LIST OF TABLES
5. CHARACTERIZATION OF VUNAC1/2 TFS IN RESPONSE TO ABIOTIC STRESSES UNDER STARVATION
5.4 DISCUSSION
5.4.2 The multifaceted VuNAC1/2 TFs are associated with a network of proteins regulating stress responses, hormonal flux, and growth signals
The functional versatility of NAC genes is accounted by the tight regulation at the transcriptional, post-transcriptional, and post-translational levels. Their specificity can be determined by the set of target genes recruited by the TFs and their coordinating partners, melding disparate biological processes at different developmental stages. Our study of co- expressed protein networks and upstream regulators suggested that the VuNAC1/2 genes might be induced and regulated by stress, nutritional and metabolic signals in a feedback loop.
Combining our findings, several modes of VuNAC regulation can be proposed (i) key stress- regulating transcription factors, (ii) ABA, auxin, and ethylene signal and homeostasis, (iii) cellular metabolism, (iv) stress stimuli, and (v) developmental signals. Thus, the plant responses can be dissected into disparate regulatory networks involving NAC TFs as central signaling molecules.
We found a cluster of TFs belonging to the NAC family and other stress-responsive groups (DREB, ERF, TCP, and WRKY), co-expressing with ATAF-subgroup (ATAF1/2, ANAC032, and ANAC102), probably involved in the co-binding to the target gene promoter. Their interaction may amalgamate diverse processes such as ethylene biosynthesis (NAC047), ABA- mediated drought response (RD26), ethylene, auxin and salt response (MYB74), cold (DREB2A), lateral root formation (NAC2), leaf senescence (NAP), ER stress response (NAC062). Multiple hormones signaling and their metabolism (ABA, ethylene, auxin, and GA) seemed intertwined with VuNAC signaling via various proteins PP2Cs, ABIs, HAB1, Aux/IAA, ERFs, and RGA1. Primary metabolites such as carbohydrates (glucose, sucrose, trehalose) and lipid, nitrate and phosphate, also seemed to undergo regulation via proteins like
CNI1, ADC2, SUS3, TPPG, etc., signaling C/N balance signal, carbon starvation, and nitrogen assimilation. The proposed upstream regulatory TFs also indicated signaling of VuNAC1/2 TF perceived by proteins like HSFA2 (heat-response), RRTF1 (oxidative stress response), COBRA (cell-expansion), MYB21 (photo-morphogenesis), ANAC025 (pollen and seed development), and flower development (PISTILLATA) (Table 5.2).
The interactome and regulator prediction stipulated the coordinated changes in the stress response, metabolic and hormone signaling underlying seed germination, seed establishment, vegetative and reproductive growth in the transgenic Arabidopsis plants. As indicated by the interactome analysis, the VuNAC1/2 TFs may interact with proteins like ABI1/2, HAB1, and PP2C49, negatively regulating the ABA signaling, UGT74E2, and Gh3.15, controlling the auxin flux, and ZAT6 regulating seed germination under stress [439, 470]. Moreover, the SnRK kinases triggering the carbon starvation response seemed to be attenuated by trehalose signaling via TPPG. After germination, the successful seedling establishment depends on the transition to autotrophic nutrition assimilation, from the heterotrophic acquire from the seed reserve. Proteins like Carbon/Nitrogen Insensitive 1 (CNI1/ATL31) ligase controlling the post- germination nutrient-signal transduction may be associated with VuNAC signaling.
Later, several proteins promoting biogenesis of structural components and cell division, such as FORMIN7, PLIM2a, GAT6, AGP1, TET8, etc., and TFs like TCP, ERF11, and ZAT6 tuning hormonal responses, may involve. Photoreceptors perceiving light can also indirectly contribute to growth by regulating metabolic programs via stem elongation, leaf expansion, seed germination, flower initiation, chloroplast development, endogenous hormone levels, and transport. Co-expressed proteins like FC1, BCS1, CYTC-2, and AOX1A regulating heme- biosynthesis, electron transfer, and photo-damage control might improve photosynthetic efficiency through better light perception by the transgenic plants. VuNAC1/2 TFs may also regulate shade tolerance under low-light conditions via BBX18 and AtMYB21. The co- expression studies implied VuNAC1/2 interaction with enzymes catalyzing the synthesis of cell-wall polysaccharides and energy generation through glycolysis, FA oxidation, and cofactor biosynthesis. APR3 and SAT1 regulating the synthesis of cysteine and PHL2 might improve sulfur and phosphate assimilation in transgenic plants. The transgenic tissues accumulated proline, glutathione, and ascorbate, even under normal conditions, indicating direct regulation of proline, glutathione, and ascorbate metabolism via VuNAC1/2 TFs. The coexpression network suggested DHAR2 and GSTs enzymes involved in ascorbate recycling and
detoxification and PAP1, ADT4, and ADT5 regulating anthocyanin production and MATE transporters [450, 464, 465].
In conclusion, our study indicated that VuNAC1/2 TFs regulate stress responses, hormonal and nutritional homeostasis, and plant development through cross-talk of photosynthesis, carbon metabolism, and ABA signaling. However, the detailed model for VuNAC1/2 regulation is still unknown. Genes like VuNAC1/2 that confer tolerance to multiple abiotic stresses and ameliorate the shrinking vegetative growth and reproductive yield should be used for sustainable drought and salinity tolerance in cash crops to accomplish dual objectives, i.e., multi-stress resilience and yield improvement. They might also play a crucial role in protection against pH-dependent phytotoxicity of aluminum and other toxic metals, which are significant constraints for root development in acidic soil [131]. As the unfavorable climatic conditions are becoming persistent, ubiquitous and constitutive expression of unique VuNAC1/2 TFs may avoid the trade-off and secure yield potential of crops. Further understanding of the underlying molecular mechanisms can bring new insights to harmonized cross-talk between stress adaptation and growth. The functional role of these TFs in native Cowpea species and related legume crops needs to be explored.