Roles of NAC transcription factors in cotton

Climate deterioration,water shortages,and abiotic stress are the main threats worldwide that seriously affect cotton growth,yield,and fiber quality.Therefore,research on improving cotton yield and tolerance to biotic and abiotic stresses is of great importance.The NAC proteins are crucial and plant-specific transcription factors(TFs)that are involved in cotton growth,development,and stress responses.The comprehensive utilization of cotton NAC TFs in the improvement of cotton varieties through novel biotechnological methods is feasible.Based on cotton genomic data,genome-wide identification and analyses have revealed potential functions of cotton NAC genes.Here,we comprehensively summarize the recent progress in understanding cotton NAC TFs roles in regulating responses to drought,salt,and Verticillium wilt-related stresses,as well as leaf senescence and the development of fibers,xylem,and glands.The detailed regulatory network of NAC proteins in cotton is also elucidated.Cotton NAC TFs directly bind to the promoters of genes associated with ABA biosynthesis and secondary cell-wall formation,participate in several biological processes by interacting with related proteins,and regulate the expression of downstream genes.Studies have shown that the overexpression of NAC TF genes in cotton and other model plants improve their drought or salt tolerance.This review elucidates the latest findings on the functions and regulation of cotton NAC proteins,broadens our understanding of cotton NAC TFs,and lays a fundamental foundation for further molecular breeding research in cotton.

Preliminary analysis of two NAC transcription factor expression patterns in Larix olgensis

The NAC transcription factor family is plant-specific with various biological functions.However,there are few studies on the NAC gene involving coniferous species.Bioinformatics research and expression analysis of NAC genes in Larix olgensis can be used to analyse the function of the NAC gene in the future.Screening of excellent genetic materials and molecular breeding have been utilized to cultivate high-quality,stress-resistant larches.According to the transcriptome data for L.olgensis,the genes Uni-gene81490 and Unigene70699 with complete ORFs(open reading frames)were obtained by conserved domain analy-sis and named LoNAC1 and LoNAC2,respectively.The cDNAs of LoNAC1 and LoNAC2 were 1971 bp and 1095 bp in length,encoding 656 and 364 amino acids,respectively.The molecular weights of the proteins encoded by the two genes were predicted to be 72.61 kDa and 41.13 kDa,and subcellular localization analysis indicated that the proteins were concentrated in the nucleus.The results of real-time quantitative PCR analysis showed that at different growth stages and in different tissues of L.olgensis,the relative expression levels of the two NAC genes were highest in the stem,and the expression differences were more obvious in non-lignified tissues.After drought,salt and alkali stress and hormone treatment,expression was induced to different degrees.The expression levels of LoNAC1 and LoNAC2 in semi-lignified L.olgensis were higher than in the other two periods(non-lignified and lignified),and expression levels significantly increased under drought and salt stress.Relative expression levels changed under hormone treatment.It is speculated that these two genes may not only be related to drought and salt stress and secondary growth but may also be induced by hormones such as abscisic acid.Overall,LoNAC1 and LoNAC2 are genetic materials that can be used for molecular breeding of larch.

A NAC transcription factor MNAC3-centered regulatory network negatively modulates rice immunity against blast disease

NAC transcription factors(TFs)are pivotal in plant immunity against diverse pathogens.Here,we report the functional and regulatory network of MNAC3,a novel NAC TF,in rice immunity.MNAC3,a transcriptional activator,negatively modulates rice immunity against blast and bacterial leaf blight diseases and pathogen-associated molecular pattern(PAMP)-triggered immune responses.MNAC3 binds to a CACG cis-element and activates the transcription of immune-negative target genes OsINO80,OsJAZ10,and OsJAZ11.The negative function of MNAC3 in rice immunity depends on its transcription of downstream genes such as OsINO80 and OsJAZ10.MNAC3 interacts with immunity-related OsPP2C41(a protein phosphatase),ONAC066(a NAC TF),and OsDjA6(a DnaJ chaperone).ONAC066 and OsPP2C41 attenuate MNAC3 transcriptional activity,while OsDjA6 promotes it.Phosphorylation of MNAC3 at S163 is critical for its negative functions in rice immunity.OsPP2C41,which plays positive roles in rice blast resistance and chitin-triggered immune responses,dephosphorylates MNAC3,suppressing its transcriptional activity on the target genes OsINO80,OsJAZ10,and OsJAZ11 and promoting the translocation of MNAC3 from nucleus to cytoplasm.These results establish a MNAC3-centered regulatory network in which OsPP2C41 dephosphorylates MNAC3,attenuating its transcriptional activity on downstream immune-negative target genes in rice.Together,these findings deepen our understanding of molecular mechanisms in rice immunity and offer a novel strategy for genetic improvement of rice disease resistance.

Structural basis of DNA binding by the NAC transcription factor ORE1,a master regulator of plant senescence

Plants use sophisticated mechanisms of gene expression to control senescence in response to environmental stress or aging.ORE1(Arabidopsis thaliana NAC092)is a master regulator of senescence that belongs to the plant-specific NAC transcription factor protein family.ORE1 has been reported to bind to multiple DNA targets to orchestrate leaf senescence,yet the mechanistic basis for recognition of the cognate gene sequence remains unclear.Here,we report the crystal structure of the ORE1-NAC domain alone and its DNA-binding form.The structure of DNA-bound ORE1-NAC revealed the molecular basis for nucleobase recognition and phosphate backbone interactions.We showthat local versatility in the DNA-binding site,in combination with domain flexibility of the ORE-NAC homodimer,is crucial for the maintenance of binding to intrinsically flexible DNA.Our results provide a platformfor understanding other plant-specific NAC protein-DNA interactions as well as insight into the structural basis of NAC regulators in plants of agronomic and scientific importance.

ORS1, an H2O2-Responsive NAC Transcription Factor, Controls Senescence in Arabidopsis thaliana

We report here that ORS1, a previously uncharacterized member of the NAC transcription factor family, controls leaf senescence in Arabidopsis thaliana. Overexpression of ORS1 accelerates senescence in transgenic plants, whereas its inhibition delays it. Genes acting downstream of ORS1 were identified by global expression analysis using transgenic plants producing dexamethasone-inducible ORSl-GR fusion protein. Of the 42 up-regulated genes, 30 （-70%） were pre- viously shown to be up-regulated during age-dependent senescence, We also observed that 32 （-76%） of the ORSl-dependent genes were induced by long-term （4 d）, but not short-term （6 h） salinity stress （150 mM NaCI）. Furthermore, expression of 16 and 24 genes, respectively, was induced after 1 and 5 h of treatment with hydrogen peroxide （H2O2）, a reactive oxygen species known to accumulate during salinity stress. ORS1 itself was found to be rapidly and strongly induced by H2O2 treatment in both leaves and roots. Using in vitro binding site selection, we determined the preferred binding motif of ORS1 and found it to be present in half of the ORSl-dependent genes. ORS1 is a paralog of ORE1/ ANACO92/AtNAC2, a previously reported regulator of leaf senescence. Phylogenetic footprinting revealed evolutionary conservation of the ORS1 and ORE1 promoter sequences in different Brassicaceae species, indicating strong positive selection acting on both genes. We conclude that ORS1, similarly to ORE1, triggers expression of senescence-associated genes through a regulatory network that may involve cross-talk with saltand H2O2-dependent signaling pathways.

Bioinformatics and Expression Analysis of NAC Transcription Factor Genes in Scutellaria baicalensis

Background:NAC,as a unique transcription factor to plants,plays important roles in multiple biological functions,such as regulation of plant growth and development,hormone levels,and responses to various kinds of stresses.However,there is a lack of research of NAC genes in Chinese herbs.Objective:The study aimed to evaluate the potential functions of NAC genes in Scutellaria baicalensis by bioinformatics and expression analysis,and provide evidence of the molecular regulation mechanism involved in flavonoid biosynthesis in S.baicalensis.Methods:The genes of NAC transcription factors in S.baicalensis were obtained from cDNA library and their functions were explored using bioinformatic methods.The NAC genes were screened from the cDNA library of S.baicalensis using BLAST comparison software.Then,the open reading frame(ORF) finder online tool was used to predict the full-length ORFs of NAC genes and their protein characteristics were explored by bioinformatic methods.The expression of NAC genes was then detected by quantitative polymerase chain reaction in different parts of S.baicalensis and different leaves treated by gibberellin GA3 treatment.Results:Six genes of NAC transcription factors were cloned,two of which had complete ORFs.NAC genes cloned in this study were mainly expressed in the flowers of S.baicalensis.The expression levels of NAC2,NAC3,NAC4,NAC5,NAC6 were increased firstly and then decreased gradually after 100 μM GA3 treatment.Meanwhile,some NACs and PAL2 in S.baicalensis showed strong correlation.Conclusion:This study suggested that NACs cloned in this study were mainly regulated the flavonoid biosynthesis in the flowers of S.baicalensis;NAC6 in S.baicalensis might be involved in the regulation of PAL2 transcription and affected the accumulation of flavonoids in the root of S.baicalensis.Our results provided a basis for further understanding the molecular regulation mechanism of flavonoid biosynthesis in S.baicalensis.

The NAC056 transcription factor confers freezing tolerance by positively regulating expression of CBFs and NIA1 in Arabidopsis

Freezing stress can seriously affect plant growth and development,but the mechanisms of these effects and plant responses to freezing stress require further exploration.Here,we identified a NAM,ATAF1/2,and CUC2(NAC)-family transcription factor(TF),NAC056,that can promote freezing tolerance in Arabidopsis.NAC056 mRNA levels are strongly induced by freezing stress in roots,and the nac056 mutant exhibits compromised freezing tolerance.NAC056 acts positively in response to freezing by directly promoting key C-repeat-binding factor(CBF)pathway genes.Interestingly,we found that CBF1 regulates nitrate assimilation by regulating the nitrate reductase gene NIA1 in plants;therefore,NAC056–CBF1–NIA1 form a regulatory module for the assimilation of nitrate and the growth of roots under freezing stress.In addition,35S::NAC056 transgenic plants show enhanced freezing tolerance,which is partially reversed in the cbfs triple mutant.Thus,NAC056 confers freezing tolerance through the CBF pathway,mediating plant responses to balance growth and freezing stress tolerance.

Cowpea NAC1/NAC2 transcription factors improve growth and tolerance to drought and heat in transgenic cowpea through combined activation of photosynthetic and antioxidant mechanisms

NAC(NAM/ATAF1/2/CUC2)transcription factors are central switches of growth and stress responses in plants.However,unpredictable interspecies conservation of function and regulatory targets makes the well-studied NAC orthologs inapt for pulse engineering.The knowledge of suitable NAC candidates in hardy pulses like cowpea(Vigna unguiculata(L.)Walp.)is still in infancy,hence warrants immediate biotechnological intervention.Here,we showed that overexpression of two native NAC genes(VuNAC1and VuNAC2)promoted germinative,vegetative,and reproductive growth and conferred multiple abiotic stress tolerance in a commercial cowpea variety.The transgenic lines displayed increased leaf area,thicker stem,nodule-rich denser root system,early flowering,higher pod production(~3.2-fold and~2.1-fold),and greater seed weight(10.3%and 6.0%).In contrast,transient suppression of VuNAC1/2 caused severe growth retardation and flower inhibition.The overexpressor lines showed remarkable tolerance to major yielddeclining terminal stresses,such as drought,salinity,heat,and cold,and recovered growth and seed production by boosting photosynthetic activity,water use efficiency,membrane integrity,Na^(+)/K^(+)homeostasis,and antioxidant activity.The comparative transcriptome study indicated consolidated activation of genes involved in chloroplast development,photosynthetic complexes,cell division and expansion,cell wall biogenesis,nutrient uptake and metabolism,stress response,abscisic acid,and auxin signaling.Unlike their orthologs,VuNAC1/2 direct synergistic transcriptional tuning of stress and developmental signaling to avoid unwanted trade-offs.Their overexpression governs the favorable interplay of photosynthesis and reactive oxygen species regulation to improve stress recovery,nutritional sufficiency,biomass,and production.This unconventional balance of strong stress tolerance and agronomic quality is useful for translational crop research and molecular breeding of pulses.

ANAC087 transcription factor positively regulates age-dependent leaf senescence through modulating the expression of multiple target genes in Arabidopsis

Leaf senescence is the final stage of leaf development and appropriate onset and progression of leaf senescence are critical for reproductive success and fitness.Although great progress has been made in identifying key genes regulating leaf senescence and elucidating the underlining mechanisms in the model plant Arabidopsis,there is still a gap to understanding the complex regulatory network.In this study,we discovered that Arabidopsis ANAC087 transcription factor(TF)positively modulated leaf senescence.Expression of ANAC087 was induced in senescing leaves and the encoded protein acted as a transcriptional activator.Both constitutive and inducible overexpression lines of ANAC087 showed earlier senescence than control plants,whereas T-DNA insertion mutation and dominant repression of the ANAC087 delayed senescence rate.A quantitative reverse transcription-polymerase chain reaction(qRT-PCR)profiling showed that the expression of an array of senescence-associated genes was upregulated in inducible ANAC087 overexpression plants including BFN1,NYE1,CEP1,RbohD,SAG13,SAG15,and VPEs,which are involved in programmed cell death(PCD),chlorophyll degradation and reactive oxygen species(ROS)accumulation.In addition,electrophoretic mobility shift assay(EMSA)and chromatin immunoprecipitation-quantitative polymerase chain reaction(ChIP-qPCR)assays demonstrated that ANAC087 directly bound to the canonical NAC recognition sequence(NACRS)motif in promoters of its target genes.Moreover,mutation of two representative target genes,BFN1 or NYE1 alleviated the senescence rate of ANAC087-overexpression plants,suggesting their genetic regulatory relationship.Taken together,this study indicates that ANAC087serves as an important regulator linking PCD,ROS,and chlorophyll degradation to leaf senescence.

TaNAC48 positively regulates drought tolerance and ABA responses in wheat(Triticum aestivum L.)

NAC family transcription factors(TFs) are important regulators in plant development and stress responses. However, the biological functions of NAC TFs in wheat are rarely studied. In this study, 43 putative drought-induced NAC genes were identified from de novo transcriptome sequencing data of wheat following drought treatment. Twelve wheat NACs along with ten known stress-related NACs from Arabidopsis and rice were clustered into Group II based on a phylogenetic analysis. Ta NAC48, which showed a higher and constitutive expression level in Group Ⅱ, was selected for further investigation.Ta NAC48 transcript was up-regulated by drought, PEG, H_(2)O_(2) and abscisic acid(ABA) treatment and encoded a nuclear localized protein. Overexpression of Ta NAC48 significantly promoted drought tolerance with increased proline content, and decreased rates of water loss, malondialdehyde(MDA), H_(2)O_(2) and O_(2)^(-) content. Root length and a stomatal aperture assay confirmed that Ta NAC48-overexpression plants increased sensitivity to ABA. Electrophoretic mobility shift assay(EMSA) and luciferase reporter analysis indicated that Ta AREB3 could bind to a cis-acting ABA-responsive element(ABRE) on Ta NAC48 promoter and activate the expression of Ta NAC48. These results suggest that Ta NAC48 is essential in mediating crosstalk between the ABA signaling pathway and drought stress responses in wheat.

Genome-wide identification of NAC gene family and expression analysis under abiotic stresses in Salvia miltiorrhiza

NAC(NAM,ATAF,CUC)is a class of transcription factors involved in plant growth regulation,abiotic stress responses,morphogenesis and metabolism.Salvia miltiorrhiza is an important Chinese medicinal herb,but the characterization of NAC genes in this species is limited.In this study,based on the Salvia miltiorrhiza genomic databases,82 NAC transcription factors were identified,which were divided into 14 groups.Meanwhile,phylogenetic analysis,gene structure,chromosomal localization and potential role of SmNACs in abiotic stress conditions were also studied.The results revealed that some SmNACs had different structures than others,which advised that these genes may have multiple/distinct functions.Real-time quantitative polymerase chain reaction(RT-qPCR)analysis showed that SmNACs exhibited differential expression patterns under salt and drought stress.The NaCl induced salinity treatments modulated the expression of several SmNAC genes more in roots compared with leaves.Conversely,under drought stress conditions,more genes were upregulated in leaves compared with roots.These results will be useful for the further study involved in the functional characteristics of SmNAC genes,especially in response to salt and drought stresses,thereby may facilitate genetic breeding in Salvia miltiorrhiza.

Transcriptome Profiling of Abscisic Acid-Related Pathways in SNAC4/9-Silenced Tomato Fruits

The NAC(NAM,ATAF,and CUC)family is considered one of the largest families of plant transcription factor,and its members are involved in fruit ripening.Abscisic acid(ABA)has been demonstrated to modulate the fruit ripening process.By applying the virus-induced gene silencing method and next-generation sequencing technology,we conducted a compara-tive analysis of the eff ects of SNAC4(SlNAC48,accession number:NM 001279348.2)and SNAC9(SlNAC19,accession number:XM 004236996.2)on tomato fruit ripening.The results of high-throughput sequencing identified 1262 significant(p<0.05)diff erentially expressed genes(DEGs)in SNAC4-silenced fruit compared to control fruit,while 655 DEGs were identified in SNAC9-silenced fruit.In addition,we selected 26 and 30 significant DEGs(p<0.05 and log 2-fold change>1.0)related to ABA in SNAC4-silenced and SNAC9-silenced tomatoes,respectively,for further analysis.The XET gene and two other genes(E8 and EXP1)were significantly down and upregulated in SNAC4-silenced tomatoes,respectively.However,the PYL9 gene and four other genes(PP2C,CYP707A2,EXPA6,and ACS6)were significantly down and upregulated in SNAC9-silenced tomatoes,respectively.In addition,ten DEGs were selected for use in tests to confirm the accuracy of the transcriptomic results by quantitative real-time polymerase chain reaction(qRT-PCR).Our results highlight the relationship between SNAC4/9 and ABA in the regulation of tomato ripening,which may help provide a theoretical basis for further research on the mechanisms of tomato fruit ripening and senescence.

Wheat leaf senescence and its regulatory gene network

Wheat leaf senescence is a developmental process that involves expressional changes in thousands of genes that ultimately impact grain protein content(GPC), grain yield(GY), and nitrogen use efficiency.The onset and rate of senescence are strongly influenced by plant hormones and environmental factors e.g. nitrogen availability. At maturity, decrease in nitrogen uptake could enhance N remobilization from leaves and stem to grain, eventually leading to leaf senescence. Early senescence is related to high GPC and somewhat low yield whereas late senescence is often related to high yield and somewhat low GPC. Early or late senescence is principally regulated by up and down-regulation of senescence associated genes. Integration of external and internal factors together with genotypic variation influence senescence associated genes in a developmental age dependent manner. Although regulation of genes involved in senescence has been studied in rice, Arabidopsis, maize, and currently in wheat, there are genotypespecific variations yet to explore. A major effort is needed to understand the interaction of positive and negative senescence regulators in determining the onset of senescence. In wheat, increasing attention has been paid to understand the role of positive senescence regulator, e.g. GPC-1, regulated gene network during early senescence time course. Recently, gene regulatory network involved early to late senescence time course revealed important senescence regulators. However, the known negative senescence regulator Ta NAC-S gene has not been extensively studied in wheat and little is known about its value in breeding. Existing data on senescence-related transcriptome studies and gene regulatory network could effectively be used for functional study in developing nitrogen efficient wheat varieties.

Overexpression of TaNAC69 Leads to Enhanced Transcript Levels of Stress Up-Regulated Genes and Dehydration Tolerance in Bread Wheat

NAC proteins are plant-specific transcription factors and enriched with members involved in plant response to drought stress. In this study, we analyzed the expression profiles of TaNAC69 in bread wheat using Affymetrix Wheat Genome Array datasets and quantitative RT-PCR. TaNAC69 expression was positively associated with wheat responses to both abiotic and biotic stresses and was closely correlated with a number of stress up-regulated genes. The functional analyses of TaNAC69 in transgenic wheat showed that TaNAC69 driven by a barley drought-inducible HvDhn4s promoter led to marked drought-inducible overexpression of TaNAC69 in the leaves and roots of transgenic lines. The HvDhn4s：Ta- NAC69 transgenic lines produced more shoot biomass under combined mild salt stress and water-limitation conditions, had longer root and more root biomass under polyethylene glycol-induced dehydration. Analysis of transgenic lines with constitutive overexpression of TaNAC69 showed the enhanced expression levels of several stress up-regulated genes. DNA-binding assays revealed that TaNAC69 and its rice homolog （ONAC131） were capable of binding to the promoter elements of three rice genes （chitinase, ZlM, and glyoxalase I） and an Arabidopsis glyoxalase I family gene, which are homologs of TaNAC69 up-regulated stress genes. These data suggest that TaNAC69 is involved in regulating stress upregulated genes and wheat adaptation to drought stress.

Global Analysis of Direct Targets of Secondary Wall NAC Master Switches in Arabidopsis

We report the genome-wide analysis of direct target genes of SND1 and VND7, two Arabidopsis thaliana NAC domain transcription factors that are master regulators of secondary wall biosynthesis in fibers and vessels, respectively. Systematic mapping of the SND1 binding sequence using electrophoretic mobility shift assay and transactivation analysis demonstrated that SND1 together with other secondary wall NACs （SWNs）, including VND6, VND7, NST1, and NST2, bind to an imperfect palindromic 19-bp consensus sequence designated as secondary wall NAC binding element （SNBE）, （T/A）NN（C/T） （TICIG）TNNNNNNNA（AIC）GN（AJCIT） （A/T）, in the promoters of their direct targets. Genome-wide analysis of direct targets of SND1 and VND7 revealed that they directly activate the expression of not only downstream transcription factors, but also a number of non-transcription factor genes involved in secondary wall biosynthesis, cell wall modification, and programmed cell death, the promoters of which all contain multiple SNBE sites. SND1 and VND7 directly regulate the expression of a set of common targets but each of them also preferentially induces a distinct set of direct targets, which is likely attributed to their differential activation strength toward SNBE sites. Complementation study showed that the SWNs were able to rescue the secondary wall defect in the sndl nstl mutant, indicating that they are functionally interchangeable. Together, our results provide important insight into the complex transcriptional program and the evolutionary mechanism underlying secondary wall biosynthesis, cell wall modification, and programmed cell death in secondary wall-containing cell types.

The OsNAC23-Tre6P-SnRK1 a feed-forward loop regulates sugar homeostasis and grain yield in rice

Tre6P(trehalose-6-phosphate)mediates sensing of carbon availability to maintain sugar homeostasis in plants,which underpins crop yield and resilience.However,how Tre6P responds to fluctuations in sugar levels and regulates the utilization of sugars for growth remains to be addressed.Here,we report that the sugar-inducible rice NAC transcription factor OsNAC23 directly represses the transcription of the Tre6P phosphatase gene TPP1 to simultaneously elevate Tre6P and repress trehalose levels,thus facilitating carbon partitioning from source to sink organs.Meanwhile,OsNAC23 and Tre6P suppress the transcription and enzyme activity of SnRK1a,a low-carbon sensor and antagonist of OsNAC23,to prevent the SnRK1a-mediated phosphorylation and degradation of OsNAC23.Thus,OsNAC23,Tre6P,and SnRK1a form a feed-forward loop to sense sugar and maintain sugar homeostasis by transporting sugars to sink organs.Importantly,plants over-expressing OsNAC23 exhibited an elevated photosynthetic rate,sugar transport,and sink organ size,which consistently increased rice yields by 13%–17%in three elite-variety backgrounds and two locations,suggesting that manipulation of OsNAC23 expression has great potential for rice improvement.Collectively,these findings enhance our understanding of Tre6P-mediated sugar signaling and homeostasis,and provide a new strategy for genetic improvement of rice and possibly also other crops.