ArNAC148 induces Acer rubrum leaf senescence by activating the transcription of the ABA receptor gene ArPYR13.

Acer rubrum, an ornamental tree known for its stunning autumn colors, has an elusive molecular mechanism that governs its leaf senescence. We performed the genome-wide analysis of NAC transcription factor genes and PYRABACTIN RESISTANCE1-LIKE (PYLs) and found that ArNAC148 and ArPYL13 were significantly upregulated in senescing leaves as compared to mature leaves. Subcellular localization studies confirmed the nuclear localization of ArNAC148 and the cytoplasmic localization of ArPYL13. Electrophoretic mobility shift assay and yeast one-hybrid assay demonstrated that ArNAC148 directly binds to the promoter of ArPYL13. Luciferase reporter assays further showed that ArNAC148 activates the transcription of ArPYL13. The transient expression of ArNAC148 and ArPYL13 in tobacco leaves promoted chlorophyll degradation, increased H2O2 level, MDA contents, and electrolyte leakage in response to abscisic acid (ABA). Moreover, the virus-induced gene silencing of ArNAC148 and ArPYL13 in A. rubrum produced results that were opposite to those observed in transient expression experiments. Our findings suggest that ArNAC148 induces leaf senescence by directly activating the transcription of ArPYL13, providing insights into the ABA-mediated regulatory mechanisms governing leaf senescence in A. rubrum. This study offers new perspectives for researchers to explore the roles of NAC and PYL genes in regulating leaf senescence in woody ornamental plants.

Overexpression of RPOTmp Being Targeted to Either Mitochondria or Chloroplasts in Arabidopsis Leads to Overall Transcriptome Changes and Faster Growth.

The transcription of Arabidopsis organellar genes is performed by three nuclear-encoded RNA polymerases: RPOTm, RPOTmp, and RPOTp. The RPOTmp protein possesses ambiguous transit peptides, allowing participation in gene expression control in both mitochondria and chloroplasts, although its function in plastids is still under discussion. Here, we show that the overexpression of RPOTmp in Arabidopsis, targeted either to mitochondria or chloroplasts, disturbs the dormant seed state, and it causes the following effects: earlier germination, decreased ABA sensitivity, faster seedling growth, and earlier flowering. The germination of RPOTmp overexpressors is less sensitive to NaCl, while rpotmp knockout is highly vulnerable to salt stress. We found that mitochondrial dysfunction in the rpotmp mutant induces an unknown retrograde response pathway that bypasses AOX and ANAC017. Here, we show that RPOTmp transcribes the accD, clpP, and rpoB genes in plastids and up to 22 genes in mitochondria.

Eggplant NAC domain transcription factor SmNST1 as an activator promotes secondary cell wall thickening.

NAC-domain transcription factors (TFs) are plant-specific transcriptional regulators playing crucial roles in plant secondary cell wall (SCW) biosynthesis. SCW is important for plant growth and development, maintaining plant morphology, providing rigid support, ensuring material transportation and participating in plant stress responses as a protective barrier. However, the molecular mechanisms underlying SCW in eggplant have not been thoroughly explored. In this study, the NAC domain TFs SmNST1 and SmNST2 were cloned from the eggplant line 'Sanyue qie'. SmNST1 and SmNST2 expression levels were the highest in the roots and stems. Subcellular localization analysis showed that they were localized in the cell membrane and nucleus. Their overexpression in transgenic tobacco showed that SmNST1 promotes SCW thickening. The expression of a set of SCW biosynthetic genes for cellulose, xylan and lignin, which regulate SCW formation, was increased in transgenic tobacco. Bimolecular fluorescence and luciferase complementation assays showed that SmNST1 interacted with SmNST2 in vivo. Yeast one-hybrid, electrophoretic mobility shift assay (EMSA) and Dual-luciferase reporter assays showed that SmMYB26 directly bound to the SmNST1 promoter and acted as an activator. SmNST1 and SmNST2 interact with the SmMYB108 promoter and repress SmMYB108 expression. Altogether, we showed that SmNST1 positively regulates SCW formation, improving our understanding of SCW biosynthesis transcriptional regulation.

Common Bean (Phaseolus vulgaris L.) NAC Transcriptional Factor PvNAC52 Enhances Transgenic Arabidopsis Resistance to Salt, Alkali, Osmotic, and ABA Stress by Upregulating Stress-Responsive Genes.

The NAC family of transcription factors includes no apical meristem (NAM), Arabidopsis thaliana transcription activator 1/2 (ATAF1/2), and cup-shaped cotyledon (CUC2) proteins, which are unique to plants, contributing significantly to their adaptation to environmental challenges. In the present study, we observed that the PvNAC52 protein is predominantly expressed in the cell membrane, cytoplasm, and nucleus. Overexpression of PvNAC52 in Arabidopsis strengthened plant resilience to salt, alkali, osmotic, and ABA stresses. PvNAC52 significantly (p < 0.05) reduced the degree of oxidative damage to cell membranes, proline content, and plant water loss by increasing the expression of MSD1, FSD1, CSD1, POD, PRX69, CAT, and P5CS2. Moreover, the expression of genes associated with abiotic stress responses, such as SOS1, P5S1, RD29A, NCED3, ABIs, LEAs, and DREBs, was enhanced by PvNAC52 overexpression. A yeast one-hybrid assay showed that PvNAC52 specifically binds to the cis-acting elements ABRE (abscisic acid-responsive elements, ACGTG) within the promoter. This further suggests that PvNAC52 is responsible for the transcriptional modulation of abiotic stress response genes by identifying the core sequence, ACGTG. These findings provide a theoretical foundation for the further analysis of the targeted cis-acting elements and genes downstream of PvNAC52 in the common bean.

Identification of Eleutherococcus senticosus NAC transcription factors and their mechanisms in mediating DNA methylation of EsFPS, EsSS, and EsSE promoters to regulate saponin synthesis.

BACKGROUND: The formation of pharmacologically active components in medicinal plants is significantly impacted by DNA methylation. However, the exact mechanisms through which DNA methylation regulates secondary metabolism remain incompletely understood. Research in model species has demonstrated that DNA methylation at the transcription factor binding site within functional gene promoters can impact the binding of transcription factors to target DNA, subsequently influencing gene expression. These findings suggest that the interaction between transcription factors and target DNA could be a significant mechanism through which DNA methylation regulates secondary metabolism in medicinal plants. RESULTS: This research conducted a comprehensive analysis of the NAC family in E. senticosus, encompassing genome-wide characterization and functional analysis. A total of 117 EsNAC genes were identified and phylogenetically divided into 15 subfamilies. Tandem duplications and chromosome segment duplications were found to be the primary replication modes of these genes. Motif 2 was identified as the core conserved motif of the genes, and the cis-acting elements, gene structures, and expression patterns of each EsNAC gene were different. EsJUB1, EsNAC047, EsNAC098, and EsNAC005 were significantly associated with the DNA methylation ratio in E. senticosus. These four genes were located in the nucleus or cytoplasm and exhibited transcriptional self-activation activity. DNA methylation in EsFPS, EsSS, and EsSE promoters significantly reduced their activity. The methyl groups added to cytosine directly hindered the binding of the promoters to EsJUB1, EsNAC047, EsNAC098, and EsNAC005 and altered the expression of EsFPS, EsSS, and EsSE genes, eventually leading to changes in saponin synthesis in E. senticosus. CONCLUSIONS: NAC transcription factors that are hindered from binding by methylated DNA are found in E. senticosus. The incapacity of these NACs to bind to the promoter of the methylated saponin synthase gene leads to subsequent alterations in gene expression and saponin synthesis. This research is the initial evidence showcasing the involvement of EsNAC in governing the impact of DNA methylation on saponin production in E. senticosus.

MpNAC1, a transcription factor from the mangrove associate Millettia pinnata, confers salt and drought stress tolerance in transgenic Arabidopsis and rice.

Pongamia (Millettia pinnata Syn. Pongamia pinnata), a mangrove associate plant, exhibits good stress tolerance, making it a treasure of genetic resources for crop improvement. NAC proteins are plant-specific transcription factors, which have been elucidated to participate in the regulation and tolerance of abiotic stresses (such as salt and drought). Here, we identified a salt-induced gene from Pongamia, MpNAC1, which encodes an NAC factor sharing five highly conserved domains with other NACs and exhibits close homology to AtNAC19/AtNAC55/AtNAC72 in Arabidopsis. MpNAC1 showed nuclear localization and transcriptional activator activity. MpNAC1-overexpressing Arabidopsis exhibited significantly stronger salt and drought tolerance compared with wild-type plants. The expression levels of stress-responsive genes were activated in transgenic Arabidopsis. Furthermore, the heterologous expression of MpNAC1 also enhanced the salt and drought tolerance of transgenic rice. The major agronomic traits, such as plant height and tiller number, panicle length, grain size, and yield, were similar between the transgenic lines and wild type under normal field growth conditions. RNA-Seq analysis revealed that MpNAC1 significantly up-regulated stress-responsive genes and activated the biosynthesis of secondary metabolites such as flavonoids, resulting in increased stress tolerance. Taken together, the MpNAC1 increased salt and drought stress tolerance in transgenic plants and did not retard the plant growth and development under normal growth conditions, suggesting the potential of MpNAC1 in breeding stress-resilient crops.

LpNAC5 positively regulates drought, salt and alkalinity tolerance of Lilium pumilum.

NACs (NAM、ATAF1/2、CUC1/2), as a large family of plant transcription factors, are widely involved in abiotic stress responses. This study aimed to isolate and clone a novel stress-responsive transcription factor LpNAC5 from Lilium pumilum bulbs. Drought, salt, alkali, and ABA stresses induced the expression of LpNAC5. Transgenic tobacco plants overexpressing LpNAC5 were generated using the Agrobacterium-mediated method to understand the role of this factor in stress response. These plants exhibited increased tolerance to drought, salt, and alkali stresses. The tobacco plants overexpressing LpNAC5 showed strong drought, salt, and alkaline tolerance. Under the three abiotic stresses, the activities of antioxidant enzymes were enhanced, the contents of proline and chlorophyll increased, and the contents of malondialdehyde decreased. The functional analysis revealed that LpNAC5 enabled plants to positively regulate drought and salt stresses. These findings not only provided valuable insights into stress tolerance mechanisms in L. pumilum but also offered a potential genetic resource for breedi.

Expression of the Arabidopsis redox-related LEA protein, SAG21 is regulated by ERF, NAC and WRKY transcription factors.

SAG21/LEA5 is an unusual late embryogenesis abundant protein in Arabidopsis thaliana, that is primarily mitochondrially located and may be important in regulating translation in both chloroplasts and mitochondria. SAG21 expression is regulated by a plethora of abiotic and biotic stresses and plant growth regulators indicating a complex regulatory network. To identify key transcription factors regulating SAG21 expression, yeast-1-hybrid screens were used to identify transcription factors that bind the 1685 bp upstream of the SAG21 translational start site. Thirty-three transcription factors from nine different families bound to the SAG21 promoter, including members of the ERF, WRKY and NAC families. Key binding sites for both NAC and WRKY transcription factors were tested through site directed mutagenesis indicating the presence of cryptic binding sites for both these transcription factor families. Co-expression in protoplasts confirmed the activation of SAG21 by WRKY63/ABO3, and SAG21 upregulation elicited by oligogalacturonide elicitors was partially dependent on WRKY63, indicating its role in SAG21 pathogen responses. SAG21 upregulation by ethylene was abolished in the erf1 mutant, while wound-induced SAG21 expression was abolished in anac71 mutants, indicating SAG21 expression can be regulated by several distinct transcription factors depending on the stress condition.

Transcription factors NtNAC028 and NtNAC080 form heterodimers to regulate jasmonic acid biosynthesis during leaf senescence in Nicotiana tabacum.

Plant senescence, as a highly integrated developmental stage, involves functional degeneration and nutrient redistribution. NAM/ATAF1/CUC (NAC) transcription factors orchestrate various senescence-related signals and mediate the fine-tuning underlying plant senescence. Previous data revealed that knockout of either NtNAC028 or NtNAC080 leads to delayed leaf senescence in tobacco (Nicotiana tabacum), which implies that NtNAC028 and NtNAC080 play respective roles in the regulation of leaf senescence, although they share 91.87% identity with each other. However, the mechanism underlying NtNAC028- and NtNAC080-regulated leaf senescence remains obscure. Here, we determined that NtNAC028 and NtNAC080 activate a putative jasmonic acid (JA) biosynthetic gene, NtLOX3, and enhance the JA level in vivo. We found that NtNAC028 and NtNAC080 interact with each other and themselves through their NA-terminal region. Remarkably, only the dimerization between NtNAC028 and NtNAC080 stimulated the transcriptional activation activity, but not the DNA binding activity of this heterodimer on NtLOX3. Metabolome analysis indicated that overexpression of either NtNAC028 or NtNAC080 augments both biosynthesis and degradation of nicotine in the senescent stages. Thus, we conclude that NtNAC028 cooperates with NtNAC080 and forms a heterodimer to enhance NtLOX3 expression and JA biosynthesis to trigger the onset of leaf senescence and impact secondary metabolism in tobacco.

Molecular mechanisms of SNAC1 (Stress-responsive NAC1) in conferring the abiotic stress tolerance.

NAC family gene - SNAC1 (Stress-responsive NAC1) is responsive to drought, salt, cold stress, and ABA. It acts as a regulator in mediating tolerance to abiotic stress through different pathways. Abiotic stress, among them drought and salinity, are adverse factors for plant growth and crop productivity. SNAC1 was an object of high interest according to the effect of improved drought and salt tolerance when overexpressed in different plant species such as rice, wheat, barley, cotton, maize, banana, or oat. SNAC1 functions by regulating the expression of genes that contain the NAC Recognized Sequence (NACRS) within their promoter region. This gene is induced by drought, specifically in guard cells. Its downstream targets have been identified. The role of SNAC1 in molecular and physiological responses during abiotic stress has been proposed, but this knowledge still needs to be expanded. Here, we describe recent advances in understanding the action of SNAC1 in adapting plants to abiotic stress.

Endosperm cell death promoted by NAC transcription factors facilitates embryo invasion in Arabidopsis.

In flowering plants, two fertilization products develop within the limited space of the seed: the embryo and the surrounding nutritive endosperm. The final size of the endosperm is modulated by the degree of embryo growth. In Arabidopsis thaliana, the endosperm expands rapidly after fertilization, but later gets invaded by the embryo that occupies most of the seed volume at maturity, surrounded by a single remaining aleurone-like endosperm layer.1,2,3,4 Embryo invasion is facilitated by the endosperm-expressed bHLH-type transcription factor ZHOUPI, which promotes weakening of endosperm cell walls.5,6 Endosperm elimination in zou mutants is delayed, and embryo growth is severely affected; the endosperm finally collapses around the dwarf embryo, causing the shriveled appearance of mature zou seeds.5,6,7 However, whether ZHOUPI facilitates mechanical endosperm destruction by the invading embryo or whether an active programmed cell death (PCD) process causes endosperm elimination has been subject to debate.2,8 Here we show that developmental PCD controlled by multiple NAC transcription factors in the embryo-adjacent endosperm promotes gradual endosperm elimination. Misexpressing the NAC transcription factor KIRA1 in the entire endosperm caused total endosperm elimination, generating aleurone-less mature seeds. Conversely, dominant and recessive higher-order NAC mutants led to delayed endosperm elimination and impaired cell corpse clearance. Promoting PCD in the zhoupi mutant partially rescued its embryo growth defects, while the endosperm in a zhoupi nac higher-order mutant persisted until seed desiccation. These data suggest that a combination of cell wall weakening and PCD jointly facilitates embryo invasion by an active auto-elimination of endosperm cells.

NACs, generalist in plant life.

Plant-specific NAC proteins constitute a major transcription factor family that is well-known for its roles in plant growth, development, and responses to abiotic and biotic stresses. In recent years, there has been significant progress in understanding the functions of NAC proteins. NAC proteins have a highly conserved DNA-binding domain; however, their functions are diverse. Previous understanding of the structure of NAC transcription factors can be used as the basis for their functional diversity. NAC transcription factors consist of a target-binding domain at the N-terminus and a highly versatile C-terminal domain that interacts with other proteins. A growing body of research on NAC transcription factors helps us comprehend the intricate signalling network and transcriptional reprogramming facilitated by NAC-mediated complexes. However, most studies of NAC proteins have been limited to a single function. Here, we discuss the upstream regulators, regulatory components and targets of NAC in the context of their prospective roles in plant improvement strategies via biotechnology intervention, highlighting the importance of the NAC transcription factor family in plants and the need for further research.

A NAC transcription factor ZaNAC93 confers floral initiation, fruit development, and prickle formation in Zanthoxylum armatum.

Zanthoxylum armatum is a dioecious prickly plant which developed apomictic reproduction. The increases in male flowers and prickle density in female plants lead to low yield and picking efficiency. However, little is known concerning the mechanisms of floral development and prickle formation. NAC is a well-known transcription factor that participates in multiple aspects of plant growth and development. Herein, we characterize the functions and regulatory mechanisms of candidate NACs controlling both traits in Z. armatum. A total of 159 ZaNACs were identified, and 16 of these were male-biased, represented by the NAP subfamily members ZaNAC93 and ZaNAC34, orthologs of AtNAC025 and AtNARS1/NAC2 respectively. Overexpression of ZaNAC93 in tomato led to modifications in flower and fruit development, including earlier flowering, increased numbers of lateral shoots and flowers, accelerated plant senescence, and reduced size and weight of fruits and seeds. In addition, the trichome density in leaves and inflorescences was dramatically reduced in ZaNAC93-OX lines. Overexpression of ZaNAC93 resulted in the up-/downregulation of genes associated with GA, ABA and JA signaling pathways, such as GAI, PYL and JAZ, as well as several TFs, including bZIP2, AGL11, FBP24 and MYB52. Yeast two-hybrid analysis revealed that ZaNAC93 protein could interact with AP1, GAI, bZIP2 and AGL11 in Z. armatum, which might contribute to floral induction, fruit growth, and trichome initiation. This work provides new insights into the molecular mechanisms of ZaNAC93 in reproductive development and prickle formation in Z. armatum.

A systematic genome-wide analysis and screening of the NAC family genes related to wood formation in Cinnamomum camphora.

Many processes, such as growth, aging, and adaptation to abiotic stress, are regulated in plants by NAC transcription factors. In woody plants, NAC transcription factors acts as a primary switch that regulates secondary xylem development by activating various downstream transcription factors and modulating expression levels of genes involved in the synthesis of the secondary cell wall. Our team had previously sequenced the whole genome of the camphor tree (Cinnamomum camphora). Here, we performed a detailed analysis of the NAC gene family of C. camphora and examined its evolutionary history. The genomic sequences of 121 NAC genes of C. camphora were identified and classified into 20 subfamilies in 2 major classes based on the phylogenetic analysis and structural features. Expansion of the CcNAC gene family occurred mainly by fragment replication and was influenced by the purifying selection. By analyzing predicted interactions of the homologous AtNAC proteins, we identified five CcNACs that potentially regulate xylem development in C. camphora. RNA sequencing revealed distinct expression profiles of CcNACs in seven different plant tissues. Subcellular localization prediction revealed that 120, 3, and 2 CcNACs have biological functions in the nucleus, cytoplasm, and chloroplast, respectively. Furthermore, we verified expression patterns of five CcNACs (CcNAC012, CcNAC028, CcNAC055, CcNAC080, and CcNAC119) in various tissue types using qRT-PCR. Our results will facilitate further in-depth studies of the molecular mechanisms by which CcNAC transcription factors regulate wood formation and other processes in C. camphora.

In silico analysis of NAC gene family in the mangrove plant Avicennia marina provides clues for adaptation to intertidal habitats.

NAC (NAM, ATAF1/2, CUC2) transcription factors (TFs) constitute a plant-specific gene family. It is reported that NAC TFs play important roles in plant growth and developmental processes and in response to biotic/abiotic stresses. Nevertheless, little information is known about the functional and evolutionary characteristics of NAC TFs in mangrove plants, a group of species adapting coastal intertidal habitats. Thus, we conducted a comprehensive investigation for NAC TFs in Avicennia marina, one pioneer species of mangrove plants. We totally identified 142 NAC TFs from the genome of A. marina. Combined with NAC proteins having been functionally characterized in other organisms, we built a phylogenetic tree to infer the function of NAC TFs in A. marina. Gene structure and motif sequence analyses suggest the sequence conservation and transcription regulatory regions-mediated functional diversity. Whole-genome duplication serves as the driver force to the evolution of NAC gene family. Moreover, two pairs of NAC genes were identified as positively selected genes of which AmNAC010/040 may be imposed on less constraint toward neofunctionalization. Quite a few stress/hormone-related responsive elements were found in promoter regions indicating potential response to various external factors. Transcriptome data revealed some NAC TFs were involved in pneumatophore and leaf salt gland development and response to salt, flooding and Cd stresses. Gene co-expression analysis found a few NAC TFs participates in the special biological processes concerned with adaptation to intertidal environment. In summary, this study provides detailed functional and evolutionary information about NAC gene family in mangrove plant A. marina and new perspective for adaptation to intertidal habitats.

Genome-wide identification and expression analysis of NAC family genes in Ginkgo biloba L.

NAC (NAM, ATAF, CUC2) transcription factors constitute one of the largest families of plant-specific transcription factors with important roles in plant growth and development and in biotic and abiotic stresses. The physicochemical properties, gene structure, cis-acting elements and expression patterns of NAC transcription factors in Ginkgo biloba were analysed using bioinformatics, and expression of this gene family was analysed via quantitative reverse transcription PCR. The family of G. biloba NAC transcription factors had 50 members, distributed on 12 chromosomes and divided into 11 groups. Members in the same group share a similar gene structure and motif distribution. Transcriptome data analysis of G. biloba showed that 35 genes were expressed in eight tissues. Correlation analysis suggested that GbNAC007 and GNAC008 might be involved in flavonoid biosynthesis. Expression levels of 12 GbNACs under cold, het, and salt stresses were analysed. Results indicate that NAC transcription factors play an important role in response to abiotic stresses. This study provides a reference for the functional analysis of the G. biloba family of NAC transcription factors, as well as a resource for studies on the involvement of this family in responses to abiotic stresses and flavonoid biosynthesis.

Transcriptome-wide identification of NAC (no apical meristem/Arabidopsis transcription activation factor/cup-shaped cotyledon) transcription factors potentially involved in salt stress response in garlic.

Soil salinity has been an increasing problem worldwide endangering crop production and human food security. It is an ideal strategy to excavate stress resistant genes and develop salt tolerant crops. NAC (no apical meristem/Arabidopsis transcription activation factor/cup-shaped cotyledon) transcription factors have been demonstrated to be involved in salt stress response. However, relevant studies have not been observed in garlic, an important vegetable consumed in the world. In this study, a total of 46 AsNAC genes encoding NAC proteins were identified in garlic plant by transcriptome data. Phylogenetic analysis showed that the examined AsNAC proteins were clustered into 14 subgroups. Motif discovery revealed that the conserved domain region was mainly composed of five conserved subdomains. Most of the genes selected could be induced by salt stress in different tissues, indicating a potential role in salt stress response. Further studies may focus on the molecular mechanisms of the AsNAC genes in salt stress response. The results of the current work provided valuable resources for researchers aimed at developing salt tolerant crops.

Orchid NAC Transcription Factors: A Focused Analysis of CUPULIFORMIS Genes.

Plant transcription factors are involved in different developmental pathways. NAC transcription factors (No Apical Meristem, Arabidopsis thaliana Activating Factor, Cup-shaped Cotyledon) act in various processes, e.g., plant organ formation, response to stress, and defense mechanisms. In Antirrhinum majus, the NAC transcription factor CUPULIFORMIS (CUP) plays a role in determining organ boundaries and lip formation, and the CUP homologs of Arabidopsis and Petunia are involved in flower organ formation. Orchidaceae is one of the most species-rich families of angiosperms, known for its extraordinary diversification of flower morphology. We conducted a transcriptome and genome-wide analysis of orchid NACs, focusing on the No Apical Meristem (NAM) subfamily and CUP genes. To check whether the CUP homologs could be involved in the perianth formation of orchids, we performed an expression analysis on the flower organs of the orchid Phalaenopsis aphrodite at different developmental stages. The expression patterns of the CUP genes of P. aphrodite suggest their possible role in flower development and symmetry establishment. In addition, as observed in other species, the orchid CUP1 and CUP2 genes seem to be regulated by the microRNA, miR164. Our results represent a preliminary study of NAC transcription factors in orchids to understand the role of these genes during orchid flower formation.

Genome-Wide Identification of the NAC Transcription Factors in Gossypium hirsutum and Analysis of Their Responses to Verticillium wilt.

The NAC transcription factors (NACs) are among the largest plant-specific gene regulators and play essential roles in the transcriptional regulation of both biotic and abiotic stress responses. Verticillium wilt of cotton caused by Verticillium dahliae (V. dahliae) is a destructive soil-borne disease that severely decreases cotton yield and quality. Although NACs constitute a large family in upland cotton (G. hirsutum L.), there is little systematic investigation of the NACs' responsive to V. dahliae that has been reported. To further explore the key NACs in response to V. dahliae resistance and obtain a better comprehension of the molecular basis of the V. dahliae stress response in cotton, a genome-wide survey was performed in this study. To investigate the roles of GhNACs under V. dahliae induction in upland cotton, mRNA libraries were constructed from mocked and infected roots of upland cotton cultivars with the V. dahliae-sensitive cultivar "Jimian 11" (J11) and V. dahliae-tolerant cultivar "Zhongzhimian 2" (Z2). A total of 271 GhNACs were identified. Genome analysis showed GhNACs phylogenetically classified into 12 subfamilies and distributed across 26 chromosomes and 20 scaffolds. A comparative transcriptome analysis revealed 54 GhNACs were differentially expressed under V. dahliae stress, suggesting a potential role of these GhNACs in disease response. Additionally, one NAC090 homolog, GhNAC204, could be a positive regulator of cotton resistance to V. dahliae infection. These results give insight into the GhNAC gene family, identify GhNACs' responsiveness to V. dahliae infection, and provide potential molecular targets for future studies for improving V. dahliae resistance in cotton.