NAM/ATAF/CUC Transcription Factors Research Articles

Functional characterization of VrNAC15 for drought resistance in mung beans

Drought stress has become an important limiting factor in mung bean production, and NAC(NAM/ATAF/CUC) transcription factors are crucial for plant growth under stress conditions, so it is important to study the regulatory role of NAC transcription factors in mung bean under drought stress. In this investigation, VrNAC15, along with its promoter, was cloned, and its structure was meticulously analyzed. Using qPCR, we examined the tissue-specific expression patterns of VrNAC15, particularly under drought stress and ABA exposure. Additionally, We performed ectopic expression of VrNAC15 in Arabidopsis to assess its function.. Gene sequence analysis revealed that VrNAC15 has a total length of 1014 bp, encoding 337 amino acids. It contains a NAM domain, localizes within the nucleus, and exhibits transcriptional activation. Promoter analysis of VrNAC15 identified essential core promoter elements and cis-acting elements related to abscisic acid, methyl jasmonate, gibberellin, adversity stress, light, and metabolism. Expression analysis demonstrated the concentration of VrNAC15 in leaves, with significant alterations following ABA and drought treatments in mung beans. Cluster analysis revealed that VrNAC15 may enhanced drought tolerance in transgenic plants through its expression. Transgenic experiments supported these findings, showing that heterologous expression of VrNAC15 led to enhanced antioxidant and osmotic adjustment capabilities in Arabidopsis plants. This resulted in the maintenance of cell membrane structural integrity during drought stress and normal physiological and biochemical metabolic reactions within cells. This research provides valuable insights into the structural and functional characteristics of the VrNAC15, setting the stage for future endeavors in molecular breeding for improved drought resistance in mung beans.

Transcriptional Control of Seed Life: New Insights into the Role of the NAC Family.

Transcription factors (TFs) regulate gene expression by binding to specific sequences on DNA through their DNA-binding domain (DBD), a universal process. This update conveys information about the diverse roles of TFs, focusing on the NACs (NAM-ATAF-CUC), in regulating target-gene expression and influencing various aspects of plant biology. NAC TFs appeared before the emergence of land plants. The NAC family constitutes a diverse group of plant-specific TFs found in mosses, conifers, monocots, and eudicots. This update discusses the evolutionary origins of plant NAC genes/proteins from green algae to their crucial roles in plant development and stress response across various plant species. From mosses and lycophytes to various angiosperms, the number of NAC proteins increases significantly, suggesting a gradual evolution from basal streptophytic green algae. NAC TFs play a critical role in enhancing abiotic stress tolerance, with their function conserved in angiosperms. Furthermore, the modular organization of NACs, their dimeric function, and their localization within cellular compartments contribute to their functional versatility and complexity. While most NAC TFs are nuclear-localized and active, a subset is found in other cellular compartments, indicating inactive forms until specific cues trigger their translocation to the nucleus. Additionally, it highlights their involvement in endoplasmic reticulum (ER) stress-induced programmed cell death (PCD) by activating the vacuolar processing enzyme (VPE) gene. Moreover, this update provides a comprehensive overview of the diverse roles of NAC TFs in plants, including their participation in ER stress responses, leaf senescence (LS), and growth and development. Notably, NACs exhibit correlations with various phytohormones (i.e., ABA, GAs, CK, IAA, JA, and SA), and several NAC genes are inducible by them, influencing a broad spectrum of biological processes. The study of the spatiotemporal expression patterns provides insights into when and where specific NAC genes are active, shedding light on their metabolic contributions. Likewise, this review emphasizes the significance of NAC TFs in transcriptional modules, seed reserve accumulation, and regulation of seed dormancy and germination. Overall, it effectively communicates the intricate and essential functions of NAC TFs in plant biology. Finally, from an evolutionary standpoint, a phylogenetic analysis suggests that it is highly probable that the WRKY family is evolutionarily older than the NAC family.

Overexpression of a Fragaria vesca NAM, ATAF, and CUC (NAC) Transcription Factor Gene (FvNAC29) Increases Salt and Cold Tolerance in Arabidopsis thaliana.

The NAC (NAM, ATAF1/2, CUC2) family of transcription factors (TFs) is a vital transcription factor family of plants. It controls multiple parts of plant development, tissue formation, and abiotic stress response. We cloned the FvNAC29 gene from Fragaria vesca (a diploid strawberry) for this research. There is a conserved NAM structural domain in the FvNAC29 protein. The highest homology between FvNAC29 and PaNAC1 was found by phylogenetic tree analysis. Subcellular localization revealed that FvNAC29 is localized onto the nucleus. Compared to other tissues, the expression level of FvNAC29 was higher in young leaves and roots. In addition, Arabidopsis plants overexpressing FvNAC29 had higher cold and high-salinity tolerance than the wild type (WT) and unloaded line with empty vector (UL). The proline and chlorophyll contents of transgenic Arabidopsis plants, along with the activities of the antioxidant enzymes like catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) under 200 mM NaCl treatment or -8 °C treatment, were higher than those activities of the control. Meanwhile, malondialdehyde (MDA) and the reactive oxygen species (ROS) content were higher in the WT and UL lines. FvNAC29 improves transgenic plant resistance to cold and salt stress by regulating the expression levels of AtRD29a, AtCCA1, AtP5CS1, and AtSnRK2.4. It also improves the potential to tolerate cold stress by positively regulating the expression levels of AtCBF1, AtCBF4, AtCOR15a, and AtCOR47. These findings suggest that FvNAC29 may be related to the processes and the molecular mechanisms of F. vesca response to high-salinity stress and LT stress, providing a comprehensive understanding of the NAC TFs.

MiR164-MhNAC1 regulates apple root nitrogen uptake under low nitrogen stress.

Nitrogen is an essential nutrient for plant growth and serves as a signaling molecule to regulate gene expression inducing physiological, growth and developmental responses. An excess or deficiency of nitrogen may have adverse effects on plants. Studying nitrogen uptake will help us understand the molecular mechanisms of utilization for targeted molecular breeding. Here, we identified and functionally validated an NAC (NAM-ATAF1/2-CUC2) transcription factor based on the transcriptomes of two apple rootstocks with different nitrogen uptake efficiency. NAC1, a target gene of miR164, directly regulates the expression of the high-affinity nitrate transporter (MhNRT2.4) and citric acid transporter (MhMATE), affecting root nitrogen uptake. To examine the role of MhNAC1 in nitrogen uptake, we produced transgenic lines that overexpressed or silenced MhNAC1. Silencing MhNAC1 promoted nitrogen uptake and citric acid secretion in roots, and enhanced plant tolerance to low nitrogen conditions, while overexpression of MhNAC1 or silencing miR164 had the opposite effect. This study not only revealed the role of the miR164-MhNAC1 module in nitrogen uptake in apple rootstocks but also confirmed that citric acid secretion in roots affected nitrogen uptake, which provides a research basis for efficient nitrogen utilization and molecular breeding in apple.

Transcription factor NtNAC56 regulates jasmonic acid-induced leaf senescence in tobacco.

Leaf senescence is a vital aspect of plant physiology and stress responses and is induced by endogenous factors and environmental cues. The plant-specific NAC (NAM, ATAF1/2, CUC2) transcription factor family influences growth, development, and stress responses in Arabidopsis (Arabidopsis thaliana) and other species. However, the roles of NACs in tobacco (Nicotiana tabacum) leaf senescence are still unclear. Here, we report that NtNAC56 regulates leaf senescence in tobacco. Transgenic plants overexpressing NtNAC56 (NtNAC56-OE) showed induction of senescence-related genes and exhibited early senescence and lower chlorophyll content compared to wild-type (WT) plants and the Ntnac56-19 mutant. In addition, root development and seed germination were inhibited in the NtNAC56-OE lines. Transmission electron microscopy observations accompanied by physiological and biochemical assays revealed that NtNAC56 overexpression triggers chloroplast degradation and reactive oxygen species accumulation in tobacco leaves. Transcriptome analysis demonstrated that NtNAC56 activates leaf senescence-related genes and jasmonic acid (JA) biosynthesis pathway genes. In addition, the JA content of NtNAC56-OE plants was higher than in WT plants, and JA treatment induced NtNAC56 expression. We performed DNA affinity purification sequencing to identify direct targets of NtNAC56, among which we focused on LIPOXYGENASE 5 (NtLOX5), a key gene in JA biosynthesis. A dual-luciferase reporter assay and a yeast one-hybrid assay confirmed that NtNAC56 directly binds to the TTTCTT motif in the NtLOX5 promoter. Our results reveal a mechanism whereby NtNAC56 regulates JA-induced leaf senescence in tobacco and provide a strategy for genetically manipulating leaf senescence and plant growth.

Rice transcription factor OsNAC2 maintains the homeostasis of immune responses to bacterial blight.

Rice (Oryza sativa) bacterial blight, caused by Xanthomonas oryzae pv. Oryzae (Xoo), threatens plant growth and yield. However, the molecular mechanisms underlying rice immunity against Xoo remain elusive. Here, we identified a NAC (NAM-ATAF-CUC) transcription factor OsNAC2 as a negative regulator in the resistance to bacterial blight disease in rice. Constitutive overexpression of OsNAC2 inhibited the expression of salicylic acid (SA) biosynthesis-related genes (i.e. isochorismate synthase 1 (OsICS1), phenylalanine ammonia lyase 3 (OsPAL3), etc.) with adverse impacts on the pathogenesis-related proteins (PRs) responses and compromised blight resistance. Moreover, OsNAC2 interacted with APETALA2/ethylene-responsive element binding protein (AP2/EREBP) transcription factor OsEREBP1 and possibly threatened its protein stability, destroying the favorable interaction of OsEREBP1-Xa21-binding protein OsXb22a in the cytoplasm during Xoo-induced infection. On the contrary, downregulation of OsNAC2 resulted in enhanced resistance to bacterial blight in rice without any growth or yield penalties. Our results demonstrated that OsNAC2 inhibits SA signaling and stably interacted with OsEREBP1 to impair disease resistance. This OsNAC2-OsEREBP1-based homeostatic mechanism provided insights into the competition between rice and bacterial pathogens, and it will be useful to improve the disease resistance of important crops through breeding.

The NAC transcription factor LuNAC61 negatively regulates fiber development in flax (Linum usitatissimum L.)

Flax is a crucial fiber crop that exhibits excellent textile properties and serves as a model plant for investigating phloem fiber development. The regulation of multiple genes significantly influences fiber development, notably involving NAC (NAM, ATAF1/2, CUC2) transcription factors in forming the fiber secondary cell wall (SCW). Overexpression of LuNAC61 in flax resulted in sparse top meristematic zone leaves and significantly reduced stem cellulose content. Scanning electron microscopy and staining observations revealed a significant reduction in fiber bundles. β-Glucuronidase (GUS) staining analysis demonstrated high activity of the LuNAC61 promoter in the bast fibers of the flax stem. Additionally, several members of the LuPLATZ and LuCesA families exhibited significant coexpression with LuNAC61. Subcellular localization indicated the presence of LuPLATZ24 protein in the nucleus and cytoplasm, LuNAC61 protein exclusively in the nucleus, and LuCesA10 in the nucleus and endoplasmic reticulum. LuPLATZ24 positively regulates LuNAC61, whereas LuNAC61 negatively affects LuCesA10, suggesting the involvement of a metabolic network in regulating flax fiber development. In conclusion, this study provides a critical opportunity for a comprehensive and in-depth analysis of the mechanisms governing flax fiber development and the potential use of biotechnology to enhance flax fiber yield.

OsNAC120 balances plant growth and drought tolerance by integrating GA and ABA signaling in rice

The crosstalk between gibberellin (GA) and abscisic acid (ABA) signaling is crucial for balancing plant growth and adaption to environmental stress. Nevertheless, the molecular mechanism of their mutual antagonism still remains to be fully clarified. In this study, we found that knockout of the rice NAC (NAM, ATAF1/2, CUC2) transcription factor gene OsNAC120 inhibits plant growth but enhances drought tolerance, whereas OsNAC120 overexpression produces the opposite results. Exogenous GA can rescue the semi-dwarf phenotype of osnac120 mutants, and further study showed that OsNAC120 promotes GA biosynthesis by transcriptionally activating the GA biosynthetic genes OsGA20ox1 and OsGA20ox3. The DELLA protein SLENDER RICE1 (SLR1) interacts with OsNAC120 and impedes its transactivation ability, and GA treatment can remove the inhibition of transactivation activity caused by SLR1. On the other hand, OsNAC120 negatively regulates rice drought tolerance by repressing ABA-induced stomatal closure. Mechanistic investigation revealed that OsNAC120 inhibits ABA biosynthesis via transcriptional repression of the ABA biosynthetic genes OsNCED3 and OsNCED4. Rice OSMOTIC STRESS/ABA-ACTIVATED PROTEIN KINASE 9 (OsSAPK9) physically interacts with OsNAC120 and mediates its phosphorylation, which results in OsNAC120 degradation. ABA treatment accelerates OsNAC120 degradation and reduces its transactivation activity. Together, our findings provide evidence that OsNAC120 plays critical roles in balancing GA-mediated growth and ABA-induced drought tolerance in rice. This research will help us to understand the mechanisms underlying the trade-off between plant growth and stress tolerance and to engineer stress-resistant, high-yielding crops.

Two adjacent NAC transcription factors regulate fruit maturity date and flavor in peach.

Although maturity date (MD) is an essential factor affecting fresh fruit marketing and has a pleiotropic effect on fruit taste qualities, the underlying mechanisms remain largely unclear. In this study, we functionally characterized two adjacent NAM-ATAF1/2-CUC2 (NAC) transcription factors (TFs), PpNAC1 and PpNAC5, both of which were associated with fruit MD in peach. PpNAC1 and PpNAC5 were found capable of activating transcription of genes associated with cell elongation, cell wall degradation and ethylene biosynthesis, suggesting their regulatory roles in fruit enlargement and ripening. Furthermore, PpNAC1 and PpNAC5 had pleiotropic effects on fruit taste due to their ability to activate transcription of genes for sugar accumulation and organic acid degradation. Interestingly, both PpNAC1 and PpNAC5 orthologues were found in fruit-producing angiosperms and adjacently arranged in all 91 tested dicots but absent in fruitless gymnosperms, suggesting their important roles in fruit development. Our results provide insight into the regulatory roles of NAC TFs in MD and fruit taste.

MaMADS1-MaNAC083 transcriptional regulatory cascade regulates ethylene biosynthesis during banana fruit ripening.

The hormone ethylene is crucial in the regulation of ripening in climacteric fruit, such as bananas. The transcriptional regulation of ethylene biosynthesis throughout banana fruit ripening has received much study, but the cascaded transcriptional machinery of upstream transcriptional regulators implicated in the ethylene biosynthesis pathway is still poorly understood. Here we report that ethylene biosynthesis genes, including MaACS1, MaACO1, MaACO4, MaACO5, and MaACO8, were upregulated in ripening bananas. NAC (NAM, ATAF, CUC) transcription factor, MaNAC083, a ripening and ethylene-inhibited gene, was discovered as a potential binding protein to the MaACS1 promoter by yeast one-hybrid screening. Further in vitro and in vivo experiments indicated that MaNAC083 bound directly to promoters of the five ethylene biosynthesis genes, thereby transcriptionally repressing their expression, which was further verified by transient overexpression experiments, where ethylene production was inhibited through MaNAC083-modulated transcriptional repression of ethylene biosynthesis genes in banana fruits. Strikingly, MaMADS1, a ripening-induced MADS (MCM1, AGAMOUS, DEFICIENS, SRF4) transcription factor, was found to directly repress the expression of MaNAC083, inhibiting trans-repression of MaNAC083 to ethylene biosynthesis genes, thereby attenuating MaNAC083-repressed ethylene production in bananas. These findings collectively illustrated the mechanistic basis of a MaMADS1-MaNAC083-MaACS1/MaACOs regulatory cascade controlling ethylene biosynthesis during banana fruit ripening. These findings increase our knowledge of the transcriptional regulatory mechanisms of ethylene biosynthesis at the transcriptional level and are expected to help develop molecular approaches to control ripening and improve fruit storability.

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.

Comprehensive Analysis of NAC Genes Reveals Differential Expression Patterns in Response to Pst DC3000 and Their Overlapping Expression Pattern during PTI and ETI in Tomato.

NAC (NAM/ATAF/CUC) transcription factors belong to a unique gene family in plants, which play vital roles in regulating diverse biological processes, including growth, development, senescence, and in response to biotic and abiotic stresses. Tomato (Solanum lycopersicum), as the most highly valued vegetable and fruit crop worldwide, is constantly attacked by Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), causing huge losses in production. Thus, it is essential to conduct a comprehensive identification of the SlNAC genes involved in response to Pst DC3000 in tomato. In this study, a complete overview of this gene family in tomato is presented, including genome localization, protein domain architectures, physical and chemical features, and nuclear location score. Phylogenetic analysis identified 20 SlNAC genes as putative stress-responsive genes, named SSlNAC 1-20. Expression profiles analysis revealed that 18 of these 20 SSlNAC genes were significantly induced in defense response to Pst DC3000 stress. Furthermore, the RNA-seq data were mined and analyzed, and the results revealed the expression pattern of the 20 SSlNAC genes in response to Pst DC3000 during the PTI and ETI. Among them, SSlNAC3, SSlNAC4, SSlNAC7, SSlNAC8, SSlNAC12, SSlNAC17, and SSlNAC19 were up-regulated against Pst DC3000 during PTI and ETI, which suggested that these genes may participate in both the PTI and ETI pathway during the interaction between tomato and Pst DC3000. In addition, SSlNAC genes induced by exogenous hormones, including indole-3-acetic acid (IAA), abscisic acid (ABA), salicylic acid (SA), and methyl jasmonic acid (MeJA), were also recovered. These results implied that SSlNAC genes may participate in the Pst DC3000 stress response by multiple regulatory pathways of the phytohormones. In all, this study provides important clues for further functional analysis and of the regulatory mechanism of SSlNAC genes under Pst DC3000 stress.

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 (VuNAC1 and 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 yield-declining 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.

Overexpression of ONAC054 Improves Drought Stress Tolerance and Grain Yield in Rice

Drought stress negatively affects plant growth and development, thus reducing plant productivity. Therefore, understanding the molecular mechanisms underlying drought stress responses is essential for crop improvement. The plant-specific NAM/ATAF1,2/CUC2 (NAC) transcription factors play important roles in the drought stress response. Here, we show that rice (Oryza sativa) ONAC054, a membrane-bound NAC transcription factor, is involved in the drought stress response. We found that onac054 mutants were sensitive, whereas ONAC054-overexpressing (ONAC054-OX) plants were tolerant to drought stress. Under drought stress conditions, several genes associated with abscisic acid (ABA) synthesis and signaling were downregulated in onac054 mutants but upregulated in ONAC054-OX plants. Among these genes, the TRANSCRIPTION FACTOR RESPONSIBLE FOR ABA REGULATION 1 (TRAB1), which encodes an ABA-inducible bZIP transcription factor, was directly activated by ONAC054. On the other hand, the expression of ONAC054 was directly activated by several ABA-responsive elements (ABRE)-binding factors (ABFs) in an ABA-dependent manner, indicating that ONAC054 acts as an enhancer of ABA-induced drought stress tolerance. Additionally, the overexpression of ONAC054 in rice greatly improved grain yield under drought stress conditions, indicating that the overexpression of ONAC054 could facilitate the improvement of drought stress tolerance in rice and other crops.

Identification of Carthamus tinctorius NAC gene family and analysis of drought stress response

The NAC(NAM/ATAF/CUC) transcription factors are members of the largest transcriptional gene family in plants and play an essential role in the response of plants to drought stress. To identify the number and function of the NAC gene family in Carthamus tinctorius, the present study adopted bioinformatics methods to identify NAC gene family members based on the whole genome data of C. tinctorius, and analyzed their physicochemical properties, chromosomal location, phylogenetic relationship, gene structure, conserved domain, and conserved motif. Meanwhile, the real-time fluorescence-based quantitative RT-PCR(qRT-PCR) was used to analyze the transcription level of four NAC genes under drought stress in different time. The results showed that C. tinctorius contained 87 NAC genes unevenly distributed on 11 chromosomes, while no NAC gene was found on chromosome 12. The encoded proteins were 103-974 amino acids and the number of CDS ranged from 3 to 9. According to the phylogenetic relationships, 87 NAC genes were clustered into17 subfamilies. The analysis of conserved domains and motifs revealed that most of the genes contained five conserved subdomains, A-E and motif2 was the most conserved among NAC genes. The expression pattern analysis showed that the transcription levels of four NAC genes related to drought resistance were all up-regulated after drought stress treatment for different time, suggesting that these four NAC genes may be related to drought resistance of C. tinctorius. This study is expected to provide a theoretical basis for further functional analysis of NAC transcription factors in C. tinctorius and references for the cultivation of drought-tolerant C. tinctorius varieties.

Bolstered plant tolerance to low temperatures by overexpressing NAC transcription factors: identification of critical variables by meta-analysis.

The potential biotechnological application of NAC overexpression has been challenged by meta-analysis, establishing a correlation between the magnitudes of several physiological and biochemical parameters and the enhanced tolerance to cold. Overexpression of various NAC (NAM/ATAF/CUC) transcription factors in different plant systems was shown to confer enhanced tolerance to low temperatures by inducing both common and distinctive stress response pathways. However, lack of consensus on the type of parameters evaluated, their magnitudes, and direction of the responses complicates drawing general conclusions on the effects of NAC expression in plant physiology. We report herein a meta-analysis summarizing the most critical response variables used to study the effect of overexpressing NAC regulators on cold stress tolerance. We found that NAC overexpression affected all of the outcome parameters in stressed plants, and one response in control conditions. Transformed plants displayed an increase of at least 40% in positive responses, while negative outcomes were reduced by at least 30%. The most reported parameters included survival, electrolyte leakage, and malondialdehyde contents, whereas the most sensitive to the treatments were the Fv/Fm parameter, survival, and the activity of catalases. We also explored how different experimental arrangements affected the magnitudes of the responses. NAC-mediated improvements were best observed after severe stress episodes and during brief treatments (ranging from 5 to 24h), especially in terms of antioxidant activities, accumulation of free proline, and parameters related to membrane integrity. Use of heterologous expression also favored several indicators of plant fitness. Our findings should help both basic and applied research on the influence of NAC expression on enhanced tolerance to cold.

Transcription factor OsNAC055 regulates GA-mediated lignin biosynthesis in rice straw

Crop straws represent enormous biomass resource that mainly contain secondary cell walls (SCWs) consisting of cellulose, hemicelluloses and lignin. Nevertheless, the regulatory mechanism of SCW biosynthesis still needs to be well understood. In this study, we identified a rice NAC (NAM, ATAF1/2, CUC2) transcription factor OsNAC055 that regulates GA-mediated lignin biosynthesis. As a nucleus-localized transcription factor, OsNAC055 exhibits the transcriptional activation activity. Overexpression of OsNAC055 increases the lignin content in rice straw. Transcriptomic analyses showed that the expression of multiple lignin biosynthetic genes was increased in OsNAC055-overexpressing plants. Further ChIP-qPCR analysis and transient transactivation assays indicated that OsNAC055 directly activates rice lignin biosynthetic genes CINNAMOYL-CoA REDUCTASE 10 (OsCCR10) and CINNAMYL ALCOHOL DEHYDROGENASE 2 (OsCAD2) by binding to their promoters. On the other hand, phytohormone measurement showed that OsNAC055 overexpression significantly increased exogenous GA3 levels in rice plants by regulating GA biosynthetic gene OsGA20ox2. Moreover, yeast two-hybrid and bimolecular fluorescence complement (BiFC) assays indicated that OsNAC055 interacts with SLENDER RICE1 (SLR1), the repressor in GA signaling. More importantly, exogenous GA treatment markedly enhanced the transcription of OsCCR10 and OsCAD2, suggesting the role of GA in lignin biosynthesis. Together, our results provide the evidence that OsNAC055 functions as an essential transcription factor to regulate the GA-mediated lignin biosynthesis, which provides a strategy for manipulating lignin production.

NAC transcription factors ATAF1 and ANAC055 affect the heat stress response in Arabidopsis

Pre-exposing (priming) plants to mild, non-lethal elevated temperature improves their tolerance to a later higher-temperature stress (triggering stimulus), which is of great ecological importance. ‘Thermomemory’ is maintaining this tolerance for an extended period of time. NAM/ATAF1/2/CUC2 (NAC) proteins are plant-specific transcription factors (TFs) that modulate responses to abiotic stresses, including heat stress (HS). Here, we investigated the potential role of NACs for thermomemory. We determined the expression of 104 Arabidopsis NAC genes after priming and triggering heat stimuli, and found ATAF1 expression is strongly induced right after priming and declines below control levels thereafter during thermorecovery. Knockout mutants of ATAF1 show better thermomemory than wild type, revealing a negative regulatory role. Differential expression analyses of RNA-seq data from ATAF1 overexpressor, ataf1 mutant and wild-type plants after heat priming revealed five genes that might be priming-associated direct targets of ATAF1: AT2G31260 (ATG9), AT2G41640 (GT61), AT3G44990 (XTH31), AT4G27720 and AT3G23540. Based on co-expression analyses applied to the aforementioned RNA-seq profiles, we identified ANAC055 to be transcriptionally co-regulated with ATAF1. Like ataf1, anac055 mutants show improved thermomemory, revealing a potential co-control of both NAC TFs over thermomemory. Our data reveals a core importance of two NAC transcription factors, ATAF1 and ANAC055, for thermomemory.

PaNAC089 is a membrane-tethered transcription factor (MTTF) that modulates flowering, chlorophyll breakdown and trichome initiation.

Flowering and senescence are essential developmental stages of green plants, which are governed by complex molecular regulatory networks. However, the connection between flowering regulation and senescence regulation in London plane tree (Platanus acerifolia ) remains unknown. In this study, we identified a gene PaNAC089 from London plane tree, which encodes a membrane-tethered transcription factor (MTTF) belonging to the NAC (NAM, ATAF1/2, CUC2) transcription factor family. We investigated the functions of PaNAC089 in the regulation of flowering and senescence through the analysis of expression profiles and transgenic phenotypes. Heterologous overexpression of ΔPaNAC089 delayed flowering and inhibited chlorophyll breakdown to produce dark green rosette leaves in Arabidopsis . In addition, the trichome density of rosette leaves was decreased in transgenic lines. In ΔPaNAC089 overexpression plants, a series of functional genes with inhibited expression were identified by quantitative real-time polymerase chain reaction (qRT-PCR), including genes that regulate flowering, chlorophyll decomposition, and trichome initiation. Furthermore, Δ PaNAC089 directly binds to the promoter of CONSTANS (CO ) and NON-YELLOWING2 (NYE2 ) in the yeast one-hybrid assay. Consistent with this, luciferase (LUC) transient expression assays also showed that Δ PaNAC089 could inhibit the activity of NYE2 . To summarise, our data suggests that PaNAC089 is an MTTF that modulates flowering, chlorophyll breakdown and trichome initiation.

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.