LIST OF TABLES
3. GENOME-WIDE ANALYSIS OF NAC TRANSCRIPTION FACTOR FAMILY IN COWPEA
3.4 DISCUSSION
However, the variable distribution of introns within the same phylogenetic groups (IV, VI, VII, and VIII) suggests both loss and gain of introns during gene rearrangement and duplication (Fig. 3.3A and Table 3.4). The membrane-tethered NTM-like genes are known to integrate biotic and abiotic stress signaling [359]. The presence of substantial membrane-tethered NAC members (12 proteins) suggested the potential involvement of these groups in the cross-talk (Fig. 3.7A). Most importantly, the high divergence of the C-terminal TAR sequences imparted novelty to the cowpea NAC family (Fig. 3.6). The presence of 27 species-specific members and 21 stress-responsive members suggested that the evolution of the cowpea NAC family occurred under unique environmental pressure (Fig. 3.1B). The promoters of these members (Group VII) were rich in ARE elements suggesting their expansion to fight anaerobic conditions like submergence (Fig. 3.9A). The over-representation of MYB/MYC binding elements specified their transcriptional regulation.
3.4.2 Multi-tier regulatory network of VuNAC TF 3.4.2.1 Unique promoter arrangement
The promoter analysis revealed the unconventional structure of the VuNAC promoters. The position of the TATA box was highly variable from +10 to +55 bp. The 5’-UTR and core region were rich in rare TC-rich elements (Fig. 3.8C). The high-expressing genes showed an inclination towards bearing the TC-rich elements, irrespective of the presence of the TATA box (Fig. 3.10B). At the same time, the presence of binding sites for NAC TFs and other multiple stress-responsive TFs like MYB/MYC, WRKY, ERF, bZIP, bHLH, etc. (Fig. 3.9B) and cis-regulatory elements regulating versatile cellular functions makes the VuNAC promoters typically unique (Table A1.8, Appendix 1). Furthermore, the protein sequences were rich in post-translational modification sites like methylation, glycosylation, and phosphorylation, along with the landscapes of amino acid repeats (Table A1.3, Appendix 1). Our analysis gave the insight that the VuNAC genes undergo a multi-tier regulation at the level of promoter- mediated expression, protein modification as well as protein-protein interaction
3.4.2.2 Transcriptome analysis and co-expression network
Recently, the comprehensive genome draft for cowpea was made available [59]. Still, the reports for the RNA-seq study of cowpea disclosing the annotation aspects of cowpea are rare.
Our study presented the first report annotating the cowpea NAC family under two different growth stages, i.e., seedling and vegetative stage, and elucidating the relationship between their
expression, promoter nature, and functional group (Fig. 3.9). NAC proteins being a TF, works together with innumerable partners. For instance, the overexpression of one of the co- expressing AtNAC TFs ANAC019/ANAC055/ANAC072) alone could not induce the ERD1 gene because the induction of ERD1 depends on the co-expression of ZFHD1 as well [94].
Thus knowing the interaction partner is crucial for the functional study of cell response in a broader way keeping the interlinked signaling cascade in consideration. Therefore, the interactome network of VuNAC TFs was explored based on the information available for Arabidopsis (Fig. 3.11). The co-expression Cluster 1 (stress-responsive) and Cluster 2 (senescence-related) were interlinked via RD26, ANAC019, DREB2A, and the other 12 genes in the network. The analysis suggested that the NAC-mediated stress response and growth processes are entangled by overlapping hormone and cell-signaling networks involved in abiotic stress, energy metabolism, and cell expansion. The senescence-associated VuNAC gene cluster potentially interacts with peroxidases (DOX1), regulating redox balance and trehalose biosynthetic genes like TPS8/TPS11, required for the onset of leaf senescence when high content of carbon is available [360]. Cluster 3 and Cluster 4 did not seem connected (Fig.
3.11B), suggesting independent regulation of the speculated basal and architectural functions.
3.4.3 Cowpea NAC family is multifarious in function 3.4.3.1 Versatile domains of VuNAC TFs
Cowpea NAC TFs harbored chimeric domains that are functional trademarks for other proteins involved in cell division, cell death, cytoskeleton synthesis, respiration, and cell signaling apart from the transcriptional motifs (Table 3.2). Furthermore, many VuNAC proteins owned the functional sequence profiles of proteins involved in pivotal processes, like folate synthesis, carbohydrate transport, lipid signaling, cell-wall binding, electron transfer, and growth regulation (Table A1.3.2, Appendix 1). This suggested that the NAC TFs have the propensity to undertake diverse cellular processes directly. In addition, the motif analysis in TAR regions and the noteworthy conservation and redundancy in the paralogous proteins advocate their functional and evolutionary importance (Fig. 3.5). Clearly, VuNAC TFs genes play crucial roles in plant processes integrating stress and growth-associated cellular metabolic phenomena. Our study revealed that 16.2% of the VuNAC TF fraction was dedicated to stress responses exclusively. 26.9% and 13.9% proteins were dedicated to cell-wall and xylem development and basal organogenesis, respectively. 22.3% of the portion was speculated to be involved in other developmental processes like flowering and aging. 20.8% of the family
having no similarity to any other species were novel to cowpea, hence a prominent candidate for further research in plant science (Table 3.5).
3.4.3.2 ATAF-like Group I members signified potential tools for crop improvement Plants continuously evolve the intricate stress-signaling pathways and their genetic determinants to sustain tolerance to the changing adverse environments by tuning innumerable biochemical, metabolic, and molecular mechanisms. Our finding regarding functional clustering of cowpea NAC TF might elaborate on understanding NAC response in development and stress response over a broad scale. The five ATAF-like members clustered in subgroup Ia held the immense potential to serve as a genetic manipulation tool to combat stress and improve crop yield (Fig. 3.1C). Because they seemed to function in coordination with other stress- responsive TFs (NAC/MYB/ERF/WRKY) and genes crucial for growth such as Dof (regulator of seed-storage protein) and TCP (cell proliferation), possibly integrated by glutathione and starch/sucrose metabolism, thus, the members could potentially boost the plant defense and basal development (Fig. 3.9B and Fig. 3.11C).