GmWRKY46, a WRKY transcription factor, negatively regulates phosphorus tolerance primarily through modifying root morphology in soybean.

Phosphorus (P) deficiency affects soybean growth and development, resulting in significant reduction of yields. However, the regulatory mechanism of P deficiency tolerance in soybean is still largely unclear. WRKY transcription factors are a family of regulators involved in a variety of abiotic stresses in plants while rarely reported in P deficiency. Here, we demonstrated that a soybean GmWRKY46 gene, belonging to group III of WRKY TF family, was involved in the regulation of P deficiency tolerance in soybean. The expression of GmWRKY46 in low P sensitive soybean varieties was significantly higher than that in tolerant soybean varieties. It was primarily expressed in roots and strongly induced by P deprivation. GmWRKY46 was localized in the nucleus. Compared with the control expressing the empty vector, overexpression of GmWRKY46 in soybean hairy roots exhibited more sensitive phenotypes to low P stress, while the RNA interfered GmWRKY46 significantly enhanced P deficiency tolerance by increasing the proliferation, elongation and P absorption efficiency of hairy roots. Expression patterns of a number of P-responsive genes (GmPht1;1, GmPht1;4, GmPTF1, GmACP1, GmPAP21 and GmExpansin-A7) were altered in both overexpression and gene silenced plants. The results provided a novel insight into how soybean responds to low P stress and new gene that may be used to improve soybean low P tolerance through gene editing approach.

Genome-wide identification of low phosphorus responsive microRNAs in two soybean genotypes by high-throughput sequencing.

MicroRNAs (miRNAs) have been reported to be correlated with various stress responses in soybean, but only a few miRNAs have been demonstrated to respond to low phosphorus (LP) stress. To unravel the response mechanisms of miRNAs to low-P stress, the roots of two representative soybean genotypes with different P efficiency, Nannong94-156 (a LP-tolerant genotype) and Bogao (a LP-sensitive genotype), were used for the construction of RNA sequencing (RNA-seq) libraries under low/normal-P treatment by high-throughput sequencing. In total, 603 existing miRNAs and 1699 novel miRNAs belonging to 248 and 1582 families in all samples were identified, respectively. Among these miRNAs, 777 miRNAs were differentially expressed (DE) across different P levels and genotypes. Furthermore, putative targets of DE miRNAs were predicted, and these miRNAs mainly targeted ERF (ethylene responsive factor), auxin response factors (ARF), zinc finger protein, MYB, and NAC domain transcription factors. Gene ontology (GO) analysis showed that targets of DE miRNAs were significantly enriched in binding, metabolic processes, biological regulation, response to stress, and phosphorus metabolic processes. In addition, the expression profiles of chosen P-responsive miRNAs and target genes were validated by quantitative real-time PCR (qRT-PCR). Our study focused on genome-wide miRNA identification in two representative soybean genotypes under low-P stress. Overall, the DE miRNAs across different P levels and genotypes and their putative target genes will provide useful information for further study of miRNAs mediating low-P response and facilitate improvements in soybean breeding.

Genome-Wide Analysis Reveals Dynamic Epigenomic Differences in Soybean Response to Low-Phosphorus Stress.

Low-phosphorus (low-P) stress has a significant limiting effect on crop yield and quality. Although the molecular mechanisms of the transcriptional level responsible for the low-P stress response have been studied in detail, the underlying epigenetic mechanisms in gene regulation remain largely unknown. In this study, we evaluated the changes in DNA methylation, gene expression and small interfering RNAs (siRNAs) abundance genome-wide in response to low-P stress in two representative soybean genotypes with different P-efficiencies. The DNA methylation levels were slightly higher under low-P stress in both genotypes. Integrative methylation and transcription analysis suggested a complex regulatory relationship between DNA methylation and gene expression that may be associated with the type, region, and extent of methylation. Association analysis of low-P-induced differential methylation and gene expression showed that transcriptional alterations of a small part of genes were associated with methylation changes. Dynamic methylation alterations in transposable element (TE) regions in the CHH methylation context correspond with changes in the amount of siRNA under low-P conditions, indicating an important role of siRNAs in modulating TE activity by guiding CHH methylation in TE regions. Together, these results could help to elucidate the epigenetic regulation mechanisms governing the responses of plants to abiotic stresses.

Up-regulating GmETO1 improves phosphorus uptake and use efficiency by promoting root growth in soybean.

Soybean is a high inorganic phosphate (Pi) demanding crop; its production is strongly suppressed when Pi is deficient in soil. However, the regulatory mechanism of Pi deficiency tolerance in soybean is still largely unclear. Here, our findings highlighted the pivotal role of the ethylene-associated pathway in soybean tolerance to Pi deficiency by comparatively studying transcriptome changes between a representative Pi-deficiency-tolerant soybean genotype NN94156 and a sensitive genotype Bogao under different Pi supplies. By further integrating high-confident linkage and association mapping, we identified that Ethylene-Overproduction Protein 1 (GmETO1), an essential ethylene-biosynthesis regulator, underlies the major quantitative trait locus (QTL) q14-2 controlling Pi uptake. GmETO1 was also the representative member of ETO1 family members that was strongly induced by Pi deficiency. Overexpressing GmETO1 significantly enhanced Pi deficiency tolerance by increasing proliferation and elongation of hairy roots, Pi uptake and use efficiency, and conversely, silencing of GmETO1 led to opposite findings. We further demonstrated that Pi-deficiency inducible genes critical for root morphological and physiological traits including GmACP1/2, Pht1;4, Expansin-A7 and Root Primordium Defective 1 functioned downstream of GmETO1. Our study provides comprehensive insight into the complex regulatory mechanism of Pi deficiency tolerance in soybean and a potential way to genetically improve soybean low-Pi tolerance.

Transcriptome-wide identification of novel circular RNAs in soybean in response to low-phosphorus stress.

Low-phosphorus (LP) stress is a major factor limiting the growth and yield of soybean. Circular RNAs (circRNAs) are novel noncoding RNAs that play a crucial role in plant responses to abiotic stress. However, how LP stress mediates the biogenesis of circRNAs in soybean remains unclear. Here, to explore the response mechanisms of circRNAs to LP stress, the roots of two representative soybean genotypes with different P-use efficiency, Bogao (a LP-sensitive genotype) and Nannong 94156 (a LP-tolerant genotype), were used for the construction of RNA sequencing (RNA-seq) libraries and circRNA identification. In total, 371 novel circRNA candidates, including 120 significantly differentially expressed (DE) circRNAs, were identified across different P levels and genotypes. More DE circRNAs were significantly regulated by LP stress in Bogao than in NN94156, suggesting that the tolerant genotype was less affected by LP stress than the sensitive genotype was; in other words, NN94156 may have a better ability to maintain P homeostasis under LP stress. Moreover, a positive correlation was observed between the expression patterns of P stress-induced circRNAs and their circRNA-host genes. Gene Ontology (GO) enrichment analysis of these circRNA-host genes and microRNA (miRNA)-targeted genes indicated that these DE circRNAs were involved mainly in defense responses, ADP binding, nucleoside binding, organic substance catabolic processes, oxidoreductase activity, and signal transduction. Together, our results revealed that LP stress can significantly alter the genome-wide profiles of circRNAs and indicated that the regulation of circRNAs was both genotype and environment specific in response to LP stress. LP-induced circRNAs might provide a rich resource for LP-responsive circRNA candidates for future studies.