NAM, ATAF, And CUC Research Articles

Phosphorylation of ZmNAC84 at Ser-113 enhances the drought tolerance by directly modulating ZmSOD2 expression in maize

NAC (NAM, ATAF1/2, and CUC2) transcription factors play vital roles in response to multiple abiotic stresses. Our previous study has demonstrated that ZmNAC84, a maize NAC transcription factor, enhanced the drought tolerance by increasing abscisic acid (ABA)-induced antioxidant enzyme activities of APX and SOD, and Ser-113, a key phosphorylation site, of ZmNAC84 played an important role in this process. However, the target gene of ZmNAC84 in this process is still unknown. Here, we found that ZmNAC84 only regulated the luciferase activity driven by ZmSOD2 promoter in tobacco. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assay showed that ZmNAC84 directly bound to the CACGTG motif of ZmSOD2 promoter. Furthermore, phosphorylation of ZmNAC84 at Ser-113 up-regulated the ZmSOD2 expression by enhancing the DNA binding ability of ZmNAC84 to ZmSOD2 promoter and improved the drought tolerance. Taken together, our results demonstrate that ZmNAC84 directly regulates ZmSOD2 expression to enhance drought tolerance and Ser-113 of ZmNAC84 is crucial in this process.

Identification and analysis of Chrysanthemum nankingense NAC transcription factors and an expression analysis of OsNAC7 subfamily members.

NAC (NAM, ATAF1-2, and CUC2) transcription factors (TFs) play a vital role in plant growth and development, as well as in plant response to biotic and abiotic stressors (Duan et al., 2019; Guerin et al., 2019). Chrysanthemum is a plant with strong stress resistance and adaptability; therefore, a systematic study of NAC TFs in chrysanthemum is of great significance for plant breeding. In this study, 153 putative NAC TFs were identified based on the Chrysanthemum nankingense genome. According to the NAC family in Arabidopsis and rice, a rootless phylogenetic tree was constructed, in which the 153 CnNAC TFs were divided into two groups and 19 subfamilies. Moreover, the expression levels of 12 CnNAC TFs belonging to the OsNAC7 subfamily were analyzed in C. nankingense under osmotic and salt stresses, and different tissues were tested during different growth periods. The results showed that these 12 OsNAC7 subfamily members were involved in the regulation of root and stem growth, as well as in the regulation of drought and salt stresses. Finally, we investigated the function of the CHR00069684 gene, and the results showed that CHR00069684 could confer improved salt and low temperature resistance, enhance ABA sensitivity, and lead to early flowering in tobacco. It was proved that members of the OsNAC7 subfamily have dual functions including the regulation of resistance and the mediation of plant growth and development. This study provides comprehensive information on analyzing the function of CnNAC TFs, and also reveals the important role of OsNAC7 subfamily genes in response to abiotic stress and the regulation of plant growth. These results provide new ideas for plant breeding to control stress resistance and growth simultaneously.

Gene Expression Correlation Analysis Reveals MYC-NAC Regulatory Network in Cotton Pigment Gland Development.

Plant NAC (NAM, ATAF1/2, and CUC2) family is involved in various development processes including Programmed Cell Death (PCD) associated development. However, the relationship between NAC family and PCD-associated cotton pigment gland development is largely unknown. In this study, we identified 150, 153 and 299 NAC genes in newly updated genome sequences of G. arboreum, G. raimondii and G. hirsutum, respectively. All NAC genes were divided into 8 groups by the phylogenetic analysis and most of them were conserved during cotton evolution. Using the vital regulator of gland formation GhMYC2-like as bait, expression correlation analysis screened out 6 NAC genes which were low-expressed in glandless cotton and high-expressed in glanded cotton. These 6 NAC genes acted downstream of GhMYC2-like and were induced by MeJA. Silencing CGF1 (Cotton Gland Formation1), another MYC-coding gene, caused almost glandless phenotype and down-regulated expression of GhMYC2-like and the 6 NAC genes, indicating a MYC-NAC regulatory network in gland development. In addition, predicted regulatory mechanism showed that the 6 NAC genes were possibly regulated by light, various phytohormones and transcription factors as well as miRNAs. The interaction network and DNA binding sites of the 6 NAC transcription factors were also predicted. These results laid the foundation for further study of gland-related genes and gland development regulatory network.

Membrane-Bound Transcriptional Activator NTL1 from Rapeseed Positively Modulates Leaf Senescence through Targeting Genes Involved in Reactive Oxygen Species Production and Programmed Cell Death.

Leaf senescence is the last stage of leaf development and is determined by various environmental and endogenous signals. Leaf senescence can determine plant productivity and fitness. Transcription factors (TFs) with the transmembrane domain constitute a special group of regulatory proteins that can translocate from the membrane system into nuclei to exert the transcriptional function upon endogenous or exogenous stimuli. Reactive oxygen species (ROSs) play an important role in numerous processes throughout the life cycle of plants including leaf senescence. Leaf senescence is characterized by massive programmed cell death (PCD) and is a type of developmental PCD. The transcriptional regulatory relationships between membrane-bound TFs and leaf senescence remain largely uncharacterized, especially in rapeseed (Brassica napus L.), an important oil crop. Here, we show that BnaNTL1 is a membrane-bound NAC (NAM, ATAF, and CUC) TF, which is predominantly expressed in senescent leaves. Expression of BnaNTL1ΔTM, a form of BnaNTL1 devoid of the transmembrane domain, can induce serious HR-like cell death symptoms and ROS accumulation in cells. Plants overexpressing BnaNTL1ΔTM show earlier leaf senescence compared with the control, accompanied by chlorophyll degradation and electrolyte leakage. Genes involved in ROS production (RbohD), PCD (VPEs and CEP1), and leaf senescence (BFN1) are significantly induced and activated by BnaNTL1ΔTM according to the quantitative reverse transcription PCR (qRT-PCR) analysis and dual luciferase reporter (Dual-LUC) assay. Moreover, electrophoretic mobility shift assay revealed that BnaNTL1 directly bound to the NTLBS elements in promoters of RbohD, γVPE, and BFN1. In conclusion, these results demonstrate that BnaNTL1 positively modulates ROS production and HR-like cell death to induce leaf senescence.

ThNAC12 from Tamarix hispida directly regulates ThPIP2;5 to enhance salt tolerance by modulating reactive oxygen species

NAC (NAM, ATAF1/2 and CUC2) transcription factors play critical roles in plant development and abiotic stress responses, and aquaporins have diverse functions in environmental stress responses. In this study, we described the salt-induced transcriptional responses of ThNAC12 and ThPIP2;5 in Tamarix hispida, and their regulatory mechanisms in response to salt stress. Using yeast one-hybrid (Y1H), chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays, we identified that ThNAC12 directly binds to the NAC recognition sequence (NACRS) of the ThPIP2;5 promoter and then activates the ThPIP2;5 expression. Subcellular localization and transcriptional activation assays demonstrated that ThNAC12 was a nuclear protein with a C-terminal transactivation domain. Compared with the corresponding control plants, transgenic plants overexpressing ThNAC12 exhibited enhanced salt tolerance and displayed increased reactive oxygen species (ROS) scavenging capability and antioxidant enzyme activity levels under salt stress. All results suggested that overexpression of ThNAC12 in plants enhanced salt tolerance through modulation of ROS scavenging via direct regulation of ThPIP2;5 expression in T. hispida.

Genome-wide identification, characterization, and expression analysis of the NAC transcription factor family in orchardgrass (Dactylis glomerata L.)

BackgroundOrchardgrass (Dactylis glomerata L.) is one of the most important cool-season perennial forage grasses that is widely cultivated in the world and is highly tolerant to stressful conditions. However, little is known about the mechanisms underlying this tolerance. The NAC (NAM, ATAF1/2, and CUC2) transcription factor family is a large plant-specific gene family that actively participates in plant growth, development, and response to abiotic stress. At present, owing to the absence of genomic information, NAC genes have not been systematically studied in orchardgrass. The recent release of the complete genome sequence of orchardgrass provided a basic platform for the investigation of DgNAC proteins.ResultsUsing the recently released orchardgrass genome database, a total of 108 NAC (DgNAC) genes were identified in the orchardgrass genome database and named based on their chromosomal location. Phylogenetic analysis showed that the DgNAC proteins were distributed in 14 subgroups based on homology with NAC proteins in Arabidopsis, including the orchardgrass-specific subgroup Dg_NAC. Gene structure analysis suggested that the number of exons varied from 1 to 15, and multitudinous DgNAC genes contained three exons. Chromosomal mapping analysis found that the DgNAC genes were unevenly distributed on seven orchardgrass chromosomes. For the gene expression analysis, the expression levels of DgNAC genes in different tissues and floral bud developmental stages were quite different. Quantitative real-time PCR analysis showed distinct expression patterns of 12 DgNAC genes in response to different abiotic stresses. The results from the RNA-seq data revealed that orchardgrass-specific NAC exhibited expression preference or specificity in diverse abiotic stress responses, and the results indicated that these genes may play an important role in the adaptation of orchardgrass under different environments.ConclusionsIn the current study, a comprehensive and systematic genome-wide analysis of the NAC gene family in orchardgrass was first performed. A total of 108 NAC genes were identified in orchardgrass, and the expression of NAC genes during plant growth and floral bud development and response to various abiotic stresses were investigated. These results will be helpful for further functional characteristic descriptions of DgNAC genes and the improvement of orchardgrass in breeding programs.

Potato NAC Transcription Factor StNAC053 Enhances Salt and Drought Tolerance in Transgenic Arabidopsis.

The NAC (NAM, ATAF1/2, and CUC2) transcription factors comprise one of the largest transcription factor families in plants and play important roles in stress responses. However, little is known about the functions of potato NAC family members. Here we report the cloning of a potato NAC transcription factor gene StNAC053, which was significantly upregulated after salt, drought, and abscisic acid treatments. Furthermore, the StNAC053-GFP fusion protein was found to be located in the nucleus and had a C-terminal transactivation domain, implying that StNAC053 may function as a transcriptional activator in potato. Notably, Arabidopsis plants overexpressing StNAC053 displayed lower seed germination rates compared to wild-type under exogenous ABA treatment. In addition, the StNAC053 overexpression Arabidopsis lines displayed significantly increased tolerance to salt and drought stress treatments. Moreover, the StNAC053-OE lines were found to have higher activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) under multiple stress treatments. Interestingly, the expression levels of several stress-related genes including COR15A, DREB1A, ERD11, RAB18, ERF5, and KAT2, were significantly upregulated in these StNAC053-overexpressing lines. Taken together, overexpression of the stress-inducible StNAC053 gene could enhance the tolerances to both salt and drought stress treatments in Arabidopsis, likely by upregulating stress-related genes.

Wood transcriptome analysis of Pinus densiflora identifies genes critical for secondary cell wall formation and NAC transcription factors involved in tracheid formation.

Although conifers have significant ecological and economic value, information on transcriptional regulation of wood formation in conifers is still limited. Here, to gain insight into secondary cell wall (SCW) biosynthesis and tracheid formation in conifers, we performed wood tissue-specific transcriptome analyses of Pinus densiflora (Korean red pine) using RNA sequencing. In addition, to obtain full-length transcriptome information, PacBio single molecule real-time iso-sequencing was carried out using RNAs from 28 tissues of P. densiflora. Subsequent comparative tissue-specific transcriptome analysis successfully pinpointed critical genes encoding key proteins involved in biosynthesis of the major secondary wall components (cellulose, galactoglucomannan, xylan and lignin). Furthermore, we predicted a total of 62 NAC (NAM, ATAF1/2 and CUC2) family transcription factor members and identified seven PdeNAC genes preferentially expressed in developing xylem tissues in P. densiflora. Protoplast-based transcriptional activation analysis found that four PdeNAC genes, homologous to VND, NST and SND/ANAC075, upregulated GUS activity driven by an SCW-specific cellulose synthase promoter. Consistently, transient overexpression of the four PdeNACs induced xylem vessel cell-like SCW deposition in both tobacco (Nicotiana benthamiana) and Arabidopsis leaves. Taken together, our data provide a foundation for further research to unravel transcriptional regulation of wood formation in conifers, especially SCW formation and tracheid differentiation.

Mutagenesis of <i>SlNAC4</i> by CRISPR/Cas9 alters gene expression and softening of ripening tomato fruit

Softening is one of the key fruit quality traits, which results from the selective expression of cell wall metabolism genes during ripening. The identification of transcription factors (TFs) that regulate fruit softening is an important field in order to understand and control fruit softening. In tomato, NAC (NAM, ATAF, and CUC) TFs members have been demonstrated to be involved in fruit ripening regulation, including NAC-NOR (non-ripening), NOR-like1, SlNAC4, SlNAC1. Here, we generated slnac4 mutant knockout (CR-SlNAC4) tomato plant by a clustered regularly interspaced short palindromic repeats genomic targeting system (CRISPR/Cas9) and SlNAC4 overexpressing (OE-SlNAC4) plant. In addition to confirming the previously reported results that SlNAC4 positively regulates fruit ripening, we found that SlNAC4 has a strong effect on tomato fruit softening. Compared with the control fruit, fruit softening was inhibited in slnac4 fruit and conversely was accelerated in OE-SlNAC4 tomato fruit. Through RNA-sequencing (RNA-seq) analysis, we found that expression levels of SlEXP1 (expansin) and SlCEL2 (endo-β-1,4 glucanase) genes involved in cell wall metabolism were significantly different in WT (wild type)/slnac4 and WT/OE-SlNAC4 fruit. Further study showed that these genes contained a NAC TF binding domain in their promoter regions. In vitro electrophoretic mobility shift assays (EMSA) and dual-luciferase reporter assays (DLR) demonstrated that these two genes were the direct targets of SlNAC4 binding and transactivation. The results enriched the function of SlNAC4 and provided a new dimension in understanding the regulation of tomato fruit softening.

NAC Transcription Factors as Positive or Negative Regulators during Ongoing Battle between Pathogens and Our Food Crops.

The NAC (NAM, ATAF1/2, and CUC2) family of proteins is one of the largest plant-specific transcription factor (TF) families and its members play varied roles in plant growth, development, and stress responses. In recent years, NAC TFs have been demonstrated to participate in crop-pathogen interactions, as positive or negative regulators of the downstream defense-related genes. NAC TFs link signaling pathways between plant hormones, including salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and abscisic acid (ABA), or other signals, such as reactive oxygen species (ROS), to regulate the resistance against pathogens. Remarkably, NAC TFs can also contribute to hypersensitive response and stomatal immunity or can be hijacked as virulence targets of pathogen effectors. Here, we review recent progress in understanding the structure, biological functions and signaling networks of NAC TFs in response to pathogens in several main food crops, such as rice, wheat, barley, and tomato, and explore the directions needed to further elucidate the function and mechanisms of these key signaling molecules.

RNA-Seq Analysis of Prickled and Prickle-Free Epidermis Provides Insight into the Genetics of Prickle Development in Red Raspberry (Rubus ideaus L.)

Red raspberry (Rubus idaeus L.) is a globally commercialized specialty crop with growing demand worldwide. The presence of prickles on the stems, petioles and undersides of the leaves complicates both the field management and harvesting of raspberries. An RNA sequencing analysis was used to identify differentially expressed genes in the epidermal tissue of prickled “Caroline” and prickle-free “Joan J.” and their segregating progeny. Expression patterns of differentially expressed genes (DEGs) in prickle-free plants revealed the downregulation of some vital development-related transcription factors (TFs), including a MIXTA-like R2R3-MYB family member; MADS-box; APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) and NAM, ATAF1/2 and CUC2 (NAC) in prickle-free epidermis tissue. The downregulation of these TFs was confirmed by qRT-PCR analysis, indicating a key regulatory role in prickle development. This study adds to the understanding of prickle development mechanisms in red raspberries needed for utilizing genetic engineering strategies for developing prickle-free raspberry cultivars and, possibly, other Rubus species, such as blackberry (Rubus sp.) and black raspberry (R. occidentalis L.).

GmNAC06, a NAC domain transcription factor enhances salt stress tolerance in soybean

Key messageWe found GmNAC06 plays an important role in salt stress responses through the phenotypic, physiological and molecular analyses of OE, VC, and Mutant composite soybean.Salinization affects 20% of all cultivated land worldwide because of the high salinity of irrigation water and the excessive use of water, and this amount is increasing daily. NAC (NAM, ATAF, and CUC) have been found to be involved in salt stress. In this study, a soybean NAC gene, GmNAC06 (Glyma06g21020.1), was cloned and functionally characterized. The results of expression analysis suggested that salt stress could influence the expression level of GmNAC06. The subcellular localization analysis results suggested that GmNAC06 may function as a transcription factor. Under salt stress, the overexpression technology combined with CRISPR-Cas9 system found that GmNAC06 could cause the accumulation of proline and glycine betaine to alleviate or avoid the negative effects of ROS; similarly, it could control the Na+/K+ ratios in hairy roots to maintain ionic homeostasis. The fresh weight of the transgenic hairy roots and the histochemical ROS staining of wild leaves suggested that transgenic hairy roots influence the function of wild leaves under salt stress conditions. Moreover, the expression levels of GmUBC2 and GmHKT1 were higher in the GmNAC06 hairy roots than in the control. Thus, the overexpression of GmNAC06 in hairy roots notably causes an entire composite plant to exhibit salt tolerance. The phenotype of composite soybean plants and transgenic Arabidopsis plants suggest that GmNAC06 plays a role in response to salt stress and could be useful in generating salt tolerant transgenic crops.

Self-transcriptional repression of the Arabidopsis NAC transcription factor ATAF2 and its genetic interaction with phytochrome A in modulating seedling photomorphogenesis.

The NAC transcription factor ATAF2 suppresses its own transcription via self-promoter binding. ATAF2 genetically interacts with the circadian regulator CCA1 and phytochrome A to modulate seedling photomorphogenesis in Arabidopsis thaliana. ATAF2 (ANAC081) is a NAC (NAM, ATAF and CUC) transcription factor (TF) that participates in the regulation of disease resistance, stress tolerance and hormone metabolism in Arabidopsis thaliana. We previously reported that ATAF2 promotes Arabidopsis hypocotyl growth in a light-dependent manner via transcriptionally suppressing the brassinosteroid (BR)-inactivating cytochrome P450 genes BAS1 (CYP734A1, formerly CYP72B1) and SOB7 (CYP72C1). Assays using low light intensities suggest that the photoreceptor phytochrome A (PHYA) may play a more critical role in ATAF2-regulated photomorphogenesis than phytochrome B (PHYB) and cryptochrome 1 (CRY1). In addition, ATAF2 is also regulated by the circadian clock. The core circadian TF CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) physically interacts with ATAF2 at the DNA-protein and protein-protein levels, and both differentially suppress BAS1- and SOB7-mediated BR catabolism. In this research, we show that ATAF2 can bind its own promoter as a transcriptional self-repressor. This self-feedback-suppression loop is a typical feature of multiple circadian-regulated genes. Additionally, ATAF2 and CCA1 synergistically suppress seedling photomorphogenesis as reflected by the light-dependent hypocotyl growth analysis of their single and double gene knock-out mutants. Similar fluence-rate response assays using ATAF2 and photoreceptor (PHYB, CRY1 and PHYA) knock-out mutants demonstrate that PHYA is required for ATAF2-regulated photomorphogenesis in a wide range of light intensities. Furthermore, disruption of PHYA can suppress the BR-insensitive hypocotyl-growth phenotype of ATAF2 loss-of-function seedlings in the light, but not in darkness. Collectively, our results provide a genetic interaction synopsis of the circadian-clock-photomorphogenesis-BR integration node involving ATAF2, CCA1 and PHYA.

NST- and SND-subgroup NAC proteins coordinately act to regulate secondary cell wall formation in cotton

Secondary cell wall (SCW) has a strong impact on plant growth and adaptation to the environments. Previous studies have shown that NAC (NAM, ATAF1/2, and CUC2) transcription factors act as key regulators of SCW biosynthesis. However, the regulatory network triggered by NAC proteins is largely unknown, especially in cotton, a model plant for SCW development studies. Here, we show that several cotton NAC transcription factors are clustered in the same group with Arabidopsis secondary wall NACs (SWNs), including secondary wall-associated NAC domain protein1 (SND1) and NAC secondary wall thickening promoting factor1/2 (NST1/2), so we name these cotton orthologs as SND1s and NST1s. We found that simultaneous silencing of SND1s and NST1s led to severe xylem and phloem developmental defect in cotton stems, however silencing either SND1s or NST1s alone had no visible phenotype. Silencing both SND1s and NST1s but not one subgroup caused decreased expression of a set of SCW-associated genes, while over-expression of cotton SWNs in tobacco leaves resulted in SCW deposition. SWNs could bind the promoter of MYB46 and MYB83, which are highly expressed in SCW-rich tissues of cotton. In total, our data provide evidence that cotton SWNs positively and coordinately regulate SCW formation.

RNA-sequencing analysis revealed genes associated drought stress responses of different durations in hexaploid sweet potato

Purple-fleshed sweet potato (PFSP) is an important food crop, as it is a rich source of nutrients and anthocyanin pigments. Drought has become a major threat to sustainable sweetpotato production, resulting in huge yield losses. Therefore, the present study was conducted to identify drought stress-responsive genes using next-generation (NGS) and third-generation sequencing (TGS) techniques. Five cDNA libraries were constructed from seedling leaf segments treated with a 30% solution of polyethylene glycol (PEG-6000) for 0, 1, 6, 12, and 48 h for second-generation sequencing. Leaf samples taken from upper third of sweet potato seedlings after 1, 6, 12, and 48 h of drought stress were used for the construction of cDNA libraries for third-generation sequencing; however, leaf samples from untreated plants were collected as controls. A total of 184,259,679 clean reads were obtained using second and third-generation sequencing and then assembled into 17,508 unigenes with an average length of 1,783 base pairs. Out of 17,508 unigenes, 642 (3.6%) unigenes failed to hit any homologs in any databases, which might be considered novel genes. A total of 2, 920, 1578, and 2,418 up-regulated unigenes and 3,834, 2,131, and 3,337 down-regulated unigenes from 1 h, 6 h, 12 h, and 48 h library were identified, respectively in drought stress versus control. In addition, after 6, 12, and 48 h of drought stress, 540 up-regulated unigenes, 486 down-regulated unigenes and 414 significantly differentially expressed unigenes were detected. It was found that several gene families including Basic Helix-loop-helix (bHLH), basic leucine zipper (bZIP), Cystein2/Histidine2 (C2H2), C3H, Ethylene-responsive transcription factor (ERF), Homo domain-leucine zipper (HD-ZIP), MYB, NAC (NAM, ATAF1/2, and CUC2), Thiol specific antioxidant and WRKY showed responses to drought stress. In total, 17,472 simple sequence repeats and 510,617 single nucleotide polymorphisms were identified based on transcriptome sequencing of the PFSP. About 96.55% of the obtained sequences are not available online in sweet potato genomics resources. Therefore, it will enrich annotated sweet potato gene sequences and enhance understanding of the mechanisms of drought tolerance through genetic manipulation. Moreover, it represents a sequence resource for genetic and genomic studies of sweet potato.

MaXB3 Modulates MaNAC2, MaACS1, and MaACO1 Stability to Repress Ethylene Biosynthesis during Banana Fruit Ripening.

Ethylene plays a critical regulatory role in climacteric fruit ripening, and its biosynthesis is fine-tuned at the transcriptional and posttranslational levels. Nevertheless, the mechanistic link between transcriptional and posttranslational regulation of ethylene biosynthesis during fruit ripening is largely unknown. This study uncovers a coordinated transcriptional and posttranslational mechanism of controlling ethylene biosynthesis during banana (Musa acuminata) fruit ripening. NAC (NAM, ATAF, and CUC) proteins MaNAC1 and MaNAC2 repress the expression of MaERF11, a protein previously known to negatively regulate ethylene biosynthesis genes MaACS1 and MaACO1 A RING E3 ligase MaXB3 interacts with MaNAC2 to promote its ubiquitination and degradation, leading to the inhibition of MaNAC2-mediated transcriptional repression. In addition, MaXB3 also targets MaACS1 and MaACO1 for proteasome degradation. Further evidence supporting the role of MaXB3 is provided by its transient and ectopic overexpression in banana fruit and tomato (Solanum lycopersicum), respectively, which delays fruit ripening via repressing ethylene biosynthesis and thus ethylene response. Strikingly, MaNAC1 and MaNAC2 directly repress MaXB3 expression, suggesting a feedback regulatory mechanism that maintains a balance of MaNAC2, MaACS1, and MaACO1 levels. Collectively, our findings establish a multilayered regulatory cascade involving MaXB3, MaNACs, MaERF11, and MaACS1/MaACO1 that controls ethylene biosynthesis during climacteric ripening.

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 comparative analysis of the effects 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 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.

Picea wilsonii NAC Transcription Factor PwNAC30 Negatively Regulates Abiotic Stress Tolerance in Transgenic Arabidopsis

NAC (NAM, ATAF1/2, and CUC2) transcription factors play important roles in the process of abiotic stress response in plants. However, little is known about the functional roles of NACs in Picea (P.) wilsonii. In this study, we functionally characterized a novel P. wilsonii NAC transcription factor PwNAC30 by heterologous expression in Arabidopsis. The results showed that PwNAC30 is mainly localized in the nucleus by transient expression in Nicotiana benthamiana and can function as a nuclear localization transcription factor. β-Glucuronidase (GUS) staining in transgenic Arabidopsis (PwNAC30-promoter-GUS) confirmed the expression pattern of PwNAC30 throughout the plant development process, which is expressed in most of the plant tissues except stamens and petals. Overexpression of PwNAC30 in Arabidopsis significantly repressed the tolerance of seedlings and mature plants to both drought and salt stresses, while exerting no influence on growth and development of plants. The accumulation of reactive oxygen species (ROS) was significantly increased in transgenic plants, and the expression of some stress-responsive genes was obviously inhibited in PwNAC30 overexpression lines. Our study reveals that PwNAC30 functions as a negative regulator of plant tolerance to drought or salt stress, which provides new insights into abiotic tolerance mechanisms in woody plants.

Ectopic expression of AmNAC1 from Avicennia marina (Forsk.) Vierh. confers multiple abiotic stress tolerance in yeast and tobacco

Abiotic factors like salinity, drought and cold affect agricultural productivity substantially worldwide. NAC (NAM, ATAF1/2 and CUC2) family transcription factors have been shown to play important roles in biotic and abiotic stress responses in many plant species. Our previous studies have shown that AmNAC1, a gene from mangrove Avicennia marina, is differentially regulated in response to salt, drought and cold stresses and salicylic acid treatment. To gain more insights into the function of AmNAC1 in abiotic stress, AmNAC1 was heterologously expressed in yeast and tobacco. Heterologous expression of AmNAC1 in yeast (Saccharomyces cerevisiae) resulted in improved tolerance to salinity (NaCl and LiCl), alkalinity (NaHCO3) stresses. In addition, transgenic yeast exhibited enhanced tolerance compared to control when subjected to high (50 °C) and freezing (− 20 °C) temperature. Similarly, overexpression of AmNAC1 in tobacco using the constitutive promoter (2xCaMV) demonstrated increased tolerance to abiotic stresses such as salinity, drought and cold. Specifically, compared to wild type (WT) and vector control (VC), AmNAC1 transgenic lines exhibited higher germination, increased root length and better survival rates at the whole plant level. Collectively, these results indicate a role for AmNAC1 in multiple abiotic stress tolerance. AmNAC1 is a stress responsive transcription factor from mangrove Avicennia marina and enhances better tolerance to NaCl, NaHCo3, LiCl, freezing and high temperature in transgenic yeast. Over expression of AmNAC1 in tobacco improved salt, cold and drought tolerance.

GUS-reporter based analysis of the promoter activity of Gossypium hirsutum NAC transcription factor, GhNAC4 that is induced by phytohormones and environmental stresses

The expression analysis of GhNAC4, a NAC (NAM, ATAF1-2, and CUC2) domain-containing transcription factor of the upland cotton, Gossypium hirsutum was carried out. Its expression was up-regulated by abscisic acid, cytokinin, methyl jasmonic acid, gibberellic acid, auxin, and ethylene. Its expression was also highly induced by drought, osmotic, oxidative, salinity, high and low-temperature stresses, and wounding. To corroborate these observations, we cloned the promoter of GhNAC4 and fused it transcriptionally with uidA (GUS) gene for studies in transgenic tobacco. Fluorometric GUS analysis of the transgenic plants revealed that it is also induced by various phytohormones and environmental stresses. The spatio-temporal analysis of the GhNAC4 promoter revealed that GUS expression was active in all stages of plant development including the reproductive organs. In the mature plant, the GhNAC4 is expressed at a higher level in vascular bundles and guard cells. We also observed intense expression in other cells upon wounding. These observations were supported by a detailed bioinformatic analysis of the GhNAC4 promoter for identifying the cis-acting elements that are associated with the regulation of gene expression in a tissue-specific and induced manner. GhNAC4 gene is induced by phytohormones and environmental stress treatments in cotton. It is expressed throughout a mature plant, including the reproductive organs, vascular bundles, and guard cells.