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Capsicum annuum S (CaS) promotes reproductive transition and is required for flower formation in pepper (Capsicum annuum) Oded Cohen, Yelena Borovsky, Rakefet David-Schwartz and Ilan Paran Institute of Plant Science, Agricultural Research Organization, The Volcani Center, PO Box 6, Bet Dagan 50250, Israel

Summary Author for correspondence: Ilan Paran Tel: +972 3 9683943 Email: [email protected] Received: 2 December 2013 Accepted: 7 January 2014

New Phytologist (2014) 202: 1014–1023 doi: 10.1111/nph.12711

Key words: flower formation, pepper (Capsicum annuum), petunia (Petunia hybrida), sympodial growth, tomato (Solanum lycopersicum), transition to flowering.

 The genetic control of the transition to flowering has mainly been studied in model species, while few data are available in crop species such as pepper (Capsicum spp.). To elucidate the genetic control of the transition to flowering in pepper, mutants that lack flowers were isolated and characterized.  Genetic mapping and sequencing allowed the identification of the gene disrupted in the mutants. Double mutants and expression analyses were used to characterize the relationships between the mutated gene and other genes controlling the transition to flowering and flower differentiation.  The mutants were characterized by a delay in the initiation of sympodial growth, a delay in the termination of sympodial meristems and complete inhibition of flower formation. Capsicum annuum S (CaS), the pepper (Capsicum annuum) ortholog of tomato (Solanum lycopersicum) COMPOUND INFLORESCENCE and petunia (Petunia hybrida) EVERGREEN, was found to govern the mutant phenotype. CaS is required for the activity of the flower meristem identity gene Ca-ANANTHA and does not affect the expression of CaLEAFY. CaS is epistatic over other genes controlling the transition to flowering with respect to flower formation.  Comparative homologous mutants in the Solanaceae indicate that CaS has uniquely evolved to have a critical role in flower formation, while its role in meristem maturation is conserved in pepper, tomato and petunia.

Introduction Flowering plants undergo major transitions in their life cycles: first a transition from the vegetative juvenile phase to the vegetative adult phase and then a transition to the reproductive phase (Huijser & Schmid, 2011). The transition from the vegetative phase to the flowering phase has been most extensively characterized in Arabidopsis (recently reviewed by Srikanth & Schmid, 2011). The shoot apical meristem (SAM) controls the aerial part of the plant, first producing the stem and leaves and then, upon the transition to flowering, producing the inflorescence meristem (IM). Plants such as Arabidopsis and Antirrhinum majus are characterized by monopodial shoot architecture in which the SAM is active throughout the plant’s life (indeterminate); that is, after the vegetative phase, flowers develop continuously on the flank of the SAM. By contrast, plants in the family Solanaceae, such as petunia (Petunia hybrida), tomato (Solanum lycopersicum) and pepper (Capsicum annuum), are characterized by sympodial shoot architecture: the SAM terminates in an IM, and plant growth continues from lateral meristems, termed sympodial meristems (SYMs), which develop in the axil of the youngest leaf below the apical inflorescence (Schmitz & Theres, 1999). The SYM develops into a shoot segment termed the sympodial unit (SU), 1014 New Phytologist (2014) 202: 1014–1023 www.newphytologist.com

consisting of vegetative and reproductive organs in a speciesspecific pattern, and new SYMs are repeatedly formed from the axils of the uppermost leaves of the preceding SU. Two of the most important genes determining inflorescence architecture and floral meristem (FM) identity in Arabidopsis are LEAFY (LFY) and TERMINAL FLOWER1 (TFL1). LFY is a transcription factor that responds to external stimuli, and directly and indirectly activates or represses a large network of genes that determine the identity of floral organs (Winter et al., 2011; Grandi et al., 2012). LFY has a role in regulating flowering in all flowering plants, and it also exists in nonflowering plants; however, its role in these latter plants is not well understood (Moyroud et al., 2010). TFL1 is a flowering repressor required to maintain the indeterminate state of the inflorescence (Bradley et al., 1997). Theoretical modeling suggests that variation in the architecture of the inflorescence may be explained by alterations in the spatio-temporal expression patterns of LFY and TFL1 (Prusinkiewicz et al., 2007). While research on the regulation of flowering and shoot architecture has focused mainly on monopodial species, in particular Arabidopsis (Srikanth & Schmid, 2011), knowledge of the genes, and their interactions, regulating these processes in sympodial plants has lagged behind. The most studied sympodial model Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

New Phytologist species are tomato and petunia. In both, generation and characterization of developmental mutants have enabled the identification of key genes controlling flowering time, inflorescence architecture, axillary branching and sympodial shoot development (reviewed by Angenent et al., 2005; Lozano et al., 2009; Castel et al., 2010). Those studies have revealed that, in most cases, genes that function in regulating these processes in sympodial plants have a related function in monopodial plants. The tomato reproductive shoot is composed of reiterated SUs each consisting of three leaves and an inflorescence. The inflorescence of tomato has a reiterated sympodial shoot structure composed of one-nodal SUs each terminated by a single flower (Lippman et al., 2008). The tomato inflorescence architecture exhibits large phenotypic variation in the degree of branching. The inflorescences of mutants such as single flower truss (sft) and uniflora (uf) consist of single flowers (Dielen et al., 2004; Lifschitz et al., 2006), while those of anantha (an), falsiflora (fa) and compound inflorescence (s) are highly branched (Lippman et al., 2008). SINGLE FLOWER TRUSS (SFT ) is the ortholog of the florigen-encoding gene FLOWERING LOCUS T (FT ) in Arabidopsis. ANANTHA (AN ) and FALSIFLORA (FA ) are the orthologs of the FM identity genes UNUSUAL FLORAL ORGANS (UFO) and LFY in Arabidopsis, respectively, and COMPOUND INFLORESCENCE (S ) encodes a Wuschelhomeobox (WOX) transcription factor that functions in the maintenance of stem cells and is not known to have a role in inflorescence architecture in Arabidopsis. After termination of the primary stem by a single flower, the petunia reproductive shoot is composed of reiterated SUs each consisting of two leaf-like organs (bracts) and a single flower (Castel, 2009). Unlike UFO in Arabidopsis, which has a limited role in regulating inflorescence architecture and IM formation, the role of the petunia ortholog DOUBLE TOP (DOT) in determining inflorescence architecture is more critical, and its expression is necessary and sufficient to induce flowering (Souer et al., 2008). In dot mutants, the inflorescence has a leafy appearance and it lacks flowers, resembling the phenotype of the tomato ortholog an. Expression of DOT is dependent on prior expression of EVERGREEN (EVG), the homolog of tomato S (Rebocho et al., 2008). DOT further activates ABERRANT LEAF AND FLOWER (ALF ), the petunia ortholog of LFY. In both tomato s and petunia evg mutants, expression of AN and DOT is delayed or reduced (Lippman et al., 2008; Rebocho et al., 2008). The delay of AN expression in s mutants is postulated to extend the period of IM indeterminacy, leading to the mutant’s highly branched inflorescence phenotype (Lippman et al., 2008). By contrast, lack of DOT activation in evg results in loss of flowering (Rebocho et al., 2008). Pepper (Capsicum spp.) is a member of the Solanaceae, having a sympodial shoot structure with solitary flowers. The distinct shoot architecture of pepper compared with other studied Solanaceae such as tomato and petunia suggests possible diversification of genes and pathways involved in controlling this trait. Therefore, to extend our understanding of the genetic control of shoot architecture in the Solanaceae, natural and induced variation in pepper mutants altered in their shoot architecture was isolated Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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and characterized. Those studies enabled the identification of key genes controlling sympodial shoot development (FASCICULATE and CaJOINTLESS), flower formation (Ca-ANANTHA) and axillary branching (CaBLIND and CaHAM) in pepper (Lippman et al., 2008; Elitzur et al., 2009; Jeifetz et al., 2011; Cohen et al., 2012; David-Schwartz et al., 2013). In the course of screening an ethyl methanesulfonate (EMS)-mutagenized population (Paran et al., 2007), allelic mutants that lack flowers were observed. The complete lack of flowers and additional associated phenotypes indicated that the disrupted gene in those mutants has a central role in regulation of shoot architecture and flower formation in pepper. The goals of the present work were to comprehensively characterize the unique nonflowering mutation, to isolate the gene disrupted by the mutation, and to compare gene function in the Solanaceae by utilizing homologous mutants in other species. We report the identification of CaS, the pepper homolog of S and EVG in tomato and petunia, respectively, as controlling the mutant phenotype. The role of CaS as a flowering promoter and its interaction with other genes controlling reproductive differentiation and shoot architecture in pepper are further described.

Materials and Methods Plant material The mutants Cas (E-327 and E-648), Cajointless (Caj), Ca-anantha (Ca-an) and E-62 were isolated from an EMSmutagenized population with Capsicum annuum cv Maor as the wild-type parent (Paran et al., 2007; Lippman et al., 2008; Cohen et al., 2012). Fasciculate (fa) is a mutant with a spontaneous natural mutation described by Elitzur et al. (2009) that was backcrossed to the ‘Maor’ background. An F2 segregating population was generated by crossing heterozygotes for the E-327 mutation to Capsicum frutescens BG 2816. Double mutants with Cas were derived from F2 populations obtained by crossing the corresponding mutants with individuals heterozygous for the Cas allele E-327, followed by self-pollination and marker-assisted selection. Mapping and isolation of CaS To map the gene governing the E-327 phenotype, the bulked segregant analysis (BSA) approach was used (Michelmore et al., 1991). Two bulks of DNA composed of 15 individuals each from mutant and wild-type plants of the F2 segregating population were constructed and screened with 400 random amplified polymorphic DNA (RAPD) primers. Polymorphic bands were recovered from an agarose gel, cloned into the pGEM-T Easy Vector system (Promega) and used as restriction fragment length polymorphism (RFLP) probes to genotype the F2 population and map the pepper genome (Rao et al., 2003). To clone CaS, tomato primers Sl-WoxF1 and Sl-WoxR1 (kindly provided by Z. Lippman; Supporting Information Table S1) were used to amplify an 834-bp fragment of CaS with pepper genomic DNA as the template. To extend the sequence of CaS toward the 5′ end of the open reading frame (ORF), inverse PCR (Ochman et al., 1988) was performed with the primers CaS-IPCRF1, New Phytologist (2014) 202: 1014–1023 www.newphytologist.com

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CaS-IPCRF2, CaS-IPCRR1 and CaS-IPCRR2 (Table S1) and genomic DNA as the template. To extend the CaS sequence toward the 3′ end of the ORF, 3′ rapid amplification of cDNA ends (RACE) was performed with a Smart RACE kit (Clontech, Palo Alto, CA, USA) and total RNA from the shoot apices. The ORF of CaS from ‘Maor’ was deposited in GenBank (accession number KC414761). Screening for Cas mutants in subsequent experiments was performed by developing a cleaved amplified polymorphic sequence (CAPS) marker using the primers CaS-DdeF and CaS-DdeR (Table S1) followed by restriction digestion with DdeI. Gene expression analysis Total RNA was extracted from shoot apices using the GeneElute Mammalian Total RNA Extraction Miniprep kit (Sigma) followed by DNaseI treatment (Sigma). Total RNA (400 ng) was used for first-strand cDNA synthesis by reverse transcription PCR (RT-PCR) using a PrimeScript RT Reagent kit (Takara Bio Inc., Otsu, Japan). For real-time quantitative PCR (qRT-PCR), three biological and two technical repeats were used for each sample. For the qRT-PCR experiments, plants were grown in a glasshouse under natural daylight during the winter season in Israel. PCR amplification was performed using the primers CaS-qRTF and CaS-qRTR for CaS, CaLFY-qRTF and CaLFY-qRTR for CaLFY and Ca-AN-qRTF and Ca-AN-qRTR for Ca-AN (Table S1). Amplified products were detected using SYBR Premix Ex Taq II (Takara) in a Rotor-Gene 6000 thermal cycler (Corbett Research, Mortlake, Australia). Results were analyzed using ROTOR-GENE 6000 SERIES SOFTWARE 1.7 (Corbett). The relative expression levels of the genes were normalized against CaUBIQUITIN (DQ975458.1) using the primers UBQ-qRTF and UBQ-qRTR.

(Ambion, Austin, TX, USA) and DIG RNA labeling mix (Roche Applied Science, Mannheim, Germany). Scanning electron microscopy (SEM) Samples for SEM were fixed directly in 70% ethanol, and critical point dried as described by Alvarez et al. (1992). SEM was performed in a Hitachi S-3500N instrument (Hitachi, Tokyo, Japan). Phylogenetic analysis Multiple sequence alignments were performed with a web-based version of CLUSTALW ( http://www.ebi.ac.uk/Tools/msa/) using the default settings. The phylogenetic tree was calculated by the neighbor-joining method and bootstrap analysis with 1000 replicates using the MEGA4 software (http://www.megasoftware.net/ mega4/mega.html). The tree was calculated from alignments of the WOX homeodomain consisting of c. 64 of the proteins’ amino acids. Accession numbers were as follows: petunia: PhEVG (EF187281) and SISTER OF EVERGREEN (Ph-SOE) (EF187282); Arabidopsis: WUSCHEL (WUS) (CAA09986), WOX1 (AAP37133), WOX2 (AAP37131), WOX3 (AAP37135), WOX4 (AAP37134), WOX5 (AAP37136), WOX6 (AAP37137), WOX8 (AAP37138), WOX9 (AAP37139), WOX11 (AAP37140), WOX12 (AAP37141) and WOX13 (AAP37142); Antirrhinum majus: Am-WUS (AAO23113); tomato: Sl-S (NP_001234072); Phaseolus coccineus: Pc-WOX9-like (ACL11801); Populus trichocarpa: Pt-WOX9 (CAJ84153); rice (Oryza sativa): WOX-like (Os07g34880) and WUS-like (Os05g48990); pepper (C. annuum): CaS (KC414761).

Results In situ hybridization In situ analysis was performed with digoxigenin (DIG)-labeled probes as previously described (Neta et al., 2011). Meristems were fixed in FAA (formaldehyde : acetic acid : 70% ethanol, 10 : 5 : 85, v/v), then dehydrated and embedded in ParaPlast (McCormick Scientific, St Louis, MO, USA). The tissue was then cut (10 lm) on a Leica RM2245 microtome (VectaMount; Vector Laboratories, Peterborough, UK) and sections were placed on SuperFrost Plus slides (Menzel-Glaser, Braunschweig, Germany) for 2 d on a 42°C hot plate. An antisense DIG-labeled RNA probe was synthesized from the 3′ end of CaS cDNA, excluding the conserved homeodomain, using the MEGAscript kit

Wild-type and mutant phenotypes After germination and during vegetative growth, the pepper SAM gives rise to a stem and leaves arranged in an alternate spiral pattern. After the development of 10.3  1 leaves on the primary stem of ‘Maor’ (Table 1), the SAM undergoes a transition to flowering by converting to an FM that subsequently develops into a flower. Further shoot growth continues from the lateral meristems which develop in the axils of the two uppermost leaves below the apical flower. The subtending leaves from the primary shoot are carried up and also carry SYMs in their axils to allow continuous growth of the shoot. The development of two

Table 1 Transition to flowering of pepper mutants

No. of leaves on primary stem until first flower

Maor

Cas

Caj

fa

E-62

Cas Caj

Cas fa

Cas E-62

10.3  1.0

21.3  1.1

17  0.6

8.6  0.5

4

61.5  1.5

20  1.5

17.7  1.5

For Cas mutants, the number of leaves on the primary stem was measured until initiation of sympodial growth, as reflected by formation of a dichasially forked shoot. Caj, Cajointless; fa, fasciculate. New Phytologist (2014) 202: 1014–1023 www.newphytologist.com

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meristems results in the typical dichasially forked shoot of wildtype pepper (Child, 1979; Fig. 1a,b). During early sympodium development, the SYM generates primordia of two leaves and terminates by forming a single flower, resulting in an SU that consists of two leaves and a flower (Fig. 1a,b). Later in development, the two leaves of the SYM grow upward and away from the flower. The next two SUs emerge at the axils of the leaves of the preceding SU and are separated by its flower. This process repeats itself and results in a shoot architecture that consists of multiple SUs arranged in a perpendicular pattern. An EMS-mutagenized population was generated using the blocky cv Maor as the wild-type parent (Paran et al., 2007) and screened for alterations in shoot architecture. Two M2 families (10 plants per family), E-327 and E-648, were identified, which segregated for a similar lack of flower phenotype in a monogenic recessive manner. No differences in the severity of the phenotype were observed between the two mutants. The two mutants were crossed (in a heterozygous state as revealed by a progeny test) and the mutant phenotype that segregated in the F1 progeny indicated that E-327 and E-648 are allelic. Further work focused only on E-327. The mutants were indistinguishable from wild-type plants during the vegetative growth phase. However, whereas

wild-type plants initiate sympodial growth and floral formation after the development of 10 leaves on the primary stem, initiation of sympodial growth in the mutant as manifested by forming the dichasially forked shoot occurs later after the development of 21.3  1.1 leaves, and the sympodial branches remain fused to each other, forming a fasciated shoot (Fig. 1c). The fasciation is released after formation of a few additional leaves, allowing separation of the shoots (Fig. 1d). Subsequent sympodial growth continues in cycles of SU formation in which the SU consists of a multiple, unfixed number of leaves and occasional fasciation of branches without formation of flowers, giving rise to the leafy phenotype of the mutant (Fig. 1e). To determine the ontogeny of the aberrations in E-327, a series of dissected apices were examined by SEM. In the wildtype, at the stage of two expanding leaves (defined as the number of leaves on the primary stem of at least 3 cm in length), the apical meristem remains vegetative (Fig. 2a). A primordial flower is observed at the six-expanding-leaf stage (Fig. 2b). At the 12-expanding-leaf stage, the terminal flower is flanked by two leaves and the subsequent SUs, each consisting of two leaves and a flower, develop in the axils of the two preceding leaves (Fig. 2c). In E-327, the SAM remains vegetative for a longer time than in

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Fig. 1 Characterization of wild-type and E-327 pepper (Capsicum annuum) plants. (a) Schematic diagram of wild-type parent ‘Maor’. At the transition to flowering, the apical flower (flower of the primary shoot, Fp) and the first two leaves (leaves of the primary shoot, Lp) originate from the primary shoot. The next leaves belong to the first two sympodial units (SUs), each consisting of two leaves (leaves of the sympodial shoot, Ls) and a flower (flower of the sympodial shoot, Fs). (b) Wild-type SU consisting of a flower (Fs) and two leaves (Ls). (c) Schematic diagram of the E-327 mutant showing stem fasciation, lack of flowers and leafy appearance. (d) Initiation of sympodial growth, primary stem fasciation and its release after formation of a few additional leaves. (e) The E-327 sympodial shoot showing stem fasciation and multiple leaves. Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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Fig. 2 Scanning electron micrographs of developing shoot apical meristem (SAM) and sympodial meristems of pepper (Capsicum annuum). (a–c) Wildtype. (a) Vegetative SAM at the two-expanding-leaf stage. P, leaf primordium. (b) SAM at the six-expanding-leaf stage with a developing apical flower (AF; red) flanked by two leaves (green). (c) Meristem at the 12-expanding-leaf stage. The terminal flower (indicated by *; red) is flanked by two leaves (L). The next two sympodial units (SUs), each consisting of two leaves (green) and a primordial flower (red), develop in the axils of the leaves from the preceding SU. (d–f) E-327. (d) Vegetative SAM at the 10-expanding-leaf stage. (e) SAM at the 14-expanding-leaf stage. The apical FM (indicated by *) is precociously terminated and is flanked by two leaves (L). Similar to the wild-type, the next two SUs, each consisting of two leaves (green) and a flower meristem (red), develop in the axils of the leaves from the preceding SU. Additional ectopic leaves develop in the flanks of the meristem. (f) Sympodial apex with multiple fused meristems. Bars, 250 lm.

the wild-type and SYMs are not observed at the 10-expandingleaf stage (Fig. 2d). At the 14-expanding-leaf stage, initiation of sympodial growth is observed with no flower formation (Fig. 2e). Similar to the wild-type, subsequent SUs are developed in the axils of the preceding leaves; however, flowers are not formed. Furthermore, the apical meristem remains vegetative, as indicated by the formation of additional ectopic leaves (Fig. 2e). At a later stage, the mutated sympodial apex is composed of several fused, flowerless, flat meristems (Fig. 2f), resulting in a fasciated shoot. CaS underlies the E-327 mutation To identify the gene governing the E-327 mutant phenotype, the mutation was mapped using the BSA approach (Michelmore et al., 1991). Screening the wild-type and mutant bulks with RAPD primers revealed that marker UBC 390 co-segregates with the mutant phenotype. This marker is linked to the RFLP marker CT277 on chromosome 2 of pepper (Rao et al., 2003). Searching for genes associated with the Solanaceae shoot architecture mapped near this marker enabled the detection of S from tomato as a candidate gene governing the pepper mutant phenotype (Lippman et al., 2008). RFLP mapping of S in an F2 segregating population of 15 mutant and 15 wild-type individuals indicated complete co-segregation with the mutant phenotype. To isolate the pepper homolog of S (CaS), a partial pepper gene was amplified using tomato primers from S and inverse-PCR and RACE techniques to isolate the complete ORF as described in the Materials and Methods section. Analysis of cDNA and genomic clones showed that CaS consists of four exons and encodes a protein of 334 amino acids (Fig. 3a). The two mutant alleles are disrupted at the beginning of the first exon: the E-327 allele is a missense mutation converting proline to leucine at position 54 of the protein. This mutation resides in the conserved homeodomain region, similar to the two tomato s missense mutations New Phytologist (2014) 202: 1014–1023 www.newphytologist.com

reported by Lippman et al. (2008) (Fig. S1). E-648 disrupts the protein-coding sequence at codon 8 by converting it into a stop codon. CaS contains a conserved homeodomain with high similarity to a subfamily of homeodomain proteins from Arabidopsis which includes WUSCHEL and 14 other related WOX proteins. CaS belongs to a subclade of WOX proteins that includes WOX9 from Arabidopsis and other WOX9-like homologs from tomato, Populus trichocarpa, Phaseolus coccineus, rice and petunia (Fig. 3b). CaS is most closely related to petunia EVG, while tomato S is most closely related to petunia SOE, the paralog of EVG that encodes a protein with a function similar to that of EVG (Rebocho et al., 2008). In contrast to petunia, no additional paralogs of S and CaS were detected in tomato and pepper, respectively. The relationships between CaS and genes controlling the transition to flowering and shoot architecture Because of the delay in the initiation of sympodial growth in the Cas mutant, the relationship between CaS and other genes affecting the transition to flowering in pepper was explored. Double mutants of Cas with other late- and early-flowering mutants were created and analyzed. We used Cajointless (Caj) disrupted at the ortholog of tomato JOINTLESS as a late-flowering mutant (Cohen et al., 2012). Early-flowering mutants included fasciculate (fa) which is disrupted at the tomato ortholog of SELF-PRUNING (Elitzur et al., 2009) and E-62, with an unknown disrupted gene. CaJ is a floral promoter in the primary and sympodial shoots of pepper; it suppresses vegetative growth on the shoot and is required for normal development of the flower. The Caj mutant is characterized by late flowering of the primary shoot (flowering after the development of 17  0.6 leaves on the primary stem), additional leaves in the SU and conversion of one of the sepals into a leaf-like organ (Fig. 4a; Cohen et al., 2012). The double Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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shortening of the SUs (Elitzur et al., 2009). These phenotypes result in a compact ‘determinate’ plant carrying condensed clusters of flowers. Double-mutant Cas fa plants are indistinguishable from Cas plants; that is, they initiate late transition to sympodial growth after the development of 20  1.5 leaves and completely lack flowers, indicating epistasis of Cas over fa. E-62 is a new monogenic recessive early-flowering mutant that was identified in the EMS-mutagenized pepper population (Paran et al., 2007). E-62 flowers after four leaves have developed on the primary stem, while no change in flowering pattern and flower structure is observed in the sympodial shoot (Fig. 4d). The double mutant Cas E-62 has the overall phenotype of Cas but its transition to sympodial growth occurs after the development of 17.7  1.5 leaves on the primary stem, an intermediate phenotype between those of the two single mutants, and it completely lacks flowers (Fig. 4e). These phenotypic characteristics suggest that, similar to the relationship with other mutants, Cas is epistatic over E-62 with respect to flower formation. However, the intermediate phenotype of transition to sympodial growth of Cas E-62 suggests an additive effect of the two genes with respect to initiation of sympodial growth (i.e. the effect of the double mutant, measured as the difference between the double mutant and the wild-type, is equal to the combined effects of the individual mutants). The relationships between CaS and FM identity genes

Fig. 3 Gene structure and phylogeny of pepper (Capsicum annuum) CaS. (a) Schematic representation of CaS structure. Introns and exons are indicated by solid lines and white boxes, respectively. Regions encoding the conserved Wuschel-homeobox (WOX)-homeodomain in exons 1 and 2 are indicated by black boxes and numbers indicate the homeodomain position on the gene sequence. Asterisks indicate the position of the mutations in E-327 and E-648. (b) Phylogenetic tree of CaS and its homologs from tomato, petunia, rice, Phaseolus coccineus, Populus trichocarpa, Antirrhinum majus and Arabidopsis. Numbers indicate percentage bootstrap support for each branch (1000 replicates).

mutant Cas Caj (Fig. 4b) has an overall phenotype that resembles that of Cas (Fig. 4c), but initiation of sympodial growth occurs very late, after the development of 61.5  1.5 leaves on the primary stem, and it lacks flowers. These phenotypes suggest that Cas is epistatic over Caj with respect to flower formation, while Cas and Caj act in a positive synergistic manner with respect to initiation of sympodial growth (i.e. the effect of the double mutant, measured as the difference between the double mutant and the wild-type, is larger than the combined effects of the individual mutants). Interestingly, heterozygous plants at the CaS locus in a Caj background initiate transition to sympodial growth very late, after the development of 47.5  2.5 leaves on the primary stem, but, unlike Cas, they do form intact flowers, further indicating an interaction between the two genes. FA is a floral repressor in the primary and sympodial shoots of pepper. The fa mutant is characterized by a reduction of flowering time (8.6  0.5 leaves developing before the first flower), and Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

To better understand the genetic hierarchy that governs flower formation in pepper, the expression patterns of CaS and the FM identity genes CaLFY and Ca-AN were analyzed in the wild-type. Expression of CaS was detected at apical meristems after floral transition but not in vegetative meristems or in more developed flower buds (Fig. 5a). CaS expression was further localized by in situ hybridization to the inner, central cells of the SYM before differentiation into a flower primordium (Fig. 5b). Low expression was also observed in an early stage of the FM (Fig. 5b). Ca-AN expression was detected in both apical meristems at floral transition and at flower buds of wild-type plants, indicating partial overlap in the timing of expression of the two genes (Fig. 5a). CaLFY transcripts were detected at various levels in all examined tissues (Fig. 5a). The expression levels of CaLFY and CaJ were not altered in the apical and sympodial meristems of the Cas mutant compared with the wild-type (Figs 5c, S2), whereas expression of Ca-AN was not detected in the Cas mutant in these meristems (Figs 5d, S2). These results suggest that CaS does not affect CaLFY expression and that the two genes probably act independently with respect to FM differentiation, while CaS is required for the expression of Ca-AN and is located upstream of it in the pepper flower formation pathway.

Discussion In the present study, the identification of CaS which controls meristem termination in pepper is reported. CaS functions in promoting the reproductive shift of the apical and sympodial shoots and is required for flower formation. New Phytologist (2014) 202: 1014–1023 www.newphytologist.com

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(e) Fig. 4 Pepper (Capsicum annuum) mutants affected in flowering time and shoot architecture and their interaction with Cas. (a) Cajointless (Caj) exhibits late flowering compared with ‘Maor’. (b) The Cas Caj double mutant exhibits extreme inhibition of initiation of sympodial growth. (c) E-327 exhibits late transition to sympodial growth and lack of flowers. (d) E-62 exhibits early transition to flowering compared with ‘Maor’. (e) Cas E-62 double mutant showing intermediate transition to sympodial growth and lack of flowers.

Role of CaS in controlling the transition to sympodial growth While initiation of sympodial growth marks the transition to flowering and flower formation in wild-type pepper, initiation of sympodial growth in Cas is associated with formation of a dichasially forked shoot, stem fasciation and lack of flowers. Cas mutants display four major pleiotropic effects: delay in termination of the apical meristem; delay in termination of sympodial meristems; fasciated sympodial shoot; and loss of flower formation. These phenotypes, as well as the expression of CaS in the apical meristem at the transition to flowering and in the SYMs (Fig. 5a,b), suggest that its primary role is to promote termination of apical and sympodial meristems. According to Park et al. (2012), the rate of meristem maturation determines inflorescence architecture in tomato and in other Solanaceae. The delay in the termination of apical and sympodial meristems in Cas results in delayed transition of the primary shoot to sympodial growth and increased vegetativeness of the meristems to a greater degree than in other known late-flowering pepper mutants (Jeifetz et al., 2011; Cohen et al., 2012). The fasciated shoot of Cas is a consequence of fusion of the SYMs, which may result because the flower that provides a physical barrier between adjacent SUs is lacking. Release of stem fasciation occurs after formation of a few subsequent SYMs, possibly because of physical constraints imposed on the apex organization. Double-mutant analyses of CaS with other genes controlling the transition to flowering showed complex patterns of relationships. CaS interacts in a positive synergistic manner with the flowering promoter CaJ, while it is epistatic to the flowering New Phytologist (2014) 202: 1014–1023 www.newphytologist.com

repressor FA and acts additively to the flowering repressor E-62. These relationships imply that the activities of CaS, CaJ and FA converge at some point in the sympodial growth transition pathway, while the gene disrupted in E-62 acts independently to CaS. Because the expression of CaJ is not affected by the Cas mutation (Fig. S2), CaJ is probably upstream of CaS. Role of CaS in controlling flower formation In wild-type pepper, termination of the apical and sympodial meristems is associated with the transition to flowering of the primary and sympodial shoot, and, for both meristems, this transition is associated with flower formation. In Cas, termination of both apical and sympodial meristems is delayed, as manifested by a delay in the initiation of sympodial growth and increased vegetativeness of the SU. The failure to produce flowers in Cas is not a consequence of the delay in meristem termination, as, in the Cas E-62 double mutant, transition to sympodial growth is enhanced relative to Cas as a result of a mutation in the flowering repressor gene of E-62; however, flowers are not formed. In petunia, expression of DOT is downstream of EVG (Rebocho et al., 2008) and is required for the formation of flowers (Souer et al., 2008). Ectopic expression of DOT in evg restores flower formation. Therefore, inhibition of flower formation in evg is interpreted as resulting from loss of DOT expression. In tomato, expression of S precedes that of AN, and the two genes promote successive stages in inflorescence and flower development (Lippman et al., 2008). Similar to petunia and tomato, expression of Ca-AN, the pepper ortholog of DOT and AN, is dependent on the activity of CaS (Figs 5d, S2). Mutation in Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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(c)

(a)

Fig. 5 Expression patterns of pepper (Capsicum annuum) floral meristem genes. (a) RT-PCR of CaS, Ca-ANANTHA (Ca-AN) and LEAFY (CaLFY) transcripts in wild-type tissues. CaUBIQUITIN (UBQ) was used as a control. Vegetative meristems were excised from apices at the two-expanding-leaf stage on the primary stem. Flower buds represent flowers 2 d before anthesis. Meristems at floral transition were excised from apices at the six-expanding-leaf stage on the primary stem. NTC, no template control. (b) Longitudinal section of wild-type shoot apex at the six-expanding-leaf stage showing detection of CaS expression by in situ hybridization in the subapical region of developing sympodial meristems (SYMs) as well as in an early stage of the flower meristem (F). Bar, 250 lm. (c, d) qRT-PCR of CaLFY (c) and Ca-AN (d) expression in wildtype and Cas apical meristems at the sixexpanding-leaf stage. Error bars denote standard error over three biological replicates.

(b)

Ca-AN results in the formation of a vegetative shoot structure that originates from the flower pedicel instead of a flower (Lippman et al., 2008). However, this phenotype does not appear in Cas; rather, there is a complete lack of flowers. This implies that, while Ca-AN is required for differentiation of the FM, CaS acts at an earlier stage of the transition to FM differentiation. This conclusion is supported by the expression patterns of the two genes (Fig. 5; Lippman et al., 2008). As a result of mutation in CaS blocking the transition phase, the signal that is required for differentiation of the FM is lacking, leading to precocious termination of the meristem without flower formation. The lack of the Ca-an phenotype in Cas may also indicate that CaS activates an unknown gene that is upstream of Ca-AN, whose function is required for FM identity. Nevertheless, the mechanism by which the complete elimination of flower formation in Cas occurs is unknown.

(d)

Comparison of Cas-homologous mutations in the Solanaceae In both tomato s and pepper Cas, it is proposed that late maturation of meristems partly causes the corresponding phenotypes. However, the two mutants have opposite consequences, as tomato s has a highly branched inflorescence, while pepper Cas completely lacks flowers (Table 2). This phenotypic difference may be attributed to the different architecture of the reproductive structures in these species, that is, the existence of an inflorescence in tomato and a solitary flower in pepper. In tomato, the IM has a sympodial structure and is bifurcated into an FM and a new IM that develops in the flank of the meristem and precedes the formation of the FM (Welty et al., 2007; Lippman et al., 2008). The delay in FM termination in s extends the duration of IM indeterminacy, resulting in

Table 2 Comparison of Cas-homologous mutants in pepper, tomato and petunia

Transition to sympodial growth Sympodium structure Inflorescence structure Flower structure Gene function

Pepper Cas

Tomato s

Petunia evg

Late

Subtle late effect

No effect

Stem fasciation, increased vegetativeness

No effect Increased branching No effect Promotes termination of flower meristem

Stem fasciation

No flower formation Promotes termination of shoot meristems and is required for flower formation

Rare flowers, no data Promotes termination of flower meristem

evg, evergreen. Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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increased branching. In pepper, the mutation in Cas results in delayed termination of the shoot meristems. However, the lack of IM does not provide a developmental window for branching. Interestingly, while in pepper Cas, termination of all shoot meristems is coordinately delayed or blocked, in tomato s, delay in meristem termination is mostly restricted to the FM as only a subtle change is observed in the timing of the transition to flowering (Quinet et al., 2006a) and no change is observed in the sympodial shoot structure. The Cas phenotype more closely resembles that of petunia evg (Rebocho et al., 2008), which is characterized by a bushy vegetative inflorescence that lacks flowers. However, compared with pepper Cas, which exhibits a delayed transition to sympodial growth and prevention of flower formation, evg switches to the flowering phase (as indicated by the appearance of bracts in the inflorescence nodes) at the same time as the wild-type and occasionally produces flowers (Table 2). Furthermore, similar to the fasciated shoot of Cas, the inflorescence stems in evg often fail to bifurcate into flower and inflorescence meristems as occurs in the wild-type, resulting in a fasciated shoot. Therefore, shoot fasciation and lack of flowers are common to both Cas and evg, although these characteristics are more strongly expressed in pepper. Furthermore, while S and EVG act predominantly in the IM and FM, CaS has a wider function in shoot meristems. The smaller phylogenetic distance between CaS and EVG than between CaS and S further supports the greater phenotypic resemblance between the pepper and petunia mutants than between the pepper and tomato ones. An additional factor that may affect diversification of gene function in different species is differential patterns of gene interactions. CaS homologs exhibit different patterns of interactions in pepper compared with tomato and petunia. In pepper, all tested double mutants involving Cas fail to undergo floral formation; that is, Cas is epistatic to other mutations in genes controlling sympodial development in affecting flower formation. By contrast, in tomato, the solitary flower mutation uniflora, as well as jointless, are epistatic to s (Quinet et al., 2006b; Thouet et al., 2012). Similarly, in petunia, the mutations extrapetals (exp) and hermit (her), having a single flower inflorescence and arrest of sympodial growth, are epistatic to evg, as double mutants with evg show the single flower phenotype (Rebocho et al., 2008). EXP is the homolog of CaJ (Table S2; Castel, 2009), while mutations in pepper homologous to uniflora and her have not been reported. Therefore, in contrast to petunia, in which the requirement of EVG for flower formation can be bypassed by altering the inflorescence architecture, this requirement of CaS is mandatory. This indicates that, while S and EVG have a primary role in specifying FM maturation (Park et al., 2014), CaS has evolved to have a critical role in flower formation in addition to its role in controlling meristem maturation. Furthermore, the opposite epistatic effects of Cas Caj homologous mutants in pepper compared with tomato and petunia indicate that different modes of gene interaction exist that may contribute to diversification of sympodial development in the Solanaceae. New Phytologist (2014) 202: 1014–1023 www.newphytologist.com

Acknowledgements We thank Saadia Nahon for technical support, Arnon Brand for graphic design and Hanita Zemach for assistance with microscopic analyses. We thank Zach Lippman (Cold Spring Harbor Laboratory) for fruitful discussions and critical reading of the manuscript, and Prof. Dani Zamir (Hebrew University) for PhD guidance of O.C. This research was supported by The Israel Science Foundation (grant no. 1349/10).

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Supporting Information Additional supporting information may be found in the online version of this article. Fig. S1 Multiple sequence alignment of CaS homologs in pepper, tomato and petunia. Fig. S2 Expression of pepper flowering-related genes in SYMs of ‘Maor’ and E-327. Table S1 Pepper primers used in this study Table S2 Comparison of Caj homologous mutants in pepper, tomato and petunia Please note: Wiley Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.

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Capsicum annuum S (CaS) promotes reproductive transition and is required for flower formation in pepper (Capsicum annuum).

The genetic control of the transition to flowering has mainly been studied in model species, while few data are available in crop species such as pepp...
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