Xiaohe Hu

Genetic control of floral zygomorphy in pea (Pisum sativum L.).

Floral zygomorphy (flowers with bilateral symmetry) has multiple origins and typically manifests two kinds of asymmetries, dorsoventral (DV) and organ internal (IN) asymmetries in floral and organ planes, respectively, revealing the underlying key regulators in plant genomes that generate and superimpose various mechanisms to build up complexity and different floral forms during plant development. In this study, we investigate the loci affecting these asymmetries during the development of floral zygomorphy in pea (Pisum sativum L.). Two genes, LOBED STANDARD 1 (LST1) and KEELED WINGS (K), were cloned that encode TCP transcription factors and have divergent functions to constitute the DV asymmetry. A previously undescribed regulator, SYMMETRIC PETALS 1 (SYP1), has been isolated as controlling IN asymmetry. Genetic analysis demonstrates that DV and IN asymmetries could be controlled independently by the two kinds of regulators in pea, and their interactions help to specify the type of zygomorphy. Based on the genetic analysis in pea, we suggest that variation in both the functions and interactions of these regulators could give rise to the wide spectrum of floral symmetries among legume species and other flowering plants.

Floral Patterning in Lotus japonicus

Floral patterning in Papilionoideae plants, such as pea (Pisum sativum) and Medicago truncatula, is unique in terms of floral organ number, arrangement, and initiation timing as compared to other well-studied eudicots. To investigate the molecular mechanisms involved in the floral patterning in legumes, we have analyzed two mutants, proliferating floral meristem and proliferating floral organ-2 (pfo-2), obtained by ethyl methanesulfonate mutagenesis of Lotus japonicus. These two mutants showed similar phenotypes, with indeterminate floral structures and altered floral organ identities. We have demonstrated that loss of function of LjLFY and LjUFO/Pfo is likely to be responsible for these mutant phenotypes, respectively. To dissect the regulatory network controlling the floral patterning, we cloned homologs of the ABC function genes, which control floral organ identity in Arabidopsis (Arabidopsis thaliana). We found that some of the B and C function genes were duplicated. RNA in situ hybridization showed that the C function genes were expressed transiently in the carpel, continuously in stamens, and showed complementarity with the A function genes in the heterogeneous whorl. In proliferating floral meristem and pfo-2 mutants, all B function genes were down-regulated and the expression patterns of the A and C function genes were drastically altered. We conclude that LjLFY and LjUFO/Pfo are required for the activation of B function genes and function together in the recruitment and determination of petals and stamens. Our findings suggest that gene duplication, change in expression pattern, gain or loss of functional domains, and alteration of key gene functions all contribute to the divergence of floral patterning in L. japonicus.

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.).

During organ development, many key regulators have been identified in plant genomes, which play a conserved role among plant species to control the organ identities and/or determine the organ size and shape. It is intriguing 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.

The REDUCED LEAFLET Genes Encode Key Components of the trans-Acting Small Interfering RNA Pathway and Regulate Compound Leaf and Flower Development in Lotus japonicus

The endogenous trans-acting small interfering RNA (ta-siRNA) pathway plays a conserved role in adaxial-abaxial patterning of lateral organs in simple-leafed plant species. However, its function in compound-leafed species is largely unknown. Using the compound-leafed species Lotus japonicus, we identified and characterized two independent mutants, reduced leaflet1 (rel1) and rel3, whose most conspicuous defects in compound leaves are abaxialized leaflets and reduction in leaflet number. Concurrent mutations in REL genes also compromise flower development and result in radial symmetric floral organs. Positional cloning revealed that REL1 and REL3 encode the homologs of Arabidopsis (Arabidopsis thaliana) SUPPRESSOR OF GENE SILENCING3 and ARGONAUTE7/ZIPPY, respectively, which are key components of the ta-siRNA pathway. These observations, together with the expression and functional data, demonstrated that the ta-siRNA pathway plays conserved yet distinct roles in the control of compound leaf and flower development in L. japonicus. Moreover, the phenotypic alterations of lateral organs in ta-siRNA-deficient mutants and the regulation of downstream targets by the ta-siRNA pathway in L. japonicus were similar to those in the monocots but different from Arabidopsis, indicating many parallels between L. japonicus and the monocots in the control of lateral organ development by the ta-siRNA pathway.

Petal Development in Lotus japonicus

Previous studies have demonstrated that petal shape and size in legume flowers are determined by two separate mechanisms, dorsoventral (DV) and organ internal (IN) asymmetric mechanisms, respectively. However, little is known about the molecular mechanisms controlling petal development in legumes. To address this question, we investigated petal development along the floral DV axis in Lotus japonicus with respect to cell and developmental biology by comparing wild-type legumes to mutants. Based on morphological markers, the entire course of petal development, from initiation to maturity, was grouped to define 3 phases or 13 stages. In terms of epidermal micromorphology from adaxial surface, mature petals were divided into several distinct domains, and characteristic epidermal cells of each petal differentiated at stage 9, while epidermal cells of all domains were observed until stage 12. TCP and MIXTA-like genes were found to be differentially expressed in various domains of petals at stages 9 and 12. Our results suggest that DV and IN mechanisms interplay at different stages of petal development, and their interaction at the cellular and molecular level guides the elaboration of domains within petals to achieve their ideal shape, and further suggest that TCP genes determine petal identity along the DV axis by regulating MIXTA-like gene expression.

PHOSPHATIDYLSERINE SYNTHASE1 is Required for Inflorescence Meristem and Organ Development in Arabidopsis

Phosphatidylserine (PS), a quantitatively minor membrane phospholipid, is involved in many biological processes besides its role in membrane structure. One PS synthesis gene, PHOSPHATIDYLSERINE SYNTHASE1 (PSS1), has been discovered to be required for microspore development in Arabidopsis thaliana L. but how PSS1 affects postembryonic development is still largely unknown. Here, we show that PSS1 is also required for inflorescence meristem and organ development in Arabidopsis. Disruption of PSS1 causes severe dwarfism, smaller lateral organs and reduced size of inflorescence meristem. Morphological and molecular studies suggest that both cell division and cell elongation are affected in the pss1-1 mutant. RNA in situ hybridization and promoter GUS analysis show that expression of both WUSCHEL (WUS) and CLAVATA3 (CLV3) depend on PSS1. Moreover, the defect in meristem maintenance is recovered and the expression of WUS and CLV3 are restored in the pss1-1 clv1-1 double mutant. Both SHOOTSTEMLESS (STM) and BREVIPEDICELLUS (BP) are upregulated, and auxin distribution is disrupted in rosette leaves of pss1-1. However, expression of BP, which is also a regulator of internode development, is lost in the pss1-1 inflorescence stem. Our data suggest that PSS1 plays essential roles in inflorescence meristem maintenance through the WUS-CLV pathway, and in leaf and internode development by differentially regulating the class I KNOX genes.

Transcriptional and Post-transcriptional Modulation of SQU and KEW Activities in the Control of Dorsal-Ventral Asymmetric Flower Development in Lotus japonicus.

In Papilionoideae legume, Lotus japonicus, the development of dorsal-ventral (DV) asymmetric flowers is mainly controlled by two TB1/CYCLOIDEA/PCF (TCP) genes, SQUARED STANDARD (SQU) and KEELED WINGS IN LOTUS (KEW), which determine dorsal and lateral identities, respectively. However, the molecular basis of how these two highly homologous genes orchestrate their diverse functions remains unclear. Here, we analyzed their expression levels, and investigated the transcriptional activities of SQU and KEW. We demonstrated that SQU possesses both activation and repression activities, while KEW acts only as an activator. They form homo- and heterodimers, and then collaboratively regulate their expression at the transcription level. Furthermore, we identified two types of post-transcriptional modifications, phosphorylation and ATP/GTP binding, both of which could affect their transcriptional activities. Mutations in ATP/GTP binding motifs of SQU and KEW lead to failure of phosphorylation, and transgenic plants bearing the mutant proteins display defective DV asymmetric flower development, indicating that the two conjugate modifications are essential for their diverse functions. Altogether, SQU and KEW activities are precisely modulated at both transcription and post-transcription levels, which might link DV asymmetric flower development to different physiological status and/or signaling pathways.

Multiple components are integrated to determine leaf complexity in Lotus japonicus.

Transcription factors and phytohormones have been reported to play crucial roles to regulate leaf complexity among plant species. Using the compound-leafed species Lotus japonicus, a model legume plant with five visible leaflets, we characterized four independent mutants with reduced leaf complexity, proliferating floral meristem (pfm), proliferating floral organ-2 (pfo-2), fused leaflets1 (ful1) and umbrella leaflets (uml), which were further identified as loss-of-function mutants of Arabidopsis orthologs LEAFY (LFY), UNUSUAL FLORAL ORGANS (UFO), CUP-SHAPED COTYLEDON 2 (CUC2) and PIN-FORMED 1 (PIN1), respectively. Comparing the leaf development of wild-type and mutants by a scanning electron microscopy approach, leaflet initiation and/or dissection were found to be affected in these mutants. Expression and phenotype analysis indicated that PFM/LjLFY and PFO/LjUFO determined the basipetal leaflet initiation manner in L. japonicus. Genetic analysis of ful1 and uml mutants and their double mutants revealed that the CUC2-like gene and auxin pathway also participated in leaflet dissection in L. japonicus, and their functions might influence cytokinin biogenesis directly or indirectly. Our results here suggest that multiple genes were interplayed and played conserved functions in controlling leaf complexity during compound leaf development in L. japonicus.

A member of the ALOG gene family has a novel role in regulating nodulation in Lotus japonicus : An ALOG gene promotes nodulation

Legumes can control the number of symbiotic nodules that form on their roots, thus balancing nitrogen assimilation and energy consumption. Two major pathways participate in nodulation: the Nod factor (NF) signaling pathway which involves recognition of rhizobial bacteria by root cells and promotion of nodulation, and the autoregulation of nodulation (AON) pathway which involves long-distance negative feedback between roots and shoots. Although a handful of genes have a clear role in the maintenance of nodule number, additional unknown factors may also be involved in this process. Here, we identify a novel function for a Lotus japonicus ALOG (Arabidopsis LSH1 and Oryza G1) family member, LjALOG1, involved in positively regulating nodulation. LjALOG1 expression increased substantially after inoculation with rhizobia, with high levels of expression in whole nodule primordia and in the base of developing nodules. The ljalog1 mutants, which have an insertion of the LORE1 retroelement in LjALOG1, had significantly fewer nodules compared with wild type, along with increased expression of LjCLE-RS1 (L. japonicus CLE Root Signal 1), which encodes a nodulation suppressor in the AON pathway. In summary, our findings identified a novel factor that participates in controlling nodulation, possibly by suppressing the AON pathway.

LjCOCH interplays with LjAPP1 to maintain the nodule development in Lotus japonicus

Legume plants develop nodules during their symbiotic interaction with rhizobia, and much progress has been made towards understanding Nod factor perception and downstream signaling pathways, while our knowledge about the maintenance of nodule organogenesis was limited. We report here the knockdown mutants of LjCOCH, an ortholog of COCHLEATA in Pisum sativum, cause severe defects in nodule organogenesis in Lotus japonicus. The mature nodule number was drastically decreased accompanied with abnormal lenticel and vascular bundle developmental defects, but not produce roots from nodules in both Ljcoch mutants and LjCOCH-RNAi transgenic hairy roots. LjAPP1, a membrane-associated soluble aminopeptidase P1, was identified to interact with LjCOCH through yeast two-hybrid screening. Unlike that of Ljcoch mutants, insertion mutants of LjAPP1 and LjAPP1-RNAi transgenic hairy roots showed increased nodule number, while the lenticel and vascular development were not affected. Gene expression analysis indicated that LjCOCH and LjAPP1 were differentially upregulated by rhizobia inoculation, and LjNF-YA1 was the major downstream target of LjCOCH and LjAPP1. Our findings suggested that LjCOCH acts as a key factor involved in determinate nodule development through direct interaction with LjAPP1 to regulate the expression of LjNF-YA1, opposite effects of LjCOCH and LjAPP1 provide a dynamic regulation of nodule development in L. japonicus.