NAM, ATAF, And CUC Research Articles

Genome-wide analysis of the rose (Rosa chinensis) NAC family and characterization of RcNAC091.

A genome-wide analysis identified 116 NAC genes in rose, including stress-related ones with different expression patterns under drought and salt stress. Silencing of RcNAC091, a member of the ATAF subfamily, decreased dehydration tolerance in rose. The NAC (NAM, ATAF, and CUC) transcription factors (TFs) are plant-specific proteins that regulate various developmental processes and stress responses. However, knowledge of the NAC TFs in rose (Rosa chinensis), one of the most important horticultural crops, is limited. In this study, 116 NAC genes were identified from the rose genome and classified into 16 subfamilies based on protein phylogenetic analysis. Chromosomal mapping revealed that the RcNAC genes were unevenly distributed on the seven chromosomes of rose. Gene structure and motif analysis identified a total of ten conserved motifs, of which motifs 1-7 were highly conserved and present in most rose NACs, while motifs 8-10 were present only in a few subfamilies. Further study of the stress-related RcNACs based on the transcriptome data showed differences in the expression patterns among the organs, at various floral developmental stages, and under drought and salt stress in rose leaves and roots. The stress-related RcNACs possessed cis-regulatory elements (CREs) categorized into three groups corresponding to plant growth and development, phytohormone response, and abiotic and biotic stress response. Reverse transcription-quantitative real-time PCR (RT-qPCR) analysis of 11 representative RcNACs revealed their differential expression in rose leaves and roots under abscisic acid (ABA), polyethylene glycol (PEG), and sodium chloride (NaCl) treatments. Furthermore, the silencing of RcNAC091 verified its role in positively regulating the dehydration stress response. Overall, the present study provides valuable insights into stress-related RcNACs and paves the way for stress tolerance in rose.

The tomato OST1-VOZ1 module regulates drought-mediated flowering.

Flowering is a critical agricultural trait that substantially affects tomato fruit yield. Although drought stress influences flowering time, the molecular mechanism underlying drought-regulated flowering in tomato remains elusive. In this study, we demonstrated that loss of function of tomato OPEN STOMATA 1 (SlOST1), a protein kinase essential for abscisic acid (ABA) signaling and abiotic stress responses, lowers the tolerance of tomato plants to drought stress. slost1 mutants also exhibited a late flowering phenotype under both normal and drought stress conditions. We also established that SlOST1 directly interacts with and phosphorylates the NAC (NAM, ATAF and CUC)-type transcription factor VASCULAR PLANT ONE-ZINC FINGER 1 (SlVOZ1), at residue serine 67, thereby enhancing its stability and nuclear translocation in an ABA-dependent manner. Moreover, we uncovered several SlVOZ1 binding motifs from DNA affinity purification sequencing analyses and revealed that SlVOZ1 can directly bind to the promoter of the major flowering-integrator gene SINGLE FLOWER TRUSS to promote tomato flowering transition in response to drought. Collectively, our data uncover the essential role of the SlOST1-SlVOZ1 module in regulating flowering in response to drought stress in tomato and offer insights into a novel strategy to balance drought stress response and flowering.

Effects of two vectors on the expression of the NbNAC1 transcription factor and preparation of its polyclonal antibody

The NAC (NAM, ATAF, and CUC) superfamily is one of the largest plant-specific families containing transcription factors. An increasing number of studies suggest that NAC1 is involved in plants response to different biotic and abiotic stimulis. Nicotiana benthamiana is a widely used system for evaluating plant-pathogen interactions. In order to study the biochemical function of NbNAC1, NbNAC1 protein and antibody are essential. Therefore, we focused on developing a prokaryotic expression system for producing the Nicotiana benthamiana NbNAC1 protein of in Escherichia coli and the preparation of its polyclonal antibody. Firstly, we constructed two different molecular weight prokaryotic expression vectors: pGE vector with GST tag (pGEX4T-1–NbNAC1) and pET expression vector with His tag (pET28a-NbNAC1). The NbNAC1 protein can be successfully expressed in both vectors. The His-tagged NbNAC1 proteins are insoluble, while the GST-tagged NbNAC1 proteins are partially soluble. We then successfully purified and enriched both proteins. The His-tagged NbNAC1 was chosen to immunize rabbits owing to an unknown protein accompanying the GST-tagged NbNAC1. The anti-NbNAC1 polyclonal antibody had good specificity and could be used in subsequent protein-related studies.

Integrative Proteomic and Phosphoproteomic Analyses of Pattern- and Effector-Triggered Immunity in Tomato.

Plants have evolved a two-layered immune system consisting of pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). PTI and ETI are functionally linked, but also have distinct characteristics. Unraveling how these immune systems coordinate plant responses against pathogens is crucial for understanding the regulatory mechanisms underlying plant defense. Here we report integrative proteomic and phosphoproteomic analyses of the tomato-Pseudomonas syringae (Pst) pathosystem with different Pst mutants that allow the dissection of PTI and ETI. A total of 225 proteins and 79 phosphopeptides differentially accumulated in tomato leaves during Pst infection. The abundances of many proteins and phosphoproteins changed during PTI or ETI, and some responses were triggered by both PTI and ETI. For most proteins, the ETI response was more robust than the PTI response. The patterns of protein abundance and phosphorylation changes revealed key regulators involved in Ca2+ signaling, mitogen-activated protein kinase cascades, reversible protein phosphorylation, reactive oxygen species (ROS) and redox homeostasis, transcription and protein turnover, transport and trafficking, cell wall remodeling, hormone biosynthesis and signaling, suggesting their common or specific roles in PTI and/or ETI. A NAC (NAM, ATAF, and CUC family) domain protein and lipid particle serine esterase, two PTI-specific genes identified from previous transcriptomic work, were not detected as differentially regulated at the protein level and were not induced by PTI. Based on integrative transcriptomics and proteomics data, as well as qRT-PCR analysis, several potential PTI and ETI-specific markers are proposed. These results provide insights into the regulatory mechanisms underlying PTI and ETI in the tomato-Pst pathosystem, and will promote future validation and application of the disease biomarkers in plant defense.

ZmSNAC13, a maize NAC transcription factor conferring enhanced resistance to multiple abiotic stresses in transgenic Arabidopsis

Abiotic stress is the main factor that severely limits crop growth and yield. NAC (NAM, ATAF1/2 and CUC2) transcription factors play an important role in dealing with various abiotic stresses. Here, we discovered the ZmSNAC13 gene in drought-tolerant maize lines by RNA-seq analysis and verified its function in Arabidopsis thaliana. First, its gene structure showed that ZmSNAC13 had a typical NAC domain and a highly variable C-terminal. There were multiple cis-acting elements related to stress in its promoter region. Overexpression of ZmSNAC13 resulted in enhanced tolerances to drought and salt stresses in Arabidopsis, characterized by a reduction in the water loss rate, a sustained effective photosynthesis rate, and increased cell membrane stability in leaves under drought conditions. Transcriptome analysis showed that a large number of differentially expressed genes regulated by overexpression of ZmSNAC13 were identified, and the main drought tolerance regulatory pathways involved were the ABA pathway and MAPK cascade signaling pathway. Overexpression of ZmSNAC13 promoted the expression of genes, such as PYL9 and DREB3, thereby enhancing tolerance to adverse environments. Adaptability, while restraining genes expression such as WRKY53 and MPK3, facilitates regulation of senescence in Arabidopsis and improves plant responses to adversity. Therefore, ZmSNAC13 is promising gene of interest for use in transgenic breeding to improve abiotic stress tolerance in crops.

Genome-Wide Identification and Analysis of NAC Transcription Factor Family in Two Diploid Wild Relatives of Cultivated Sweet Potato Uncovers Potential NAC Genes Related to Drought Tolerance.

NAC (NAM, ATAF1/2, and CUC2) proteins play a pivotal role in modulating plant development and offer protection against biotic and abiotic stresses. Until now, no systematic knowledge of NAC family genes is available for the food security crop, sweet potato. Here, a comprehensive genome-wide survey of NAC domain-containing proteins identified 130 ItbNAC and 144 ItfNAC genes with full length sequences in the genomes of two diploid wild relatives of cultivated sweet potato, Ipomoea triloba and Ipomoea trifida, respectively. These genes were physically mapped onto 15 I. triloba and 16 I. trifida chromosomes, respectively. Phylogenetic analysis divided all 274 NAC proteins into 20 subgroups together with NAC transcription factors (TFs) from Arabidopsis. There were 9 and 15 tandem duplication events in the I. triloba and I. trifida genomes, respectively, indicating an important role of tandem duplication in sweet potato gene expansion and evolution. Moreover, synteny analysis suggested that most NAC genes in the two diploid sweet potato species had a similar origin and evolutionary process. Gene expression patterns based on RNA-Seq data in different tissues and in response to various hormone, biotic or abiotic treatments revealed their possible involvement in organ development and response to various biotic/abiotic stresses. The expression of 36 NAC TFs, which were upregulated in the five tissues and in response to mannitol treatment, was also determined by real-time quantitative polymerase chain reaction (RT-qPCR) in hexaploid cultivated sweet potato exposed to drought stress. Those results largely corroborated the expression profile of mannitol treatment uncovered by the RNA-Seq data. Some significantly up-regulated genes related to drought stress, such as ItbNAC110, ItbNAC114, ItfNAC15, ItfNAC28, and especially ItfNAC62, which had a conservative spatial conformation with a closely related paralogous gene, ANAC019, may be potential candidate genes for a sweet potato drought tolerance breeding program. This analysis provides comprehensive and systematic information about NAC family genes in two diploid wild relatives of cultivated sweet potato, and will provide a blueprint for their functional characterization and exploitation to improve the tolerance of sweet potato to abiotic stresses.

Genome-Wide Identification and Analysis of NAC Transcription Factor Family in Two Diploid Wild Relatives of Cultivated Sweet Potato Uncovers Potential NAC Genes Related to Drought Tolerance

NAC (NAM, ATAF1/2, and CUC2) proteins play a pivotal role in modulating plant development and offer protection against biotic and abiotic stresses. Until now, no systematic knowledge of NAC family genes is available for the food security crop, sweet potato. Here, a comprehensive genome-wide survey of NAC domain-containing proteins identified 130 ItbNAC and 144 ItfNAC genes with full length sequences in the genomes of two diploid wild relatives of cultivated sweet potato, Ipomoea triloba and Ipomoea trifida, respectively. These genes were physically mapped onto 15 I. triloba and 16 I. trifida chromosomes, respectively. Phylogenetic analysis divided all 274 NAC proteins into 20 subgroups together with NAC transcription factors (TFs) from Arabidopsis. There were 9 and 15 tandem duplication events in the I. triloba and I. trifida genomes, respectively, indicating an important role of tandem duplication in sweet potato gene expansion and evolution. Moreover, synteny analysis suggested that most NAC genes in the two diploid sweet potato species had a similar origin and evolutionary process. Gene expression patterns based on RNA-Seq data in different tissues and in response to various hormone, biotic or abiotic treatments revealed their possible involvement in organ development and response to various biotic/abiotic stresses. The expression of 36 NAC TFs, which were upregulated in the five tissues and in response to mannitol treatment, was also determined by real-time quantitative polymerase chain reaction (RT-qPCR) in hexaploid cultivated sweet potato exposed to drought stress. Those results largely corroborated the expression profile of mannitol treatment uncovered by the RNA-Seq data. Some significantly up-regulated genes related to drought stress, such as ItbNAC110, ItbNAC114, ItfNAC15, ItfNAC28, and especially ItfNAC62, which had a conservative spatial conformation with a closely related paralogous gene, ANAC019, may be potential candidate genes for a sweet potato drought tolerance breeding program. This analysis provides comprehensive and systematic information about NAC family genes in two diploid wild relatives of cultivated sweet potato, and will provide a blueprint for their functional characterization and exploitation to improve the tolerance of sweet potato to abiotic stresses.

Genome-Wide Identification of NAC Transcription Factor Family in Juglans mandshurica and Their Expression Analysis during the Fruit Development and Ripening.

The NAC (NAM, ATAF and CUC) gene family plays a crucial role in the transcriptional regulation of various biological processes and has been identified and characterized in multiple plant species. However, genome-wide identification of this gene family has not been implemented in Juglans mandshurica, and specific functions of these genes in the development of fruits remain unknown. In this study, we performed genome-wide identification and functional analysis of the NAC gene family during fruit development and identified a total of 114 JmNAC genes in the J. mandshurica genome. Chromosomal location analysis revealed that JmNAC genes were unevenly distributed in 16 chromosomes; the highest numbers were found in chromosomes 2 and 4. Furthermore, according to the homologues of JmNAC genes in Arabidopsis thaliana, a phylogenetic tree was constructed, and the results demonstrated 114 JmNAC genes, which were divided into eight subgroups. Four JmNAC gene pairs were identified as the result of tandem duplicates. Tissue-specific analysis of JmNAC genes during different developmental stages revealed that 39 and 25 JmNAC genes exhibited upregulation during the mature stage in walnut exocarp and embryos, indicating that they may serve key functions in fruit development. Furthermore, 12 upregulated JmNAC genes were common in fruit ripening stage in walnut exocarp and embryos, which demonstrated that these genes were positively correlated with fruit development in J. mandshurica. This study provides new insights into the regulatory functions of JmNAC genes during fruit development in J. mandshurica, thereby improving the understanding of characteristics and evolution of the JmNAC gene family.

Silencing GhJUB1L1 (JUB1-like 1) reduces cotton (Gossypium hirsutum) drought tolerance.

Drought stress massively restricts plant growth and the yield of crops. Reducing the deleterious effects of drought is necessary for agricultural industry. The plant-specific NAC (NAM, ATAF1/2 and CUC2) transcription factors (TFs) are widely involved in the regulation of plant development and stress response. One of the NAC TF, JUNGBRUNNEN1 (JUB1), has been reported to involve in drought resistance in Arabidopsis. However, little is known of how the JUB1 gene respond to drought stress in cotton. In the present study, we cloned GhJUB1L1, a homologous gene of JUB1 in upland cotton. GhJUB1L1 is preferentially expressed in stem and leaf and could be induced by drought stress. GhJUB1L1 protein localizes to the cell nucleus, and the transcription activation region of which is located in the C-terminal region. Silencing GhJUB1L1 gene via VIGS () reduced cotton drought tolerance, and retarded secondary cell wall (SCW) development. Additionally, the expression of some drought stress-related genes and SCW synthesis-related genes were altered in the GhJUB1L1 silencing plants. Collectively, our findings indicate that GhJUB1L1 may act as a positive regulator in response to drought stress and SCW development in cotton. Our results enriched the roles of NAC TFs in cotton drought tolerance and laid a foundation for the cultivation of transgenic cotton with higher drought tolerance.

A tomato NAC transcription factor, SlNAM1, positively regulates ethylene biosynthesis and the onset of tomato fruit ripening.

Fruit ripening in tomato (Solanum lycopersicum) is the result of selective expression of ripening-related genes, which are regulated by transcription factors (TFs). The NAC (NAM, ATAF1/2, and CUC2) TF family is one of the largest families of plant-specific TFs and members are involved in a variety of plant physiological activities, including fruit ripening. Fruit ripening-associated NAC TFs studied in tomato to date include NAC-NOR (non-ripening), SlNOR-like1 (non-ripening like1), SlNAC1, and SlNAC4. Considering the large number of NAC genes in the tomato genome, there is little information about the possible roles of other NAC members in fruit ripening, and research on their target genes is lacking. In this study, we characterize SlNAM1, a NAC TF, which positively regulates the initiation of tomato fruit ripening via its regulation of ethylene biosynthesis. The onset of fruit ripening in slnam1-deficient mutants created by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9) technology was delayed, whereas fruit ripening in OE-SlNAM1 lines was accelerated compared with the wild type. The results of RNA-sequencing (RNA-seq) and promoter analysis suggested that SlNAM1 directly binds to the promoters of two key ethylene biosynthesis genes (1-aminocyclopropane-1-carboxylate synthase: SlACS2 and SlACS4) and activates their expression. This hypothesis was confirmed by electrophoretic mobility shift assays and dual-luciferase reporter assay. Our findings provide insights into the mechanisms of ethylene production and enrich understanding of the tomato fruit ripening regulatory network.

Genome-wide identification and comprehensive analysis of the NAC transcription factor family in sunflower during salt and drought stress

The NAC (NAM, ATAF1/2, and CUC2), is a large family of plant-specific transcription factors (TFs) that exert crucial regulatory roles in various physiological processes and abiotic stresses. There is scanty information on the role of the NAC family in sunflower (Helianthus annuus L.). In this study, we conducted a genome-wide survey and expression analysis of the NAC family in sunflower. A total of 150 HaNACs were identified in sunflower. Phylogenetic analysis to compare HaNACs with Arabidopsis NACs generated 15 clusters. Among them, eight membrane-bound NAC TFs with transmembrane helixes were found (designated as NTLs), which were suggested to be localized in the membrane and transferred to the nucleus through proteolysis. Notably, 12 HaNACs were potentially regulated via miR164 cleavage or translational inhibition. By analyzing RNA-seq data from Sequence Read Archive (SRA), the expression of HaNACs showed tissue specificity and strong response to drought stress. Additionally, phylogenetic analysis of 150 HaNACs with the previously reported NACs related to abiotic stress revealed that 75% of the abiotic stress-related NACs were clustered into the SNAC (abiotic stress-related NAC) group, and only 25% were in the Non-SNAC group. qRT-PCR further demonstrated that about 75% of the HaNACs in the SNAC subgroup were induced by salt and drought stress, and the expression of some HaNACs showed tissue specificity. These findings provide valuable information that can deepen the understanding of how NAC TFs in sunflower respond to abiotic stress.

The NAC transcription factor ClNAC68 positively regulates sugar content and seed development in watermelon by repressing ClINV and ClGH3.6

NAC (NAM, ATAF1/2, and CUC2) transcription factors play important roles in fruit ripening and quality. The watermelon genome encodes 80 NAC genes, and 21 of these NAC genes are highly expressed in both the flesh and vascular tissues. Among these genes, ClNAC68 expression was significantly higher in flesh than in rind. However, the intrinsic regulatory mechanism of ClNAC68 in fruit ripening and quality is still unknown. In this study, we found that ClNAC68 is a transcriptional repressor and that the repression domain is located in the C-terminus. Knockout of ClNAC68 by the CRISPR-Cas9 system decreased the soluble solid content and sucrose accumulation in mutant flesh. Development was delayed, germination was inhibited, and the IAA content was significantly decreased in mutant seeds. Transcriptome analysis showed that the invertase gene ClINV was the only gene involved in sucrose metabolism that was upregulated in mutant flesh, and expression of the indole-3-acetic acid-amido synthetase gene ClGH3.6 in the IAA signaling pathway was also induced in mutant seeds. EMSA and dual-luciferase assays showed that ClNAC68 directly bound to the promoters of ClINV and ClGH3.6 to repress their expression. These results indicated that ClNAC68 positively regulated sugar and IAA accumulation by repressing ClINV and ClGH3.6. Our findings provide new insights into the regulatory mechanisms by which NAC transcription factors affect fruit quality and seed development.

SbNAC2 enhances abiotic stress tolerance by upregulating ROS scavenging activities and inducing stress-response genes in sorghum

Major abiotic stresses such as drought and salinity affect the growth and yield of crops. NAC (NAM, ATAF1/2, and CUC2) is a plant-specific gene family that has been demonstrated to contribute to responses and adaption to abiotic stress in plants. The specific NAC transcription factors (TFs) that play important roles in the response to abiotic stress in sorghum are unknown. Here, we characterized a sorghum TF, SbNAC2, that can be strongly induced by abiotic stresses, including polyethylene glycol (PEG)-simulated dehydration stress treatment, salt treatment, and 4 °C chilling. Knockdown of SbNAC2 in sorghum seedlings by virus-induced gene silencing (VIGS) weakened the drought tolerance of plants. Heterologous overexpression of SbNAC2 in Arabidopsis and rice can induce plant antioxidant enzyme activity under abiotic stress conditions. Furthermore, SbNAC2 directly activates the expression of a stress responsive AP2/ERF type TF gene, SbAP37, by binding to the TTNCGTA motif of its promoter. These results indicate that SbNAC2 functions as a potentially important regulator in response to abiotic stresses and that it may possibly be used for molecular breeding to enhance crop yields under adverse environmental conditions.

Transcriptomics Analysis of Heat Stress-Induced Genes in Pepper (Capsicum annuum L.) Seedlings

Pepper (Capsicum annuum L.) is one of the most economically important crops worldwide. Heat stress (HS) can significantly reduce pepper yield and quality. However, changes at a molecular level in response to HS and the subsequent recovery are poorly understood. In this study, 17-03 and H1023 were identified as heat-tolerant and heat-sensitive varieties, respectively. Their leaves’ transcript abundance was quantified using RNA sequencing to elucidate the effect of HS and subsequent recovery on gene expression. A total of 11,633 differentially expressed genes (DEGs) were identified, and the differential expression of 14 randomly selected DEGs was validated using reverse-transcription polymerase chain reaction. Functional enrichment analysis revealed that the most enriched pathways were metabolic processes under stress and photosynthesis and light harvesting during HS and after recovery from HS. The most significantly enriched pathways of 17-03 and H1023 were the same under HS, but differed during recovery. Furthermore, we identified 38 heat shock factors (Hsps), 17 HS transcription factors (Hsfs) and 38 NAC (NAM, ATAF1/2, and CUC2), and 35 WRKY proteins that were responsive to HS or recovery. These findings facilitate a better understanding of the molecular mechanisms underlying HS and recovery in different pepper genotypes.

Identification and Characterization of Secondary Wall-Associated NAC Genes and Their Involvement in Hormonal Responses in Tobacco (Nicotiana tabacum).

Secondary wall-associated NAC (SWN) genes are a subgroup of NAC (NAM, ATAF, and CUC) transcription factors (TF) that play a key role in regulating secondary cell wall biosynthesis in plants. However, this gene family has not been systematically characterized, and their potential roles in response to hormones are unknown in Nicotiana tabacum. In this study, a total of 40 SWN genes, of which 12 from Nicotiana tomentosiformis, 13 from Nicotiana sylvestris, and 15 from Nicotiana tabacum, were successfully identified. The 15 SWNs from Nicotiana tabacum were further classified into three groups, namely, vascular-related NAC domain genes (NtVNDs), NAC secondary wall thickening promoting factor genes (NtNSTs), and secondary wall-associated NAC domain genes (NtSNDs). The protein characteristic, gene structure, and chromosomal location of 15 NtSWNs (also named Nt1 to Nt15) were also analyzed. The NtVND and NtNST group genes had five conserved subdomains in their N-terminal regions and a motif (LP[Q/x] L[E/x] S[P/A]) in their diverged C- terminal regions. Some hormones, dark and low-temperature related cis-acting elements, were significantly enriched in the promoters of NtSWN genes. A comprehensive expression profile analysis revealed that Nt4 and Nt12 might play a role in vein development. Others might be important for stem development. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) revealed that in the NtNST group, genes such as Nt7, Nt8, and Nt13 were more sensitive than the genes in NtVND and NtSND groups under abiotic stress conditions. A transactivation assay further suggested that Nt7, Nt8, and Nt13 showed a significant transactivation activity. Overall, SWN genes were finally identified and characterized in diploid and tetraploid tobacco, revealing new insights into their evolution, variation, and homology relationships. Transcriptome, cis-acting element, qRT-PCR, and transactivation assay analysis indicated the roles in hormonal and stress responses, which provided further resources in molecular mechanism and genetic improvement.

Temporal expression profiling of GhNAC transcription factor genes in cotton cultivars under abiotic stresses

Gossypium sp. is a significant natural fiber source for the textile industry around the world. The crop yield is unfavorably affected as a result of various stress factors. The plant-specific NAC (NAM, ATAF and CUC) proteins form a major transcription factor (TF) family prominent for their roles in diverse developmental processes. We examined cotton NAC TF within the plant TF database, wherein we found 18 distinctive phylogenetically segregated subfamilies comprising 78 NAC domain-containing proteins. Here, we report previously uncharacterized cotton NAC transcription factors which are diversified structurally. Six upland cotton cultivars were selected for the study and a quantitative RT-PCR shown the expressions of 17 GhNAC genes in leaves and roots which were regulated in cold, heat, salt and drought stresses. In cold stress, the expression profile of GhNAC2 showed more than 100-log fold increase in all the cultivars studied and subsequent analysis revealed that GhNAC9 was found to be highly expressed during the heat stress among all the cultivars. GhNAC15 expression during salinity recorded more than 100-log fold increase in all the cultivars except KC-3. Genes GhNAC2, GhNAC9, and GhNAC11 were found to be upregulated during drought stress induced by mannitol and dehydration. These results confirmed that reputed functions of several GhNAC TFs, which in turn will contribute towards for their functional characterization and applications for genetic and agricultural cotton crop improvement.

Characterization of NAC family genes in Salvia miltiorrhiza and NAC2 potentially involved in the biosynthesis of tanshinones

The NAC (NAM, ATAF, and CUC) family members are specific transcription factors in plants. The large family is involved in many plant growth and developmental processes, as well as in abiotic/biotic stress responses. It has been well studied in the genomes of various plants, including Arabidopsis thaliana, tomato, and quinoa. However, identification and functional studies of NAC family members in medicinal Salvia miltiorrhiza are limited. Here, we systematically identified 84 NAC genes and named them according to their gene IDs in the recently sequenced genome. The phylogeny of NAC family protein sequences was analyzed using bioinformatics methods, which divided them into nine subfamilies. Then, their chromosomal locations, gene structures and conserved domains were analyzed comprehensively. To further investigate the regulatory functions of NACs in S. miltiorrhiza, we analyzed the response of 10 selected NAC genes to methyl jasmonate and used NAC2 for transgenic experiments. The overexpression of Sm-NAC2 decreased the tanshinone I and IIA contents by 56% and 62%, respectively. However, Sm-NAC2-RNAi promoted the accumulation of four tanshinones, tanshinone I, tanshinone IIA, cryptotanshinone, and dihydrotanshinone I, which increased 3.68-, 4.1-, 3.13- and 5.9- fold, respectively, compared with wild type. In the tanshinone biosynthetic pathways, the overexpression of Sm-NAC2 down-regulated CYP76AH1, and the silencing of Sm-NAC2 up-regulated the expression levels of HMGR1, DXS2, KSL2, and CYP76AH1. This study provides information on the evolution of Sm-NAC genes and their possible functions, and it lays a foundation for further research into the NAC family-associated regulation of tanshinone biosynthesis.

Senescence-Associated Glycine max (Gm)NAC Genes: Integration of Natural and Stress-Induced Leaf Senescence.

Leaf senescence is a genetically regulated developmental process that can be triggered by a variety of internal and external signals, including hormones and environmental stimuli. Among the senescence-associated genes controlling leaf senescence, the transcriptional factors (TFs) comprise a functional class that is highly active at the onset and during the progression of leaf senescence. The plant-specific NAC (NAM, ATAF, and CUC) TFs are essential for controlling leaf senescence. Several members of Arabidopsis AtNAC-SAGs are well characterized as players in elucidated regulatory networks. However, only a few soybean members of this class display well-known functions; knowledge about their regulatory circuits is still rudimentary. Here, we describe the expression profile of soybean GmNAC-SAGs upregulated by natural senescence and their functional correlation with putative AtNAC-SAGs orthologs. The mechanisms and the regulatory gene networks underlying GmNAC081- and GmNAC030-positive regulation in leaf senescence are discussed. Furthermore, new insights into the role of GmNAC065 as a negative senescence regulator are presented, demonstrating extraordinary functional conservation with the Arabidopsis counterpart. Finally, we describe a regulatory circuit which integrates a stress-induced cell death program with developmental leaf senescence via the NRP-NAC-VPE signaling module.

Genome-wide Identification and Expression Analysis of NAC Transcription Factor Family Genes during Fruit and Kernel Development in Siberian Apricot

The NAC (NAM, ATAF1/2, and CUC2) family is a group of plant-specific transcription factors that have vital roles in the growth and development of plants, and especially in fruit and kernel development. This study aimed to identify members of the NAC gene (PsNACs) family and investigate their functions in siberian apricot (Prunus sibirica). A total of 102 predicted PsNAC proteins (PsNACs) were divided into 14 clades and the genes were mapped to the eight chromosomes in siberian apricot. The PsNACs of the same clade had similar structures. A synteny analysis showed that the PsNACs had close relationships with the NAC genes of japanese apricot (Prunus mume). An expression pattern analysis of the PsNACs revealed many differences in various tissues and at different stages of fruit and kernel development. All eight PsNACs in clade XI have crucial roles in fruit and kernel development. Seven PsNACs (PsNACs 18, 64, 23, 33, 9, 4, and 50) in clades I, III, VI, VII, and XIII are related to fruit development. Eight PsNACs (PsNACs 6, 13, 46, 51, 41, 67, 37, and 59) in clades I, II, V, VIII, and XIII are involved in fruit ripening. Five PsNACs (PsNACs 6, 94, 41, 32, and 17) in clades I, IV, V, VII, and XI regulated the rapid growth of the kernel. Four PsNACs (PsNACs 50, 4, 67, and 84) in clades I, III, V, and XIII affected the hardening of the kernel. Four PsNACs (PsNACs 17, 82, 13, and 51) in clades II, XI, and IX acted on kernel maturation. We have characterized the NAC genes in siberian apricot during this study. Our results will provide resources for future research of the biological roles of PsNACs in fruit and kernel development in siberian apricot.

NAC Transcription Factor PwNAC11 Activates ERD1 by Interaction with ABF3 and DREB2A to Enhance Drought Tolerance in Transgenic Arabidopsis

NAC (NAM, ATAF1/2, and CUC2) transcription factors are ubiquitously distributed in eukaryotes and play significant roles in stress response. However, the functional verifications of NACs in Picea (P.) wilsonii remain largely uncharacterized. Here, we identified the NAC transcription factor PwNAC11 as a mediator of drought stress, which was significantly upregulated in P. wilsonii under drought and abscisic acid (ABA) treatments. Yeast two-hybrid assays showed that both the full length and C-terminal of PwNAC11 had transcriptional activation activity and PwNAC11 protein cannot form a homodimer by itself. Subcellular observation demonstrated that PwNAC11 protein was located in nucleus. The overexpression of PwNAC11 in Arabidopsis obviously improved the tolerance to drought stress but delayed flowering time under nonstress conditions. The steady-state level of antioxidant enzymes’ activities and light energy conversion efficiency were significantly increased in PwNAC11 transgenic lines under dehydration compared to wild plants. PwNAC11 transgenic lines showed hypersensitivity to ABA and PwNAC11 activated the expression of the downstream gene ERD1 by binding to ABA-responsive elements (ABREs) instead of drought-responsive elements (DREs). Genetic evidence demonstrated that PwNAC11 physically interacted with an ABA-induced protein—ABRE Binding Factor3 (ABF3)—and promoted the activation of ERD1 promoter, which implied an ABA-dependent signaling cascade controlled by PwNAC11. In addition, qRT-PCR and yeast assays showed that an ABA-independent gene—DREB2A—was also probably involved in PwNAC11-mediated drought stress response. Taken together, our results provide the evidence that PwNAC11 plays a dominant role in plants positively responding to early drought stress and ABF3 and DREB2A synergistically regulate the expression of ERD1.