NAM/ATAF/CUC Research Articles

A heat stress responsive NAC transcription factor heterodimer plays key roles in rice grain filling.

High temperature often leads to failure of grain filling in rice (Oryza sativa) causing yield loss, but the underlying mechanisms are still not elucidated. Here, we report that two genes encoding seed-specific NAM/ATAF/CUC (NAC) domain transcription factors, ONAC127 and ONAC129, are responsive to heat stress and involved in the grain filling process of rice. ONAC127 and ONAC129 are dominantly expressed in the pericarp and can form a heterodimer during rice grain filling. CRISPR/Cas9 induced mutants and overexpression lines were then generated to investigate the function of these two transcription factors. Interestingly, both knock-out and overexpression plants showed incomplete grain filling and shrunken grains, which became more severe under heat stress. Transcriptome analysis revealed that ONAC127 and ONAC129 mainly regulate stimulus response and nutrient transport. ChIP-seq analysis identified that the direct target genes of ONAC127 and ONAC129 in developing rice seeds include monosaccharide transporter gene OsMST6, sugar transporter gene OsSWEET4, calmodulin-like protein gene OsMSR2 and AP2/ERF factor gene OsEATB. These results suggest that ONAC127 and ONAC129 regulate grain filling by affecting sugar transportation and abiotic stress responses. Overall, this study demonstrates a transcriptional regulatory network with ONAC127 and ONAC129 coordinating multiple pathways to modulate seed development and heat stress responses at rice reproductive stages.

Ectopic overexpression of a membrane-tethered transcription factor gene NAC60 from oilseed rape positively modulates programmed cell death and age-triggered leaf senescence.

Senescence is an integrative final stage of plant development that is governed by internal and external cues. The NAM, ATAF1/2, CUC2 (NAC) transcription factor (TF) family is specific to plants and membrane-tethered NAC TFs (MTTFs) constitute a unique and sophisticated mechanism in stress responses and development. However, the function of MTTFs in oilseed rape (Brassica napus L.) remains unknown. Here, we report that BnaNAC60 is an MTTF associated with the endoplasmic reticulum (ER) membrane. Expression of BnaNAC60 was induced during the progression of leaf senescence. Translocation of BnaNAC60 into nuclei was induced by ER stress and oxidative stress treatments. It binds to the NTLBS motif, rather than the canonical NAC recognition site. Overexpression of BnaNAC60 devoid of the transmembrane domain, but not the full-length BnaNAC60, induces significant reactive oxygen species (ROS) accumulation and hypersensitive response-like cell death in both tobacco (Nicotiana benthamiana) and oilseed rape protoplasts. Moreover, ectopic overexpression of BnaNAC60 devoid of the transmembrane domain, but not the full-length BnaNAC60, in Arabidopsis also induces precocious leaf senescence. Furthermore, screening and expression profiling identified an array of functional genes that are significantly induced by BnaNAC60 expression. Further it was found that BnaNAC60 can activate the promoter activities of BnaNYC1, BnaRbohD, BnaBFN1, BnaZAT12, and multiple BnaVPEs in a dual-luciferase reporter assay. Electrophoretic mobility shift assay and chromatin immunoprecipitation coupled to quantitative PCR assays revealed that BnaNAC60 directly binds to the promoter regions of these downstream target genes. To summarize, our data show that BnaNAC60 is an MTTF that modulates cell death, ROS accumulation, and leaf senescence.

Transgenic soybean overexpressing GmNAC085 enhanced expression of important genes under salinity stress condition

Climate change has made abiotic stresses such as drought and salinity bigger threats to ecosystem and global food security. In response to unfavorable conditions, physiological, biochemical and molecular activities in plants are altered. Particularly, various members of NAC (NAM, ATAF1/2, CUC2) transcriptional factor family have been reported to be the key regulators in modulating multiple biological processes of plant responses to osmotic stress conditions caused by drought and salinity. Previously, transgenic studies have shown the positive regulatory role of GmNAC085, a NAC transcription factor from soybean (Glycine max), in plant resistance against drought stress. Therefore, in this study, we extended the investigation on its contribution to salinity stress. According to our RT-qPCR analyses, expression of several important stress-related genes was significantly induced in the transgenic soybean plants in comparison with the wild-type plants, including antioxidant enzyme-encoding genes (GmSOD, GmAPX and GmCAT), sodium-proton antiporter encoding gene (GmNHX1) and proline metabolic gene (GmP5CS). Furthermore, biochemical results were also in agreement with the molecular data, with enhanced antioxidant enzyme activities of peroxidase and catalase, and in couple with lower cellular content of the reactive oxygen species hydrogen peroxide in the transgenic plants. Taken these altogether, the transgenic plants might acquire advantages in dealing with oxidative stress using enzymes and proline, as well as cellular Na+ removal under salinity stress conditions. Therefore, underlying mechanisms of GmNAC085 associated with salinity stress should be fully elaborated to find out its potential utility in crop improvement.

Strawberry FaNAC2 Enhances Tolerance to Abiotic Stress by Regulating Proline Metabolism.

The quality and yields of strawberry plants are seriously affected by abiotic stress every year. NAC (NAM, ATAF, CUC) transcription factors are plant-specific, having various functions in plant development and response to stress. In our study, FaNAC2 from strawberry (Fragaria × ananassa, cultivar “Benihoppe”) was isolated and found to be a member of the ATAF sub-family, belonging to the NAC family of transcription factors. FaNAC2 was strongly expressed in the shoot apical meristem and older leaves of strawberries, and was induced by cold, high salinity, and drought stress. To investigate how FaNAC2 functions in plant responses to abiotic stress, transgenic Nicotiana benthamiana plants ectopically overexpressing FaNAC2 were generated. The transgenic plants grew better under salt and cold stress, and, during simulated drought treatment, these transgenic lines not only grew better, but also showed higher seed germination rates than wild-type plants. Gene expression analysis revealed that key genes in proline biosynthesis pathways were up-regulated in FaNAC2 overexpression lines, while its catabolic pathway genes were down-regulated and proline was accumulated more with the overexpression of FaNAC2 after stress treatments. Furthermore, the gene expression of abscisic acid biosynthesis was also promoted. Our results demonstrate that FaNAC2 plays an important positive role in response to different abiotic stresses and may be further utilized to improve the stress tolerance of strawberry plants.

Genome-Wide Analysis of the Role of NAC Family in Flower Development and Abiotic Stress Responses in Cleistogenes songorica.

Plant-specific NAC (NAM, ATAF, CUC) transcription factor (TF) family plays important roles in biological processes such as plant growth and response to stress. Nevertheless, no information is known about NAC TFs in Cleistogenes songorica, a prominent xerophyte desert grass in northwestern China. In this study, 162 NAC genes were found from the Cleistogenes songorica genome, among which 156 C. songorica NAC (CsNAC) genes (96.3%) were mapped onto 20 chromosomes. The phylogenetic tree constructed by CsNAC and rice NAC TFs can be separated into 14 subfamilies. Syntenic and Ka/Ks analyses showed that CsNACs were primarily expanded by genomewide replication events, and purifying selection was the primary force driving the evolution of CsNAC family genes. The CsNAC gene expression profiles showed that 36 CsNAC genes showed differential expression between cleistogamous (CL) and chasmogamous (CH) flowers. One hundred and two CsNAC genes showed differential expression under heat, cold, drought, salt and ABA treatment. Twenty-three CsNAC genes were commonly differentially expressed both under stress responses and during dimorphic floret development. Gene Ontology (GO) annotation, coexpression network and qRT-PCR tests revealed that these CsNAC genes may simultaneously regulate dimorphic floret development and the response to stress. Our results may help to characterize the NAC transcription factors in C. songorica and provide new insights into the functional research and application of the NAC family in crop improvement, especially in dimorphic floret plants.

Comparative biochemical and transcriptome analysis provides insights into the regulatory mechanism of striped leaf albinism in arecanut (Areca catechu L.)

Frequent striped leaf albinism occurring in young seedlings leads to production loss of arecanut (Areca catechu L.) industry. Previous efforts has proved that this albinism phenotype was determined by genetic factors. However, the mechanism underlying albinism in arecanut remains unclear. In this study, to elucidate the regulatory mechanism of striped leaf albinism, comparison analysis was performed between leaves of normal and albinotic seedlings at physiological, biochemical and transcriptomic levels in arecanut. The albinotic leaves showed obvious degeneration in chloroplast, low chlorophyll content and reduced photosynthetic capacity. More than 5000 DEGs (differentially-expressed genes) was identified between normal and albinotic leaves. Among them, genes involved into the biosynthesis of chlorophyll, carotenoid and anthocyanin were significantly down-regulated in albinotic leaves. On the contrary, genes involved in leaf senescence and ABA (abscisic acid) synthesis were up-regulated in albinotic leaves. Unexpectedly, most genes involved in chloroplast development were up-regulated in albinotic leaves, indicating different regulatory mechanism of albinism in arecanut. Furthermore, transcription factor family genes, such as AP2/ERF-ERF (APETALA2/ethylene responsive factor), NAC (NAM/ATAF/CUC) and WRKY, were identified as DEGs between normal and albinotic leaves, which also indicated the senescence of albinotic leaves. The results indicated that the albinism mechanism of arecanut is different with those reported in other plants. Thus our data provides new insights into the mechanism underlying albinism. The genes related to albinism of arecanut identified in this study would be important targets for genetic modification or molecular markers development, which will be critical for improving the cultivation efficiency of this cash crop.

Transcriptome Analysis Reveals Potential Roles of Abscisic Acid and Polyphenols in Adaptation of Onobrychis viciifolia to Extreme Environmental Conditions in the Qinghai-Tibetan Plateau.

A detailed understanding of the molecular mechanisms of plant stress resistance in the face of ever-changing environmental stimuli will be helpful for promoting the growth and production of crop and forage plants. Investigations of plant responses to various single abiotic or biotic factors, or combined stresses, have been extensively reported. However, the molecular mechanisms of plants in responses to environmental stresses under natural conditions are not clearly understood. In this study, we carried out a transcriptome analysis using RNA-sequencing to decipher the underlying molecular mechanisms of Onobrychis viciifolia responding and adapting to the extreme natural environment in the Qinghai-Tibetan Plateau (QTP). The transcriptome data of plant samples collected from two different altitudes revealed a total of 8212 differentially expressed genes (DEGs), including 5387 up-regulated and 2825 down-regulated genes. Detailed analysis of the identified DEGs uncovered that up-regulation of genes potentially leading to changes in hormone homeostasis and signaling, particularly abscisic acid-related ones, and enhanced biosynthesis of polyphenols play vital roles in the adaptive processes of O. viciifolia. Interestingly, several DEGs encoding uridine diphosphate glycosyltransferases, which putatively regulate phytohormone homeostasis to resist environmental stresses, were also discovered. Furthermore, numerous DEGs encoding transcriptional factors, such as members of the myeloblastosis (MYB), homeodomain-leucine zipper (HD-ZIP), WRKY, and nam-ataf1,2-cuc2 (NAC) families, might be involved in the adaptive responses of O. viciifolia to the extreme natural environmental conditions. The DEGs identified in this study represent candidate targets for improving environmental stress resistance of O. viciifolia grown in higher altitudes of the QTP, and can provide deep insights into the molecular mechanisms underlying the responses of this plant species to the extreme natural environmental conditions of the QTP.

Multilayered Regulation of Membrane-Bound ONAC054 Is Essential for Abscisic Acid-Induced Leaf Senescence in Rice.

In most plants, abscisic acid (ABA) induces premature leaf senescence; however, the mechanisms of ABA signaling during leaf senescence remain largely unknown. Here, we show that the rice (Oryza sativa) NAM/ATAF1/2/CUC2 (NAC) transcription factor ONAC054 plays an important role in ABA-induced leaf senescence. The onac054 knockout mutants maintained green leaves, while ONAC054-overexpressing lines showed early leaf yellowing under dark- and ABA-induced senescence conditions. Genome-wide microarray analysis showed that ABA signaling-associated genes, including ABA INSENSITIVE5 (OsABI5) and senescence-associated genes, including STAY-GREEN and NON-YELLOW COLORING1 (NYC1), were significantly down-regulated in onac054 mutants. Chromatin immunoprecipitation and protoplast transient assays showed that ONAC054 directly activates OsABI5 and NYC1 by binding to the mitochondrial dysfunction motif in their promoters. ONAC054 activity is regulated by proteolytic processing of the C-terminal transmembrane domain (TMD). We found that nuclear import of ONAC054 requires cleavage of the putative C-terminal TMD. Furthermore, the ONAC054 transcript (termed ONAC054α) has an alternatively spliced form (ONAC054β), with seven nucleotides inserted between intron 5 and exon 6, truncating ONAC054α protein at a premature stop codon. ONAC054β lacks the TMD and thus localizes to the nucleus. These findings demonstrate that the activity of ONAC054, which is important for ABA-induced leaf senescence in rice, is precisely controlled by multilayered regulatory processes.

Genome-wide analysis of NAC transcription factors and their response to abiotic stress in celery (Apium graveolens L.)

In plants, NAC (NAM, ATAF, CUC) is a class of transcription factors (TFs) involved in growth regulation and is associated with abiotic stress, morphogenesis, and metabolism. Celery (Apium graveolens L.) is an important leafy vegetable and its yield and quality are considerably influenced by environmental factors. Currently, the characterization of NAC genes in celery is still limited. In this study, a total of 111 putative NAC TFs were determined based on the celery transcriptome and genome database. They were divided into 18 subfamilies on the basis of their NAC domain. NAC TFs in celery account for a moderate number compared with other species, similar to that in carrot. Real-time quantitative PCR (RT-qPCR) showed that some AgNAC genes were differentially expressed under adverse conditions (heat, cold, drought, and salt). AgNAC63 (ortholog of ANAC072/RD26) was highly induced by heat, cold, and salt conditions. The expression levels of AgNAC47 in leaf blades were 105.25- and 123.14-fold those of petioles and roots, respectively. AgNAC63 and AgNAC47 showed significant tissue specificity, high expression in leaves, and varying degrees of response under the four treatments. This study provides a basis for the improved investigation of the structure and function of AgNAC TFs and celery stress resistance.

Physiological and Transcriptional Responses of Industrial Rapeseed (Brassica napus) Seedlings to Drought and Salinity Stress

Abiotic stress greatly inhibits crop growth and reduces yields. However, little is known about the transcriptomic changes that occur in the industrial oilseed crop, rapeseed (Brassica napus), in response to abiotic stress. In this study, we examined the physiological and transcriptional responses of rapeseed to drought (simulated by treatment with 15% (w/v) polyethylene glycol (PEG) 6000) and salinity (150 mM NaCl) stress. Proline contents in young seedlings greatly increased under both conditions after 3 h of treatment, whereas the levels of antioxidant enzymes remained unchanged. We assembled transcripts from the leaves and roots of rapeseed and performed BLASTN searches against the rapeseed genome database for the first time. Gene ontology analysis indicated that DEGs involved in catalytic activity, metabolic process, and response to stimulus were highly enriched. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that differentially expressed genes (DEGs) from the categories metabolic pathways and biosynthesis of secondary metabolites were highly enriched. We determined that myeloblastosis (MYB), NAM/ATAF1-2/CUC2 (NAC), and APETALA2/ethylene-responsive element binding proteins (AP2-EREBP) transcription factors function as major switches that control downstream gene expression and that proline plays a role under short-term abiotic stress treatment due to increased expression of synthesis and decreased expression of degradation. Furthermore, many common genes function in the response to both types of stress in this rapeseed.

Ectopic Expression of Glycine maxGmNAC109 Enhances Drought Tolerance and ABA Sensitivity in Arabidopsis.

The NAC (NAM, ATAF1/2, CUC2) transcription factors are widely known for their various functions in plant development and stress tolerance. Previous studies have demonstrated that genetic engineering can be applied to enhance drought tolerance via overexpression/ectopic expression of NAC genes. In the present study, the dehydration- and drought-inducible GmNAC109 from Glycine max was ectopically expressed in Arabidopsis (GmNAC109-EX) plants to study its biological functions in mediating plant adaptation to water deficit conditions. Results revealed an improved drought tolerance in the transgenic plants, which displayed greater recovery rates by 20% to 54% than did the wild-type plants. In support of this finding, GmNAC109-EX plants exhibited lower water loss rates and decreased endogenous hydrogen peroxide production in leaf tissues under drought, as well as higher sensitivity to exogenous abscisic acid (ABA) treatment at germination and early seedling development stages. In addition, analyses of antioxidant enzymes indicated that GmNAC109-EX plants possessed stronger activities of superoxide dismutase and catalase under drought stress. These results together demonstrated that GmNAC109 acts as a positive transcriptional regulator in the ABA-signaling pathway, enabling plants to cope with adverse water deficit conditions.

Transcription factor CaNAC1 regulates low-temperature-induced phospholipid degradation in green bell pepper.

Phospholipids constitute the main component of biomembranes. During low-temperature storage and transportation of harvested bell peppers (Capsicum annuum), chilling injury participates in their decay. A primary cause of this chilling injury is phospholipid degradation. In this study, three genes encoding phospholipase D (PLD) were identified from bell peppers and their activities were examined under cold stress. Low temperature (4 °C) induced strong accumulation of the CaPLDα4 transcript, suggesting that it is associated with the phenomenon of phospholipid degradation and destruction of cell membranes. Low temperature also significantly induced increased amounts of NAM-ATAF1/2-CUC2 (NAC) domain transcription factors. CaNAC1 was found to interact with the promoter of CaPLD4 in a yeast one-hybrid screen. Electrophoretic mobility shift and ß-glucuronidase reporter assays demonstrated that CaNAC1 binds to the CTGCAG motif in the CaPLDα4 promoter, thereby activating its transcription and controlling phospholipid degradation. The ubiquitination sites of the CaNAC1 protein were characterized by liquid chromatography-tandem mass spectrometry. We conclude that CaNAC1 is a transcriptional activator of CaPLDα4 and suggested that it participates in the degradation of membrane lipids in bell peppers when they are stored at low temperature.

The Methylation Patterns and Transcriptional Responses to Chilling Stress at the Seedling Stage in Rice.

Chilling stress is considered the major abiotic stress affecting the growth, development, and yield of rice. To understand the transcriptomic responses and methylation regulation of rice in response to chilling stress, we analyzed a cold-tolerant variety of rice (Oryza sativa L. cv. P427). The physiological properties, transcriptome, and methylation of cold-tolerant P427 seedlings under low-temperature stress (2–3 °C) were investigated. We found that P427 exhibited enhanced tolerance to low temperature, likely via increasing antioxidant enzyme activity and promoting the accumulation of abscisic acid (ABA). The Methylated DNA Immunoprecipitation Sequencing (MeDIP-seq) data showed that the number of methylation-altered genes was highest in P427 (5496) and slightly lower in Nipponbare (Nip) and 9311 (4528 and 3341, respectively), and only 2.7% (292) of methylation genes were detected as common differentially methylated genes (DMGs) related to cold tolerance in the three varieties. Transcriptome analyses revealed that 1654 genes had specifically altered expression in P427 under cold stress. These genes mainly belonged to transcription factor families, such as Myeloblastosis (MYB), APETALA2/ethylene-responsive element binding proteins (AP2-EREBP), NAM-ATAF-CUC (NAC) and WRKY. Fifty-one genes showed simultaneous methylation and expression level changes. Quantitative RT-PCR (qRT-PCR) results showed that genes involved in the ICE (inducer of CBF expression)-CBF (C-repeat binding factor)—COR (cold-regulated) pathway were highly expressed under cold stress, including the WRKY genes. The homologous gene Os03g0610900 of the open stomatal 1 (OST1) in rice was obtained by evolutionary tree analysis. Methylation in Os03g0610900 gene promoter region decreased, and the expression level of Os03g0610900 increased, suggesting that cold stress may lead to demethylation and increased gene expression of Os03g0610900. The ICE-CBF-COR pathway plays a vital role in the cold tolerance of the rice cultivar P427. Overall, this study demonstrates the differences in methylation and gene expression levels of P427 in response to low-temperature stress, providing a foundation for further investigations of the relationship between environmental stress, DNA methylation, and gene expression in rice.

OsWRKY5 Promotes Rice Leaf Senescence via Senescence-Associated NAC and Abscisic Acid Biosynthesis Pathway.

The onset of leaf senescence is triggered by external cues and internal factors such as phytohormones and signaling pathways involving transcription factors (TFs). Abscisic acid (ABA) strongly induces senescence and endogenous ABA levels are finely tuned by many senescence-associated TFs. Here, we report on the regulatory function of the senescence-induced TF OsWRKY5 TF in rice (Oryza sativa). OsWRKY5 expression was rapidly upregulated in senescing leaves, especially in yellowing sectors initiated by aging or dark treatment. A T-DNA insertion activation-tagged OsWRKY5-overexpressing mutant (termed oswrky5-D) promoted leaf senescence under natural and dark-induced senescence (DIS) conditions. By contrast, a T-DNA insertion oswrky5-knockdown mutant (termed oswrky5) retained leaf greenness during DIS. Reverse-transcription quantitative PCR (RT-qPCR) showed that OsWRKY5 upregulates the expression of genes controlling chlorophyll degradation and leaf senescence. Furthermore, RT-qPCR and yeast one-hybrid analysis demonstrated that OsWRKY5 indirectly upregulates the expression of senescence-associated NAM/ATAF1/2/CUC2 (NAC) genes including OsNAP and OsNAC2. Precocious leaf yellowing in the oswrky5-D mutant might be caused by elevated endogenous ABA concentrations resulting from upregulated expression of ABA biosynthesis genes OsNCED3, OsNCED4, and OsNCED5, indicating that OsWRKY is a positive regulator of ABA biosynthesis during leaf senescence. Furthermore, OsWRKY5 expression was suppressed by ABA treatment. Taken together, OsWRKY5 is a positive regulator of leaf senescence that upregulates senescence-induced NAC, ABA biosynthesis, and chlorophyll degradation genes.

KNAT2/6b, a class I KNOX gene, impedes xylem differentiation by regulating NAC domain transcription factors in poplar.

Wood formation is the terminal differentiation of xylem mother cells derived from cambial initials, and negative regulators play important roles in xylem differentiation. The molecular mechanism of the negative regulator of xylem differentiation PagKNAT2/6b was investigated. PagKNAT2/6b is an ortholog of Arabidopsis KNAT2 and KNAT6 that is highly expressed in phloem and xylem. Compared to nontransgenic control plants, transgenic poplar plants overexpressing PagKNAT2/6b present with altered vascular patterns, characterized by decreased secondary xylem with thin cell walls containing less cellulose, xylose and lignin. RNA sequencing analyses revealed that differentially expressed genes are enriched in xylem differentiation and secondary wall synthesis functions. Expression of NAM/ATAF/CUC (NAC) domain genes including PagSND1-A1, PagSND1-A2, PagSND1-B2 and PagVND6-C1 is downregulated by PagKNAT2/6b, while PagXND1a is directly upregulated. Accordingly, the dominant repression form of PagKNAT2/6b leads to increased xylem width per stem diameter through downregulation of PagXND1a. PagKNAT2/6b can inhibit cell differentiation and secondary wall deposition during wood formation in poplar by modulating the expression of NAC domain transcription factors. Direct activation of PagXND1a by PagKNAT2/6b is a key node in the negative regulatory network of xylem differentiation by KNOXs.

Selection and verification of candidate reference genes for gene expression by quantitative RT-PCR in Hibiscus hamabo Sieb.et Zucc.

Ten candidate reference genes were examined in Hibiscus hamabo Sieb. et Zucc. ACT and SKIP are proposed as good reference genes for gene studies in Hibiscus hamabo Sieb. et Zucc. Because of its sensitivity and rapidness, quantitative real-time PCR (qRT-PCR) is currently extensively used to analyze gene-expression patterns. Selecting suitable reference genes to normalize qRT-PCR results is essential. Hibiscus hamabo Sieb. et Zucc. (H. hamabo) is a semi-mangrove plant that is widely used for the ecological restoration of saline-alkali land and coastal afforestation owing to its excellent salt tolerance. However, suitable reference genes used for the normalization of H. hamabo qRT-PCR data have not been selected or verified. Here, we tested the expression stabilities of ten candidate reference genes in different H. hamabo tissues under a set of abiotic stresses (salt, drought, high temperature, and low temperature) and hormonal treatments (methyl jasmonate, abscisic acid, and salicylic acid) using three statistical algorithms, i.e., NormFinder, geNorm, and BestKeeper. Actin (ACT) and ski-interacting protein (SKIP) can be regarded as good choices as reference genes in studying gene expression of H. hamabo. In addition, the qRT-PCR analysis of the NAC (NAM/ATAF1/2/CUC2) target gene’s expression pattern under NaCl-treated conditions confirmed the suitability of selected reference genes. Here, we used qRT-PCR technology to provide a stable reference gene list for H. hamabo gene-expression studies.

A GmSIN1/GmNCED3s/GmRbohBs Feed-Forward Loop Acts as a Signal Amplifier That Regulates Root Growth in Soybean Exposed to Salt Stress.

Abscisic acid (ABA) and reactive oxygen species (ROS) act as key signaling molecules in the plant response to salt stress; however, how these signals are transduced and amplified remains unclear. Here, a soybean (Glycine max) salinity-induced NAM/ATAF1/2/CUC2 (NAC) transcription factor encoded by SALT INDUCED NAC1 (GmSIN1) was shown to be a key component of this process. Overexpression of GmSIN1 in soybean promoted root growth and salt tolerance and increased yield under salt stress; RNA interference-mediated knockdown of GmSIN1 had the opposite effect. The rapid induction of GmSIN1 in response to salinity required ABA and ROS, and the effect of GmSIN1 on root elongation and salt tolerance was achieved by boosting cellular ABA and ROS contents. GmSIN1 upregulated 9-cis-epoxycarotenoid dioxygenase coding genes in soybean (GmNCED3s, associated with ABA synthesis) and Respiratory burst oxidase homolog B genes in soybean (GmRbohBs, associated with ROS generation) by binding to their promoters at a site that has not been described to date. Together, GmSIN1, GmNCED3s, and GmRbohBs constitute a positive feed-forward system that enables the rapid accumulation of ABA and ROS, effectively amplifying the initial salt stress signal. These findings suggest that the combined modulation of ABA and ROS contents enhances soybean salt tolerance.

Molecular cloning and characterization of NAC genes from four foxtail millet genotypes

Transcription factor gene family of NAC (NAM, ATAF, CUC) is tightly involved in plant development and in the response to stresses. In this study, we reported the isolation and the characterization of NAC gene homolog from four foxtail millet genotypes. Band with approximately 1300 bp size was successfully amplified from the genomic DNA of four foxtail millet genotypes (ICERI-4, ICERI-5, ICERI-6 and ICERI-10) using gene specific primer. The fragment was designated as SiNAC065 after showing high similarity with NAC gene homologs in the GenBank. Sequence analysis results showed that the SiNAC065 genes isolated from the four genotypes were 1265 bp in length with one intron and two exons. The two exons encode 325 amino acids with the conserved domain located between amino acid 19-325. The SiNAC065 protein identified in this study have 8 conserved motives in the conserved region which categorized them as SNAC (stress responsive NACs) orthologs that are involved in the abiotic stress responses. Different features of SiNAC065 isolated from the tolerant- and the sensitive-genotypes should provide information of the gene’s role in salinity tolerance mechanism of foxtail millet.

Transcriptome analysis reveals regulatory framework for salt and osmotic tolerance in a succulent xerophyte

BackgroundZygophyllum xanthoxylum is a succulent xerophyte with remarkable tolerance to diverse abiotic stresses. Previous studies have revealed important physiological mechanisms and identified functional genes associated with stress tolerance. However, knowledge of the regulatory genes conferring stress tolerance in this species is poorly understood.ResultsHere, we present a comprehensive analysis of regulatory genes based on the transcriptome of Z. xanthoxylum roots exposed to osmotic stress and salt treatments. Significant changes were observed in transcripts related to known and obscure stress-related hormone signaling pathways, in particular abscisic acid and auxin. Significant changes were also found among key classes of early response regulatory genes encoding protein kinases, transcription factors, and ubiquitin-mediated proteolysis machinery. Network analysis shows a highly integrated matrix formed by these conserved and novel gene products associated with osmotic stress and salt in Z. xanthoxylum. Among them, two previously uncharacterized NAC (NAM/ATAF/CUC) transcription factor genes, ZxNAC083 (Unigene16368_All) and ZxNAC035 (CL6534.Contig1_All), conferred tolerance to salt and drought stress when constitutively overexpressed in Arabidopsis plants.ConclusionsThis study provides a unique framework for understanding osmotic stress and salt adaptation in Z. xanthoxylum including novel gene targets for engineering stress tolerance in susceptible crop species.

The NAC family transcription factor GmNAC42\u20131 regulates biosynthesis of the anticancer and neuroprotective glyceollins in soybean

BackgroundGlyceollins are isoflavonoid-derived pathogen-inducible defense metabolites (phytoalexins) from soybean (Glycine max L. Merr) that have important roles in providing defense against pathogens. They also have impressive anticancer and neuroprotective activities in mammals. Despite their potential usefulness as therapeutics, glyceollins are not economical to synthesize and are biosynthesized only transiently and in low amounts in response to specific stresses. Engineering the regulation of glyceollin biosynthesis may be a promising approach to enhance their bioproduction, yet the transcription factors (TFs) that regulate their biosynthesis have remained elusive. To address this, we first aimed to identify novel abiotic stresses that enhance or suppress the elicitation of glyceollins and then used a comparative transcriptomics approach to search for TF gene candidates that may positively regulate glyceollin biosynthesis.ResultsAcidity stress (pH 3.0 medium) and dehydration exerted prolonged (week-long) inductive or suppressive effects on glyceollin biosynthesis, respectively. RNA-seq found that all known biosynthetic genes were oppositely regulated by acidity stress and dehydration, but known isoflavonoid TFs were not. Systemic acquired resistance (SAR) genes were highly enriched in the geneset. We chose to functionally characterize the NAC (NAM/ATAF1/2/CUC2)-family TF GmNAC42–1 that was annotated as an SAR gene and a homolog of the Arabidopsis thaliana (Arabidopsis) indole alkaloid phytoalexin regulator ANAC042. Overexpressing and silencing GmNAC42–1 in elicited soybean hairy roots dramatically enhanced and suppressed the amounts of glyceollin metabolites and biosynthesis gene mRNAs, respectively. Yet, overexpressing GmNAC42–1 in non-elicited hairy roots failed to stimulate the expressions of all biosynthesis genes. Thus, GmNAC42–1 was necessary but not sufficient to activate all biosynthesis genes on its own, suggesting an important role in the glyceollin gene regulatory network (GRN). The GmNAC42–1 protein directly bound the promoters of biosynthesis genes IFS2 and G4DT in the yeast one-hybrid (Y1H) system.ConclusionsAcidity stress is a novel elicitor and dehydration is a suppressor of glyceollin biosynthesis. The TF gene GmNAC42–1 is an essential positive regulator of glyceollin biosynthesis. Overexpressing GmNAC42–1 in hairy roots can be used to increase glyceollin yields > 10-fold upon elicitation. Thus, manipulating the expressions of glyceollin TFs is an effective strategy for enhancing the bioproduction of glyceollins in soybean.