Soybean steroids improve crop abiotic stress tolerance and increase yield.

Sterols have long been associated with diverse fields, such as cancer treatment, drug development, and plant growth; however, their underlying mechanisms and functions remain enigmatic. Here, we unveil a critical role played by a GmNF-YC9-mediated CCAAT-box transcription complex in modulating the steroid metabolism pathway within soybeans. Specifically, this complex directly activates squalene monooxygenase (GmSQE1), which is a rate-limiting enzyme in steroid synthesis. Our findings demonstrate that overexpression of either GmNF-YC9 or GmSQE1 significantly enhances soybean stress tolerance, while the inhibition of SQE weakens this tolerance. Field experiments conducted over two seasons further reveal increased yields per plant in both GmNF-YC9 and GmSQE1 overexpressing plants under drought stress conditions. This enhanced stress tolerance is attributed to the reduction of abiotic stress-induced cell oxidative damage. Transcriptome and metabolome analyses shed light on the upregulation of multiple sterol compounds, including fucosterol and soyasaponin II, in GmNF-YC9 and GmSQE1 overexpressing soybean plants under stress conditions. Intriguingly, the application of soybean steroids, including fucosterol and soyasaponin II, significantly improves drought tolerance in soybean, wheat, foxtail millet, and maize. These findings underscore the pivotal role of soybean steroids in countering oxidative stress in plants and offer a new research strategy for enhancing crop stress tolerance and quality from gene regulation to chemical intervention.

SiMYB19 from Foxtail Millet (Setaria italica) Confers Transgenic Rice Tolerance to High Salt Stress in the Field.

Salt stress is a major threat to crop quality and yield. Most experiments on salt stress-related genes have been conducted at the laboratory or greenhouse scale. Consequently, there is a lack of research demonstrating the merit of exploring these genes in field crops. Here, we found that the R2R3-MYB transcription factor SiMYB19 from foxtail millet is expressed mainly in the roots and is induced by various abiotic stressors such as salt, drought, low nitrogen, and abscisic acid. SiMYB19 is tentatively localized to the nucleus and activates transcription. It enhances salt tolerance in transgenic rice at the germination and seedling stages. SiMYB19 overexpression increased shoot height, grain yield, and salt tolerance in field- and salt pond-grown transgenic rice. SiMYB19 overexpression promotes abscisic acid (ABA) accumulation in transgenic rice and upregulates the ABA synthesis gene OsNCED3 and the ABA signal transduction pathway-related genes OsPK1 and OsABF2. Thus, SiMYB19 improves salt tolerance in transgenic rice by regulating ABA synthesis and signal transduction. Using rice heterologous expression analysis, the present study introduced a novel candidate gene for improving salt tolerance and increasing yield in crops grown in saline-alkali soil.

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

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

Salt-Induced Stability of SR1/CAMTA3 mRNA Is Mediated by Reactive Oxygen Species and Requires the 3' End of Its Open Reading Frame.

Soil salinity, a prevalent abiotic stress, causes enormous losses in global crop yields annually. Previous studies have shown that salt stress-induced reprogramming of gene expression contributes to the survival of plants under this stress. However, mechanisms regulating gene expression in response to salt stress at the posttranscriptional level are not well understood. In this study, we show that salt stress increases the level of Signal Responsive 1 (SR1) mRNA, a member of signal-responsive Ca2+/calmodulin-regulated transcription factors, by enhancing its stability. We present multiple lines of evidence indicating that reactive oxygen species generated by NADPH oxidase activity mediate salt-induced SR1 transcript stability. Using mutants impaired in either nonsense-mediated decay, XRN4 or mRNA decapping pathways, we show that neither the nonsense-mediated mRNA decay pathway, XRN4 nor the decapping of SR1 mRNA is required for its decay. We analyzed the salt-induced accumulation of eight truncated versions of the SR1 coding region (∼3 kb) in the sr1 mutant background. This analysis identified a 500-nt region at the 3' end of the SR1 coding region to be required for the salt-induced stability of SR1 mRNA. Potential mechanisms by which this region confers SR1 transcript stability in response to salt are discussed.

Overexpression of soybean DREB1 enhances drought stress tolerance of transgenic wheat in the field.

Drought-response-element binding (DREB)-like transcription factors can significantly enhance plant tolerance to water stress. However, most research on DREB-like proteins to date has been conducted in growth chambers or greenhouses, so there is very little evidence available to support their practical use in the field. In this study, we overexpressed GmDREB1 from soybean in two popular wheat varieties and conducted drought-tolerance experiments across a range of years, sites, and drought-stress regimes. We found that the transgenic plants consistently exhibited significant improvements in yield performance and a variety of physiological traits compared with wild-type plants when grown under limited water conditions in the field, for example showing grain yield increases between 4.79-18.43%. Specifically, we found that the transgenic plants had reduced membrane damage and enhanced osmotic adjustment and photosynthetic efficiency compared to the non-transgenic controls. Three enzymes from the biosynthetic pathway of the phytohormone melatonin were up-regulated in the transgenic plants, and external application of melatonin was found to improve drought tolerance. Together, our results demonstrate the utility of transgenic overexpression of GmDREB1 to improve the drought tolerance of wheat in the field.

Continuous salt stress-induced long non-coding RNAs and DNA methylation patterns in soybean roots.

BACKGROUND: Environmental stimuli can activate a series of physiological and biochemical responses in plants accompanied by extensive transcriptional reprogramming. Long non-coding RNAs (lncRNAs), as versatile regulators, control gene expression in multiple ways and participate in the adaptation to biotic and abiotic stresses. RESULTS: In this study, soybean seedlings were continuously cultured for 15 days with high salinity solutions started from seed germination. Strand-specific whole transcriptome sequencing and stringent bioinformatic analysis led to the identification of 3030 long intergenic non-coding RNAs (lincRNAs) and 275 natural antisense transcripts (lncNATs) in soybean roots. In contrast to mRNAs, newly identified lncRNAs exhibited less exons, similar AU content to UTRs, even distribution across the genome and low evolutionary conservation. Remarkably, more than 75% of discovered lncRNAs that were activated or up-regulated by continuous salt stress mainly targeted proteins with binding and catalytic activities. Furthermore, two DNA methylation maps with single-base resolution were generated by using reduced representation bisulfite sequencing, offering a genome-wide perspective and important clues for epigenetic regulation of stress-associated lncRNAs and protein-coding genes. CONCLUSIONS: Taken together, our findings systematically demonstrated the characteristics of continuous salt stress-induced lncRNAs and extended the knowledge of corresponding methylation profiling, providing valuable evidence for a better understanding of how plants cope with long-term salt stress circumstances.

Enhancing the CRISPR/Cas9 system based on multiple GmU6 promoters in soybean.

Small guide RNA (sgRNA) is an important component of the CRISPR/Cas9 system. The gene editing efficiency of the CRISPR/Cas9 system could be enhanced by using highly active U6 promoters to drive the expression of sgRNA. Therefore, we constructed various expression vectors based on the 11 GmU6 promoters predicted and cloned in the whole soybean genome. The expression of truncated GUS driven by 11 GmU6 promoters was tested in hairy roots and by Arabidopsis thaliana transformation. The results indicated that higher transcriptional levels were driven by 5 GmU6 promoters (GmU6-4, GmU6-7, GmU6-8, GmU6-10 and GmU6-11) in both soybean hairy roots and Arabidopsis thaliana. In addition, three genes, Glyma03g36470, Glyma14g04180 and Glyma06g136900, were selected as targets to detect the transcriptional levels of multiple GmU6 promoters. Mutations in these three genes were detected in soybean hairy roots after Agrobacterium rhizogenes infection, indicating efficient target gene editing, including nucleotide insertion, deletion, and substitution. Mutation efficiencies differed among the 11 GmU6 promoters, ranging from 2.8% to 20.6%, and markedly higher efficiencies were obtained with all three genes using the GmU6-8 (20.3%) and GmU6-10 (20.6%) promoters. These two GmU6 promoters also showed higher ability to drive truncated GUS transcription in both soybean hairy roots and transformed Arabidopsis thaliana. These results will help to construct an efficient CRISPR-Cas9 gene editing system and promote the application of the CRISPR-Cas9 genome editing system in soybean molecular breeding.

iTRAQ-based quantitative proteomic analysis of wheat roots in response to salt stress.

Salinity is a major abiotic stress that affects plant growth and development. Plant roots are the sites of salt uptake. Here, an isobaric tag for a relative and absolute quantitation based proteomic technique was employed to identify the differentially expressed proteins (DEPs) from seedling roots of the salt-tolerant genotype Han 12 and the salt-sensitive genotype Jimai 19 in response to salt treatment. A total of 121 NaCl-responsive DEPs were observed in Han 12 and Jimai 19. The main DEPs were ubiquitination-related proteins, transcription factors, pathogen-related proteins, membrane intrinsic protein transporters and antioxidant enzymes, which may work together to obtain cellular homeostasis in roots and to determine the overall salt tolerance of different wheat varieties in response to salt stress. Functional analysis of three salt-responsive proteins was performed in transgenic plants as a case study to confirm the salt-related functions of the detected proteins. Taken together, the results of this study may be helpful in further elucidating salt tolerance mechanisms in wheat.

Global selection on sucrose synthase haplotypes during a century of wheat breeding.

Spike number per unit area, number of grains per spike, and thousand kernel weight (TKW) are important yield components. In China, increases in wheat (Triticum aestivum) yields are mainly due to increases in grain number per spike and TKW. TKW mainly depends on starch content, as starch accounts for about 70% of the grain endosperm. Sucrose synthase catalysis is the first step in the conversion of sucrose to starch, that is, the conversion of sucrose to fructose and UDP-glucose by the wheat sucrose synthase genes (TaSus1 and TaSus2) that are located on chromosomes 7A/7B/7D and 2A/2B/2D, respectively. A total of 1,520 wheat accessions were genotyped at the six loci. Two, two, five, and two haplotypes were identified at the TaSus2-2A, TaSus2-2B, TaSus1-7A, and TaSus1-7B loci, respectively. Their main variations were detected within the introns. Significant differences between the haplotypes correlated with TKW differences among 348 modern Chinese cultivars from the core collection. Frequency changes for favored haplotypes showed gradual increases in cultivars released since beginning of the last century in China, Europe, and North America. Geographic distributions and time changes of favored haplotypes were characterized in six major wheat production regions worldwide. Strong selection bottlenecks to haplotype variations occurred at polyploidization and domestication and during breeding of wheat. Genetic-effect differences between haplotypes at the same locus influence the selection time and intensity. This work shows that the endosperm starch synthesis pathway is a major target of indirect selection in global wheat breeding for higher yield.

A yield-associated gene TaCWI, in wheat: its function, selection and evolution in global breeding revealed by haplotype analysis.

KEY MESSAGE: Wheat anther-specific invertase genes were haplotyped in wheat. Strong allelic selection occurred during wheat polyploidization, domestication and breeding because of their association with yield traits. Plant invertase hydrolyzes sucrose into glucose and fructose. Cell wall invertase (CWI), one of the three types of invertase, is essential for plant development. Based on isolated TaCWI genes from chromosomes 4A, 5B and 5D, two SNPs were detected in the promoter region of TaCWI-4A, and four SNPs and two Indels were present in the TaCWI-5D gene. No polymorphism was detected in TaCWI-5B coding or promoter regions. CAPS markers caps4A and caps5D were developed to discriminate haplotypes of TaCWI-4A and TaCWI-5D. Marker/trait association analysis indicated that Hap-5D-C at TaCWI-5D was significantly associated with higher thousand kernel weight (TKW) in 348 Chinese modern cultivars grown in multiple environments. Geographic distributions and changes over time of favored haplotypes showed that Hap-5D-C was the most frequent haplotype in modern cultivars and was strongly positively selected in six major wheat production regions worldwide. However, selection for haplotypes at TaCWI-4A was not so evident, possibly due to balancing effects of the two haplotypes on TKW and grain number per spike (GN). In rainfed production regions, Hap-4A-C was favored because it brought more seeds, but in well irrigated conditions, Hap-4A-T was favored in modern breeding because of higher TKW. Evolutionary analysis among wheat and its relatives showed that genetic diversity of TaCWI genes on chromosomes 4A and 5D declined dramatically in progression from the diploid level to modern polyploid cultivars. There was strong allelic selection during polyploidization, domestication and breeding.

GmNFYA3, a target gene of miR169, is a positive regulator of plant tolerance to drought stress.

Nuclear factor Y (NF-Y) is a heterotrimeric transcription factor composed of NF-YA, NF-YB and NF-YC proteins. In this study, we identified and characterized a gene, GmNFYA3, which encodes the NF-YA subunit of the NF-Y complex in soybeans (Glycine max L.). Real time RT-PCR analysis indicated that GmNFYA3 was induced by abscisic acid (ABA) and abiotic stresses, such as polyethylene glycol, NaCl and cold. Subcellular localization analysis suggested that GmNFYA3 may activate its specific targets in the nucleus. Histochemical ß-glucuronidase (GUS) staining revealed that the expression of the GUS gene driven by the GmNFYA3 promoter occurred in various transgenic Arabidopsis tissues. Coexpression in Nicotiana benthamiana and 5' RACE assays indicated that miR169 directs GmNFYA3 mRNA cleavage in vivo. Overexpression of GmNFYA3 resulted in Arabidopsis with reduced leaf water loss and enhanced drought tolerance. In addition, the transgenic Arabidopsis exhibited increased sensitivity to high salinity and exogenous ABA. Moreover, the transcript levels of ABA biosynthesis (ABA1, ABA2), ABA signaling (ABI1, ABI2) and stress-responsive genes, including RD29A and CBF3, were generally higher in GmNFYA3 plants than in wild-type controls under normal conditions. These results suggest that the GmNFYA3 gene functions in positive modulation of drought stress tolerance and has potential applications in molecular breeding to enhance drought tolerance in crops.

Analyses of a Glycine max degradome library identify microRNA targets and microRNAs that trigger secondary siRNA biogenesis.

Plant microRNAs (miRNAs) regulate gene expression mainly by guiding cleavage of target mRNAs. In this study, a degradome library constructed from different soybean (Glycine max (L.) Merr.) tissues was deep-sequenced. 428 potential targets of small interfering RNAs and 25 novel miRNA families were identified. A total of 211 potential miRNA targets, including 174 conserved miRNA targets and 37 soybean-specific miRNA targets, were identified. Among them, 121 targets were first discovered in soybean. The signature distribution of soybean primary miRNAs (pri-miRNAs) showed that most pri-miRNAs had the characteristic pattern of Dicer processing. The biogenesis of TAS3 small interfering RNAs (siRNAs) was conserved in soybean, and nine Auxin Response Factors were identified as TAS3 siRNA targets. Twenty-three miRNA targets produced secondary small interfering RNAs (siRNAs) in soybean. These targets were guided by five miRNAs: gma-miR393, gma-miR1508, gma-miR1510, gma-miR1514, and novel-11. Multiple targets of these secondary siRNAs were detected. These 23 miRNA targets may be the putative novel TAS genes in soybean. Global identification of miRNA targets and potential novel TAS genes will contribute to research on the functions of miRNAs in soybean.

DNA-Binding Activity of CAMTA3 Is Essential for Its Function: Identification of Critical Amino Acids for Its Transcriptional Activity.

Calmodulin-binding transcription activators (CAMTAs), a small family of highly conserved transcription factors, function in calcium-mediated signaling pathways. Of the six CAMTAs in Arabidopsis, CAMTA3 regulates diverse biotic and abiotic stress responses. A recent study has shown that CAMTA3 is a guardee of NLRs (Nucleotide-binding, Leucine-rich repeat Receptors) in modulating plant immunity, raising the possibility that CAMTA3 transcriptional activity is dispensable for its function. Here, we show that the DNA-binding activity of CAMTA3 is essential for its role in mediating plant immune responses. Analysis of the DNA-binding (CG-1) domain of CAMTAs in plants and animals showed strong conservation of several amino acids. We mutated six conserved amino acids in the CG-1 domain to investigate their role in CAMTA3 function. Electrophoretic mobility shift assays using these mutants with a promoter of its target gene identified critical amino acid residues necessary for DNA-binding activity. In addition, transient assays showed that these residues are essential for the CAMTA3 function in activating the Rapid Stress Response Element (RSRE)-driven reporter gene expression. In line with this, transgenic lines expressing the CG-1 mutants of CAMTA3 in the camta3 mutant failed to rescue the mutant phenotype and restore the expression of CAMTA3 downstream target genes. Collectively, our results provide biochemical and genetic evidence that the transcriptional activity of CAMTA3 is indispensable for its function.

Overexpression of lncRNA77580 Regulates Drought and Salinity Stress Responses in Soybean.

Emerging evidence indicates that long non-coding RNAs (lncRNAs) play important roles in diverse biological processes. However, the biological functions of most plant lncRNAs are still unknown. We previously discovered a soybean abiotic-stress-related lncRNA, lncRNA77580, and cloned the entire full-length sequence. Here, in order to fully identify the function of lncRNA77580 in soybean stress response, we created transgenic soybean lines overexpressing lncRNA77580. Compared with the wild type, overexpression of lncRNA77580 enhances the drought tolerance of soybean. However, the transgenic plants exhibit increased sensitivity to high salinity at the seedling stage. We found that lncRNA77580 modulates the transcription of different gene sets during salt and drought stress response. Under water deficit at the reproductive stage, lncRNA77580 overexpression increases the seed yield by increasing the seed number per plant. These results provide insight into the role of lncRNA77580 in soybean stress response.

Foxtail millet MYB-like transcription factor SiMYB16 confers salt tolerance in transgenic rice by regulating phenylpropane pathway.

R2R3-MYB transcription factors play an important role in the synthesis of phenylpropanoid-derived compounds, which in turn provide salt tolerance in plant. In this study, we found that the expression of foxtail millet R2R3-MYB factor SiMYB16 can be induced by salt and drought. SiMYB16 is localized in the nucleus and acts as a transcriptional activator. Phylogenetic analysis indicates that SiMYB16 belongs to the R2R3-MYB transcription factor family subgroup 24. Transgenic rice expressing SiMYB16 (OX16) had a higher survival rate, lower malondialdehyde content, and heavier fresh weight compared with type (WT) under salt stress conditions. The transgenic plants also had a higher germination rate in salt treatment conditions and higher yield in the field compared with wild-type plants. Transcriptome analysis revealed that the up-regulated differential expression genes in the transgenic rice were mainly involved in phenylpropanoid biosynthesis, fatty acid elongation, phenylalanine metabolism, and flavonoid biosynthesis pathways. Quantitative real-time PCR analysis also showed that the genes encoding the major enzymes in the lignin and suberin biosynthesis pathways had higher expression level in SiMYB16 transgenic plants. Correspondingly, the content of flavonoid and lignin, and the activity of fatty acid synthase increased in SiMYB16 transgenic rice compared with wild-type plants under salt stress treatment. These results indicate that SiMYB16 gene can enhance plant salt tolerance by regulating the biosynthesis of lignin and suberin.

Overexpression of gma-MIR394a confers tolerance to drought in transgenic Arabidopsis thaliana.

MicroRNAs, key posttranscriptional regulators of eukaryotic gene expression, play important roles in plant development and response to stress. In this study, a soybean gma-MIR394a gene was functionally characterized, especially with regard to its role in drought stress resistance. Expression analysis revealed that gma-MIR394a was expressed differentially in various soybean tissues and was induced by drought, high salinity, low temperature stress, and abscisic acid treatment in leaves. One target gene of gma-miR394a, Glyma08g11030, was predicted and verified using a modified 5' RLM-RACE (RNA ligase-mediated rapid amplification of 5' cDNA ends) assay. Overexpression of gma-MIR394a resulted in plants with lowered leaf water loss and enhanced drought tolerance. Furthermore, overexpression of gma-MIR394a in Arabidopsis reduced the transcript of an F-box gene (At1g27340) containing a miR394 complementary target site. These results suggest that the gma-MIR394a gene functions in positive modulation of drought stress tolerance and has potential applications in molecular breeding to enhance drought tolerance in crops.

The wheat (T. aestivum) sucrose synthase 2 gene (TaSus2) active in endosperm development is associated with yield traits.

Sucrose synthase catalyzes the reaction sucrose + UDP → UDP-glucose + fructose, the first step in the conversion of sucrose to starch in endosperm. Previous studies identified two tissue-specific, yet functionally redundant, sucrose synthase (SUS) genes, Sus1 and Sus2. In the present study, the wheat Sus2 orthologous gene (TaSus2) series was isolated and mapped on chromosomes 2A, 2B, and 2D. Based on sequencing in 61 wheat accessions, three single-nucleotide polymorphisms (SNPs) were detected in TaSus2-2B. These formed two haplotypes (Hap-H and Hap-L), but no diversity was found in either TaSus2-2A or TaSus2-2D. Based on the sequences of the two haplotypes, we developed a co-dominant marker, TaSus2-2B ( tgw ), which amplified 423 or 381-bp fragments in different wheat accessions. TaSus2-2B ( tgw ) was located between markers Xbarc102.2 and Xbarc91 on chromosome 2BS in a RIL population from Xiaoyan 54 × Jing 411. Association analysis suggested that the two haplotypes were significantly associated with 1,000 grain weight (TGW) in 89 modern wheat varieties in the Chinese mini-core collection. Mean TGW difference between the two haplotypes over three cropping seasons was 4.26 g (varying from 3.71 to 4.94 g). Comparative genomics analysis detected major kernel weight QTLs not only in the chromosome region containing TaSus2-2B (tgw), but also in the collinear regions of TaSus2 on rice chromosome 7 and maize chromosome 9. The preferred Hap-H haplotype for high TGW underwent very strong positive selection in Chinese wheat breeding, but not in Europe. The geographic distribution of Hap-H was perhaps determined by both latitude and the intensity of selection in wheat breeding.

Large DNA fragment deletion in lncRNA77580 regulates neighboring gene expression in soybean (Glycine max).

Long non-coding RNAs (lncRNAs) affect gene expressions via a wide range of mechanisms and are considered important regulators of numerous essential biological processes, including abiotic stress responses. However, the biological functions of most lncRNAs are yet to be determined. Moreover, to date, no effective methods have been developed to study the function of plant lncRNAs. We previously discovered a salt stress-related lncRNA, lncRNA77580 in soybean (Glycine max L.). In this study, we cloned the full-length lncRNA77580 and found that it shows nuclear-specific localisation. Furthermore, we employed CRISPR/Cas9 technology to induce large DNA fragment deletions in lncRNA77580 in soybean using a dual-single guide RNA/Cas9 design. As a result, we obtained deletion mutant soybean roots with targeted genomic fragment deletion in lncRNA77580. Deletion and overexpression of lncRNA77580 were found to alter the expression of several neighboring protein-coding genes associated with the response to salt stress. The longer the deleted DNA fragment in lncRNA77580, the greater the influence on the expression of lncRNA77580 itself and neighboring genes. Collectively, the findings of this study revealed that large DNA fragment deletion in lncRNAs using the CRISPR/Cas9 system is a powerful method to obtain functional mutations of soybean lncRNAs that could benefit future research on lncRNA function in soybean.

Genome-wide identification of microRNAs and phased siRNAs in soybean roots under long-term salt stress.

BACKGROUND: Salinity stress, as the key limiting factor for agricultural productivity, can activate a series of molecular responses and alter gene expression in plants. Endogenous regulatory small RNAs, such as microRNAs (miRNAs) and phased siRNAs (phasiRNAs), play crucial roles during stress adaptation and prevent the injury from environmental circumstances. OBJECTIVE: To identify long-term salt stress responsive miRNAs and phasiRNAs as well as their associated genes and pathways in soybean roots. METHODS: Small RNA and degradome sequencing strategies were applied to genome widely investigate miRNAs and phasiRNAs in soybean roots under control and long-term salt stress conditions. RESULTS: In this study, stringent bioinformatic analysis led to the identification of 253 conserved and 38 novel miRNA candidates. Results of expression profiling, target and endogenous target mimics predictions provided valuable clues to their functional roles. Furthermore, 156 genes were identified to be capable of generating 21 nt and 24 nt phasiRNAs, in which 37 candidates were confirmed by degradome data for miRNA-directed cleavage. Approximately 90% of these phasiRNA loci were protein coding genes. And GO enrichment analysis pointed to "signal transduction" and "ADP binding" entries and reflected the functional roles of identified phasiRNA genes. CONCLUSION: Taken together, our findings extended the knowledge of salt responsive miRNAs and phasiRNAs in soybean roots, and provided valuable information for a better understanding of the regulatory events caused by small RNAs underlying plant adaptations to long-term salt stress.

Overexpression of soybean miR169c confers increased drought stress sensitivity in transgenic Arabidopsis thaliana.

The miR169 family, a large-scale microRNA gene family conserved in plants, is involved in stress responses, although how soybean miR169 functions in response to drought stress remains unclear. We show that gma-miR169c exerts a negative regulatory role in the response to drought stress by inhibiting the expression of its target gene, nuclear factor Y-A (NF-YA). A real-time RT-PCR analysis indicated that gma-miR169c is widely expressed in soybean tissues and induced by polyethylene glycol (PEG), high salt, cold stress and abscisic acid (ABA). Histochemical ß-glucuronidase (GUS) staining showed that the gma-miR169c promoter drives GUS reporter gene expression in various transgenic Arabidopsis tissues, and the stress-induced pattern was confirmed in transgenic Arabidopsis and transgenic soybean hairy roots. Arabidopsis overexpressing gma-miR169c is more sensitive to drought stress, with reduced survival, accelerated leaf water loss, and shorter root length than wild-type plants. We identified a precise cleavage site for 10 gma-miR169c targets and found reduced transcript levels of the AtNFYA1 and AtNFYA5 transcription factors in gma-miR169c-overexpressing Arabidopsis and reduced expression of the stress response genes AtRD29A, AtRD22, AtGSTU25 and AtCOR15A. These results indicate that gma-miR169c plays a negative regulatory role in drought stress and is a candidate miRNA for improving plant drought adaptation.