NAM, ATAF, And CUC Research Articles

Genome-wide identification of the NAC family in Hemerocallis citrina and functional analysis of HcNAC35 in response to abiotic stress in watermelon.

NAC (NAM, ATAF, and CUC) transcription factor family, one of the important switches of transcription networks in plants, functions in plant growth, development, and stress resistance. Night lily (Hemerocallis citrina) is an important horticultural perennial monocot plant that has edible, medicinal, and ornamental values. However, the NAC gene family of night lily has not yet been analyzed systematically to date. Therefore, we conducted a genome-wide study of the HcNAC gene family and identified a total of 113 HcNAC members from the Hemerocallis citrina genome. We found that 113 HcNAC genes were unevenly distributed on 11 chromosomes. Phylogenetic analysis showed that they could be categorized into 16 instinct subgroups. Proteins clustering together exhibited similar conserved motifs and intron-exon structures. Collinearity analysis indicated that segmental and tandem duplication might contribute to the great expansion of the NAC gene family in night lily, whose relationship was closer with rice than Arabidopsis. Additionally, tissue-specific pattern analysis indicated that most HcNAC genes had relatively higher expression abundances in roots. RNA-Seq along with RT-qPCR results jointly showed HcNAC genes expressed differently under drought and salinity stresses. Interestingly, HcNAC35 was overexpressed in watermelon, and the stress resilience of transgenic lines was much higher than that of wild-type watermelon, which revealed its wide participation in abiotic stress response. In conclusion, our findings provide a new prospect for investigating the biological roles of NAC genes in night lily.

Genome-Wide Identification of NAC Family Genes and Their Expression Analyses in Response to Osmotic Stress in Cannabis sativa L.

NAC (NAM, ATAF1/2, and CUC2) transcription factors are unique and essential for plant growth and development. Although the NAC gene family has been identified in a wide variety of plants, its chromosomal location and function in Cannabis sativa are still unknown. In this study, a total of 69 putative CsNACs were obtained, and chromosomal location analysis indicated that the CsNAC genes mapped unevenly to 10 chromosomes. Phylogenetic analyses showed that the 69 CsNACs could be divided into six subfamilies. Additionally, the CsNAC genes in group IV-a are specific to Cannabis sativa and contain a relatively large number of exons. Promoter analysis revealed that most CsNAC promoters contained cis-elements related to plant hormones, the light response, and abiotic stress. Furthermore, transcriptome expression profiling revealed that 24 CsNAC genes in two Cannabis sativa cultivars (YM1 and YM7) were significantly differentially expressed under osmotic stress, and these 12 genes presented differential expression patterns across different cultivars according to quantitative real-time PCR (RT-qPCR) analysis. Among these, the genes homologous to the CsNAC18, CsNAC24, and CsNAC61 genes have been proven to be involved in the response to abiotic stress and might be candidate genes for further exploration to determine their functions. The present study provides a comprehensive insight into the sequence characteristics, structural properties, evolutionary relationships, and expression patterns of NAC family genes under osmotic stress in Cannabis sativa and provides a basis for further functional characterization of CsNAC genes under osmotic stress to improve agricultural traits in Cannabis sativa.

Genome-Wide Identification of NAC Gene Family Members of Tree Peony (Paeonia suffruticosa Andrews) and Their Expression under Heat and Waterlogging Stress.

An important family of transcription factors (TFs) in plants known as NAC (NAM, ATAF1/2, and CUC2) is crucial for the responses of plants to environmental stressors. In this study, we mined the NAC TF family members of tree peony (Paeonia suffruticosa Andrews) from genome-wide data and analyzed their response to heat and waterlogging stresses in conjunction with transcriptome data. Based on tree peony's genomic information, a total of 48 PsNAC genes were discovered. Based on how similar their protein sequences were, these PsNAC genes were divided into 14 branches. While the gene structures and conserved protein motifs of the PsNAC genes within each branch were largely the same, the cis-acting elements in the promoter region varied significantly. Transcriptome data revealed the presence of five PsNAC genes (PsNAC06, PsNAC23, PsNAC38, PsNAC41, PsNAC47) and one PsNAC gene (PsNAC37) in response to heat and waterlogging stresses, respectively. qRT-PCR analysis reconfirmed the response of these five PsNAC genes to heat stress and one PsNAC gene to waterlogging stress. This study lays a foundation for the study of the functions and regulatory mechanisms of NAC TFs in tree peony. Meanwhile, the NAC TFs of tree peony in response to heat and waterlogging stress were excavated, which is of great significance for the selection and breeding of new tree peony varieties with strong heat and waterlogging tolerance.

Transcription factor NAC78 cooperates with NAC78 INTERACTING PROTEIN6 to confer drought tolerance in rice.

NAC (NAM, ATAF1/2, and CUC2) family transcription factors are involved in several cellular processes, including responses to drought, salinity, cold, and submergence. However, whether or how certain NAC proteins regulate drought tolerance in rice (Oryza sativa) remain unclear. In this study, we show that over-expression of OsNAC78 enhanced rice resistance to drought treatment, whereas Osnac78 mutant plants were susceptible to drought stress. We further characterized the OsNAC78 interacting protein, named NAC78 interacting protein6 (OsNACIP6), and found that it conferred rice drought tolerance. Our results demonstrate that OsNACIP6 enhanced transcription of OsNAC78 and promoted the expression of its downstream target OsGSTU37, encoding a glutathione reductase. The ABRE4 cis-element in the promoter region of OsNACIP675-1-127 conferred significant up-regulation of OsNACIP6 expression and initiated the OsNACIP6/OsNAC78-OsGSTU37 module that facilitates rice growth under drought conditions. Together, our results uncover a transcriptional module composed of OsNACIP6, OsNAC78, and OsGSTU37 and provide insights into the molecular mechanisms underlying the drought stress response in rice.

Pathogen resistance was negatively regulated by the NAC transcription factor ScATAF1 in sugarcane

The NAC (NAM, ATAF, and CUC) is one of the largest transcription factor gene families in plants. In this study, 180, 141, and 131 NAC family members were identified from Saccharum complex, including S. officinarum, S. spontaneum, and Erianthus rufipilus. The Ka/Ks ratio of ATAF subfamily was all less than 1. Besides, 52 ATAF members from 12 representative plants were divided into three clades and there was only a significant expansion in maize. Surprisingly, ABA and JA cis-elements were abundant in hormonal response factor, followed by transcriptional regulator and abiotic stressor. The ATAF subfamily was differentially expressed in various tissues, under low temperature and smut pathogen treatments. Further, the ScATAF1 gene, with high expression in leaves, stem epidermis, and buds, was isolated. The encoded protein, lack of self-activation activity, was situated in the cell nucleus. Moreover, SA and JA stresses down-regulated the expression of this gene, while ABA, NaCl, and 4°C treatments led to its up-regulation. Interestingly, its expression in the smut susceptible sugarcane cultivars was much higher than the smut resistant ones. Notably, the colors presented slight brown in tobacco transiently overexpressing ScATAF1 at 1 d after DAB staining, while the symptoms were more obvious at 3 d after inoculation with Ralstonia solanacearum, with ROS, JA, and SA signaling pathway genes significantly up-regulated. We thus speculated ScATAF1 gene could negatively mediate hypersensitive reactions and produce ROS by JA and SA signaling pathways. These findings lay the groundwork for in-depth investigation on the biological roles of ATAF subfamily in sugarcane.

Multiomic analyses reveal key sectors of jasmonate-mediated defense responses in rice.

The phytohormone jasmonate (JA) plays a central role in plant defenses against biotic stressors. However, our knowledge of the JA signaling pathway in rice (Oryza sativa) remains incomplete. Here, we integrated multiomic data from three tissues to characterize the functional modules involved in organizing JA-responsive genes. In the core regulatory sector, MYC2 transcription factor transcriptional cascades are conserved in different species but with distinct regulators (e.g. bHLH6 in rice), in which genes are early expressed across all tissues. In the feedback sector, MYC2 also regulates the expression of JA repressor and catabolic genes, providing negative feedback that truncates the duration of JA responses. For example, the MYC2-regulated NAC (NAM, ATAF1/2, and CUC2) transcription factor genes NAC1, NAC3, and NAC4 encode proteins that repress JA signaling and herbivore resistance. In the tissue-specific sector, many late-expressed genes are associated with the biosynthesis of specialized metabolites that mediate particular defensive functions. For example, the terpene synthase gene TPS35 is specifically induced in the leaf sheath and TPS35 functions in defense against oviposition by brown planthoppers and the attraction of this herbivore's natural enemies. Thus, by characterizing core, tissue-specific, and feedback sectors of JA-elicited defense responses, this work provides a valuable resource for future discoveries of key JA components in this important crop.

GmNAC3 acts as a key regulator in soybean against drought stress

The NAC (NAM, ATAF and CUC) family is one of the largest transcription factor (TF) families in plant that are involved in the regulatory mechanisms of plant growth and development as well as responses to abiotic stresses. However, the underlying molecular mechanism of drought-responsive NAC family members in soybean still remains inexplicit. In this study, a total of 179 GmNAC genes were identified in the soybean genome. We discovered that the majority of GmNAC members have more than three exons and share a gene and motif structure that is mostly conserved at the N-terminus. Phylogenetic analysis suggested that soybean GmNAC proteins were divided into 10 separate groups. The analysis of cis-elements highlighted the potential role of GmNAC genes in various hormonal and defense related activities. In addition, most of the GmNAC genes showed notable expression in roots and leaves, suggesting their likely role in abiotic stress adaptation. The overexpression of GmNAC3-OE in Arabidopsis increased tolerance to drought stress. Similarly, the GmNAC3-OE plants displayed better survival rates, root length and antioxidant activities. Enhanced expression of stress specific genes in GmNAC3-OE was also recorded. Our findings revealed the potential role of GmNAC3 gene role in regulating soybean response to drought stress and could be used as a potential marker to generate stress resilient plants.

The miR6445-NAC029 module regulates drought tolerance by regulating the expression of glutathione S-transferase U23 and reactive oxygen species scavenging in Populus.

MicroRNAs are essential in plant development and stress resistance, but their specific roles in drought stress require further investigation. Here, we have uncovered that a Populus-specific microRNAs (miRNA), miR6445, targeting NAC (NAM, ATAF, and CUC) family genes, is involved in regulating drought tolerance of poplar. The expression level of miR6445 was significantly upregulated under drought stress; concomitantly, seven targeted NAC genes showed significant downregulation. Silencing the expression of miR6445 by short tandem target mimic technology significantly decreased the drought tolerance in poplar. Furthermore, 5' RACE experiments confirmed that miR6445 directly targeted NAC029. The overexpression lines of PtrNAC029 (OE-NAC029) showed increased sensitivity to drought compared with knockout lines (Crispr-NAC029), consistent with the drought-sensitive phenotype observed in miR6445-silenced strains. PtrNAC029 was further verified to directly bind to the promoters of glutathione S-transferase U23 (GSTU23) and inhibit its expression. Both Crispr-NAC029 and PtrGSTU23 overexpressing plants showed higher levels of PtrGSTU23 transcript and GST activity while accumulating less reactive oxygen species (ROS). Moreover, poplars overexpressing GSTU23 demonstrated enhanced drought tolerance. Taken together, our research reveals the crucial role of the miR6445-NAC029-GSTU23 module in enhancing poplar drought tolerance by regulating ROS homeostasis. This finding provides new molecular targets for improving the drought resistance of trees.

Regulatory loops between rice transcription factors OsNAC25 and OsNAC20/26 balance starch synthesis.

Several starch synthesis regulators have been identified, but these regulators are situated in the terminus of the regulatory network. Their upstream regulators and the complex regulatory network formed between these regulators remain largely unknown. A previous study demonstrated that NAM, ATAF, and CUC (NAC) transcription factors, OsNAC20 and OsNAC26 (OsNAC20/26), redundantly and positively regulate the accumulation of storage material in rice (Oryza sativa) endosperm. In this study, we detected OsNAC25 as an upstream regulator and interacting protein of OsNAC20/26. Both OsNAC25 mutation and OE resulted in a chalky seed phenotype, decreased starch content, and reduced expression of starch synthesis-related genes, but the mechanisms were different. In the osnac25 mutant, decreased expression of OsNAC20/26 resulted in reduced starch synthesis; however, in OsNAC25-overexpressing plants, the OsNAC25-OsNAC20/26 complex inhibited OsNAC20/26 binding to the promoter of starch synthesis-related genes. In addition, OsNAC20/26 positively regulated OsNAC25. Therefore, the mutual regulation between OsNAC25 and OsNAC20/26 forms a positive regulatory loop to stimulate the expression of starch synthesis-related genes and meet the great demand for starch accumulation in the grain filling stage. Simultaneously, a negative regulatory loop forms among the 3 proteins to avoid the excessive expression of starch synthesis-related genes. Collectively, our findings demonstrate that both promotion and inhibition mechanisms between OsNAC25 and OsNAC20/26 are essential for maintaining stable expression of starch synthesis-related genes and normal starch accumulation.

IbNIEL-mediated degradation of IbNAC087 regulates jasmonic acid-dependent salt and drought tolerance in sweet potato.

Sweet potato (Ipomoea batatas [L.] Lam.) is a crucial staple and bioenergy crop. Its abiotic stress tolerance holds significant importance in fully utilizing marginal lands. Transcriptional processes regulate abiotic stress responses, yet the molecular regulatory mechanisms in sweet potato remain unclear. In this study, a NAC (NAM, ATAF1/2, and CUC2) transcription factor, IbNAC087, was identified, which is commonly upregulated in salt- and drought-tolerant germplasms. Overexpression of IbNAC087 increased salt and drought tolerance by increasing jasmonic acid (JA) accumulation and activating reactive oxygen species (ROS) scavenging, whereas silencing this gene resulted in opposite phenotypes. JA-rich IbNAC087-OE (overexpression) plants exhibited more stomatal closure than wild-type (WT) and IbNAC087-Ri plants under NaCl, polyethylene glycol, and methyl jasmonate treatments. IbNAC087 functions as a nuclear transcriptional activator and directly activates the expression of the key JA biosynthesis-related genes lipoxygenase (IbLOX) and allene oxide synthase (IbAOS). Moreover, IbNAC087 physically interacted with a RING-type E3 ubiquitin ligase NAC087-INTERACTING E3 LIGASE (IbNIEL), negatively regulating salt and drought tolerance in sweet potato. IbNIEL ubiquitinated IbNAC087 to promote 26S proteasome degradation, which weakened its activation on IbLOX and IbAOS. The findings provide insights into the mechanism underlying the IbNIEL-IbNAC087 module regulation of JA-dependent salt and drought response in sweet potato and provide candidate genes for improving abiotic stress tolerance in crops.

Transcriptional regulatory mechanisms of flavonoid metabolism from flower bud to flower in Hemerocallis citrina

The flower buds of Hemerocallis citrina are well-known functional foods in East Asian countries for their promoting intelligence and forgetting worries, in which their nutrients, especially flavonoids, play an important role. However, they will be discarded when the buds develop into flowers, part of the reason is the type and content of flavonoids change considerably. At present, the developmental mechanism responsible for these differences is still unclear. Based on "multi-omics" technologies, the results of this study revealed that 18 flavonoids were significantly differential, including five flavonoids, eight flavonols and three flavanones, one isoflavone and one anthocyanin, and their biosynthesis involved 50 differential genes and 25 differential enzyme proteins. Among them, nine genes encoding four key enzymes (chalcone isomerase, isoflavone 2′-hydroxylase, flavonol-3-O-l-rhamnoside-7-O-glucosyltransferase and phlorizin synthase) were targeted by seven conserved miRNA (microRNA) families, covering miR394, miR477, miR396, miR528, miR164, miR395 and miR447. Furthermore, 10 genes encoding five transcription factor families (bZIP (basic leucine zipper), NAC (NAM, ATAF1/2 and CUC2), GRAS (GAI, RGA and SCR), TCP (TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR) and MADS (MCM1/AGAMOUS/DEFICIENS/SRF)) were targeted by seven conserved miRNA families, including miR393, miR166, miR171, miR477, miR164, miR528 and miR447. They are jointly involved in the metabolic regulation of flavone and flavonol biosynthesis, isoflavonoid biosynthesis, and flavonoid biosynthesis from flower buds to flowering development of H. citrina.

Membrane-Bound Transcription Factor ZmNAC074 Positively Regulates Abiotic Stress Tolerance in Transgenic Arabidopsis.

Maize (Zea mays L.) is one of the most widely cultivated crops for humans, making a vital contribution to human nutrition and health. However, in recent years, due to the influence of external adverse environments, the yield and quality of maize have been seriously affected. NAC (NAM, ATAF1/2 and CUC2) transcription factors (TFs) are important plant-unique TFs, which are crucial for regulating the abiotic stress response of plants. Therefore, it is of great biological significance to explore the underlying regulatory function of plant NAC TFs under various abiotic stresses. In this study, wild-type and ZmNAC074-overexpressed transgenic Arabidopsis were used as experimental materials to dissect the stress-resistant function of ZmNAC074 in transgenic Arabidopsis at phenotypic, physiological and molecular levels. The analyses of seed germination rate, survival rate, phenotype, the content of chlorophyll, carotenoids, malondialdehyde (MDA), proline and other physiological indexes induced by distinct abiotic stress conditions showed that overexpression of ZmNAC074 could confer the enhanced resistance of salt, drought, and endoplasmic reticulum (ER) stress in transgenic Arabidopsis, indicating that ZmNAC074 plays an important regulatory role in plant response to abiotic stress, which provides an important theoretical foundation for further uncovering the molecular regulation mechanism of ZmNAC074 under abiotic stresses.

MaSPL16 positively regulates fruit ripening in bananas via the direct transcriptional induction of MaNAC029

Fruit ripening (FR) is attributed to the selective expression of several genes precisely governed by various specific transcription factors (TFs). The NAC (NAM, ATAF, and CUC) TF, MaNAC029, positively regulated banana ripening by directly inducing ethylene biosynthesis and transcription of fruit quality-related genes. However, its upstream regulatory mechanism still needs to be clarified. Herein, yeast one-hybrid screening revealed that a SQUAMOSA promoter binding protein-like (SPL) TF, MaSPL16, was a potentially upstream regulator of Musa acuminata NAC (NAM, ATAF, CUC) 029 (MaNAC029). Furthermore, gel mobility shift assay revealed that MaSPL16 can directly bound with the “GTAC” element of the MaNAC029 promoter. The gene expression and promoter activity assays demonstrated that Musa acuminata SPL (SQUAMOSA promoter binding protein-like) 16 (MaSPL16) expression was inducible by ethylene and ripening. MaSPL16 was localized to the nucleus, displayed a potenial capacity for transcriptional activation of MaNAC029. More critically, the transient expression of MaSPL16 in bananas accelerated FR via the upregulation of MaNAC029 and its downstream genes. Collectively, the mechanistic basis of a regulatory cascade involving MaSPL16-MaNAC029 that governed ethylene biosynthesis and fruit quality throughout the entire process of banana fruit ripening was unveiled. These outcomes increase the understanding of the gene-transcriptional regulatory mechanisms in FR. They are envisaged to help devise molecular techniques to regulate maturation and improve future fruit quality.

The Carbon Flow Shifts from Primary to Secondary Metabolism during Xylem Vessel Cell Differentiation inArabidopsis thaliana.

Xylem vessel cell differentiation is characterized by the deposition of a secondary cell wall (SCW) containing cellulose, hemicellulose and lignin. VASCULAR-RELATED NAC-DOMAIN7 (VND7), a plant-specific NAC (NAM, ATAF1/2, and CUC2) transcription factor, is a master regulator of xylem vessel cell differentiation in Arabidopsis (Arabidopsis thaliana). Previous metabolome analysis using the VND7-inducible system in tobacco BY-2 cells successfully revealed significant quantitative changes in primary metabolites during xylem vessel cell differentiation. However, the flow of primary metabolites is not yet well understood. Here, we performed a metabolomic analysis of VND7-inducible Arabidopsis T87 suspension cells. Capillary electrophoresis-time-of-flight mass spectrometry quantified 57 metabolites, and subsequent data analysis highlighted active changes in the levels of UDP-glucose and phenylalanine, which are building blocks of cellulose and lignin, respectively. In a metabolic flow analysis using stable carbon 13 (13C) isotope, the 13C-labeling ratio specifically increased in 3-phosphoglycerate after 12 h of VND7 induction, followed by an increase in shikimate after 24 h of induction, while the inflow of 13C into lactate from pyruvate was significantly inhibited, indicating an active shift of carbon flow from glycolysis to the shikimate pathway during xylem vessel cell differentiation. In support of this notion, most glycolytic genes involved in the downstream of glyceraldehyde 3-phosphate were downregulated following the induction of xylem vessel cell differentiation, whereas genes for the shikimate pathway and phenylalanine biosynthesis were upregulated. These findings provide evidence for the active shift of carbon flow from primary metabolic pathways to the SCW polymer biosynthetic pathway at specific points during xylem vessel cell differentiation.

Genome-Wide Identification and Characterization of the NAC Gene Family and Its Involvement in Cold Response in Dendrobium officinale.

The NAC (NAM, ATAF1/2 and CUC2) gene family is one of the largest plant-specific transcription factor families, functioning as crucial regulators in diverse biological processes such as plant growth and development as well as biotic and abiotic stress responses. Although it has been widely characterized in many plants, the significance of the NAC family in Dendrobium officinale remained elusive up to now. In this study, a genome-wide search method was conducted to identify NAC genes in Dendrobium officinale (DoNACs) and a total of 110 putative DoNACs were obtained. Phylogenetic analysis classified them into 15 subfamilies according to the nomenclature in Arabidopsis and rice. The members in the subfamilies shared more similar gene structures and conversed protein domain compositions. Furthermore, the expression profiles of these DoNACs were investigated in diverse tissues and under cold stress by RNA-seq data. Then, a total of five up-regulated and five down-regulated, cold-responsive DoNACs were validated through QRT-PCR analysis, demonstrating they were involved in regulating cold stress response. Additionally, the subcellular localization of two down-regulated candidates (DoNAC39 and DoNAC58) was demonstrated to be localized in the nuclei. This study reported the genomic organization, protein domain compositions and expression patterns of the NAC family in Dendrobium officinale, which provided targets for further functional studies of DoNACs and also contributed to the dissection of the role of NAC in regulating cold tolerance in Dendrobium officinale.

Genome-wide analysis of the NAC gene family and functional verification of the DcNAC043s in Dendrobium catenatum

The NAC (NAM, ATAF1/2 and CUC2) domain protein plays an important role in plant growth and development. The function of members of NAC gene family has been deeply studied in many plants. However, the evolutionary relationships and characteristics of NAC family genes in Dendrobium catenatum (D. candidum) unclear. In this study, we identified 33 NAC genes in D.catenatum, all contain NAM conservative domain. Subcellular localization predictions indicated that all the DcNAC proteins are localized to the nucleus. Phylogenetic analysis suggested that the DcNAC gene family could be divided into four groups. Then, the amino-acid composition, physicochemical properties, gene structure, motif, and promoter cis-acting elements were analyzed, the evolutionarily conservative gene DcNAC043-1 and DcNAC043-2 were found. Using qRT-PCR and phloroglucinol staining experiments, it was demonstrated that DcNAC043s can respond to drought stress in Dendrobium. Within a certain range, the longer the duration of drought stress, the higher the expression level of DcNAC043s, and the stronger the degree of plant lignification. We supplemented the relevant information of NAC gene family in D. catenatum. At the same time, the gene function of DcNAC043s and its contribution to the response of Dendrobium to drought stress were verified. These results provide a comprehensive evolutionary history of NAC genes in D. catenatum, and insight into the biological functions of DcNAC043s genes in response to drought stress.

The AP2/ERF transcription factor PtoERF15 confers drought tolerance via JA-mediated signaling in Populus.

Drought stress is one of the major limiting factors for the growth and development of perennial trees. Xylem vessels act as the center of water conduction in woody species, but the underlying mechanism of its development and morphogenesis under water-deficient conditions remains elucidation. Here, we identified and characterized an osmotic stress-induced ETHYLENE RESPONSE FACTOR 15 (PtoERF15) and its target, PtoMYC2b, which was involved in mediating vessel size, density, and cell wall thickness in response to drought in Populus tomentosa. PtoERF15 is preferentially expressed in differentiating xylem of poplar stems. Overexpression of PtoERF15 contributed to stem water potential maintaining, thus promoting drought tolerance. RNA-Seq and biochemical analysis further revealed that PtoERF15 directly regulated PtoMYC2b, encoding a switch of JA signaling pathway. Additionally, our findings verify that three sets of homologous genes from NAC (NAM, ATAF1/2, and CUC2) gene family: PtoSND1-A1/A2, PtoVND7-1/7-2, and PtoNAC118/120, as the targets of PtoMYC2b, are involved in the regulation of vessel morphology in poplar. Collectively, our study provides molecular evidence for the involvement of the PtoERF15-PtoMYC2b transcription cascade in maintaining stem water potential through the regulation of xylem vessel development, ultimately improving drought tolerance in poplar.

A Transcription Factor SlNAC4 Gene of Suaeda liaotungensis Enhances Salt and Drought Tolerance through Regulating ABA Synthesis.

The NAC (NAM, ATAF1/2 and CUC2) transcription factors are ubiquitously distributed in plants and play critical roles in the construction of plant organs and abiotic stress response. In this study, we described the cloning of a Suaeda liaotungensis K. NAC transcription factor gene SlNAC4, which contained 1450 bp, coding a 331 amino acid. We found that SlNAC4 was highly expressed in stems of S. liaotungensis, and the expression of SlNAC4 was considerably up-regulated after salt, drought, and ABA treatments. Transcription analysis and subcellular localization demonstrated that the SlNAC4 protein was located both in the nucleus and cytoplasm, and contained a C-terminal transcriptional activator. The SlNAC4 overexpression Arabidopsis lines significantly enhanced the tolerance to salt and drought treatment and displayed obviously increased activity of antioxidant enzymes under salt and drought stress. Additionally, transgenic plants overexpressing SlNAC4 had a significantly higher level of physiological indices. Interestingly, SlNAC4 promoted the expression of ABA metabolism-related genes including AtABA1, AtABA3, AtNCED3, AtAAO3, but inhibited the expression of AtCYP707A3 in overexpression lines. Using a yeast one-hybrid (Y1H) assay, we identified that the SlNAC4 transcription factor could bind to the promoters of those ABA metabolism-related genes. These results indicate that overexpression of SlNAC4 in plants enhances the tolerance to salt and drought stress by regulating ABA metabolism.

MdNAC104 positively regulates apple cold tolerance via CBF-dependent and CBF-independent pathways.

Low temperature is the main environmental factor affecting the yield, quality and geographical distribution of crops, which significantly restricts development of the fruit industry. The NAC (NAM, ATAF1/2 and CUC2) transcription factor (TF) family is involved in regulating plant cold tolerance, but the mechanisms underlying these regulatory processes remain unclear. Here, the NAC TF MdNAC104 played a positive role in modulating apple cold tolerance. Under cold stress, MdNAC104-overexpressing transgenic plants exhibited less ion leakage and lower ROS (reactive oxygen species) accumulation, but higher contents of osmoregulatory substances and activities of antioxidant enzymes. Transcriptional regulation analysis showed that MdNAC104 directly bound to the MdCBF1 and MdCBF3 promoters to promote expression. In addition, based on combined transcriptomic and metabolomic analyses, as well as promoter binding and transcriptional regulation analyses, we found that MdNAC104 stimulated the accumulation of anthocyanin under cold conditions by upregulating the expression of anthocyanin synthesis-related genes, including MdCHS-b, MdCHI-a, MdF3H-a and MdANS-b, and increased the activities of the antioxidant enzymes by promoting the expression of the antioxidant enzyme-encoding genes MdFSD2 and MdPRXR1.1. In conclusion, this study revealed the MdNAC104 regulatory mechanism of cold tolerance in apple via CBF-dependent and CBF-independent pathways.

ClNAC100 Is a NAC Transcription Factor of Chinese Fir in Response to Phosphate Starvation

Phosphate (Pi) deficiency is one of the most limiting factors for Chinese fir growth and production. Moreover, continuous cultivation of Chinese fir for multiple generations led to the reduction of soil nutrients, which hindered the yield of Chinese fir in southern China. Although NAC (NAM, ATAF, and CUC) transcription factors (TFs) play critical roles in plant development and abiotic stress resistance, it is still unclear how they regulate the response of Chinese fir to phosphate (Pi) starvation. Based on Pi-deficient transcriptome data of Chinses fir root, we identified a NAC transcription factor with increased expression under Pi deficiency, which was obtained by PCR and named ClNAC100. RT-qPCR confirmed that the expression of ClNAC100 in the root of Chinese fir was induced by phosphate deficiency and showed a dynamic change with time. It was positively regulated by ABA and negatively regulated by JA, and ClNAC100 was highly expressed in the roots and leaves of Chinese fir. Transcriptional activation assay confirmed that ClNAC100 was a transcriptional activator. The promoter of ClNAC100 was obtained by genome walking, which was predicted to contain a large number of stress, hormone, and growth-related cis-elements. Tobacco infection was used to verify the activity of the promoter, and the core promoter was located between −1519 bp and −589 bp. We identified 18 proteins bound to the ClNAC100 promoter and 5 ClNAC100 interacting proteins by yeast one-hybrid and yeast two-hybrid, respectively. We speculated that AHL and TIFY family transcription factors, calmodulin, and E3 ubiquitin ligase in these proteins might be important phosphorus-related proteins. These results provide a basis for the further study of the regulatory mechanism and pathways of ClNAC100 under Pi starvation.