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• gma-miR169c is widely expressed in soybean tissues and induced by polyethylene glycol, high salt levels, cold stress and abscisic acid. • Arabidopsis overexpressing gma-miR169c is more sensitive to drought stress, with reduced survival, accelerated leaf water loss, and shorter root length than the wild-type plants. • gma-miR169c negatively regulates the drought stress response by inhibiting the expression of the targets AtNFYA1 and AtNFYA5 and reducing the transcript levels of the stress response genes AtRD29A , AtRD22 , AtGSTU25 and AtCOR15A. 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. [ABSTRACT FROM AUTHOR]

Background: Natural antisense transcripts (NATs) are a class of RNAs that contain a sequence complementary to other transcripts. NATs occur widely in eukaryotes and play critical roles in post-transcriptional regulation. Soybean NAT sequences are predicted in the PlantNATsDB, but detailed analyses of these NATs remain to be performed. Results: A total of 26,216 NATs, including 994 cis-NATs and 25,222 trans-NATs, were predicted in soybean. Each sense transcript had 1-177 antisense transcripts. We identified 21 trans-NATs using RT-PCR amplification. Additionally, we identified 179 cis-NATs and 6,629 trans-NATs that gave rise to small RNAs; these were enriched in the NAT overlapping region. The most abundant small RNAs were 21, 22, and 24 nt in length. The generation of small RNAs was biased to one stand of the NATs, and the degradation of NATs was biased. High-throughput sequencing of the degradome allowed for the global identification of NAT small interfering RNAs (nat-siRNAs) targets. 446 target genes for 165 of these nat-siRNAs were identified. The nat-siRNA target could be one transcript of a given NAT, or from other gene transcripts. We identified five NAT transcripts containing a hairpin structure that is characteristic of pre-miRNA. We identified a total of 86 microRNA (miRNA) targets that had antisense transcripts in soybean. Conclusions: We globally identified nat-siRNAs, and the targets of nat-siRNAs in soybean. It is likely that the cis-NATs, trans-NATs, nat-siRNAs, miRNAs, and miRNA targets form complex regulatory networks. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Drought seriously affects the yield and quality of soybean. Previous studies have shown that the gma-miR394a / GmFBX176 module regulates the response of Arabidopsis to drought stress. However, whether the gma-miR394a / GmFBX176 module is involved in the regulation of the soybean drought stress response remains unclear. Here, the function of the gma-miR394a / GmFBX176 module in the soybean drought stress response was evaluated. In soybean hairy roots, drought stress induced the transcription of gma-miR394a and inhibited the transcription of GmFBX176. GUS histochemical staining showed that transgenic GmFBX176p:GUS soybean hairy root staining was weak and that GUS transcript levels decreased under drought stress. A transient expression experiment in tobacco showed that gma-miR394a inhibited GmFBX176 transcription. Under drought stress, composite soybean plants overexpressing gma-miR394a showed increased drought resistance compared with control K599 composite soybean plants (K599); their survival rate and peroxidase activity were higher than those of K599, and their malondialdehyde content was lower. In contrast, composite soybean plants overexpressing GmFBX176m3 (gma-miR394a complement site mutation) presented lower drought resistance than K599 plants. Transcriptomic sequencing showed that the gma-miR394a / GmFBX176 module affected the transcript levels of stress response genes and transcription factors. These results indicate that the gma-miR394a / GmFBX176 module can be used to improve the drought resistance of soybean. • gma-miR394a is upregulated and its target GmFBX176 is downregulated in 10% PEG-simulated drought-stressed soybean hairy roots. • Overexpression of gma-miR394a increased the tolerance of composite soybean plants to drought stress, while overexpression of GmFBX176m3 decreased the tolerance of composite soybean plants to drought stress. • Under PEG treatment, the gma-MIR394a / GmFBX176 module regulates the transcript levels of stress-related genes. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) have important functions in the plant salt stress response. However, the functions of nonconserved miRNAs in soybean salt stress adaptation remain unclear. In this study, the soybean nonconserved miRNA gma-miR4359b and its target mRNA F-box gene GmFBX193 were functionally identified in response to salt stress. gma-miR4359b responded to salt stress in soybean hairy roots, stems, and leaves. Under salt stress, the root length ratio, root surface area ratio, root volume ratio, root tip number, survival rate, chlorophyll content, soluble sugar content, proline content, superoxide dismutase activity, and peroxidase enzyme activity of soybean hairy roots overexpressing gma-miR4359b (MIR4359b) were increased compared to those of the K599 (control) soybean hairy roots, while the relative electrolyte leakage and malondialdehyde content of the MIR4359b soybean hairy roots decreased. In contrast, soybean hairy roots in which target mimicry (MIM4359b) and short tandem target mimic (STTM4359b) inhibited gma-miR4359b exhibited a salt-sensitive phenotype. The transcription level of GmFBX193 was decreased in MIR4359b soybean hairy roots, while the transcription level of GmFBX193 was increased in MIM4359b and STTM4359b soybean hairy roots. GmFBX193 responded to salt stress in soybean roots, and GmFBX193 was identified as a nuclear-localized protein. Under salt stress, soybean hairy roots overexpressing GmFBX193 had a similar phenotype to MIM4359b and STTM4359b soybean hairy roots. Compared with K599 (control) soybean hairy roots, MIR4359b soybean hairy roots had 172 differentially expressed genes (DEGs), and MIM4359b soybean hairy roots had 2129 DEGs. Some known stress-related transcription factors were included among the DEGs. Taken together, these results suggest that gma-miR4359b plays a positive role in the soybean salt stress response by negatively regulating GmFBX193. • The nonconserved soybean miRNA gma-miR4359b and its target mRNA F-box gene GmFBX193 responded to salt stress. gma-miR4359b positively regulates the soybean response to salt stress by targeting GmFBX193. • gma-miR4359b is a positive regulator of salt tolerance in soybean. • GmFBX193 negatively regulates soybean tolerance to salt stress. • Transcriptome sequencing showed that gma-miR4359b could affect the transcription levels of some transcription factors related to the salt stress response. [ABSTRACT FROM AUTHOR]

• The GmNF-YA10 gene encodes an NF-YA transcription factor located in the nucleus that responds to PEG and ABA treatment at the transcription level. • Overexpression of the GmNF-YA10 gene increases the drought resistance of transgenic Arabidopsis by accumulating osmotic substances, slowing membrane lipid peroxidation, enhancing photosynthesis, regulating ABA-mediated stomatal closure, reducing the leaf water loss rate, and affecting the transcript levels of stress-related genes. • GmNF-YA10 gene participates in Arabidopsis response to drought stress through two pathways, ABA-dependent and ABA-independent. Nuclear factor (NF)-Y is composed of three subunits, NF-YA, NF-YB and NF-YC, and plays important roles in the regulation of plant abiotic stress response gene expression. However, the function of individual soybean NF-YA proteins in response to drought stress is unclear. In this study, the soybean NF-YA subunit gene GmNF-YA10 was cloned, and its function in response to drought stress was identified. GmNF-YA10 is broadly expressed in soybean tissues and is specifically located in the nucleus. In soybean roots, the transcription level of the GmNF-YA10 gene was induced by polyethylene glycol (PEG)-simulated drought stress and abscisic acid (ABA) treatment. Under mannitol stress, the germination rate of transgenic Arabidopsis overexpressing GmNF-YA10 was higher than that of wild-type (WT) Arabidopsis. After drought stress, the survival rate, relative water content, ABA content, proline content and chlorophyll content of transgenic Arabidopsis overexpressing GmNF-YA10 were higher than those of WT Arabidopsis while the rate of water loss and the contents of malondialdehyde and sucrose were lower than those of WT Arabidopsis , indicating that transgenic Arabidopsis overexpressing GmNF-YA10 has better drought resistance than WT Arabidopsis. Under the ABA treatment, the root length of transgenic Arabidopsis overexpressing GmNF-YA10 was shorter than that of WT Arabidopsis and the degree of stomatal closure was greater than that of WT Arabidopsis , indicating that transgenic Arabidopsis overexpressing GmNF-YA10 was more sensitive to ABA at the post-germination stage. Transcriptome sequencing and real-time quantitative PCR analysis showed that GmNF-YA10 affected the expression of both ABA-dependent and ABA-independent signalling pathway genes. These results indicate that GmNF-YA10 significantly improves the drought resistance of transgenic Arabidopsis through ABA-dependent and ABA-independent signal transduction pathways. [ABSTRACT FROM AUTHOR]

• GmFBX176 encodes a nuclear-localized F-box protein that responds to PEG treatment, ABA treatment, and salt stress at the transcriptional level. • Stress tolerance analysis of heterologous GmFBX176-overexpressing Arabidopsis revealed that GmFBX176 is a negative regulatory protein that improves tolerance to drought and salt stress. • GmFBX176-overexpressing Arabidopsis displayed reduced sensitivity to ABA. • Constitutive heterologous overexpression of the GmFBX176 gene affects stress-related physiological indicators and the transcript levels of stress response genes in transgenic Arabidopsis. As key components of the E3 ubiquitin ligase in the Skp1/Cullin or CDC53/F-box (SCF) complex, F-box proteins are involved in plant abiotic stress responses. However, the functions of F-box proteins in soybean in response to abiotic stress remain unclear. In this study, the soybean F-box protein-encoding gene GmFBX176 was cloned and functionally analyzed. GmFBX176 localized to the nucleus, was widely expressed in tissues (with the highest level in the roots), and responded to polyethylene glycol (PEG) treatment, abscisic acid (ABA) treatment, and salt stress at the transcriptional level. GmFBX176 -overexpressing Arabidopsis had lower survival rates; faster water loss rates; lower proline, sucrose and chlorophyll contents; greater malondialdehyde (MDA) contents; greater relative electrical conductivity (REC); and greater drought stress sensitivity than did wild-type (WT) Arabidopsis. Moreover, GmFBX176 -overexpressing Arabidopsis had lower survival rates, lower chlorophyll content, lower superoxide dismutase (SOD) activity, greater sensitivity to salt stress, longer roots, greater stomatal opening rates, and lower sensitivity to ABA than did WT Arabidopsis. Transcriptome sequencing and quantitative real-time PCR (qPCR) analyses revealed that GmFBX176 affects the transcript levels of stress response genes. These results indicate that GmFBX176 regulates ABA-mediated responses to drought and salt stress. [ABSTRACT FROM AUTHOR]