Role of VILAMBIT Genes Controlling Flowering Time and Jasmonic Acid Signaling in Arabidopsis

The transition to flowering is an important decision for plants since seed-setting and the survival of the progeny depend on the environmental conditions prevalent during this transition. Therefore, to ensure maximum reproductive success, plants have evolved several regulatory mechanisms to enable them flower at the most appropriate time. Environmental parameters such as light, temperature and nutrient availability as well as endogenous factors such as age and hormonal status of the plant profoundly affect floral transition (Boss et al., 2004; Srikanth and Schmid, 2011). Studies in Arabidopsis and other model plant species have identified several distinct genetic pathways that integrate the information from the endogenous and environmental cues to regulate flowering (Boss et al., 2004; Srikanth and Schmid, 2011). Many components and gene regulatory networks identified in Arabidopsis are conserved in other commercially important species including rice, maize, sorghum, potato and tomato. Therefore, it is important to understand the basic mechanisms that modulate the flowering response in model plants such as Arabidopsis thaliana, the knowledge from which can be used to develop better adapted and high-yielding varieties of crop plants in the wake of challenges like global warming and increasing food demand. In the present study, we have studied the function of VLB1 and VLB2, genes that code for plant-specific Zn-finger transcription factors. Previous studies from our laboratory (Pratibha Choudhary, Ph.D thesis, 2011) and by other research groups have reported that VLBs redundantly promote flowering in A. thaliana (Yasui et al., 2012; Celesnik et al., 2013). However, the underlying mechanism of this regulation is not well understood. Our data suggests that VLBs redundantly promote the transition to flowering specifically in the photoperiod pathway, the major floral induction pathway in A. thaliana. CO, which is the 93 key regulatory gene in this pathway, is regulated by various factors at the transcriptional as well as post-transcriptional level (Suarez-Lopez et al., 2001; Yanovsky and Kay, 2002; Srikanth and Schmid, 2011). Using genetics, we show that VLBs and CO function together to promote flowering in the photoperiod pathway. Further, our BiFC results reveal that VLBs and CO interact physically. Nevertheless, the physical interaction between VLBs and CO needs to be further validated by in vitro and in vivo by co-immunoprecipitation experiments. We hypothesize that the interaction between VLBs and CO is important to regulate FT expression and hence, flowering. However, whether VLBs interact with CO and promote the CO-stability, or facilitates its recruitment to the FT promoter region, still needs to be determined. Apart from its role in flowering, VLBs have been recently shown to regulate biotic and abiotic responses in Arabidopsis (Nakai et al., 2013a; Nakai et al., 2013b). Also, even though it has been demonstrated that VLBs code for transcription factors, no direct targets of VLBs have been reported till date. We performed a whole genome trancriptome-profiling and found that several important classes of genes including WRKY, RLPs, NBS-LRR and JAZs were affected suggesting that, in addition to their role in floral transition, VLBs have important functions in other plant processes as well. In fact, vlb1vlb2 mutant showed an early senescence phenotype and many senescence-associated genes (SAGs) were up-regulated in our microarray experiments, which was further validated by qRT-PCR analysis. By comparing the differentially-regulated genes and PatMatch analysis, we have identified 82 putative direct targets of VLBs in the Arabidopsis genome which need to be validated by chromatin immunoprecipitation (ChIP) assay and functional studies. 94 Results of global transcriptome analysis revealed that the expression of several JA-signaling and response genes was significantly down-regulated. JA is an important phytohormone involved in plant defense and other developmental processes such as stamen development, root growth and senescence (Wasternack, 2007). Results from the JA-induced expression analysis and root inhibition assay confirmed that JA-signaling and response are indeed compromised in the vlb1vlb2 double mutant. Moreover, in vitro DNA-binding assay showed that MYC2, the key transcriptional regulator of JA-responsive gene expression, is a direct transcriptional target of VLB2. A recent study reported that loss-of-function of VLB genes impairs plant defense while their overexpression confers biotic stress tolerance in Arabidopsis (Nakai et al., 2013a; Nakai et al., 2013b). Compromised JA signaling in the vlb1vlb2 double mutant might partly explain this reduced tolerance to pathogens. However, whether VLBs are associated with the MYC2 promoter in planta needs to be tested by performing ChIP and other in vivo assays. In conclusion, our study shows that VLBs have important regulatory roles in diverse processes including control of flowering time, senescence and JA signaling in Arabidopsis. The validation and functional characterization of the direct targets of VLBs will shed more light on the role of VLBs. Since VLBs are conserved in vascular plants, it will be interesting to see if the function of VLBs is also conserved across species and what might be its ancestral function in evolution