LMADS4.

3.3. Phylogeny

SEP genes form a well-supported clade within the MADS box gene phylogeny that can

be further subdivided into three clades: a mixed eudicot and monocot clade containing the

Arabidopsis thaliana SEP3 gene, a eudicot clade containing the Arabidopsis SEP1,

SEP2, and AGL genes, and a clade comprised solely of monocot sequences that is not

homologous to either the SEP3 or the SEP1/SEP2/AGL3 clade [36]. The phylogenetic

relationships of the CsatSEP3-like protein with members of the SEP lineage, together

with the AGL6 and AP1/FUL lineages are shown in Fig. 3. The tree was generated by

the Neighbor-Joining method using the p-distance correction. The crocus gene fall

into a branch together with the Asparagus officinalis (Asparagales) AOM1 SEP-like

sequence. This branch belongs in an SEP3 clade that also includes other SEP3-like

monocot as well as eudicot sequences.

3.4. Patterns of domain composition are conserved between Crocus sativus and

Arabidopsis thaliana

The current sequencing of SEP-like genes extends the variety of MIKCc-type MADS-

proteins from crocus and enables to perform a relatively comprehensive analysis of

the domain composition in this family of transcription factors in crocus. In a previous

study the domain composition of MADS-box subfamilies from Arbidopsis was

determined by identifying the PRODOM-domain content of the corresponding

proteins [33]. The MADS-box domain (PRODOM domain PD000256) and the K-box

domain (PRODOM domain PD000423) were shown to be highly conserved across all

subfamilies, whereas the domain compositions along the C-terminal part are

conserved within but not across subfamilies. Here, we repeated the analysis in crocus,

while using all of the available sequences of MADS-box proteins (Figure 3). All of

the crocus MADS-box proteins are of the MIKCC-type, i.e., in addition to the MADS-

box domain these proteins possess I, K, and C domains [38]. In accordance with the

results from Arabidopsis, we observe that the MADS-box domain and the K-box

domain are found in all subfamilies, whereas the domain compositions along the

C-terminal part are conserved within but not across subfamilies. Similar to

Arabidopsis, the only two subfamilies which share a common third domain are the

SEP-like and AGL6 subfamilies, most likely reflecting the comparatively high

relatedness of theses subfamilies [33].

It is interesting to note that in 4 out of the 6 subfamilies studied (AG, AP3, AGL6,

and SEP-like) the overall domain pattern, including the C-terminus of the proteins, is

conserved between crocus (a monocot) and Arabidopsis (a eudicot). This is surprising

since the C-terminus of the MIKCC-type MADS-proteins was suggested to be

significantly less conserved than the MADS-box and K-box domains [33, 39].

Moreover, the commonly distributed PRODOM domain PD000423 corresponds only

to the first 2 of the 3 predicted α-helices of the K domain [40], helices that were

shown to be more significance to the formation of dimmers than the third helix [41].

The high conservation of the domain composition within subfamilies and across

relatively distant species (Arabidopsis and crocus), found here, indicates that the c-

terminus part of MIKC-type proteins is more conserved than previously suggested.

Considering the high level of conservation of the interaction partners found within the

different subfamilies [33], our results suggest that the nature of the domain

composition in the C-terminus has a role in specifying the nature of interactions.

3.5. Expression analysis of Csat SEP-like in wild type and mutant flowers.

The expression pattern of the isolated CsatSEP-like genes was compared in

different tissues by RT-PCR. Experiments revealed the presence of the transcripts

mainly in flowers and not in leaves (although a weak amplification product was

observed in leaves for CsatSEP3b and CsatSEP3c). The expression pattern of the

CsatSEP3-like genes was also examined in different flower tissues. As shown in

Figure 4 the RT-PCR experiment performed with cDNA synthesized from sepals,

petals, stamens and carpels resulted in the identification of all four transcripts in all

mature flower parts. No differences in expression were observed in the mutant flowers

examined (Figure 5).

4. Discussion

SEP-like genes play an important role in flower development encoding one

member of the transcriptional regulatory complex controlling organ formation in the

three inner whorls of mature flower in most plants. The class E Arabidopsis SEP

genes encode related proteins that are redundantly required to specify petals, stamens

and carpels, as sep1sep2sep3 triple mutant flowers contain only sepals. SEP4 is

required redundantly with the other three SEP genes to confer sepal identity and

contributes to the development of the other three organ types. SEP MADS box gene

phylogeny revealed major duplication events during angiosperm evolution [37, 42]. Thus, SEP

phylogeny is subdivided into a mixed eudicot-monocot clade containing the Arabidopsis

SEP3 gene where all four isolated crocus genes reported here also belong (Figure 2), a

eudicot clade containing the Arabidopsis SEP1, SEP2, and SEP4 genes, and a clade

comprised solely of monocot sequences [36]. Despite our extensive search only SEP3 type

genes were isolated from the monocot crocus flower. All lineages have a conserved C-terminus

common internal motif, but have diverse motifs at the 3’end. The alignment of CsatSEP3

deduced amino acid C-terminal sequences with other SEP-like proteins revealed a

C-terminal motif (YMPGWLQ) typical for members of the SEP3 lineage [35] present

in CsatSEP3A and CsatSEP3B, while a similar but modified (YTPGWFP) motif was

present in CsatSEP3C and CsatSEP3D sequences (Figure 1). Similar motifs are

present in Arabidopsis SEP3, antirrhinum EFH72, and Lilium longiflorum LMADS3

and LMADS4. Our analysis indicates that the domain composition in the SEP-like

protein subfamily, as in three other subfamilies, remains conserved between

Arabidopsis and crocus (Figure 3). In Arabidopsis, the conservation of intra-

subfamily domain composition is correlated with the conservation of the functional

role of the family member – i.e., the conservation of their interactions partner [33].

Unlike the functional-structural conservation, the expression pattern of SEP-like

proteins differs between monocots and eudicots. Within monocots, SEP-like genes

have been most intensively studied in grasses, including the important cereals maize

and rice. SEP-like genes in grasses show relatively heterogeneous expression patterns,

strongly suggesting that they are also functionally not a homogeneous class of genes

[43]. In maize, there are at least eight different SEP-like genes with distinguishable

expression patterns that have been suggested to be involved in determining the

alternative identity of spikelet primordial, the upper vs. the lower floret within each

spikelet primordium, or conferring determinacy to the spikelet or upper floret

meristem [7, 38, 44]. Similar observations have been reported for rice plants in which

OSMADS1, the putative ortholog of the maize ZMM8, plays an important role in floral

meristem determination during the early development of rice florets [45]. Two AGL2-

like MADS-box genes, later renamed LMADS3 and LMADS4 from lily (Lilium longiflorum),

were expressed in the inflorescence meristem, and in floral buds of different developmental

stages. LMADS4 mRNA is also expressed in vegetative leaf and in the inflorescence stem

where LMADS3 expression is absent [46]. Expression of the isolated SEP-like genes of

crocus was detected in all four whorls of flower organs. This pattern of extended

expression of E-type genes together with B-type genes reported previously [22, 21] is

compatible with tepal formation in whorl 1 of crocus. Muscari armeniacum is another

member of Asparagales that has petaloid organs in the outer two whorls and expression

of class B genes extended to whorl 1, similarly to crocus [47]. This extended expression

of class B and class E genes to whorl 1 fits the modified ABCE model proposed to

explain tepal formation in tulip and other nongrass monocots [48-50]. However,

expression of B-type and E-type genes in whorl 4 observed in crocus and other nongrass

monocts is a result difficult to explain [50]. One possibility is that although gene

expression is observed in whorl 4, active protein is not synthesized. This may occur

when expression levels are not adequate for accumulation of translated products or post-

transcriptional mechanisms are involved to restrict ectopic protein synthesis. In the lily

Lilium longiflorum, for example, a class B gene (LMADS1) is strongly expressed in the

second and third and weakly expressed in the first and fourth whorls but the protein is

synthesized only in whorls 2 and 3, suggesting that the expression of LMADS1 is regulated

post-transcriptionaly [51]. It would be very informative to examine protein accumulation of

crocus B- and E-type genes in whorl 4 in order to verify their presence at this site.

In summary, four crocus SEP-like genes have been isolated and their phylogenetic

relationships and expression patterns have been characterized. All four isolated genes

belong to the SEP3 mixed eudicot-monocot subclade of MADS-box proteins and although

presence of other crocus SEP-like genes cannot be ruled out, these were the only genes

isolated after extensive search with different methods. The four genes were expressed in all

flower organs and in the mutant flowers indicating that the CsatSEP genes were not involved

in conferring the mutant phenotypes. These data suggest that the inferred role of the isolated

genes is compatible with that of class E MADS-box transcription factors.

Figure legends

Figure 1. Alignment of the four CsatSEP deduced amino acid sequences C-

terminus regions with selected representatives of SEP-like proteins and the outgroup

lineage AGL6-like proteins using ClustalW (Thompson et al., 1994). Three highly

conserved C-terminal motifs, SEP-motif I, SEP/AGL6-motif, and SEP-motif II

designated according to Kanno et al. (2006) are boxed and residues that are highly

conserved in these regions are shaded.

Figure 2. Phylogenetic relationships of the four CsatSEP proteins with

members of SEP-like MADS-box proteins in other plants. Condensed branches of the

AP1/FUL and AGL6 clades are shown. The tree was generated by the Neighbor-

Joining method using the p-distance correction. Numbers next to the nodes are

bootstrap values from 1000 replications. Scale indicated amino acid substitutions.

Figure 3. Domain conservation and divergence across the MADS proteins and

subfamilies. Full boxes highlight similarities. The phylogeneetic tree was constructed

according to CLUSTALW multiple alignment. The figure is adaptation to crocus of a

figure from (Veron et al, 2007) describing the domain conservation in Arabidopsis.

Figure 4. PCR amplification products on cDNA from Leaves (L) and Flowers

(F), Sepals (S), Petals (P), Stamens (ST) and Carpels (CA) using the CsSEP3a-,b-,c-

,d-and β-actin-gene specific primers. MW indicates the λHindIII/ΦX174HaeIII

molecular weight marker.

Figure 5 Gene expression analysis with RT-PCR in carpels of wild-type (WT)

flower, a mutant (M) crocus flower with no stamens and a double flower (DF) using

CsatSEP3a-, b-, c-, d- gene specific primers.

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SEP motif I SEP/AGL6 motif SEP motif II

SEP1 A. thaliana AHHQAQSQGLYQPLEC---NPTLQMGYD--NPVCSEQITATTQAQ---AQQGNGYIPGWML------SEP3 A. thaliana HQQQQHSQAFFQPLECE---PILQIGY-------QGQQDGMGAGP----SVNNY-MLGWLPYDTNSISEP4-AGL3 A. thaliana SNPPIQEAGFFKPLQG---NVALQMSSHYNHNPANATNSATTSQN------VNGFFPGWMV------CsatSEP3a C. sativus QANQ-QEEEFYQPLDCQ---PTLQIGF-------QAD---QMAGP----SVTN-YMPGWLQ------CsatSEP3b C. sativus QANQ-QREEFYQPLDCQ---PTLHIGF-------QGD---QMAGP----SVTT-YMPGWLQ------CsatSEP3d C. sativus QQTQPQVGEFFHPLACQ---PTLQMGF-------QTE---QLSGP----SAST-YTPGWFP------CsatSEP3c C. sativus QQTQPQVGEFFHPLACQ---PTLQMGF-------QTE---QLSGP----SAST-YTPGWFP------AoMADS1 A. officinalis QPNQPHGDQFFHPLECQ---PTLQIGF-------QPD---QMPGP----SVSN-YMPGWLA------AoMADS2 A. officinalis QPSQPQGEEFFHPLECQ---PTLQIGF-------QPD---QMPGP----SASS-FMPGWLQ------DEFH72 A. majus AAQPQG-DGFFHPLECE---PTLQMGF-------QSEITVGAAGP----SVNNYNMTGWLP------SEP-like T. aestivum QAPHHGGNGFFHPLDP-TTEPTLQIGY-------TQE---QINNA----CVAASFMPTWLP------AOM4 A. officinalis QPSQPQGEEFFHPLECQ---PSLGG---------------QC-------------------------

AGL6-like A. praecox VFSMHPS—-HSSAMECE---PTLQIGY---HQLVQPEG-SLPRN----SGGENNFMLGWVL------AOM3 A. officinalis AFSMHPS--QSSAMDCE---PTLQIGY---HHLVQPEA-ALPRS----SGGENNFMLGWVL------CsatAGL6a C. sativus AFPIHPS--QSSAMDCE---PTLQIGY---HHLVQPET-ALPRN----SAGENNFMLGWVL------CsatAGL6b C. sativus AFPFHPS--QSSAMDCE---PTLQIGY---HHLVQPET-VLPRI----SEGENNFMVGWVL------AGL6-like R. bulbosus NYSGHPS--HSSSMDCE---PTLQIGY---HQYVSADGGPIQRN----NAGENNFIQGWEL------

SE

P-l

ike

A

GL

6-l

ike

Figure 1

Figure(s)

CsatSEP3a

CsatSEP3b

AOM4 Asparagus officinalis

CsatSEP3c

CsatSEP3d

SEP-like Triticum aestivum

SEP3 Arabidopsis thaliana

DEFH72 Antirrhinum majus

Mixed monocot-eudicotSEP clade

SEP1 Arabidopsis thaliana

SEP2 Arabidopsis thaliana

SEP4-AGL3 Arabidopsis thaliana

LHS1 Aristida longiseta

OsMADS1 Oryza sativa

LHS1 Avena sativa

OsMADS5 Oryza sativa

AP1/FUL

AGL6

100

72

99

100

100

100

92

99

99

100

94

90

96

88

97

0.000.050.100.150.200.25

Monocot SEP clade

Eudicot SEP clade

Figure 2

Figure 3

MW L F S P St Ca

CsatSEP3a

CsatSEP3b

CsatSEP3c

CsatSEP3d

actin

Figure 4

CsatSEP3a CsatSEP3b CsatSEP3c CsatSEP3dMW WT M DF WT M DF MW WT M DF WT M DF

Figure 5