Genetic and molecular analysis of two new loci controlling flowering in garden pea

Flowering is one of the key developmental process associated with the life cycle of plant and it is regulated by different environmental factors and endogenous cues. In the model species Arabidopsis thaliana a mobile protein, FLOWERING LOCUS T (FT) plays central role to mediate flowering time and expression of FT is regulated by photoperiod. While flowering mechanisms are well-understood in A. thaliana, knowledge about this process is limited in legume (family Fabaceae) which are the second major group of crops after cereals in satisfying the global demand for food and fodder. Due to its short generation time, ability to reproduce via self or cross-pollination and availability of diverse lines, garden pea (Pisum sativum) serves as a model legume species for flowering time studies. Isolation and characterization of mutants have been a key research strategy in order to identify genes responsible for flowering in pea. The current study involved investigation of two novel EMS mutants namely late3 and late4 in the background of a cultivated pea line NGB5839 which are extremely late flowering indicating that LATE3 and LATE4 are essential for normal promotion of flowering in pea. Detailed phenotypic characterization carried out in the present study showed that both the loci positively regulate various vegetative, reproductive and yield related traits across different growth stages strongly suggesting potential global regulatory role for the underlying genes. Prior to the present study, synteny between pea and a closely related species, M. truncatula was exploited in order to roughly map LATE3 and LATE4 at the middle of pea LGIII and bottom of LGV respectively. These positions were significantly refined in the present study as LATE3 and LATE4 were mapped within a narrow genetic intervals in the syntenic regions of M. truncatula consisted of 62 (chromosome 3) and 54 (chromosome 7) genes respectively. Usage of the high-throughput RNA sequencing technology along with phylogenetic, co-segregation analysis and direct sequencing assisted in determining LATE3 and LATE4 genes as the pea homologue of Cyclin Dependent Kinase 8 (PsCDK8) and Cyclin C (PsCYCC1). CDK8 and CYCC1 are components of highly conserved CDK8 module (along with MED12 and MED13) of eukaryotic mediator complex that negatively regulate transcription of various genes involved in different biological processes. Analysis of the entire mediator complex revealed that this complex is highly conserved in A. thaliana, M. truncatula and pea. Phenotypic characterization of loss-of-function mutant of novel flowering gene CYCC1 in A. thaliana exhibited delayed flowering similar to already known similar mutants of AtCDK8, AtMED12 and AtMED13 which was consistent with the notion that these four genes act together to carry out the same regulatory process. Genetic interaction and yeast two hybrid assay unveiled complementarity and strong physical interaction between LATE3 and LATE4 which strongly suggested that these two genes act in the same regulatory process in a likely inter-dependent manner. Further genetic and regulatory interaction studies involving already known key pea flowering loci showed that LATE3 and LATE4 mediate flowering by regulating expression of known genes such as FTa1, FTc, PIM (PsAP1), VEG1(PsFULc), UNI (PsLFY)and VEG2 (PsFD) and LF (PsTF1c). The present study also undertook a systems biology approach for generating relevant hypothesis about the function and regulation of LATE3 and LATE4 genes. To this end, STRING v10.5 predicted the potential interactome network of AtCDK8 and AtCYCC1 consisted of members from cell cycle and mediator complex indicating a conserved role for them. In addition, predicted functions of MtCDK8 and MtCYCC1 genes obtained from AraNET v2.0 database was similar to that of PsCDK8, PsCYCC1, AtCYCC1 genes, thus giving hints of potential conservation of the function of both the genes in plant system. Moreover, PlantTFDB v4.0 database was used to forecast the transcription factors that may regulate the function of CDK8 and CYCC1 genes in M. truncatula and pea. As various mediator complex genes in A. thaliana are known to be involved in the regulatory processes driving response to different environmental factors, therefore relevant experimentation have shown that both LATE3 and LATE4 genes are important for controlling response to general light/darkness, ambient temperature, UVB, heat, mechanical wound and salt stress in pea. Overall, the present study provided significant understanding about the role of LATE3/PsCDK8 and LATE4/PsCYCC1 in mediating diverse range of reproductive, developmental and adaptive characteristics in pea where flowering time was the most important trait.