LIST OF TABLES
2. INTRODUCTION
2.1 NAC transcription factor: a potential integrator of stress tolerance, growth, and yield traits in cowpea
2.1.3 Potential utilization of NAC genes for abiotic stress tolerance in food crops
Several NAC genes from Arabidopsis, staple cereal crops (rice, wheat, maize, millet, etc.), legumes (soybean, chickpea, and peanut), vegetables, and fruits, have been characterized by overexpression or knockdown to identify their functional roles (Table 2.2). The studies present substantial evidence to support the potential of NAC TF as a crop improvement tool. The utilization of suitable NAC genes may potentially rescue crop yield from loss due to environmental stress. However, the functional conservation of NAC TFs across species and their response to a particular stress type is still debatable, mainly due to the partial divergent structure of NAC proteins and their complex regulation. Though enough information is available for the soybean NAC family, unlike cereal crops, the functional study of NAC in legume species is still preliminary.
Table 2.2 NAC role in stress response of food crops and model plants
NAC TF Function Reference
ATAF1 (Arabidopsis) Overexpression enhanced tolerance to drought, ABA biosynthesis, sensitivity to ABA, salt, oxidative stress, and necrotrophic fungus, and promoted senescence in Arabidopsis; Overexpression increased salt and cold tolerance and ABA insensitivity in rice;
[27, 28, 56, 57]
ATAF2 Overexpression increased age-dependent and dark-induced senescence [137]
ANAC032 Overexpression enhanced age-dependent and stress-induced senescence, repressed anthocyanin accumulation in stress in Arabidopsis
[138, 139]
ANAC102 Overexpression promoted seed germination under hypoxia-stress [128]
ANAC019, ANAC055, ANAC072
Overexpression of ANAC019/055/072 increased drought tolerance and chlorophyll degradation during senescence in Arabidopsis; Overexpression of ANAC019 improved reproductive recovery in drought stress
[140]
[141]
ANAC096 Mutation decreased tolerance to dehydration and osmotic stress; [142]
ANAC016 Mutation delayed senescence and improved drought tolerance in Arabidopsis [143, 144]
ANAC047/SHYG Overexpression increased water-logging tolerance by inducing hyponastic leaf movement
[129]
ANAC029/AtNAP Overexpression promoted leaf and fruit senescence and reduced salt tolerance in Arabidopsis
[145-147]
ANAC092/AtNAC2 Overexpression promoted senescence, lateral root development under salt stress in Arabidopsis, and improved tolerance to drought and salt in peanut
[148-150]
ANAC046 Overexpression promoted senescence and chlorophyll degradation [151]
NTL9/CBNAC Overexpression promoted senescence in Arabidopsis [152]
ANAC042/JUB1 Overexpression delayed senescence to extend longevity and enhanced tolerance to heat stress in Arabidopsis
[153, 154]
OsNAC14 (Rice) Overexpression improved drought tolerance in rice [155]
ONAC066 Overexpression increased tolerance to drought, oxidative stress, and ABA sensitivity in rice
[126]
ONAC095 Overexpression increases drought susceptibility and cold tolerance in rice [156]
ONAC096 Mutation increased panicle number and delayed senescence in rice [157]
ONAC022 Overexpression increased tolerance to drought, salinity stress, and ABA sensitivity in rice
[158]
ONAC106 Overexpression inhibited leaf senescence and increased salt tolerance in rice [159]
ONAC011 Overexpression promoted leaf senescence in rice [160]
OsNAP Overexpression promoted leaf senescence in rice [109]
OsNAC45 Overexpression increased salt tolerance in rice, whereas the knockout had the opposite effect
[161, 162]
OsNAC2 Overexpression reduced tolerance to drought and salinity; promoted leaf senescence via ABA biosynthesis in rice
[163, 164]
OsNAC3 Overexpression improved tolerance to drought and heat through ROS scavenging [165]
OsNAC5 Overexpression improved tolerance drought, salt, and cold stress in rice and root diameter
[166]
OsNAC10 Overexpression increased tolerance to drought and grain yield in rice [167]
OsNAC6/SNAC2 Overexpression improved salt and cold tolerance and reduced growth in rice [30, 168]
OsNAC9/SNAC1 Overexpression enhanced tolerance to drought and salt stress in rice, wheat, and cotton
[169-171]
TaRNAC1 (Wheat) Overexpression enhanced tolerance to drought in wheat [172]
TaNAC47 Overexpression enhanced tolerance to drought, salt, and freezing stresses and ABA hypersensitivity in Arabidopsis
[173]
TaNAC29 Overexpression enhanced drought and salt tolerance in Arabidopsis [174, 175]
TaNAC2L Overexpression enhanced heat tolerance in Arabidopsis [176]
TaNAC2 Overexpression enhanced tolerance to drought, salt, and freezing stresses in Arabidopsis; enhanced drought tolerance in tobacco and wild wheat;
[177-179]
TaNAC69 Overexpression enhanced tolerance to drought and water-use efficiency in wheat [179, 180]
ZmNAC55 (Maize) Overexpression enhanced dehydration tolerance in Arabidopsis [181]
ZmNAC111 Overexpression enhanced tolerance to drought and water-use efficiency in maize and Arabidopsis
[182]
ZmSNAC1 Overexpression enhanced drought tolerance in Arabidopsis [183]
PgNAC21 (Millet) Overexpression enhanced salt stress tolerance in Arabidopsis [184]
EcNAC67 (Millet) Overexpression enhanced tolerance to dehydration and salt stress in rice [185]
HvSNAC1 (Barley) Overexpression enhanced dehydration tolerance in barley [186]
HvNAC005 (Barley) Overexpression resulted in stunted growth and early senescence in barley [187]
GmNAC109 (Soybean) Overexpression improved drought and salt tolerance in Arabidopsis [116, 188]
GmNAC019 Overexpression improved drought tolerance in Arabidopsis [189]
GmSNAC49 Overexpression improved drought tolerance in Arabidopsis [190]
GmNAC085 Overexpression improved drought tolerance in Arabidopsis [124, 191]
GmSNAC1 Overexpression improved drought tolerance in Arabidopsis [190]
GmNTL1 Overexpression improved drought tolerance in Arabidopsis [192]
GmNAC20, GmNAC11 Overexpression of GmNAC20 improved salt and freezing tolerance, and GmNAC11 improved salt tolerance in Arabidopsis
[166]
GmNAC2 Overexpression increased hypersensitivity towards drought, salt, and cold in tobacco
[33]
GmNAC1, GmNAC5, GmNAC6
Transient expression in enhanced senescence in tobacco [193]
AhNAC2 (Peanut) Overexpression enhanced salt and drought resistance in Arabidopsis [194]
AhNAC3 Overexpression enhanced drought tolerance in tobacco [195]
AhNAC4 Overexpression enhanced drought tolerance in tobacco [196]
MuNAC4 (Horse gram) Overexpression enhanced drought tolerance in peanut [197]
CarNAC6 (Chickpea) Overexpression enhanced drought tolerance in Arabidopsis [198]
CarNAC4 Overexpression enhanced drought and salt tolerance in Arabidopsis [199]
CarNAC3 Overexpression enhanced drought and salt tolerance in poplar [200]
SlNAC11 (Tomato) Silencing reduced drought and salt tolerance in tomato [201]
SlNAM1 Overexpression enhanced chilling stress in tobacco [202]
SlJUB1 Silencing reduced drought tolerance in tomato [203]
2.1.3.1 NAC-mediated stress-response in the developmental model: Arabidopsis
Arabidopsis, a dicotyledonous species from the mustard family, serves as a developmental biology model. Most of the knowledge of NAC TFs has been stemmed from reverse genetics and genome-wide expression study in this model. In this species, the ATAF group is discovered as a bona fide regulator of various biotic and abiotic stress responses, consisting of four genes, ATAF1, ATAF2, ANAC032, and ANAC102. Numerous gene expression surveys have implicated ATAF-like members as wide-range stress regulators. The early studies reported early and local induction of ATAF1/2 (Arabidopsis transcription activation Factor 2) in wound response [204]. Later, it was found that ATAF2/ANAC081 is involved in auxin biosynthesis through NIT2 (nitrlase2) expression [205]. It also regulates photo-morphogenesis and brassinosteroids (BR) deficiency-mediated dwarfism by suppressing BR catabolic genes like BAS1 (CYP734A1) and SOB7 (CYP72C1) that control hypocotyl elongation and root growth via transcriptional feed-back regulation and DNA-protein and protein-protein interaction with CCA1 (circadian clock associated 1) [29, 206]. In addition, ATAF2 regulates age-dependent and dark-induced leaf senescence by expressing ANAC092/ORE1 [137]. Similarly, ATAF1/ANAC002 is also a negative regulator of various pathogenic responses, but it plays a positive role in abiotic stress tolerance through the ABA-dependent pathway. ATAF1 overexpression led to dwarf and short primary root phenotypes, increasing sensitivity to ABA, salt, and oxidative stress but enhanced tolerance towards drought response correlated with enhanced expression of stress-responsive marker genes such as ADH1(alcohol dehydrogenase 1), RD22/RD29A (responsive to desiccation 22/29A), COR47 (cold regulated 47), and genes involved in ABA biosynthesis and transport, such as NCED3 (nine-cis-epoxycarotenoid dioxygenase 3), and ABCG40 (ABC transporter G family member 40) [27, 56]. ATAF1 positively regulates chloroplast maintenance and senescence cascade by expressing GLK1 (golden2-like 1) and ORE1 (ORESARA1/ANAC092) [57]. At the protein level, ATAF1 interacts
with AKIN10 and AKIN11, catalytic subunits of SnRK1 protein (SNF1-related protein kinase 1), a key integrator in stress and glucose signal transduction, to coordinate metabolic, hormonal, and developmental signaling pathways [58]. Contrary to the behavior in Arabidopsis, ATAF1 overexpression in rice increased salt tolerance and ABA insensitivity [28].
Whereas, ANAC102 regulates stage-specific hypoxia stress by enhancing the viability of seed germination under low-oxygen [128]. ANAC032 has been characterized as a positive regulator of age-dependent and stress-induced leaf senescence by promoting the production of H2O2
under high light, sucrose, auxin and salt stress, and expression of AtNYE1 (non-yellowing 1), SAG113, and SAUR36 (small auxin-up RNA genes/SAG201), involved in chlorophyll degradation, auxin and ABA-mediated onset of senescence [138]. Moreover, ANAC032 represses the production of anthocyanin pigment under various stress by downregulating DFR (dihydroflavonol 4-reductase), ANS (anthocyanidin synthase)/LDOX, TT8 (transparent testa 8), AtMYBL2, and SPL9 (squamosa promoter-binding-like protein 9) [139]. ANAC032 inhibits ROS-mediated root cell elongation through MYB30 regulation [207]. ATAF1 and ANAC032 are induced in carbon starvation [57, 58]. The transcriptome and physiological analysis revealed that ANAC032 inhibits photosynthetic genes, induces ROS accumulation and carbon starvation by directing expression of TRE1 (trehalase1) that triggers sugar and amino acid catabolism to maintain energy supply [208].
Three closely related genes, ANAC019, ANAC055/AtNAC3, and ANAC072/RD26, that bind to ERD1 gene promoter, respond to diverse stress and hormone stimulus such as dehydration, high salinity, freezing, wounding, ABA, JA, etc. through an overlapping network.
Overexpression of these homologous genes significantly improved drought tolerance, possibly by regulating the expression of glyoxalase I involved in glutathione-based detoxification but played an antagonist role in ABA signaling and ionic osmotic stress [140]. Y1H study identified potential overlapping upstream regulators, such as ABA-responsive genes (ABF3, ABF4, and ABI4) and a cluster of MYB TFs (MYB2, MYB21, MYB108, MYB112, and MYB116), implicated in stress responses and ABA/JA signaling, interacting with the promoters of ANAC019, ANAC055, and ANAC072, whereas CBF1-4 (C-repeat binding factor 1-4) binds ANAC072 promoter only [111]. Finally, it was found that MYC2 and ANAC019 interact physically and synergistically to regulate the NYE1 involved in degreening through chlorophyll catabolism [209]. In addition, the distinct regulators justify the differential role of the gene set, i.e., the involvement of ANAC072 in cold, desiccation, and ANAC019 and ANAC055 in JA and/or ethylene mediated pathogen response.
ANAC092/AtNAC2, preferentially expressed in roots and flowers, regulates salt stress tolerance through modulation of lateral root architecture, serving as a downstream target of auxin and ethylene signaling [148]. When expressed in peanut (Arachis hypogaea), AtNAC2 showed enhanced tolerance to drought and salinity with improved yield [149]. In contrast, ANAC029/AtNAP, a senescence regulator induced by NaCl, mannitol, and ABA treatments, negatively regulates salt-response by repressing genes such as AREB1(ABA-responsive element binding protein 1), RD20, and RD29B [147]. In addition, ANAC016, a senescence regulator, negatively regulates drought tolerance by repressing AREB1 [144]. In addition, ANAC096 cooperates with the bZIP type TFs encoded by ABF2 and ABF4 synergistically to activate RD29A and many other ABA-responsive genes. Mutation of ANAC096 resulted in ABA hyposensitivity displaying impaired ABA-induced stomatal closure and increased transpiration and sensitivity to dehydration and osmotic [142]. In contrast to senescence, ANAC042/JUB1 regulates longevity, also imparting thermo-tolerance by tuning DREB2A, HFSFA2, and Glutathione S-transferases, resulting in lower H2O2 level and elevated proline and trehalose content [154].
2.1.3.2 NAC-mediated stress-response in cereal model: Rice
Fang et al., 2008 reported that 20 out of 140 ONAC genes were significantly induced in drought and/or salt, with five genes induced by drought only, 19 genes by salt only, and 16 genes by cold [210]. The SNAC (stress-responsive NAC) group of rice is well documented for its involvement in stress regulation. Overexpression of NAM-like SNAC1/OsNAC1 significantly enhanced drought tolerance and higher seed setting (22–34%) under severe dehydration imposed at the reproductive stage and improved salt tolerance at the vegetative stage [169]. Also, the gene ameliorated drought and salt tolerance and elevated ABA sensitivity in wheat by tuning stress-related genes such as FAB1B (1-phosphatidylinositol-3-phosphate- 5-kinase), SPS (sucrose phosphate synthase), type 2C protein phosphatases, and regulatory components of ABA receptors, resulting in higher leaf-water and chlorophyll content [170].
In addition, SNAC1 expression improved tolerance to drought and high salinity in cotton through vigorous root development and reduced transpiration rate [171]. Whereas, OsNAC2, also belonging to the NAM subfamily, negatively regulated drought and tolerance, probably by downregulating stress marker genes, such as OsLEA3 (late embryogenesis abundant 3) and OsSAPK1 (stress-activated protein kinases 1) [163].
Two rice genes, OsNAC6/SNAC2 and OsNAC5, belonging to the ATAF family, are induced by cold, salt, drought, wounding, ABA and JA, integrating signals from abiotic and biotic stresses in rice. The transgenic plants overexpressing OsNAC6 exhibited tolerance to drought, high salinity, cold, and blast disease. OsNAC6 overexpression improved plant vigor during freezing conditions and enhanced germination and growth rate under salt stress. Although the constitutive expression of OsNAC6 retarded growth and caused yield penalty, which was compensated by using stress-inducible LIP9 and OsNAC6 promoters were used [32, 168].
However, OsNAC5 expression not only improved drought tolerance but also improved and root diameter and grain yield under both drought (22-63%) and normal conditions (9-23%), when expressed in roots, regulating genes implicated in root growth and development such as GLP (germin-like protein 3-5), PDX1-like protein 4, MERI5 (meristem protein 5) and OMT (O- methyl transferase) [30, 31]. Microarray analysis of the transgenic plants showed upregulation of genes such as PRX46 (peroxidase 46), OsOAT (ornithine aminotransferase), HMA (heavy metal-associated protein), NHE3 (sodium/hydrogen exchanger 3), HSP (heat shock protein), GDSL-like lipase, and OsPAL (phenylalanine ammonia lyase) [168]. Like OsNAC5, OsNAC10 and OsNAC9 also increased drought endurance through improved root architecture. Root specific expression of OsNAC10 increased tolerance and grain yield under drought (by 22- 42%) and normal conditions (4-14%), while the constitutive expression gave a similar yield to that of control [167]. Similarly, root-specific expression of OsNAC9 (similar to SNAC1) increased yield under drought (28-72%) and normal conditions (13-18%), and the constitutive expression increased 13-12% yield under normal conditions [211].
Furthermore, ONAC022 is a positive regulator of drought and salt tolerance that control transcriptional water loss, Na+ accumulation, and contents of proline and soluble sugars through increased ABA biosynthesis and signaling mediated by target genes such as OsNCED, OsPSY, OsPP2C02, OsPP2C49, OsPP2C68, OsbZIP23, OsAP37, OsDREB2a, OsMYB2, OsRAB21, OsLEA3 and OsP5CS1 [158]. Recently, ONAC66 was reported to be induced by PEG, NaCl, H2O2, and ABA treatment to improve drought and oxidative stress tolerance by increasing ABA sensitivity and decreasing ROS accumulation [126]. Another SNAC gene, SNAC3/ONAC003, induced by multiple abiotic stress and ABA treatment, enhanced tolerance to high temperature, drought, and oxidative stress when overexpressed in rice by modulating ROS homeostasis through increased expression of ROS scavengers like OsCATA, OsAPX8, and OsRbohF. The overexpressor and silenced lines exhibited no change in ABA signaling genes, indicating that SNAC3 exerts its function through an ABA-independent mechanism,
unlike most NAC TFs [165]. A rice gene OsNAC14, predominantly expressed at the meiosis stage, resulted in the vegetative stage drought endurance, higher panicle count, and filling rate, by interacting with OsRAD51A1, a vital component of the DNA repair system, when overexpressed [155]. OsNAP, a senescence-associated gene, conferred ABA-dependent tolerance to high salinity, drought, and cold during the vegetative stage and improved yield under drought stress at the flowering stage [212]. ONAC106, a negative regulator of senescence, positively regulated salt-stress response by tuning salt-signaling through OsBREB2A, OsLEA3, and OsbZIP23 [159].
2.1.3.3 NAC-mediated responses in other cereals and legumes
In wheat (Triticum aestivum), a staple cereal food, several NAC TFs have been identified to regulate disparate stress responses. The expression of two NAC genes, TaNAC2 and TaNAC47, resulted in enhanced tolerance to drought, salt, and freezing in Arabidopsis by inducing DREB2A, RD22, RD29A/B, ABI1/2/5, and Rab18 [173, 178]. Another gene, TaNAC2L, similar to TaNAC2, activated the heat tolerance in Arabidopsis [176]. TaNAC29, predominantly expressed in senesced leaves, boosted drought and salt tolerance, accompanied with ABA hypersensitivity, delayed bolting, and flowering in transgenic Arabidopsis [174].
Overexpression of TaNAC69 improved drought adaptation and biomass in wheat [180].
HvSNAC1, a close homolog of TaNAC2, enhanced drought tolerance and photosynthetic activity in barley (Hordeum vulgare) [186]. SbSNAC1 of sorghum (Sorghum bicolor), induced by dehydration, salinity, and ABA, improved drought tolerance in Arabidopsis [213]. In maize (Zea mays), two genes ZmSNAC1 and ZmNAC55 conferred dehydration tolerance when expressed in Arabidopsis, but increased hypersensitivity to ABA and osmotic stress at the germination stage [181, 183]. ZmNAC111 overexpression enhanced drought endurance in maize seedlings [182]. EcNAC67 from Finger millet (Eleusine coracana) improved drought and salt endurance in rice by retaining grain yield and biomass under exhibiting better post- stress recovery [185]. Another millet gene, PgNAC21 from Pearl millet (Pennisetum glaucum), conferred salt tolerance in transgenic Arabidopsis by expressing COR47, RD20, and GSTF6 (Glutathione S-transferase F6) [184].
In soybean (Glycine max), a vital legume, 31 GmNAC genes were cloned and analyzed for response in drought, salinity, cold, and ABA, indicating their diverse role, as per early reports [214]. GmNAC2, an ATAF-like gene, is a negative regulator of major abiotic stresses in tobacco, repressing the ROS scavenging genes [33]. Two genes GmNAC11 and GmNAC20,
differentially regulate stress response. GmNAC11 overexpression imparts salt tolerance, whereas GmNAC20 confers salt and freezing tolerance in transgenic soybean through DREB/CBF-COR pathway [166]. As per recent reports, ectopic expression of GmNAC109 augmented drought tolerance in Arabidopsis, probably through stronger superoxide mutase and catalase activities and increased ABA sensitivity, displaying a 20-54% greater recovery rate [188]. Similarly, GmNAC085 positively regulates drought tolerance by elevating GSH/GSSG (reduced/oxidized glutathione) ratio in transgenic Arabidopsis via glutathione-dependent detoxification of ROS and methylglyoxal, however, caused growth retardation [191]. Also, GmSNAC49 expression increased drought tolerance in Arabidopsis [190].
Three drought-responsive genes have been isolated from peanut (Arachis hypogaea) and characterized in model plants. An ATAF-like gene, AhNAC4, isolated from immature peanut seeds, conferred drought tolerance in tobacco by regulating stomatal closure and improving water-use-efficiency [196]. Another gene, AhNAC3, showed hyper-resistance to dehydration in tobacco by accumulating osmoprotectants and ROS scavengers due to induction of mainly four genes, i.e., SOD, LEA, ERD10C, and P5SC [195]. AhNAC2, when expressed in Arabidopsis, resulted in ABA hypersensitivity to attenuate root growth, seed germination, and stomatal closure, implying a role in positive ABA signaling [194]. Moreover, the expression of horse gram MuNAC4, improved long-term desiccation tolerance in peanut, accompanied by increased lateral root and improved osmotic adjustment and antioxidant activity [197].
CarNAC3, an AtNAP like gene from chickpea (Cicer arietinum), conferred drought and salt tolerance in poplar, but reduced plant height [200]. Two other genes, CarNAC4 and CarNAC6, improved drought and salt endurance along with root growth in Arabidopsis [198, 199].
2.1.4 Potential utilization of NAC genes to improve seed quality, pod-yield, and growth