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LATHYROIDES, encoding a WUSCHEL-related homeobox1 transcription factor, controls organ lateral growth and regulates tendril and dorsal petal identities in garden pea (Pisum sativum L.) Authors: LiLi Zhuang1, Mike Ambrose2, Catherine Rameau3, Lin Weng4, Jun Yang4, XiaoHe Hu5, Da Luo 5* and Xin Li5* 1 School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China 2 Department of Crop Genetics, John Innes Centre, Colney, Norwich NR4 7UH, United Kingdom 3 Station de Génétique et d'Amélioration des Plantes, Institut J.P. Bourgin, UR 254 INRA, Versailles, France 4 National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China 5 State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, 135 West Xin-gang Road, Guangzhou, 510275, China * Co-corresponding authors: Tel: +86 (0)20 3994 3512; Fax: +86 (0)20 3994 3513; E-mail: [email protected] ; [email protected] ABSTRACT During organ development, many key regulators have been identified in plant genomes, which play conserved role among plant species to control the organ identities and/or determine the organ size and shape. It is intriguing that whether these key regulators can acquire diverse function and be integrated into different molecular pathways among different species, giving rise to the immense diversity of organ forms in nature. In this study, we have characterized and cloned LATHYROIDES (LATH), a classical locus in pea, whose mutation displays pleiotropic alteration of lateral growth of organs and predominant effects on tendril and dorsal petal development. LATH encodes a WUSCHEL-related homeobox1 (WOX1) transcription factor, which has a conserved function in determining organ lateral growth among different plant species. Furthermore, we showed that LATH regulated the expression level of TENDRIL-LESS (TL), a key factor in the control of tendril development in compound leaf, and LATH genetically interacted with LOBED STANDARD (LST), a floral dorsal factor, to affect the dorsal petal identity. Thus, LATH plays multiple roles during organ development in pea: it maintains a conserved function controlling organ lateral outgrowth, and modulates organ identities in compound leaf and zygomorphic flower development, respectively. Our data indicated that a key regulator can play important roles in different aspects of organ development and dedicate to the complexity of the molecular mechanism in the control of organ development so as to create distinct organ forms in different species. Key words: Pea, LATH, LST, TL, Lateral growth, Organ identity INTRODUCTION Organ formation plays an important role in plant development which gives rise to enormous diversity of plant forms. Leaves and petals, two types of the most conspicuous plant organs, are developed from specific primordia that initiate continuously on the flanks of the shoot apical meristem (SAM) or in the second whorl of floral meristem (FM), respectively, where actively dividing cells are recruited and serve as founder initials. After initiation at the right position, the organ identities are determined and the distinct organ forms are developed, which are characteristic of shape and size in different species (Ingram and Waites, 2006; Veit, 2006). A number of key regulators in the control of organ size and shape have been identified from model plants, and the developmental and environmental signals such as phytohormones have been shown to integrate into several conserved pathways to regulate organ growth (Krizek, 2009; Johnson and Lenhard, 2011). Completely differentiated organ forms are thought to be driven by interconnected cell proliferation and cell expansion at the cellular level along three axes: proximodistal, mediolateral and adaxial-abaxial (Byrne et al., 2001; Golz and Hudson, 2002). Although adaxial-abaxial differentiation is thought to be a prerequisite for lamina outgrowth (Waites and Hudson, 1995), the presence of mutants in Arabidopsis that are affected only in proximodistal or mediolateral growth suggests that different mechanisms of cell proliferation and expansion patterns may exist to facilitate growth in length and width directions (Byrne, 2005; Tsukaya, 2006). It has been shown that the WOX transcription factors serve as master switches and control organ lateral growth along mediolateral axis (Scanlon et al., 1996; Matsumoto and Okada, 2001; Nardmann et al., 2004; Shimizu et al., 2009; Vandenbussche et al., 2009; Tadege et al., 2011). Two mutants in maize, narrowsheath1 and 2 (NS1 and NS2 are duplicated WOX3-like genes) lead to a reduction of leaf blade in the ns1 ns2 double mutant (Scanlon et al., 1996; Nardmann et al., 2004). In Arabidopsis, the knockout of PRESSED FLOWER (PRS/WOX3) gene leads to defects in lateral sepals, petals, and stipules (Matsumoto and Okada, 2001; Shimizu et al., 2009). In Arabidopsis, the wox1 single mutant has no visible phenotype but the prs wox1 double mutant has been shown to cause lamina reduction (Vandenbussche et al., 2009). In Petunia, a mutation of the WOX1-like gene, maewest (maw), can cause a narrowing of the lamina and defective petal fusion phenotype (Vandenbussche et al., 2009). Recent studies show that, STENOFOLIA (STF) and LAM1, WOX1-like genes in Medicago truncatula and Nicotiana sylvestris respectively, are required for blade outgrowth and leaf vascular patterning (Tadege et al., 2011). The stf mutant produces narrow leaves where mediolateral outgrowth of the blade is severely curtailed while proximodistal growth and trifoliate identity remain unaffected (Tadege et al., 2011). Garden pea, with a large collection of mutations in germplasm due to the long research history, possesses compound leaves and conspicuous petals, and presents a good system for exploring the key regulators in the control of organ formation. A typical compound leaf of pea is composed of a basal pair of foliaceous stipules, one or more pairs of proximal leaflets, one or more pairs of distal tendrils and a single terminal tendril. Stipule, leaflet and tendril primordia are initiated in an acropetal manner on the compound leaf primordium which is genetically controlled by a number of key regulators (Meicenheimer et al., 1983; Marx, 1987; Villani and DeMason, 1997; Gourlay et al., 2000). Some appear to affect only blade development and others to affect only stipule development. In pea, the inactivation of UNIFOLIATA (UNI), conditions a simple leaf, primarily with one single terminal leaflet (Hofer et al., 1997). AFILA (AF) and TENDRIL-LESS (TL) control the identities of proximal leaflets and distal tendrils, respectively: proximal leaflets are transformed into branched tendrils in af mutant; and, the distal and terminal tendrils are transformed into leaflets in tl mutant (Villani and DeMason, 1997; Gourlay et al., 2000, Mishra et al., 2009). In the typical zygomorphic flower of pea, three types of petals are arranged along a dorsoventral (DV) axis: a single large dorsal petal (the standard), two lateral petals (the wings) and two small ventral petals (the two petals fuse to form a keel) (Tucker, 2003). The DV differentiations among different types of petals can be defined by their different shape and size. It has been found that LST and Keeled Wings (K), two CYCLOIDEA-like TCP genes in pea, confer the dorsal and lateral identities, respectively. The mutants at the LST locus result in the abnormal shape and size of the dorsal petals whereas the mutations at the K locus give rise to a homeotic transformation phenotype where that the lateral petals mimic the ventral in shape and size (Winfield, 1987; Ambrose, 2003; Wang et al., 2008). Furthermore, the identities of petals can be defined at the cellular levels and the epidermal cells with characteristic different forms on different petals of legume flowers have been used as micromorphological markers for the dorsal, lateral and ventral petal identities (Feng et al., 2006; Wang et al., 2008; Ojeda et al., 2009; Weng et al., 2011). In this study, we analyzed a classical mutation lathyroides (Berdnikov et al., 1997) in which the lateral growth of stipules, leaflets and petals was reduced, and the development of tendril and dorsal petal was predominantly affected. LATH was found to encode a WOX1 transcription factor, consisting with its conserved function in the control of organ lateral growth. Our data further showed that LATH was involved in the control of organ identities by regulating the expression level of TL, a master gene in the controlling of tendril development, and by interacting with LST to affect the dorsal petal development in pea. All the data indicated that LATH could be integrated into the control of different aspects of organ development. RESULTS Identification of lath mutants To investigate key components controlling organ morphogenesis during compound leaf and flower development, we screened mutants with altered organ shape and size which were isolated from two mutagenesis experiment on pea (Wang et al., 2008; Li et al., 2010). Six recessive mutants with substantially altered leaves and petals in pea were identified from two genetic backgrounds, Terese (one mutant) and JI2822 (five mutants), respectively (Figure 1A–1D and Supplemental Table 1), which mimic the phenotype of a classical pea mutant lathyroides (lath) (Berdnikov et al., 1997). Genetic analyses were conducted and it was found that these mutants were allelic (See Material and Methods). One recessive mutant narrow organs1 (nao1) with similar phenotype was isolated from a mutagenesis population of Lotus japonicus (Figure 1E). Comparative mapping data showed that lath in chromosome VII of pea and nao1 in chromosome IV of L. japonicus shared a good synteny (See below), indicating lath and nao1 could be homologous. In lath mutants, lateral outgrowth of the leaflet blade and stipules was severely reduced (Figure 1C). The mutation reduced the width of the leaflets by 50% and the width of the stipules was reduced to a lesser extent (Figure 1C and 1D). Compared with the wild type compound leaf, narrower leaflets were found in place of tendrils in lath mutants (Figure 1C and Supplemental Figure 1B). The most striking effect of lath mutation however was on the dorsal petal, which was reduced to a narrow strip (Figure 1A, 1B and Supplemental Figure 1A). Apart from petals, other floral structures were less affected, but after fertilization the carpel developed into an abnormal reduced pod without seeds and with the dorsal suture opened. No obvious developmental defects were observed in the stamens of lath mutants. The weak allele (lath1-6) was the only male and female fertile mutant. All other alleles were male fertile but female sterile. The floral development of lath was characterized by using scanning electronic microscopy (SEM) in different developmental stages according to the previous studies (Tucker, 1989; Wang et al., 2008). It showed that there was no visible difference in the early floral developmental stages between wild type and lath mutant. The earliest visible variation in lath mutant commenced at floral developmental stage six (Figure 2A-2I), when the vascular tissues are developed in petals, and the asymmetric shape of both lateral and ventral petals becomes obvious (Wang et al., 2008). The lateral expansion of the dorsal petal primordium was strongly reduced in lath mutant compared with the wild type at floral developmental stage seven (Figure 2E and 2J). Molecular characterization of LATH The comparative genomics approach was conducted to clone LATH in pea. lath locus was mapped in the linkage group VII between molecular marker Pgm and Fabtin (Figure 3A). In a parallel experiment in L. japonicus, mapping of nao1 was conducted by constructing an F2 population (number of mutants=1683). DNA pools, each of which consisted of fifteen randomly-selected mutant plants were examined to scan the whole genome of L. japonicus. More than thirty-six SSR markers in different chromosomes were tested (Chen et al., 2006) and nao1 was found to be linked with an SSR marker TM0069 in the long arm of chromosome IV. Chromosome walking was conducted to map and clone the nao1 locus and nao1 was located to a 250 kb region of chromosome IV between SSR marker TM0042 and TM0445, where a complete contig TM2003 and the DNA sequence were subsequently obtained (Figure 3A). Comparative genomics showed that this region had synteny among genomes of pea, M. truncatula and L. japonicus (Choi et al, 2004; Cannon et al., 2006). Based on the similar phenotype and synteny, the lath and nao1 could have resulted from loss function of ortholog in pea and L. japonicus respectively (Figure 3A). Sequence analysis revealed a candidate gene LjWOX1 within the contig of L. japonicus and MtWOX1 (STF) within the syntenic contig of M. trunctula, which involved in leaf blade development and vascular patterning (Tadege et al., 2011). When the gene structure of LjWOX1 in nao1 was analyzed, a one base substitution was found in the nao1 mutant, which should give rise to a stop codon and resulted in a putative truncated protein without the C-terminal (containing the WUS box and STF box) (Figure 3B). PsWOX1, the homologue of LjWOX1 in pea, was cloned by using degenerate oligoes. Cosegregation analysis confirmed that PsWOX1 was located inside the region containing lath locus. When the gene structure of PsWOX1 was analyzed, various mutations were detected in all the lath mutants. Complete deletions of PsWOX1 were detected in both lath1-2 and lath1-4. The other alleles, lath1-1, lath1-3, lath1-5 and lath1-6, were found to contain variation in the sequence of PsWOX1. A deletion of two base pairs at the beginning of the second exon of PsWOX1 was detected in lath1-1, which should cause a frame-shift and disrupt all the conserved domains from the homeodomain onward (Figure 3C). lath1-3 and lath1-5 carried deletions at the end of second and third exons, respectively (the remaining coding sequences of lath1-3 and lath1-5 were 509 bp and 657 bp respectively), giving rise to the putative truncate proteins without the C-terminal (including the WUS box and STF box) (Figure 3C). The weak allele lath1-6 carried a 54-bp deletion at the end of last exon, which would truncate the C-terminal by 18 amino acids including STF box (Figure 3C). Sequences alignment and phylogenetic analysis showed that NAO1 and LATH encoded homeodomain (HD) protein with high similarity to the WOX1 subgroup of the WOX transcription factors (Figure 3D and Supplemental Figure 2A). Thus, we concluded that the classical locus LATH was allelic to PsWOX1, which encodes a putative WOX1-like protein with high similarity to STF and MAW (Figure 3D). LATH expression pattern The developmental defects could be detected in young emerging dorsal petal primordium of lath mutants, indicating that LATH was already active during the early stages of organ development. A semi-quantitative RT-PCR experiment showed that LATH was expressed in all the aerial organs with the highest levels of LATH expression being found in the floral buds (Figure 4A). To examine the temporal and spatial LATH expression pattern in more detail, we performed RNA in situ hybridization experiments. It was found that LATH was absent from SAM (Figure 4B). In young leaf primordia, LATH expression was detected in the margin of leaflets and tendrils, as well as in the central regions of leaflets and stipules where the provascular tissues should initiate (Figure 4B and 4C). In developing flower buds, LATH transcripts were found in all emerging primordia (Figure 4D-4G). Expression in petals and stamens was predominantly detected at the lateral margins (Figure 4D-4F). During later stages of flower development, expression levels remained high in the pistil and on the adaxial side of the two carpels where fusion occurred (Figure 4G). LATH genetically interacts with LST to regulate the dorsal petal development Since lath possesses a strip-like dorsal petal and displays a strong effect on the dorsal petal development (Figure 5A and 5B), we tested its genetic interaction with LST and K, the two CYC-like TCP genes controlling the dorsal and lateral petal identities in pea, respectively (Wang et al., 2008). lath was introduced into k and lst genetic backgrounds, respectively. The lath k double mutant appeared to display an additive phenotype where the dorsal petal of the double mutant resembled that of lath and the flower bore slightly narrower keeled lateral and ventral petals (Figure 5B-5D). In contrast, the lath lst double mutant showed an unexpected phenotype in that the dorsal petal of the double mutant bore partially developed blade, and was somewhat similar to the lst single mutant (Figure 1B; Supplemental Figure 1A; Figure 5E and 5F). Characteristic epidermal cells on different types of petals were used to monitor the petal identities of the mutants (Feng et al., 2006; Wang et al., 2008; Ojeda et al., 2009; Weng et al., 2011). In wild type, a representative cell type of dorsal petals is a long strip with wavy margins, one of the lateral petals is jigsaw puzzle-like, and the one of the ventral petals is ripple-like (Figure 5G). The dorsal petals of lst mutant exhibited the abnormal epidermal cells which mimicked the ones with lateral petal identity in the wild type (Figure 5G), indicating the defect of dorsal activity in lst. In lath mutant, the dorsal petals lost the blade where the typical dorsal epidermal cells developed. However, in the lath lst double mutant, the epidermal cells of the abnormal dorsal petals possessed the shape and size, which resembled that of the dorsal ones in the wild type (Figure 5G). The representative epidermal cells of lateral and ventral petals showed no difference between wild type and mutants. All the data indicated that LATH genetically interacted with LST in the control of the dorsal petal development. We compared the LST and K expression level in the three types of petals by quantitative real-time PCR. The results showed that LST was only expressed in the dorsal petals and there was no obvious alteration of expression level between wild type and lath mutant (Supplemental Figure 3A). Vice versa, LATH expression level did not have obvious change in lst mutant (Supplemental Figure 3B). K was expressed in the dorsal and lateral petals and there was no obvious alteration of expression level between wild type and lath mutant (Supplemental Figure 3C). Vice versa, LATH expression level did not have obvious change in k mutant (Supplemental Figure 3D). These results suggested that LATH and PsCYCs (LST and K) did not appear to regulate each other’s transcript levels. In addition, there was no direct interaction between LATH and LST in the yeast two-hybrid assays (data not shown). Transcription factors being regulated by LATH To further analyze how LATH was involved in the regulation of organ development, the expression levels of some transcription factors in the control of organ development were analyzed. Recent study has shown that TL, encoding a Class I homeodomain leucine zipper (HD-ZIP) transcription factor, controls the tendril identity and regulates tendril development (Hofer et al., 2009;DeMason and Chetty, 2011). In the compound leaves of lath mutant, the defect tendril development was reminiscent of the phenotype of tl mutant and a large portion of tendrils was transformed into leaflets (Supplemental Figure 1B). Consistently, real-time PCR analysis showed that the expression level of TL was decreased about 4 fold in lath mutants in comparison with the wild type (Figure 6A). MIXTA-like genes encoding MYB-like transcription factors have been reported to control the petal epidermal cell differentiation (Noda et al. 1994; Glover et al. 1998; Perez-Rodriguez et al. 2005; Baumann et al. 2007; Weng et al., 2011). Two MIXTA-like genes, PsMYB1 and PsMYB2 were identified from the pea Transcriptome Shotgun Assembly (TSA) database (Supplemental Figure 2D) (Franssen et al., 2011). It was found that PsMYB1 was predominantly expressed at the dorsal petals, but PsMYB2 was evenly expressed in all the petals (data not shown), suggesting the expression level of PsMYB1 could be regulated by the dorsal regulators. The dorsal petals of wild type and mutants were sampled from young floral buds for qRT-PCR analysis. PsMYB1 was upregulated 2 fold in the lath mutant and downregulated 10 fold in the lst mutant. Interestingly, the transcription level of PsMYB1 in the lath lst double mutant was 1.5 fold compared to the wild type (Figure 6B). In contrast, PsMYB2 exhibited no obvious changes in transcript levels between the wild type and mutants (Figure 6B). Thus, the expression of PsMYB1 is upregulated by LST but downregulated by LATH, indicating that PsMYB1 could be a downstream target of both LATH and LST. Compared to the wild type, the vascular pattern of dorsal petals showed alterations in lath and lst mutants. In wild type, the venation of the dorsal petal was constituted of several orders of veins (primary, secondary, tertiary and quaternary veins and veinlets) (Supplemental Figure 4A). In lath mutant, the tertiary veins, quaternary veins and veinlets were poorly developed (Supplemental Figure 4B) while in lst mutant and lath lst double mutant, the development of the higher order of veins (quaternary veins and veinlets) was reduced to a less extent (Supplemental Figure 4C and 4D). As described in previous study, AtHB8 and APL are the procambial identity marker and the phloem developmental marker in Arabidopsis respectively (Baima et al., 2001; Bonke et al., 2003). Their homologues in pea, PsHB8 and PsAPL, were cloned (Supplemental Figure 2B and 2C) and the expression levels were analyzed. PsHB8 was downregulated 10 fold in the lath mutant and 2.5 fold in the lst mutant while its transcript levels in the lath lst double mutant was close to that in the lst mutant (Figure 6C). As a comparison, PsAPL exhibited the similar lower expression levels among the single and double mutants than the one of the wild type (Figure 6C). Discussion In recent years, there has been a significant progress in legume genomics due to the genome sequencing projects of several model legume plants, such as M. truncatula, L. japonicus and soybean. Studies indicate that different species in the Papilionoideae subfamily exhibit extensive genome conservation and share micro-syntenic blocks (Choi et al. 2004; Kaló et al. 2004; Cannon et al. 2006). A major challenge for legume comparative genomics is to utilize the incomplete genome information gained from a few model legume species efficiently so as to map and clone important loci in grain legumes such as pea and peanut, whose genome information is less extensive (Varshney et al. 2009; Young and Udvardi 2009). Recent progress in legume genome projects has now made it feasible to map genetic loci in pea efficiently by using a comparative genomic approach (Li et al., 2010).The LATH comparative map was constructed and the syntenic region was anchored to the contig in chromosome 4 of L. japonicas. Consequently, LATH was identified and cloned. It demonstrates the efficiency of the comparative genomic approach to deal with the map-based cloning work in the complex legume genome like pea. Sequence analysis revealed that LATH encoded a protein with high similarity to the WOX1-like transcription factors. In the term of protein structure, LATH can be grouped with STF from M. trunctula and MAW from Petunia (Figure 3D and Supplemental Figure 2A). They shared conserved motifs at the N-terminal and C-terminal regions, in addition to the homeodomain, WUS box and STF box (Tadege et al., 2011). Six alleles of lath in pea were isolated and characterized in this study. The lath alleles which either lost the whole gene due to radiation-induced deletions or whose homeodomain and WUS box were truncated or disrupted, displayed the similar strong lathyroides phenotype. In contrast, lath1-6 encoding a putative truncate protein without the STF box, displayed a much weaker phenotype. The allelic variants suggested that the homeodomain and WUS box are critically important for LATH function, while the truncate protein without STF box should be still partially functional. It has been found that in different plant species, the lateral outgrowth of plant organs is affected when the function of WOX1-like factors is disrupted (Vandenbussche et al., 2009; Tadege et al., 2011). Consistently, it was found that LATH encoding a WOX1-like transcription factor which was predominantly expressed at the lateral margins, and lath mutants exhibited a reduced lateral growth of stipules, leaflets and petals, demonstrating that LATH played a conserved role in the control of lateral outgrowth during organ development. Similarly to the function of STF in Medicago, LATH was also involved in the vascular patterning. In lath mutant, the number of high order of vein in the dorsal petals was reduced. Consistently, the expression levels of PsPHB8 and PsAPL, the vascular key regulator were down regulated in the mutants. Furthermore, our study indicated that LATH should be involved in different aspects of organ development in pea. This is evident in the compound leaf development. The mature leaf in wild type consists of the basal stipules, proximal leaflets, distal tendrils and terminal tendril. In lath mutants, the outgrowth of blade in stipules and leaflets was reduced while the tendrils were replaced by narrower leaflets (Figure 1C and 1D; Supplemental Figure 1B). Consistently, the expression level of TL, a key regulator to determine the tendril development, was dramatically low in the lath mutant. Therefore, LATH is essential for the activation and/or maintenance of TL expression so as to control the tendril identity, although it is unclear yet how LATH regulates the expression level of TL. More evidences for LATH being involved in different aspects of organ development came from the analysis of petal development. While the lateral outgrowth of all petals was affected in lath mutants, the strongest effect occurred at the dorsal petal (Figure 1A and 1B; Supplemental Figure 1A). Similarly, the dorsal petal of stf mutants showed the strongest reduction in lateral expansion in Medicago (Tadege et al., 2011). In a comparison, Petunia also possesses conspicuous petals in its actinomorphic flowers. In the maw mutants, the malfunction of WOX1-like factor caused defect of lateral outgrowth of all petals: the petal fusion (to form the tube) was partially disrupted and each petal became narrower in the similar scale (Vandenbussche et al., 2009). In the maw chsu double mutant, the petal fusion was completely eliminated and only limited petal outgrowth occurred (Vandenbussche et al., 2009). Interestingly, in Arabidopsis, although there was no visible phenotype in wox1 mutants but there were very narrow petals in wox1 prs double mutants (Vandenbussche et al., 2009). There are other evidences to confirm LATH being involved in the dorsal petal development. When lst, the mutated form of dorsal factor, was introduced into the lath background, the defect of blade development in dorsal petals was partially recovered (Figure 5F). Furthermore, shape and size of representative epidermal cells of dorsal petals in the lath lst double mutant resembled the ones in the wild type, indicating that loss of dorsal identity in epidermal cells of lst could also be partially recovered by knockout of LATH (Figure 5G). In contrast, only additive phenotype was observed in the lath k double mutant (Figure 5D). Efforts have been made to investigate the molecular basis for the genetic interaction between lath and lst. So far, there was no indication for the direct interaction between LATH and LST at transcriptional level and protein level, based on the expression analysis and yeast two hybrid assay. It has been shown that the Arabidopsis WUS and several rice WOX proteins bind to TTAATGG sequence (Lohmann et al., 2001; Kamiya et al., 2003; Leibfried et al., 2005; Dai et al., 2007; Zhao et al., 2009) while TCP transcription factors bind to the core sequence GGNCCC (Kosugi and Ohashi, 2002; Li et al., 2005; Aggarwal et al., 2010). Thus, it is probable that LATH and LST could bind to different cis-elements in the promoter regions of the some common targets and coordinately regulate their expression in the dorsal petal. Indeed, the expression levels of a few potential downstream regulators, such as PsMYB1 and PsHB8, appeared to be regulated by LATH and LST. In the case of PsMYB1, LATH and LST regulated its expression level antagonistically, while both regulated the expression level of PsHB8 positively. These suggest that possibly the regulation of different downstream factors by LATH and LST should be involved in complex regulatory network. Recently it has become feasible to produce transcriptomic resources by Second Generation sequencing (2GS) at reasonable cost (Metzker, 2010). Franssen and the colleagues used 2GS with the Roche/454 platform and generated libraries from different tissues and treatments comprising a total of 450 megabases which serves well as a first comprehensive reference set for pea transcriptom (Franssen et al., 2011). At present, transcriptome analysis of the wild type, lath and lst single mutants, as well as the lath lst double mutant, is being conducted to identify the common targets of LATH and LST, and this should shield light on how LATH and LST interplay to control the dorsal petal development in pea. In conclusion, LATH encodes a WOX1-like factor and plays an important role in the different aspects of organ development. LATH maintains a conserved role to control the organ lateral growth, and is involved in the regulation of tendril and dorsal petal identities, respectively. Although only a limited number of potential downstream regulators have been investigated, our data strongly indicate that LATH has acquired diverse function by recognizing different targets and by genetically interacting with other factors during organ development. Since tendrils and the conspicuous dorsal petals in pea are considered as very specialized organs, the involvement of LATH in the control of their identities could be a unique event in pea, presenting a special case for the evolution of the molecular mechanism in the control of distinct organ development in legumes. Materials and methods Plant Material and Growth Conditions The nao1 mutant was isolated from EMS mutagenesis M2 population in L. japonicus ecotype Gifu B-129 generated in Shanghai. All pea lines used in this study were obtained from the John Innes Pisum Collection (http://www.jic.ac.uk/germplas/pisum/), except lath1-1 which was identified from fast neutron mutagenesis in the Terese genetic background. The lath1-2, lath1-3, lath1-4, lath1-5 and lath1-6 mutant were identified from the JI2822 genetic background. An allelic test was conducted by crossing lath1-2 with the heterozygous plants of lath1-1. In the F1 generation, the segregation of the wild type and mutant plants (12 wild-type plants and 12 mutants) fitted a 1:1 ratio. We crossed lath1-2 plants with lath1-6 (partial fertile) and all F1 plants showed mutant phenotype similar to lath1-6. Allelism was confirmed by crosses among lath1-2, lath1-3, lath1-4 and lath1-5 using heterozygote female parents because the mutant is female sterile (Mike Ambrose did the test in John Innes Centre). lst (JI3158) mutant was obtained by fast neutron mutagenesis in JI2822 genetic background (Li et al., 2008). The k (JI17) is a classic marker line (Pellew and Sverdrup, 1923) and backcrossed with JI2822 three times for genetic analysis in this study. The alleles of K and LST used for genetic interaction analysis are complete deletions. All plants were grown at 20 °C to 22 °C with a 16-h-light/8-h-dark photoperiod at 150 μmol m-2 s-1. Microscopy and Statistical Analysis SEM of mature petals was performed on plastic replicas as described by Green et al., (1990). SEM of floral buds was prepared as described by Chen et al., (2000). Samples were examined in JEOL JSM-6360LV (JEOL) at 10 to 15 kV of acceleration voltage. The measurements of petals and leaflets characters (Length, Width and Area) were conducted from the digital pictures by Image-Pro Plus 6.0. For analysis of the percentage of tendrils and leaflets in JI2822 and lath1-2, we counted total number of leaflets and tendrils of three compound leaves from fifth to seventh nodes in wild type and mutant respectively (Twenty plants for each genotype). Comparative Mapping and Gene Cloning NAO1 was mapped as described by Chen et al., (2006). The cosegregation of LjWOX1 with NAO1 was performed in a population consisted of 1683 mutants from nao1 x MG20 F2 population by marker using primers SL4933/SL4934. Three markers, AA90, Pgm and FabatinL were used to locate lath in linkage group Ⅶ of pea using 400 mutants from lath1-1 x JI992 F2 population (Aubert et al., 2006; Loridon et al., 2005). Primer sets used for amplifying PsWOX1 (SL4935/SL5577) gene was designed by sequence analysis of WOX1 CDS from M. truncatula and L. japonicus. Allelic variants were analyzed by PCR and the 3’ Rapid Amplification of cDNA Ends (RACE) PCR. Degenerate primer sets used for amplifying PsHB8 (SL7035/SL7040) and PsAPL (SL7042/7045) genes were designed by sequence analysis of Medicago and soybean, and Vitis vinifera respectively. Primers used for mapping and cloning see Supplemental Table 2. Phylogenetic Analysis Homologous alignments were performed by using the ClustalX program (version 1.83) (Thompson et al., 1994). Phylogenetic tree was constructed using the MEGA (version 4.0) by neighbor-joining method (JFF Matrix model) with 1000 bootstrap replications (Tamura et al., 2007). Real-time RT-PCR and Semi-quantitative RT-PCR Analysis of Gene Expression 5mm floral buds were dissected by eliminating the outermost sepals and dorsal, lateral and ventral petals were collected in RNase-free tubes stored in -80℃ freezer. This was done in triplicate (biological replicates). Total RNA was extracted by using the Trizol reagent (Invitrogen) following the manufacturer’s instructions. Samples were then treated with RNase free DNase (Promega) for 30 minutes. For real-time PCR, 2µg of total RNA was used to synthesize first strand cDNA using First Strand cDNA Synthesis Kit (Fermentas) following the manufacturer’s protocols. Real-time RT-PCR analysis was performed in triplicate (technical replicate) in 96-well plates using SYBR® Premix Ex Taq™ (Takara), and on an ABI StepOnePlus machine according to the manufacturer’s protocol (Applied Biosystems). The relative quantification method (Delta-Delta CT) was used to evaluate quantitative variation between the replicates examined. The amplification of PsActin was used as an internal control to normalize all data. For Semi-quantitative RT-PCR, cDNA preparation was the same for real-time RT-PCR. Primer pairs SL5355/SL5317 were used for amplification of the lath transcripts. PsEF1α was amplified with primers SL3128/SL3129 as an internal control. For the primers used for real-time RT-PCR and semi-quantitative RT-PCR see Supplemental Table 2. RNA in Situ Hybridization For in situ probes, lath transcript was generated with primer sets SL5355/SL5317 and SL4935/SL5577 (see Supplemental Table 2), from cDNA fragments. Nonradioactive in situ hybridization was performed essentially as described by Coen et al., (1990). Yeast Two-Hybrid Assay The Matchmaker Gold System (Clontech) was used in yeast two-hybrid assays. The coding sequences of LATH and LST were PCR amplified from cDNA templates, cloned into the pGBKT7 vector, and used as the bait. Coding sequences of LATH and LST were PCR amplified from cDNA templates, cloned into the pGADT7 vector, and used as prey. Positive clones grown on double selection media (SD-Leu-Trp) were tested on quadruple media (SD-Leu-Trp-His-Ade + X-α-gal) for protein–protein interactions following the manufacturer’s instructions (Clontech). All primers used for PCR amplification are listed in Supplemental Table 2. Accession Number Sequence data from this article can be found in the GenBank data libraries under accession number: JQ291249 (LATH); JQ291250 (NAO1); JQ291251 (PsHB8); JQ291252 (PsAPL). MIXTA (X79108); AmMYBML1 (CAB43399); AmMYBML2 (AAV70655); AmMYBML3 (AAU13905); AmVENOSA (ABB838280); AmROSEA1 (ABB83826); AmROSEA2 (ABB83827); AtMYB16 (NM_121535); AtMYB106 (NM_110979); AtMYB17 (NM_115989); PhMYB1 (CAA78386); LjMYB1 (JF754926); LjMYB2 (JF754927); AtHB8 (NP_195014); AtHB15 (NP_175627); AtHB14 (NP_181018); AtHB9 (NP_174337); IFL (NP_200877); GLABRA2 (NP_565223); GmHB8a (XP_003522716) GmHB8b (XP_003526496); MtHB8 (XP_003603630); VvAPL (XP_002265800); APL (AT1G79430); AT3G12730; AT4G13640; AT3G24120; AT1G69580. Funding This work was supported by National Natural Science Foundation of China (Grants nos. 30930009) and Science and Technology Planning Project of Guangdong Province, China (Grants nos. 2011A020201008). Acknowledgments We thank Shusei Sato and Satoshi Tabata for providing Lotus sequence information in the gene cloning. We acknowledge Z. Xu, H. Yin, P. Gao, S. He, H. Xia, X. Han, and Z. Li for the critical comments on the manuscript. Figure legends Figure 1. Phenotypic analysis of lath and nao1 mutants (A) Front and side views of flowers and flattened petals of wild type pea JI2822 and lath1-2 mutant. Red arrows indicate the narrow strip of dorsal petal in lath mutant compared with wild type. DP, dorsal petal; LP, lateral petal; VP, ventral petal. (B) Measurements of dorsal, lateral and ventral petal size of JI2822, lath1-2, lst1-2 and lath1-2 lst1-2. This graph presents all petal size in terms of the dorsal petal size. n =12, error bars indicate ±SD. (C) Compound leaf phenotype of JI2822 and lath1-2. Red arrows indicate the tendrils are transformed into leaflets in lath mutant compared with wild type. Spl, stipule; FL, first pair of leaflets; SL, second pair of leaflets; FT, first pair of tendrils; ST, second pair of tendrils; TT, terminal tendril. (D) Measurements of the first pair of leaflets length and width in JI2822 and lath1-2 mutant. n=10, error bars indicate ±SD. LL, leaflet length; LW, leaflet width. (E) Front and side views of flowers and flattened petals of wild type L. japonicus Gifu and nao1 mutant. Red arrows indicate the narrow strip of dorsal petal in nao1 mutant compared with wild type. (A), (C), (E) Scale bar = 1 cm. Figure 2. Ontology of floral organ and petal development of wild type and lath mutant (A) Floral organs at stage 4 in wild type. Scale bar= 50 μm. (B) Floral organs at early stage 5 in wild type. Scale bar= 50 μm. (C) Floral organs at late stage 5 in wild type. Scale bar= 100 μm. (D) Floral organs at stage 6 in wild type. Scale bar= 100 μm. (E) Floral organs at stage 7 in wild type. Scale bar= 200 μm. (F) Floral organs at stage 4 in lath mutant. Scale bar= 50 μm (G) Floral organs at early stage 5 in lath mutant. Scale bar= 50 μm (H) Floral organs at late stage 5 in lath mutant. Scale bar= 100 μm. (I) Floral organs at stage 6 in lath mutant. Red arrow indicates a narrower, pointed standard petal is found in mutant. Scale bar= 100 μm. (J) Floral organs at stage 7 in the lath mutant. Red arrow indicates the DP which has become more divergent from wild type. Scale bar= 200 μm. Sad, dorsal sepal; CPad, dorsal common primordium; CPl, lateral common primordium; CPab, ventral common primordium; Ca, carpel; DP, dorsal petal; LP, lateral petal; VP, ventral petal; St, stamen. Figure 3. Comparative genomic-based cloning of LATH. (A) Syntenic analysis of LATH locus in Pea, Medicago and Lotus. The LATH locus was mapped in garden pea and found to locate on the long arm of linkage group VII. Dotted lines indicate the homologous makers or genes.Then LATH was projected to the genome of Medicago and the syntenic region was identified. NAO1 was anchored in a 250 kb contig of L. japonicus. (B) The gene structure of NAO1. nao1 carried a C to T mutation. Gene structure represented as light green boxes for exons and black lines for introns. The red box indicates the homeodomain, the light blue box indicates the WUS box, and the purple red box indicates the STF box. (C) LATH gene structure showing the position of the lath mutation site in six independent mutant lines. lath1-2 and lath1-4 are complete deletion. Black arrows show the starting sites of deletions in lath1-3, lath1-5 and lath1-6 mutants. (D) Sequence alignment of LATH, NAO1, STF and MAW proteins using full-length amino acid sequences. The homeodomain, WUS box and STF box is underlined in red, light blue and purple red respectively. Figure 4. LATH expression pattern (A) Expression patterns of LATH in wild type. R, root; S, stems; L, leaves; VA, vegetative apex; IM, inflorescence meristem; FB, floral buds. (B) - (G) LATH expression pattern in vegetative and floral apices by RNA in situ hybridization. Sections were hybridized with a digoxigenin-labeled antisense LATH RNA probe. Black arrows indicate the expression pattern. Scale bar = 200 μm. (B) LATH expression in 14 days-old vegetative shoot apex viewed in longitudinal sections. LATH is absent from the central zone of SAM (white star). (C) LATH expression in the margin of leaflets and tendrils as well as in the central narrow zone corresponding to the provascular tissue in young leaf primordia. (D) LATH is strongly expressed in lateral margins in stamens. (E), (F) LATH expression in petals with the strongest expression in the lateral margin of floral organ primordium. (G) LATH expression in a carpel viewed in longitudinal section. (H) RNA in situ hybridization in the flower using LATH sense probe as negative control. Scale bar= 200 μm. Spl, stipule; l, leaflet; tl, tendril; DP, dorsal petal; LP, lateral petal; VP, ventral petal; St, stamen; Ca, carpel. Figure 5. Interaction between LATH and LST during the dorsal petal development. (A) - (F) Flattened petals of wild type and mutant flowers are shown. Scale bar = 1 cm. (G) Representative epidermal cells on the adaxial side of mature petals. One of the cells in different background petals was marked with red or yellow by Adobe Photoshop CS to show dorsal and lateral representative cell type. Selected parts of the petals for SEM were shown in black rectangular box in Figure 5A, 5E and 5G. The representative epidermal cells in ventral petal showed no difference between wild type and mutants. Scale bar = 500 μm. Figure 6. Expression analysis of some transcription factors being regulated by LATH. (A) Relative expression levels of TL in the vegetative apex of wild type and mutants. Error bars indicate ±SD. (B) Relative expression levels of PsMYB1 and PsMYB2 in the dorsal petal of wild type and mutants. Error bars indicate ±SD. (C) Relative expression levels of PsHB8 and PsAPL in the dorsal petal of wild type and mutants. Error bars indicate ±SD. Error bars represent SD calculated from three technical replicates done on three biological replicates. 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