Functional Analysis Of LATHYROIDES In Legumes

During organ development, many key regulators have been identified in plantgenomes, which play conserved role among plant species to control the organidentities and/or determine the organ size and shape. It is intriguing thatwhether these key regulators can acquire diverse function and be integratedinto different molecular pathways among different species, giving rise to theimmense diversity of organ forms in nature. In this study, we havecharacterized and cloned LATHYROIDES (LATH), a classical locus in pea,whose mutation displays pleiotropic alteration of lateral growth of organs andpredominant effects on tendril and dorsal petal development. LATH encodesa WUSCHEL-related homeobox1(WOX1) transcription factor, which has aconserved function in determining organ lateral growth among different plant species. Furthermore, we studied the function of LATH by genetics, cellbiology and molecular biology methods and found that LATH interplay withmultiple developmental factors to regulate development of flower andcompound leaf.1. Isolation and characterization of lath and nao1mutants from pea andLotusOne narrow organ1(nao1) and6allelic mutants (lath) similar tolathyroide mutant were isolated from EMS mutagenesis in Lotus and fastneutron mutagenesis in pea respectively. Both nao1and lath had pleiotropiceffect including narrower leaves and flowers especially dorsal petal from abig petal to a narrow strip, and all the mutants except the weaker allelelath1-6were female sterile. Genetic analysis showed that nao1and lath wereboth controlled by single recessive locus.2. Molecular cloning the LATH/NAO1geneWe fine mapped NAO1to a250K region in the end of the long arm inchromosome4of Lotus. This region covered three BACs which overlappedto form CM0042contig and shared microsynteny with AC174277andAC137078in the end of chromosome4of Medicago truncatula. LATHexactly was primarily mapped to the counterpart of the region.Bioinformatics analysis found that there was a WUSCHEL RELATED HOMEOBOX1(WOX1) homologous gene in the region. Squences analysisshowed that nao1mutant carried a single base substitution in LjWOX1which putatively resulted in forming a truncated protein and PsWOX1mutated in all the six allelic mutants in pea.3. LATH expression patternRT-PCR analysis showed that LATH expressed in aerial organs andshowed the most high expression level in floral buds. But strikingly, therewas almost no or very low expression level in roots even with high cycles ofamplification. RNA in situ hybridization analysis showed that LATH mainlyexpressed in central vascular bundle in stipules and leaflets, and in the marginof leaflets and tendrils. As in floral development, LATH mainly expressed inlateral parts of five petals and early stage stamens. LATH also expressed inthe dorsal fusion region of the carpal. LATH showed no expression pattern inshoot apical meristerm (SAM).4. Genetic interaction among LATH and PsCYCsIn order to investigate the interaction between LATH and PsCYCs, wecarried out a series of genetic cross. lath k double mutant showed an additivephenotype. the lath lst double mutant showed an unexpected phenotype inthat the dorsal petal of the double mutant bore partially developed blade, andwas somewhat similar to the lst single mutant. These results suggested that LATH genetic interact with LST to regulate petal development. Narroweddorsal petal of lath losed subdomain with dorsalized epidermal cell, andlobed dorsal petal of lst altered dorsal identity with lateralized epidermal cell.However dorsal petal of lath lst somehow reaquired dorsal identiy as judgedby appearance of the dorsalized epidermal cell types.Fluorescence real-time PCR analysis showed that there was noregulation between LATH and LST in transcription level. Yeast two hybridassay also showed there was no direct interaction between LATH and LST inyeast.5. LATH regulated Tendril-less to affect tendril development in peaThe distal part of the compound leaves showed a tendril converting tonarrow leaflets phenotype in lath mutant which reminiscent of tendril-lessmutant which also showed increased leaflets but not change the complexityof the compound leaves. Fluorescence real-time PCR analysis showed thatTL was downregulated in lath mutants. This showed that LATH controlledtendril development through regulating TL gene.