LIST OF TABLES
5. CHARACTERIZATION OF VUNAC1/2 TFS IN RESPONSE TO ABIOTIC STRESSES UNDER STARVATION
5.3 RESULTS
5.3.6 Elucidation of orthologous interactome indicated VuNAC1/2 interaction with regulators of stress responses, hormone balance, metabolism, and growth
Integrative gene regulatory networks (iGRN) covering orthologous genes of model plants can provide a foundational framework for understanding and inferring the gene regulation of plants with unknown mechanisms [430]. The upstream regulators of the VuNAC1/2 TFs predicted based on the Arabidopsis homologs indicated that they perceive the signal of several other proteins such as HSFA2 (combined stress-response and ascorbate signaling) [431], RRTF1 (oxidative stress response) [432], COBRA (cell-expansion), MYB21 (photo- morphogenesis), ANAC025 (pollen and seed development) [104], flower development (PISTILLATA) [433], and PHL1 and HRS1 (nitrogen, phosphate and iron starvation and breaking of ABA-mediated seed dormancy) [434-436] (Table 5.2). Further, we generated a gene interactome for ATAF subgroups of Arabidopsis, bearing close homology with the VuNAC1/2 TFs, by convoluting experimental data of multiple co-expression studies under different developmental and environmental conditions, including PPIs (protein-protein interactions). The analysis predicted possible interacting partners of the VuNAC TFs (Fig.
5.11, Table 5.3, and Table A3.2), corroborating with the functional phenotypes of the transgenic plants and providing supporting evidence. As discussed below, the orthologous interactome suggested the interaction of VuNAC1/2 TFs with many stress and hormone signaling proteins, transporters, and metabolic enzymes. Combining the upstream regulatory model and coordinating gene network, suggested that VuNAC1/2 might be induced by stress, hormone, nutrition, light, and developmental signals in a feedback loop.
5.3.1.1 Interaction with signaling proteins
The protein network comprised PROTEIN PHOSPHATASES TYPE 2C (PP2Cs), ABA INSENSITIVE (ABIs), and HYPERSENSITIVE TO ABA1 (HAB1) that negatively regulates ABA signaling by inhibiting SNF1-RELATED PROTEIN KINASE1 (SnRK1). PP2CG1 signals salt tolerance, HIGHLY ABA-INDUCED 1 (HA1) regulates drought acclimatization via dehydrins and LEA proteins, ZINC-INDUCED FACILITATOR PROTEIN (ZIFL1) controls polar auxin transport and drought tolerance, UDP GLYCOSYLTRANSFERASE
74E2 (UGT74E2) and GH3 protein (Gh3.15) regulate plant architecture by perturbing auxin homeostasis. The interactome also included NAC and other family TFs involved in the developmental and stress-associated processes, such as NAC2, RD26, NAC062, DREB2A, GBF3, regulating drought response [299, 437, 438], NAP and NAC047 tuning senescence and ethylene biosynthesis during hypoxia, Ras-related proteins (RAP2.4 and RAP2.6L), and ERF members (ERF1, ERF4, and ERF6) modulating cross-talk between ethylene, auxin, and ABA responses [129, 439-442]. Proteins like MYB74, CCX2, and CFZ1 regulating salt-response and osmo-tolerance also co-expressed, suggesting possible interaction. Other stress-responsive proteins in the network were bZIP60 and NAC062, mitigating ER stress, DJ1A activating cytosolic superoxide dismutase, G2-like flavonoid regulator (GFR) involved in cold response and flavonoid metabolism, NAP and MKK9 playing a crucial role in senescence regulation and several proteins from MATE family regulating detoxification processes.
5.3.1.2 Interaction with stress-responsive proteins
The interactome included NAC and other TFs involved in the developmental and stress- responses, such as NAC2, RD26, NAC062, DREB2A, GBF3, regulating drought response [148, 299, 437, 438]. Proteins like MYB74, CCX2, and CFZ1 regulating salt-response and osmo-tolerance also co-expressed, suggesting possible interaction [443-445]. Other stress- responsive proteins in the network were bZIP60 and NAC062, mitigating endoplasmic reticulum (ER) stress [446], DJ1A activating cytosolic superoxide dismutase, G2-like flavonoid regulator (GFR) involved in cold response and flavonoid metabolism, NAP and MKK9 regulating senescence [146, 447-449], and several proteins from MATE (Multidrug And Toxic Compound Extrusion) family regulating detoxification [450].
5.3.1.3 Interaction with growth-regulating proteins
Several signaling proteins associated with growth and developmental processes were found in the network, such as Carbon/Nitrogen Insensitive 1 (CNI1/ATL31) ligase controlling post- germination nutrient signaling [451], ZAT6 regulating germination under salt and osmotic stress, as well as cadmium tolerance [452], ERF6/11 stimulating growth through GA-mediated responses [453]. MADS-box proteins (AGL12/16) and CYCLIC DOF FACTOR 2 (CDF2) controls flowering time and floral transition [454, 455]. TCP proteins TCP7/14/15/23 are involved in perceiving endogenous signals (circadian clock, plant hormones) and environmental (pathogens, light, nutrients), forming an interface between environmental cues and plant growth [456-458]. Moreover, proteins like BBX18 regulate seedling photo-
morphogenesis by preventing hypocotyl inhibition [459], ALTERNATIVE OXIDASE 1A (AOX1A) protecting against oxidative damage by intense light [460], BCS1 and CYTC-2 forming the mitochondrial electron transport chain, were also included.
5.3.1.4 Interaction with metabolism-associated and signaling proteins
Metabolic enzymes such as CINNAMOYL COA REDUCTASE (CCR), CINNAMYL- ALCOHOL DEHYDROGENASE (CCD), FRK1, and BAM1 synthesizing structural components (cellulose, starch, and lignin), ARABINOGALACTAN POLYSACCHARIDES 1 (AGP1), and GAT6 synthesizing arabinogalactan polysaccharides essential for foliage growth were also found in the interactome [461, 462]. ADENOSINE 5'-PHOSPHOSULFATE REDUCTASE (APR3) and serine acetyltransferase (SAT1) catalyzing sulfur assimilation in cysteine and methionine, THIAMINE PYROPHOSPHATE KINASE 1 (TPK1) kinase controlling TPP activity required for fuelling photosynthesis and cellular metabolism [463], DEHYDROASCORBATE REDUCTASE (DHAR2) involved in ascorbate recycling to maintain redox homeostasis [464], PAP1, ADT4, and ADT5 regulating anthocyanin production and radical scavenging, GLUTATHIONE S-TRANSFERASES (GSTs) mediating cellular detoxification and stress tolerance through flavonoid metabolism [465], and PHL2 mediating phosphate starvation comprising the network, indicated cross-talk between growth and stress signaling [466]. Transporters regulating nucleotide, folate, carnitine, and other solute transport [467, 468], and calcium signaling proteins (CBL1, CPK32, CML37), and other signal transducers like MAPKKK14/18, PERK9, LYK5 linked the interactome nodes [469].
Table 5.2 Upstream regulators of VuNAC1/2 TFs predicted by RnR database
Gene ID Gene ID P.R. Score Function
COBRA At5g60920 99.4 necessary for oriented cell expansion
MYB103 At1g63910 99.2 involved in tapetum development and exine formation in anthers MYB28 At5g61420 98.7 major regulator glucosinolate biosynthesis protects against insects PISTILLATA At5g20240 98.4 required for petals and stamen development
ANAC025 At1g61110 98.3 anther, pollen and seed development HsFA2 At2g26150 98.2 heat-stress recovery
PHL1 At5g29000 98.2 involved phosphate starvation response;
RGA1 At2g01570 97.8 represses GA-induced vegetative growth and floral initiation MYB48 At3g46130 97.8 regulate flavonol biosynthesis primarily in cotyledons DIV2 At5g04760 96.7 negatively regulates salt response
RRTF1 At4g34410 100 regulates redox homeostasis SAP6 At3g52800 99.4 stress response
ATHB-8 At4g32880 99.2 involved in the regulation of vascular development ATMYB21 At3g27810 99.1 involved in photomorphogenesis in the light
HRS1 At1g13300 97.3 nitrate and phosphate starvation responses and adaptation of root architecture CYP79B2 At4g39950 99.5 a precursor for tryptophan-derived glucosinolates and indole-3-acetic acid (IAA) Source: http://webs2.kazusa.or.jp/kagiana/rnr0912/indexff.html
Table 5.3 List of orthologous proteins in the putative VuNAC1/2 interactome
Gene ID Description Supportability Rank Function
Hormone AFP1 ABI five binding protein 3 7.1 ABA signaling
HAB1 HYPERSENSITIVE TO ABA1 2 5.6 ABA signaling [470]
PP2CG1 Protein phosphatase 2C family protein 1 5.5 ABA signalling [440]
ABI2 Protein phosphatase 2C family protein 2 5 ABA signaling HAI1 PP2C protein (Clade A protein phosphatases type 2C) 3 6.4 ABA signaling [471]
MAPKKK18 mitogen-activated protein kinase kinase kinase 18 1 6 ABA signaling IAGLU indole-3-acetate beta-D-glucosyltransferase 2 5.4 Auxin metabolism SAUR-like SAUR-like auxin-responsive protein family 1 5.2 Auxin signaling ZIFL1 zinc induced facilitator-like 1 2 5.8 Auxin signaling [441]
ACS6 1-aminocyclopropane-1-carboxylic acid (acc) synthase 6 3 5
Ethylene biosynthesis [472]
RAP2-13 Integrase-type DNA-binding superfamily protein 1 5.7
Ethylene biosynthesis [472]
ERF1 ethylene-responsive element binding factor 1 3 8.1 Ethylene signaling [442]
JAZ1 jasmonate-zim-domain protein 1 3 5 JA signalling
Abiotic Stress MYB74 myb domain protein 74 0 6.7 Salt stress response [443]
STZ salt tolerance zinc finger 3 5.6 Salt stress response
SZF1 salt-inducible zinc finger 1 3 5.3 Salt stress response
CZF1 zinc finger (CCCH-type) family protein 3 8 Salt stress response TSPO TSPO (outer membrane tryptophan-rich sensory protein)-like
protein 3 5.5 Salt stress response [444]
GBF3 G-box binding factor 3 2 5.7 Drought response
RDUF1 zinc finger (C3HC4-type RING finger) family protein 3 5.9 Drought response
LEA4-5 Late Embryogenesis Abundant 4-5 3 5.8 Desiccation
BZIP60 basic region/leucine zipper motif 60 3 6 ER stress
NAC062 NAC domain containing protein 62 3 7.7 ER Stress tolerance [446]
HSFB2A heat shock transcription factor B2A 3 6 Heat stress RHL41 C2H2-type zinc finger family protein 3 6 Light stress DJ1A
Class I glutamine amidotransferase-like
superfamily protein 3 6 Oxidative stress [447]
PMSR1 peptidemethionine sulfoxide reductase 1 1 5 Oxidative stress
OPR2 12-oxophytodienoate reductase 2 2 5.1 Detoxification
MKK9 MAP kinase kinase 9 3 5 Senescence [449]
AT2G28400
senescence regulator (Protein of unknown
function: DUF584) 2 5.4 Senescence
SAP9 A20/AN1-like zinc finger family protein 1 6.1 Stress response
DREB2A DRE-binding protein 2A 0 5.9 Stress response
UGT73B3 UDP-glucosyl transferase 73B3 3 5.5 Stress response AGC2-1 AGC (cAMP-dependent: cGMP-dependent and protein kinase C)
kinase family protein 3 5.6 Stress signaling
S6K2 serine/threonine protein kinase 2 0 5.3 Stress signaling CAD1 MAC/Perforin domain-containing protein 3 5.1 PCD
Biotic Stress BAP1 BON association protein 1 2 5.7 Defense signaling
EXO70B1 exocyst subunit exo70 family protein B1 3 5.4 Defense signaling
NHL3 NDR1/HIN1-like 3 3 5.2 Defense signaling
NPR3 NPR1-like protein 3 2 5.8 Defense signaling
TIP TCV-interacting protein 3 5.3 Defense signaling
RIPK Protein kinase superfamily protein 3 5.1 Disease resistance AT5G54165 Avr9/Cf-9 rapidly elicited protein 2 5.2 Pathogen resistance
WRKY33 WRKY DNA-binding protein 33 3 6.5 Pathogen resistance
WRKY40 WRKY DNA-binding protein 40 3 6.3 Pathogen resistance
Growth &
Development ZAT6 C2H2-type zinc finger family protein 3 5
Phospahte homeostasis [452]
OCT5 organic cation/carnitine transporter5 0 5
Lateral root development [467]
ZF2 zinc-finger protein 2 3 5.8 Flower abscission
CYP81D8 cytochrome P450: family 81: subfamily D: polypeptide 8 2 5.1 Photosynthesis
BCS1 cytochrome BC1 synthesis 3 5.7 Photosynthesis
CNI1 carbon/nitrogen insensitive 1 3 5 C/N tolerance [451]
VQ22 VQ motif-containing protein 2 7.2 Growth regulator
TET8 tetraspanin8 3 5.5 Cell differentiation
Metabolism ADC2 arginine decarboxylase 2 1 5.8 Arginine metabolism
SUS3 sucrose synthase 3 3 5 Sugar Metabolism
BAM1 beta-amylase 1 2 6.5 Sugar Metabolism
BGLU11 beta glucosidase 11 2 5.6 Sugar Metabolism
GPX2 glutathione peroxidase 2 1 5.2 Glutathione metabolism
FC1 ferrochelatase 1 3 5.5 Heme biosynthesis
GATL10 galacturonosyltransferase-like 10 3 5 Pectin metabolism GSTU24 glutathione S-transferase TAU 24 3 7 Reseveratrol metabolism CGS1 CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase 3 5.6 Phospholipid metabolism GLTP Glycolipid transfer protein (GLTP) family protein 3 5.7 Lipid transfer
Fig. 5.11. Proposed regulatory network (A) The orthologous (ATAF-subgroup network). (B) The interactome of co-expressing genes (connected by grey lines) and protein-protein interactions (connected by red lines), predicted the regulon for VuNAC1 and VuNAC2 (C) The gene ontology (GO) analysis of the network annotated the cellular, molecular and biological function associated with VuNAC1/2 TFs.