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Summary MYB transcription factors (TFs) have been reported to regulate the biosynthesis of secondary metabolites, as well as to mediate plant adaption to abiotic stresses, including drought. However, the roles of MYB TFs in regulating plant architecture and yield potential remain poorly understood. Here, we studied the roles of the dehydration‐inducible GmMYB14 gene in regulating plant architecture, high‐density yield and drought tolerance through the brassinosteroid (BR) pathway in soybean. GmMYB14 was shown to localize to nucleus and has a transactivation activity. Stable GmMYB14‐overexpressing (GmMYB14‐OX) transgenic soybean plants displayed a semi‐dwarfism and compact plant architecture associated with decreased cell size, resulting in a decrease in plant height, internode length, leaf area, leaf petiole length and leaf petiole angle, and improved yield in high density under field conditions. Results of the transcriptome sequencing suggested the involvement of BRs in regulating GmMYB14‐OX plant architecture. Indeed, GmMYB14‐OX plants showed reduced endogenous BR contents, while exogenous application of brassinolide could partly rescue the phenotype of GmMYB14‐OX plants. Furthermore, GmMYB14 was shown to directly bind to the promoter of GmBEN1 and up‐regulate its expression, leading to reduced BR content in GmMYB14‐OX plants. GmMYB14‐OX plants also displayed improved drought tolerance under field conditions. GmBEN1 expression was also up‐regulated in the leaves of GmMYB14‐OX plants under polyethylene glycol treatment, indicating that the GmBEN1‐mediated reduction in BR level under stress also contributed to drought/osmotic stress tolerance of the transgenic plants. Our findings provided a strategy for stably increasing high‐density yield and drought tolerance in soybean using a single TF‐encoding gene.

Cystathionine-β-synthase (CBS) domain-containing proteins (CDCPs) constitute a large family in plants, and members of this family have been implicated in a variety of biological processes. However, the precise functions and the underlying mechanisms of most members of this family in plants remain to be elucidated. CBSDUF proteins belong to the CDCP superfamily, which contains one domain of unknown function (DUF21) and an N terminus that is adjacent to two intracellular CBS domains. In this study, a comprehensive genome database analysis of soybean was performed to investigate the role(s) of these CBSDUFs and to explore their nomenclature, classification, chromosomal distribution, exon–intron organization, protein structure, and phylogenetic relationships; the analysis identified a total of 18 putative CBSDUF genes. Using specific protein domains and phylogenetic analysis, the CBSDUF gene family was subdivided into eight groups. The soybean CBSDUF genes showed an uneven distribution on 12 chromosomes of Glycine max. RNA-seq transcriptome data from different tissues in public databases revealed tissue-specific and differential expression profiles of the GmCBSDUFs, and qPCR analysis revealed that certain groups of soybean CBSDUFs are likely involved in specific stress responses. In addition, GmCBSDUF3 transgenic Arabidopsis was subjected to phenotypic analysis under NaCl, PEG, and ABA stress treatments. The overexpression of GmCBSDUF3 could enhance tolerance to drought and salt stress in Arabidopsis. This study presents a first comprehensive look at soybean CBSDUF proteins and provides valuable resources for functionally elucidating this protein subgroup within the CBS domain-containing protein family. Electronic supplementary material The online version of this article (10.1007/s10528-020-09991-w) contains supplementary material, which is available to authorized users.

Seed vigor plays an essential role in soybean production. Soybean seed is sensitive to environment and easy to get seed deterioration. Generally soybean seed vigor can be maintained for less than one year and have to be multiplied every year. Varieties with good seed vigor are essential for maintaining planting population and stable yield. In this research, 419 germplasms from three eco-regions, South China, Huanghuai region and Northeast China, were evaluated for seed vigor by assessing germination ratio, germination potential and germination index. About 34.8% of tested germplasm had germination ratio equal or more than 85%, suggested by the national soybean seed quality standard of China. Only 9 from 82 tested cultivars met the seed standard. Seed germination ratio distribution had obvious regional feature. Landraces performed better seed vigor than cultivars. Germplasms from South China had stronger seed vigor compared to those from Huanghuai and Northeast regions. Seed vigor has no significant relationship with maturity and 100-seed weight. Totally 21 germplasms with strong seed vigor, favor maturation and seed size were identified and can be used in future breeding program.

Soybean (Glycine max) is a legume crop with great economic value that provides rich protein and oil for human food and animal feed. In order to cope with the ever-increasing need for soybean products and the changing environment, soybean genetic improvement needs to be accelerated. In recent years, the rapid developed genome editing technologies, such as zinc finger nuclease (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR associated protein (CRISPR/Cas), have shown broad application prospects in gene function research and improvement of important agronomic traits in many crops, and has also brought opportunities for soybean breeding. Here we systematically reviewed recent advances in genome editing technology. We also summarized the significances, current applications, challenges and future perspectives in soybean genome editing, which could provide references for exerting the feature and advantage of this technology to better soybean improvement.

Legume nodule development and senescence directly affect nitrogen fixation efficiency and involve a programmed series of molecular events. These molecular events are carried out synchronously by legumes and rhizobia. The characteristics and molecular mechanisms of nitrogen fixation at soybean important developmental stages play critical roles in soybean cultivation and fertilizer application. Although the gene expression of soybean were analyzed in nodules at five important soybean developmental stages, information on the expression of rhizobial genes in these nodule samples is limited. In the present study, we investigated the expression of Bradyrhizobium diazoefficiens 113-2 genes in the nodule samples from five developmental stages of soybean (Branching stage, flowering stage, fruiting stage, pod stage and harvest stage). Similar gene expression patterns of B. diazoefficiens 113-2 were existed during optimal symbiotic functioning, while different expression patterns were found among early nodule development, nitrogen fixation progress and nodule senescence. Besides, we identified 164 important different expression genes (DEGs) associated with nodule development and senescence. These DEGs included those encoding nod, nif, fix proteins and T3SS secretion system-related proteins, as well as proteins involved in nitrogen metabolism, ABC transporters and two-component system pathways. Gene Ontology, KEGG pathway and homology analysis of the identified DEGs revealed that most of these DEGs are uncharacterized genes associated with nodule development and senescence, and they are not core genes among the rhizobia genomes. Our results provide new clues for the understanding of the genetic determinants of soil rhizobia in nodule development and senescence, and supply theoretical basis for the creation of high efficiency soybean cultivation technology.

Crop yield has been maintaining its attraction for researchers because of the demand of global population growth. Mutation of flowering activators, such as florigen, increases plant biomass at the expense of later flowering, which prevents crop maturity in the field. As a result, it is difficult to apply flowering activators in agriculture production. Here, we developed a strategy to utilize florigen to significantly improve soybean yield in the field. Through the screening of transgenic lines of RNAi-silenced florigen homologs in soybean (Glycine-max-Flowering Locus T Like, GmFTL), we identified a line, GmFTL-RNAi#1, with minor changes in both GmFTL expression and flowering time but with notable increase in soybean yield. As expected, GmFTL-RNAi#1 matured normally in the field and exhibited markedly high yield over multiple locations and years, indicating that it is possible to reach a trade-off between flowering time and high yield through the fine-tuning expression of flowering activators. Further studies uncovered an unknown mechanism by which GmFTL negatively regulates photosynthesis, a substantial source of crop yield, demonstrating a novel function of florigen. Thus, because of the highly conserved functions of florigen in plants and the classical RNAi approach, the findings provide a promising strategy to harness early flowering genes to improve crop yield.

Delaying the nodule senescence of legume crops can prolong the time of nitrogen fixation and attenuate the lack of fertilizer in the later stage of legume crop cultivation, resulting in improved crop yield and reduced usage of nitrogen fertilizer. However, effective measures to delay the nodule senescence of legume crops in agriculture are relatively lacking. In the present review, we summarized the structural and physiological characteristics of nodule senescence, as well as the corresponding detection methods, providing technical support for the identification of nodule senescence phenotype. We then outlined the key genes currently known to be involved in the regulation of nodule senescence, offering the molecular genetic information for breeding varieties with delayed nodule senescence. In addition, we reviewed various abiotic factors affecting nodule senescence, providing a theoretical basis for the interaction between molecular genetics and abiotic factors in the regulation of nodule senescence. Finally, we briefly prospected research foci of nodule senescence in the future.

Background Plant papain-like cysteine proteases (PLCPs) are a large class of proteolytic enzymes and play important roles in root nodule symbiosis (RNS), while the whole-genome studies of PLCP family genes in legume are quite limited, and the roles of Glycine max PLCPs (GmPLCPs) in nodulation, nodule development and senescence are not fully understood. Results In the present study, we identified 97 GmPLCPs and performed a genome-wide survey to explore the expansion of soybean PLCP family genes and their relationships to RNS. Nineteen paralogous pairs of genomic segments, consisting of 77 GmPLCPs, formed by whole-genome duplication (WGD) events were identified, showing a high degree of complexity in duplication. Phylogenetic analysis among different species showed that the lineage differentiation of GmPLCPs occurred after family expansion, and large tandem repeat segment were specifically in soybean. The expression patterns of GmPLCPs in symbiosis-related tissues and nodules identified RNS-related GmPLCPs and provided insights into their putative symbiotic functions in soybean. The symbiotic function analyses showed that a RNS-related GmPLCP gene (Glyma.04G190700) really participate in nodulation and nodule development. Conclusions Our findings improved our understanding of the functional diversity of legume PLCP family genes, and provided insights into the putative roles of the legume PLCPs in nodulation, nodule development and senescence.

In the present study, we sequenced the complete genome of Bradyrhizobium diazoefficiens 113-2. The genomic characteristics of six selected rhizobial strains (two fast-growing rhizobia, two medium-slow-growing rhizobia and two slow-growing rhizobia) with four different legume hosts were analyzed by comparative genomic analysis. Genomes of B. diazoefficiens 113-2 and B. diazoefficiens USDA110 were found to share a large synteny blocks and a high ANI value, supporting 113-2 as a strain of B. diazoefficiens. 5,455 singletons and 11,656 clusters were identified among the six rhizobia genomes, and most of the pair-wise comparisons clusters were shared by the two genomes of strains in the same genus. Similar genus-specific gene numbers in the assigned COG functional terms were present in the two strains of the same genus, while the numbers were decreased with the increase of growth rate in most of the COG terms. KEGG pathway analysis of B. diazoefficiens 113-2 suggested that the rhizobial genes in ABC transporters and Two-Component system were mainly species-specific. Besides, the candidate genes related to secretion system and surface polysaccharides biosynthesis in the genomes of the six strains were explored and compared. 39 nodulation gene families, 12 nif gene families and 10 fix gene families in the genomes of these six strains were identified, and gene classes in most of gene families and the types and total gene numbers of gene families were substantially different among these six genomes. We also performed synteny analyses for above-mentioned nod, nif, and fix gene groupings, and selected NodW, NolK, NoeJ, NifB, FixK, and FixJ gene families to perform phylogeny analyses. Our results provided valuable molecular insights into species specificity and host specificity. The genetic information responsible for host specificity will play important roles in expanding the host range of rhizobia among legumes, which might provide new clues for the understanding of the genetic determinants of non-legume-rhizobium symbiosis.

As the second most destructive disease in soybean (Glycine max (L.) Merr), Phytophthora root or stem-rot (PRR) of soybean, which was caused by Phytophthora sojae (P. sojae), could lead to a 10–40% or complete yield loss annually. Researches were needed to perform to identify the P. sojae-resistant germplasm and to better understand the genetic basis of P. sojae resistance in soybean. A total of 225 diverse soybean cultivars and 109 recombinant inbred lines (RILs) derived from crossing ‘DongnongL-28’ (resistant to P. sojae race 1) and ‘Hefeng 25’ (susceptible to P. sojae race 1) were used to evaluate P. sojae race 1 resistance. These 225 soybean cultivars were sequenced using the Specific Locus Amplified Fragment Sequencing (SLAF-seq) approach, and 28,722 single-nucleotide polymorphisms (SNPs) were obtained from the mapping of resistant loci through genome-wide association. Eight quantitative trait nucleotides (QTNs) were associated with resistance to P. sojae race 1. The QTN on Chr.03 was colocalised according to linkage mapping of the RILs. A total of 18 candidate genes were predicted in the flanking region of the colocalised locus on Chr.03. There were stress response-related motifs, such as cis-acting regulatory elements involved in salicylic acid or MeJA responsiveness, in the 1-kb upstream region of sixteen genes. Quantitative RT-PCR showed that the Glyma.03G033700 was induced by P. sojae race 1. Association analysis for Rps loci showed that Glyma.03G033700 and Glyma.03G033800 were the candidates near peak SNP. The identified loci along with the candidate genes could be valuable for studying the molecular mechanisms underlying soybean resistance to P. sojae race 1 and breeding resistant varieties.

Chlorophyll plays an essential role in photosynthetic light harvesting and energy transduction in green tissues of higher plants and is closely related to photosynthesis and crop yield. Identification of transcription factors (TFs) involved in regulating chlorophyll biosynthesis is still limited in soybean (Glycine max), and the previously identified GmGATA58 is suggested to potentially modulate chlorophyll and nitrogen metabolisms, but its complete function is still unknown. In this study, subcellular localization assay showed that GmGATA58 was localized in the nucleus. Histochemical GUS assay and qPCR assay indicated that GmGATA58 was mainly expressed in leaves and responded to nitrogen, light and phytohormone treatments. Overexpression of GmGATA58 in the Arabidopsis thaliana ortholog AtGATA21 (gnc) mutant complemented the greening defect, while overexpression in Arabidopsis wild-type led to increasing chlorophyll content in leaves through up-regulating the expression levels of the large of chlorophyll biosynthetic pathway genes, but suppressing plant growth and yield, although the net photosynthetic rate was slightly improved. Dual-luciferase reporter assay also supported that GmGATA58 activated the transcription activities of three promoters of key chlorophyll biosynthetic genes of soybean in transformed protoplast of Arabidopsis. It is concluded that GmGATA58 played an important role in regulating chlorophyll biosynthesis, but suppressed plant growth and yield in transgenic Arabidopsis.

DPPH assay of the in-house marine-derived fungi uncovered that the EtOAc extract of the cultured fungus Aspergillus europaeus WZXY-SX-4-1, which was isolated from the marine sponge Xestospongia testudinaria, possesses radical scavenging activity. Chromatographic separation of the bioactive extract resulted in the isolation of 20 polyketide derivatives, including six new compounds namely eurobenzophenones A-C (1-3), euroxanthones A-B (4-5), and (+)1-O-demethylvariecolorquinones A (6). The structures of new compounds were determined on the basis of the analyses of spectroscopic data, including the Snatzke method for the configurational assignment. Benzophenones 3, 9 and 10 exhibited potent radical scavenging activity against DPPH. All polyketides were evaluated for the inhibitory effects toward the LPS induced nitric oxide (NO) production in mouse microglia BV2 cells and the NF-κB activation in human colon carcinoma cell line SW480. Compound 9 with the significant DPPH radical scavenging activity is corresponded to the potent inhibition against NF-κB in SW480 cells induced by LPS. Compounds 2, 4, 16-18 exerted remarked down-regulation of NF-κB in LPS-induced SW480 cells with weak inhibitory effects against NO production and the DPPH radical scavenging activity.

Five new methylsuccinimide-based sulfur-bearing compounds, namely violaceimides A–E (1–5), were isolated from the sponge-associated fungal strain Aspergillus violaceus WZXY-m64-17. Their structures were determined by analyses of the HRESIMS, 1D and 2D NMR data, along with the Mosher’s and Snatzke’s methods, experimental and calculated ECD data, as well as chemical conversion for the configurational assignment. The structures of 1–5 featured the fusion of methylsuccinimide with a modified cysteine to incorporate sulfur element, which was found from nature for the first time. The biogenetic relationship of the sulfur-containing compounds was postulated. Violaceimides A and B exerted selective inhibition against the growth of human leukemia U937 and human colorectal cancer cell HCT-8 with low cytotoxicity toward Vero cells, whereas violaceimide E showed moderate activity to inhibit U937 cells. The preliminary structure-activity relationship was discussed.

As major environment factors, drought or high salinity affect crop growth, development and yield. Transgenic approach is an effective way to improve abiotic stress tolerance of crops. In this study, we comparatively analyzed gene structures, genome location, and the evolution of syntaxin proteins containing late embryogenesis abundant (LEA2) domain. GmSYP24 was identified as a dehydration-responsive gene. Our study showed that the GmSYP24 protein was located on the cell membrane. The overexpression of GmSYP24 (GmSYP24ox) in soybean and heteroexpression of GmSYP24 (GmSYP24hx) in Arabidopsis exhibited insensitivity to osmotic/drought and high salinity. However, wild type soybean, Arabidopsis, and the mutant of GmSYP24 homologous gene of Arabidopsis were sensitive to the stresses. Under the abiotic stresses, transgenic soybean plants had greater water content and higher activities of POD, SOD compared with non-transgenic controls. And the leaf stomatal density and opening were reduced in transgenic Arabidopsis. The sensitivity to ABA was decreased during seed germination of GmSYP24ox and GmSYP24hx. GmSYP24hx induced up-regulation of ABA-responsive genes. GmSYP24ox alters the expression of some aquaporins under osmotic/drought, salt, or ABA treatment. These results demonstrated that GmSYP24 played an important role in osmotic/drought or salt tolerance in ABA signal pathway.

Background The plant architecture has significant effects on grain yield of various crops, including soybean (Glycine max), but the knowledge on optimization of plant architecture in order to increase yield potential is still limited. Recently, CRISPR/Cas9 system has revolutionized genome editing, and has been widely utilized to edit the genomes of a diverse range of crop plants. Results In the present study, we employed the CRISPR/Cas9 system to mutate four genes encoding SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors of the SPL9 family in soybean. These four GmSPL9 genes are negatively regulated by GmmiR156b, a target for the improvement of soybean plant architecture and yields. The soybean Williams 82 was transformed with the binary CRISPR/Cas9 plasmid, assembled with four sgRNA expression cassettes driven by the Arabidopsis thaliana U3 or U6 promoter, targeting different sites of these four SPL9 genes via Agrobacterium tumefaciens-mediated transformation. A 1-bp deletion was detected in one target site of the GmSPL9a and one target site of the GmSPL9b, respectively, by DNA sequencing analysis of two T0-generation plants. T2-generation spl9a and spl9b homozygous single mutants exhibited no obvious phenotype changes; but the T2 double homozygous mutant spl9a/spl9b possessed shorter plastochron length. In T4 generation, higher-order mutant plants carrying various combinations of mutations showed increased node number on the main stem and branch number, consequently increased total node number per plants at different levels. In addition, the expression levels of the examined GmSPL9 genes were higher in the spl9b-1 single mutant than wild-type plants, which might suggest a feedback regulation on the expression of the investigated GmSPL9 genes in soybean. Conclusions Our results showed that CRISPR/Cas9-mediated targeted mutagenesis of four GmSPL9 genes in different combinations altered plant architecture in soybean. The findings demonstrated that GmSPL9a, GmSPL9b, GmSPL9c and GmSPL9 function as redundant transcription factors in regulating plant architecture in soybean. Electronic supplementary material The online version of this article (10.1186/s12870-019-1746-6) contains supplementary material, which is available to authorized users.

Drought tolerance level of 136 soybean genotypes, the correlations among traits were evaluated, and several important drought-tolerant genotypes, traits, SNPs and genes were possibly useful for soybean genetic breeding. Drought is an adverse environmental factor affecting crops growth, development, and yield. Promising genotypes and genes with improved tolerance to drought are probably effective ways to alleviate the situation. In this study, our main task was to determine drought tolerance level of 136 soybean genotypes, the correlations among physiological and agronomic traits under drought, and drought-tolerant single nucleotide polymorphism (SNPs) and genes. In this study, twenty-six varieties were identified as excellent tolerant genotypes to stress among which S14, S93 and S135 with high drought-tolerant index (DTI > 1.3) and yield (Y > 300 kg). Fourteen varieties were identified as drought-sensitive genotypes, such as S25, S45 and S58, with low drought-tolerant index (DTI

A R2R3-MYB transcription factor EsAN2 was isolated from Epimedium sagittatum and functionally characterized to regulate the anthocyanin biosynthetic pathway. Epimedium plants are used widely both as traditional Chinese medicinal herbs and ornamental perennials. Anthocyanins, acting as major contributors to plant color diversity, their biosynthesis are regulated by a series of transcription factors, including MYB, bHLH and WD40 protein. Previously, a MYB transcription factor involved in regulation of the anthocyanin pathway from Epimedium sagittatum, EsMYBA1 has been isolated, but was found to be expressed mostly in leaves. In this research, another MYB transcription factor, designated as EsAN2, was isolated from flowers by the screening of E. sagittatum EST database. Preferential expression of EsAN2 in flowers and flower buds was found. Ectopic expression of EsAN2 in tobacco significantly enhanced the anthocyanin biosynthesis and accumulation, both in leaves and flowers. Most structural genes of the anthocyanin biosynthetic pathway were strongly upregulated, as well as two bHLH regulators (NtAn1a and NtAn1b) in old leaves of tobacco overexpressing EsAN2, compared to the control plants. While only three structural genes, chalcone synthase (CHS), chalcone isomerase (CHI) and anthocyanidin synthase (ANS), were upregulated by EsAN2 ectopic expression in tobacco flowers. Yeast two-hybrid assay showed that EsAN2 was capable of interacting with four bHLH regulators of the anthocyanin biosynthetic pathway. These results suggest that EsAN2 is involved in regulation of the anthocyanin biosynthesis in Epimedium flowers. Identification and characterization of EsAN2 provide insight into the coloration of Epimedium flowers and a potential candidate gene for metabolic engineering of flavonoids in the future.

Association analysis techniques were used to identify and verify twelve single nucleotide polymorphisms (SNPs) associated with Fusarium graminearum resistance. Two novel candidate genes were obtained. Fusarium graminearum causes seed and root rot and seedling damping-off of soybean, leading to severe yield loss. Presently, the genetic basis of resistance to F. graminearum is elucidated in only four soybean accessions, which is not sufficient for resistance improvement. The objective of the present study was to identify the genome-wide genetic architecture of resistance to F. graminearum in landraces and cultivated soybeans based on a growth room evaluation. The resistance levels of 314 diverse accessions were tested, and 22,888 single nucleotide polymorphisms (SNPs) with a minor allele frequency of > 0.05 were developed using the specific-locus amplified fragment sequencing (SLAF-seq) approach. Twelve SNPs were identified as associated with F. graminearum resistance, and these SNPs were located at 12 genomic regions on eight chromosomes (Chr.) and could explain 5.53–14.71% of the observed phenotypic variation. One SNP, rs9479021, located on Chr.6, overlapped with qRfg_Gm06, the known QTL for resistance to F. graminearum. The other SNPs were novel and associated with resistance to F. graminearum. Nine novel candidate genes were predicted to contribute to resistance to F. graminearum according to the haplotype and transcript abundance analysis of the candidate genes. The identified markers and resistant cultivars are valuable for the improvement of resistance to F. graminearum.

Root nodule symbiosis (RNS) is one of the most productive and economical systems for nitrogen fixation, and previous studies have shown that several nodule-specific C2H2-zinc finger proteins (ZFPs) play important roles in symbiosis establishment and nodule function. However, C2H2-ZFPs are the most widespread ZFPs in eukaryotes, and a great variation of structure and function exist among the family members. It remains largely unclear whether or not special types of C2H2-ZF genes participate in symbiosis, especially in soybean. In the present study, we performed a genome-wide survey of soybean C2H2-ZF genes, and 321 soybean C2H2-ZF genes were identified and classified into 11 clearly distinguishable subsets (Gm-t1-SF, Gm-t2-SF, Gm-1i-Q-SF, Gm-1i-M-SF, Gm-1i-Z-SF, Gm-1i-D-SF, Gm-2i-Q-SF, Gm-2i-M-SF, Gm-2i-Mix-SF, Gm-3i-SF, and Gm-4i-SF) based on the arrangements, numbers, and types of C2H2-ZF domains. Phylogenetic and gene ontology analyses were carried out to assess the conserved sequence and GO function among these subsets, and the results showed that the classification of soybean C2H2-ZFPs was reasonable. The expression profile of soybean C2H2-ZFPs in multiple tissues showed that nearly half of soybean C2H2-ZFPs within different subsets had expressions in nodules, including a clustering branch consisting of 11 Gm-1i-Q-SF genes specifically expressed in symbiotic-relative tissues. RNA-Seq was used to identify symbiosis-related soybean C2H2-ZFPs, and the expression pattern of the soybean C2H2-ZFPs in roots and nodules at different development stages showed that soybean C2H2-ZFPs mainly played roles in nodule development or nodule function rather than nodulation signal transduction, and nearly half of these genes had high expressions and/or different expression patterns during soybean nodule development, especially for the six clustering branches of genes consisting of different subsets of C2H2-ZFPs. Furthermore, the selected symbiosis-related soybean C2H2-ZFPs might function in legume-rhizobium symbiosis through regulating or interacting with other key proteins. Taken together, our findings provided useful information for the study on classification and conservative function of C2H2-ZFPs, and offered solid evidence for investigation of rhizobium symbiosis-related C2H2-ZFPs in soybean or other legumes.

OBJECTIVE The aim of this study was to test the hypothesis that exercise training promotes motor recovery after stroke by facilitating axonal remodeling via inhibition of the Nogo-A/NgR1 and Rho-A pathway. DESIGN A distal middle cerebral artery occlusion model was generated in stroke-prone renovascular hypertensive rats. Stroke-prone renovascular hypertensive rats were randomly divided into a control group, an exercise training group, and a sham group. Motor function was measured using the grip strength test. Axon and myelin remodeling markers, growth-associated protein 43, myelin basic protein, Tau, and amyloid precursor protein were detected by immunofluorescence. The expression of Nogo-A, NgR1, and Rho-A was demonstrated by immunofluorescence and Western blotting in the peri-infarction area at 7, 14, 28, and 52 days after distal middle cerebral artery occlusion. RESULTS Grip strength was higher in the exercise training group (P < 0.05). Exercise training increased the expression of growth-associated protein 43, myelin basic protein (at 7, 14, and 28 days), and Tau (at 7 and 14 days), and decreased the expression of axonal damage amyloid precursor protein (at 7 and 14 days), compared with the control group. The protein levels of Nogo-A (at 7 and 14 days), NgR1 (at 7, 14, and 28 days), and Rho-A (at 14 and 28 days) were reduced after exercise training. CONCLUSIONS Exercise training promotes axonal recovery, which is associated with functional improvement after cerebral infarction. Down-regulation of the Nogo-A/NgR1/Rho-A may mediate the axonal remodeling induced by exercise training.

Identifying a superior soybean variety with high defoliator resistance is important to avoid yield loss. Cotton bollworm (Helicoverpa armigera Hubner) is one of the major defoliators of soybean (Glycine max [L.] Merr.) worldwide. In this study, we evaluated the effect of H. armigera larvae on ED059, a wild soybean (Glycine soja Sieb. et Zucc.), and three cultivated soybean varieties: Tianlong 2, PI 535807, and PI 533604, in choice and no-choice assays. The percentage of ED059 leaflets consumed by H. armigera was lower than that of the three cultivated soybeans. Larvae that fed on ED059 exhibited low weight gain and high mortality rate. Waldbauer nutritional indices suggested that ED059 reduced the growth, consumption, and frass production of H. armigera larvae. Larvae that fed on ED059 showed lower efficiency of conversion of ingested and of digested food than those that fed on Tianlong 2 and PI 533604. However, they showed statistically similar consumption index and approximate digestibility compared with those fed on the three cultivated soybeans. Quantitative real-time PCR analysis revealed that 24 h after insect attack, ED059 had higher transcript levels of Kunitz trypsin inhibitor 3, Cysteine proteinase inhibitor 2, and Nerolidol synthase 1 but a lower transcript level of Pathogenesis-related protein 1 than Tianlong 2. The gene expression results were consistent with the presence of higher levels of jasmonic acid (JA) and transcript levels of the JA biosynthesis enzyme allene oxide cyclase 3 in ED059 than in Tianlong 2. Our findings indicate that ED059 is a superior soybean line with strong insect resistance that may be mediated via the JA pathway.

Neural stem cells (NSCs) transplanted is one of the hottest research to treat Alzheimer’s disease (AD), but cholinergic neurons from stem cells were also susceptible to cell death which Heat shock protein 70 (HSP70) was affirmed to reverse. Related to cognitive impairment, cholinergic nervous cells should be investigated and ginsenoside Rg1 (G-Rg1) was considered to increase them. We chose tert-butyl hydroperoxide (t-BHP) damage model to study in vitro. Functional properties of our recombination plasmid pEGFP-C2-HSP70 were affirmed by SH-SY5Y cells. To opposite the transitory appearance of HSP70, NSCs used as the vectors of HSP70 gene overexpressed HSP70 for at least 7 days in vitro. After transfection for 3 days, G-Rg1 pretreatment for 4 hours, and coculture for 3 days, the expression of acetylcholinesterase (ChAT), synaptophysin, and the ratio of NeuN and GFAP were assessed by western blot; Morphological properties were detected by 3D reconstruction and immunofluorescence. ChAT was markedly improved in the groups contained G-Rg1. In coculture system, the ratio of neurons/astrocytes and the filaments of neurons were increased; apoptosis cells were decreased, compared to monotherapy (P<0.05). In conclusion, we demonstrated that, as a safe cotreatment affirmed in vitro, overexpression of HSP70 in NSCs plus G-Rg1 promoted nervous cells regeneration from chronic oxidative damage.

Lodging is one of the major influencing factors of yield and quality in soybean [Glycine max (L.) Merr.] and other crops. To dissect the genetic basis of lodging in soybean, a recombinant inbred line population consisting of 165 lines was used to evaluate lodging percentage and eight related traits (branch number, internode length, number of nodes, plant height, stem diameter, stem strength, root length, and root weight) in three environments. Regression analysis indicated that plant height and root weight, which explain more than 55% of the variation in lodging percentage, might be the key factors influencing lodging in soybean. Nine consensus quantitative trait locus (QTLs) of lodging percentage were detected in one to three environments. Of which, eight consensus QTLs were colocated with 16 consensus QTLs of lodging-related traits by meta-analysis. In addition, seven candidate genes with the biological functions of shoot branching, root development, internode elongation, and lignin biosynthesis were identified on four pleiotropic QTL regions (oq.13-1, oq.13-2, oq.19-2, and oq.19-3) for lodging percentage and related traits. These findings showed that the consensus QTLs of lodging percentage might result from the pleiotropic QTLs affecting the lodging-related traits. Soybean lodging is determined by the cumulative effect of many traits/processes of growth and development. The combination of MAS, statistical model, and phenotypic selection will provide a powerful breeding strategy for lodging resistance in soybean.

l-Ascorbic acid (vitamin C, AsA), is an essential component for collagen biosynthesis and the major antioxidant in human, mainly obtained from the diet. Strawberry as fruit with higher AsA concentration processes a distinct AsA biosynthesis pathway from tomato (Solanum lycopersicum) which dominants in d-mannose/l-galactose pathway. Firstly, the activity of d-galacturonic acid reductase (GalUR; EC = 1.1.1.19) parallel to AsA accumulation in crude extract of tomato leaves and fruits was detected. Subsequently, transgenic tomato lines overexpressing strawberry FaGalUR gene resulted in twofold and 1.6-fold increase in AsA level in tomato fruit and leaf, respectively, which correlated positively with FaGalUR transcriptional abundance and GalUR activity. Furthermore, FaGalUR-overexpressing plants showed enhanced tolerance to abiotic stresses induced by oxidization (methyl viologen), salt (NaCl) and cold as compared to the wild-type plants. Taken together, the present findings suggest that tomato might share the alternative d-galacturonate pathway for ascorbate biosynthesis, and abiotic stress tolerance as well as AsA accumulation in tomato can be enhanced by regulating strawberry GalUR gene.

The Dof (DNA binding with One Finger) family encoding single zinc finger proteins has been known as a family of plant-specific transcription factors. These transcription factors are involved in a variety of functions of importance for different biological processes in plants. In the current study, we identified 34 Dof family genes in tomato, distributed on 11 chromosomes. A complete overview of SlDof genes in tomato is presented, including the gene structures, chromosome locations, phylogeny, protein motifs and evolution pattern. Phylogenetic analysis of 34 SlDof proteins resulted in four classes constituting six clusters. In addition, a comparative analysis between these genes in tomato, Arabidopsis and rice was also performed. The tomato Dof family expansion has been dated to recent duplication events, and segmental duplication is predominant for the SlDof genes. Furthermore, the SlDof genes displayed differential expression either in their transcript abundance or in their expression patterns under normal growth conditions. This is the first step towards genome-wide analyses of the Dof genes in tomato. Our study provides a very useful reference for cloning and functional analysis of the members of this gene family in tomato and other species.

Real-time quantitative reverse transcription PCR is a sensitive and widely used technique to quantify gene expression. To achieve a reliable result, appropriate reference genes are highly required for normalization of transcripts in different samples. In this study, 9 previously published reference genes (60S, Fbox, ELF1A, ELF1B, ACT11, TUA5, UBC4, G6PD, CYP2) of soybean [Glycine max (L.) Merr.] were selected. The expression stability of the 9 genes was evaluated under conditions of biotic stress caused by infection with soybean mosaic virus, nitrogen stress, across different cultivars and developmental stages. ΔCt and geNorm algorithms were used to evaluate and rank the expression stability of the 9 reference genes. Results obtained from two algorithms showed high consistency. Moreover, results of pairwise variation showed that two reference genes were sufficient to normalize the expression levels of target genes under each experimental setting. For virus infection, ELF1A and ELF1B were the most stable reference genes for accurate normalization. For different developmental stages, Fbox and G6PD had the highest expression stability between two soybean cultivars (Tanlong No. 1 and Tanlong No. 2). ELF1B and ACT11 were identified as the most stably expressed reference genes both under nitrogen stress and among different cultivars. The results showed that none of the candidate reference genes were uniformly expressed at different conditions, and selecting appropriate reference genes was pivotal for gene expression studies with particular condition and tissue. The most stable combination of genes identified in this study will help to achieve more accurate and reliable results in a wide variety of samples in soybean.

Nodulation, nodule development and senescence directly affects nitrogen fixation efficiency, and previous studies have shown that inhibition of some cysteine proteases delay nodule senescence, so their nature inhibitors, cystatin genes, are very important in nodulation, nodule development and senescence. Although several cystatins are actively transcribed in soybean nodules, their exact roles and functional diversities in legume have not been well explored in genome-wide survey studies. In this report, we performed a genome-wide survey of cystatin family genes to explore their relationship to nodulation and nodule development in soybean and identified 20 cystatin genes that encode peptides with 97~245 amino acid residues, different isoelectric points (pI) and structure characteristics, and various putative plant regulatory elements in 3000 bp putative promoter fragments upstream of the 20 soybean cystatins in response to different abiotic/biotic stresses, hormone signals and symbiosis signals. The expression profiles of these cystatin genes in soybean symbiosis with rhizobium strain Bradyrhizobium japonicum strain 113-2 revealed that 7 cystatin family genes play different roles in nodulation as well as nodule development and senescence. However, these genes were not root nodule symbiosis (RNS) - specific and did not encode special clade cystatin protein with structures related to nodulation and nodule development. Besides, only two of these soybean cystatins were not upregulated in symbiosis after ABA treatment. The functional analysis showed that a candidate gene Glyma.15G227500 (GmCYS16) was likely to play a positive role in soybean nodulation. Besides, evolutionary relationships analysis divided the cystatin genes from Arabidopsis thaliana, Nicotiana tabacum, rice, barley and four legume plants into three groups. Interestingly, Group A cystatins are special in legume plants, but only include one of the above-mentioned 7 cystatin genes related to nodulation and nodule development. Overall, our results provide useful information or clues for our understanding of the functional diversity of legume cystatin family proteins in soybean nodulation and nodule development and for finding nodule-specific cysteine proteases in soybean.

Flavonols as plant secondary metabolites with vital roles in plant development and defense against UV light, have been demonstrated to be the main bioactive components in the genus Epimedium plants, several species of which are used as materials for Herba Epimedii, an important traditional Chinese medicine. The flavonol biosynthetic pathway genes had been already isolated from E. sagittatum, but a R2R3-MYB transcription factor regulating the flavonol synthesis has not been functionally characterized so far in Epimedium plants. In this study, we isolated and characterized the R2R3-MYB transcription factor EsMYBF1 involved in regulation of the flavonol biosynthetic pathway from E. sagittatum. Sequence analysis indicated that EsMYBF1 belongs to the subgroup 7 of R2R3-MYB family which contains the flavonol-specific MYB regulators identified to date. Transient reporter assay showed that EsMYBF1 strongly activated the promoters of EsF3H (flavanone 3-hydroxylase) and EsFLS (flavonol synthase), but not the promoters of EsDFRs (dihydroflavonol 4-reductase) and EsANS (anthocyanidin synthase) in transiently transformed Nicotiana benthamiana leaves. Both yeast two-hybrid assay and transient reporter assay validated EsMYBF1 to be independent of EsTT8, or AtTT8 bHLH regulators of the flavonoid pathway as cofactors. Ectopic expression of EsMYBF1 in transgenic tobacco resulted in the increased flavonol content and the decreased anthocyanin content in flowers. Correspondingly, the structural genes involved in flavonol synthesis were upregulated in the EsMYBF1 overexpression lines, including NtCHS (chalcone synthase), NtCHI (chalcone isomerase), NtF3H and NtFLS, whereas the late biosynthetic genes of the anthocyanin pathway (NtDFR and NtANS) were remarkably downregulated, compared to the controls. These results suggest that EsMYBF1 is a flavonol-specific R2R3-MYB regulator, and involved in regulation of the biosynthesis of the flavonol-derived bioactive components in E. sagittatum. Thus, identification and functional characterization of EsMYBF1 provide insight into understanding the biosynthesis and regulation of the flavonol-derived bioactive components in Epimedium plants, and also provide an effective tool gene for genetic manipulation to improve the flavonol synthesis.

The root nodule symbiosis (RNS) between legume plants and rhizobia is the most efficient and productive source of nitrogen fixation, and has critical importance in agriculture and mesology. Soybean (Glycine max), one of the most important legume crops in the world, establishes a nitrogen-fixing symbiosis with different types of rhizobia, and the efficiency of symbiotic nitrogen fixation in soybean greatly depends on the symbiotic host-specificity. Although, it has been reported that rhizobia use surface polysaccharides, secretion proteins of the type-three secretion systems and nod factors to modulate host range, the host control of nodulation specificity remains poorly understood. In this report, the soybean roots of two symbiotic systems (Bradyrhizobium japonicum strain 113-2-soybean and Sinorhizobium fredii USDA205-soybean)with notable different nodulation phenotypes and the control were studied at five different post-inoculation time points (0.5, 7–24 h, 5, 16, and 21 day) by RNA-seq (Quantification). The results of qPCR analysis of 11 randomly-selected genes agreed with transcriptional profile data for 136 out of 165 (82.42%) data points and quality assessment showed that the sequencing library is of quality and reliable. Three comparisons (control vs. 113-2, control vs. USDA205 and USDA205 vs. 113-2) were made and the differentially expressed genes (DEGs) between them were analyzed. The number of DEGs at 16 days post-inoculation (dpi) was the highest in the three comparisons, and most of the DEGs in USDA205 vs. 113-2 were found at 16 dpi and 21 dpi. 44 go function terms in USDA205 vs. 113-2 were analyzed to evaluate the potential functions of the DEGs, and 10 important KEGG pathway enrichment terms were analyzed in the three comparisons. Some important genes induced in response to different strains (113-2 and USDA205) were identified and analyzed, and these genes primarily encoded soybean resistance proteins, NF-related proteins, nodulins and immunity defense proteins, as well as proteins involving flavonoids/flavone/flavonol biosynthesis and plant-pathogen interaction. Besides, 189 candidate genes are largely expressed in roots and\or nodules. The DEGs uncovered in this study provides molecular candidates for better understanding the mechanisms of symbiotic host-specificity and explaining the different symbiotic effects between soybean roots inoculated with different strains (113-2 and USDA205).

Trichomes are universal biological structures originating from the aerial epidermis, which serve as an excellent model to study plant differentiation at the cell level. Although the pathway regulating trichome formation in the Rosids has been well characterized, only very recently a few genes were identified for trichome initiation in the Asterids. In this study, we cloned Woolly ( Wo ), essential for trichome formation in tomato. Transgenic experiments revealed that the woolly phenotype is caused by the mutation in Wo which encodes a homeodomain protein containing a bZIP motif and a START domain. We identified three alleles of Wo and found that each allele contains a missense mutation, which respectively results in an amino acid substitution at the C terminus. Microarray and expression analysis showed that the expression of a B-type cyclin gene, SlCycB2 , is possibly regulated by Wo , which also participates in trichome formation. Suppression of Wo or SlCycB2 expression by RNAi decreased the number of type I trichomes, and direct protein–protein interaction was detected between them, implying that both proteins may work together in the regulation of this type of trichome formation. Cytological observation and Wo transcript analysis in the developing seeds showed that embryo development was also correlated with Wo .

Reduced l -ascorbic acid can be oxidized by ascorbate peroxidase (APX) in ascorbate metabolism pathway. In present study, a mitochondrial APX (mitAPX) was cloned and suppressed by RNA interference in tomato via Agrobacterium -mediated transformation. The existence of the RNA interference T-DNA in transformed plants and their progenies were confirmed by polymerase chain reaction amplification and Southern blotting. Decreased mitAPX expression in homozygous transgenic lines was detected by semi-quantitative reverse transcriptase-polymerase chain reaction, accompanied by decreasing cytosolic APX activity by 0.4- to 0.8-fold of the untransformed control. The mitochondrial APX enzyme activity in RNAi lines decreased to 29–45% of the wild type plants. The down-regulated mitAPX expression and APX enzyme activity lead to 1.4- to 2.2-fold increase in ascorbic acid content in tomato fruits compared with wild-type control. The APX expression and APX enzyme activity were found to be correlated with increase in the amount of ascorbic acid in tomato fruits. This report demonstrates the modulation of ascorbic acid metabolic pathway by RNA interference method, and provides a feasible approach to improve ascorbic acid content in horticultural plants by suppressing genes related to ascorbic acid metabolism.

Reduced ascorbic acid (AsA) can be converted into oxidized state by ascorbate oxidase (AO). In present study, AO gene was down-regulated by RNA interference in tomato via Agrobacterium-mediated transformation. The existence of the RNA interference T-DNA in transformed plants and their progenies were confirmed by polymerase chain reaction amplification and Southern blotting. AO expression levels in transgenic lines were monitored by semi-quantitative reverse transcriptase-polymerase chain reaction. The decreased AO enzyme activity and significantly improved AsA content in tomato fruit were observed to be correlated with AO gene suppression. Transgenic plants with AO suppression and AsA improvement exhibited higher photosynthesis capacity than untransformed control under drought stress. This report demonstrates the AsA oxidation pathway by RNA interference, and provides a feasible approach to improve AsA content in horticultural plants by regulating genes related to AsA metabolism.

GDP-Mannose 3',5'-epimerase (GME; EC 5.1.3.18) catalyses the conversion of GDP-D-mannose to GDP-L-galactose, an important step in the ascorbic acid (AsA) biosynthesis pathway in higher plants. In this study, two members of the GME gene family were isolated from tomato (Solanum lycopersicum). Both SlGME genes encode 376 amino acids and share a 92% similarity with each other. Semi-quantitative RT-PCR indicated that SlGME1 was constantly expressed in various tissues, whereas SlGME2 was differentially expressed in different tissues. Transient expression of fused SlGME1-GFP (green fluorescent protein) and SlGME2-GFP in onion cells revealed the cytoplasmic localisation of the two proteins. Transgenic plants over-expressing SlGME1 and SlGME2 exhibited a significant increase in total ascorbic acid in leaves and red fruits compared with wild-type plants. They also showed enhanced stress tolerance based on less chlorophyll content loss and membrane-lipid peroxidation under methyl viologen (paraquat) stress, higher survival rate under cold stress, and significantly higher seed germination rate, fresh weight, and root length under salt stress. The present study demonstrates that the overexpression of two members of the GME gene family resulted in increased ascorbate accumulation in tomato and improved tolerance to abiotic stresses.

To unravel the molecular mechanisms of drought responses in tomato, gene expression profiles of two drought-tolerant lines identified from a population of Solanum pennellii introgression lines, and the recurrent parent S. lycopersicum cv. M82, a drought-sensitive cultivar, were investigated under drought stress using tomato microarrays. Around 400 genes identified were responsive to drought stress only in the drought-tolerant lines. These changes in genes expression are most likely caused by the two inserted chromosome segments of S. pennellii, which possibly contain drought-tolerance quantitative trait loci (QTLs). Among these genes are a number of transcription factors and signalling proteins which could be global regulators involved in the tomato responses to drought stress. Genes involved in organism growth and development processes were also specifically regulated by drought stress, including those controlling cell wall structure, wax biosynthesis, and plant height. Moreover, key enzymes in the pathways of gluconeogenesis (fructose-bisphosphate aldolase), purine and pyrimidine nucleotide biosynthesis (adenylate kinase), tryptophan degradation (aldehyde oxidase), starch degradation (beta-amylase), methionine biosynthesis (cystathionine beta-lyase), and the removal of superoxide radicals (catalase) were also specifically affected by drought stress. These results indicated that tomato plants could adapt to water-deficit conditions through decreasing energy dissipation, increasing ATP energy provision, and reducing oxidative damage. The drought-responsive genes identified in this study could provide further information for understanding the mechanisms of drought tolerance in tomato.

Isopentenyl diphosphate isomerase (IPI; EC5.3.3.2) catalyzes isomerization between isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), both of which are essential precursors for terpenoid biosynthesis. A novel gene encoding isopentenyl diphosphate isomerase (designated as SlIPI) was isolated from tomato based on tentative consensus (TC183769) and unigene SGN-U569721 sequences. The SlIPI cDNA contained a 708-bp open reading frame (ORF) encoding a 235-amino-acid protein. The deduced SlIPI protein had an isoelectric point of 5.06 and molecular weight of about 27.18 kDa. Amino acid sequence comparison analysis showed 83-95% similarity to IPIs from other plant species. Phylogenetic analysis revealed that SlIPI had the closest relationship to IPI from Nicotiana tabacum. The SlIPI was likely to be localized in cytoplasm; while, SlIPI2 contained a chloroplast transit peptide. A three dimensional structure modeling revealed that the structure of SlIPI was similar to that of SlIPI2. Tissue expression analysis indicated that SlIPI was constitutively expressed, with the highest expression level detected in the root. Heterologous expression of the recombinant SlIPI in engineered Escherichia coli resulted in the production and accumulation of carotenoid in E. coli, thus confirming that the SlIPI was a functional gene.

Lutein injection is a possible therapeutic approach for retinal diseases, but the molecular mechanism of its neuroprotective effect remains to be elucidated. The aim of this study was to investigate its protective effects in retinal ganglion cells (RGCs) against N-methyl-D-aspartate (NMDA)-induced retinal damage in vivo. Retinal damage was induced by intravitreal NMDA injection in rats. Each animal was given five daily intraperitoneal injections of Lutein or vehicle along with intravitreal NMDA injections. Electroretinograms were recorded. The number of viable RGCs was quantified using the retinal whole-mount method by immunofluorescence. Proteins were measured by Western blot assays. Lutein reduced the retinal damage and improved the response to light, as shown by an animal behavior assay (the black-and-white box method) in rats. Furthermore, Lutein treatment prevented the NMDA-induced reduction in phNR wave amplitude. Lutein increased RGC number after NMDA-induced retina damage. Most importantly, Bax, cytochrome c, p-p38 MAPK, and p-c-Jun were all upregulated in rats injected with NMDA, but these expression patterns were reversed by continuous Lutein uptake. Bcl-2, p-GSK-3β, and p-Akt in the Lutein-treated eyes were increased compared with the NMDA group. Lutein has neuroprotective effects against retinal damage, its protective effects may be partly mediated by its anti-excitability neurotoxicity, through MAPKs and PI3K/Akt signaling, suggesting a potential approach for suppressing retinal neural damage.

GATA transcription factors are transcriptional regulatory proteins that contain a characteristic type-IV zinc finger DNA-binding domain and recognize the conserved GATA motif in the promoter sequence of target genes. Previous studies demonstrated that plant GATA factors possess critical functions in developmental control and responses to the environment. To date, the GATA factors in soybean (Glycine max) have yet to be characterized. Thus, this study identified 64 putative GATA factors from the entire soybean genomic sequence. The chromosomal distributions, gene structures, duplication patterns, phylogenetic tree, tissue expression patterns, and response to low nitrogen stress of the 64 GATA factors in soybean were analyzed to further investigate the functions of these factors. Results indicated that segmental duplication predominantly contributed to the expansion of the GATA factor gene family in soybean. These GATA proteins were phylogenetically clustered into four distinct subfamilies, wherein their gene structure and motif compositions were considerably conserved. A comparative phylogenetic analysis of the GATA factor zinc finger domain sequences in soybean, Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa) revealed four major classes. The GATA factors in soybean exhibited expression diversity among different tissues; some of these factors showed tissue-specific expression patterns. Numerous GATA factors displayed upregulation or downregulation in soybean leaf in response to low nitrogen stress, and two GATA factors GATA44 and GATA58 were likely to be involved in the regulation of nitrogen metabolism in soybean. Overexpression of GmGATA44 complemented the reduced chlorophyll phenotype of the Arabidopsis ortholog AtGATA21 mutant, implying that GmGATA44 played an important role in modulating chlorophyll biosynthesis. Overall, our study provides useful information for the further analysis of the biological functions of GATA factors in soybean and other crops.

In this study, Rpp6907, a novel resistance gene/allele to Phakopsora pachyrhizi in soybean, was mapped in a 111.9-kb region, including three NBS-LRR type predicted genes, on chromosome 18. Soybean rust caused by Phakopsora pachyrhizi Sydow has been reported in numerous soybean-growing regions worldwide. The development of rust-resistant varieties is the most economical and environmentally safe method to control the disease. The Chinese soybean germplasm SX6907 is resistant to P. pachyrhizi and exhibits immune reaction compared with the known Rpp genes. These characteristics suggest that SX6907 may carry at least one novel Rpp gene/allele. Three F2 populations from the crosses of SX6907 (resistant) and Tianlong 1, Zhongdou40, and Pudou11 (susceptible) were used to map the Rpp gene. Three resistance responses (immune, red-brown, and tan-colored lesion) were observed from the F2 individuals. The segregation follows a ratio of 1(resistance):2(heterozygous):1(susceptible), indicating that the resistance in SX6907 is controlled by a single incomplete dominant gene (designated as Rpp6907). Results showed that Rpp6907 was mapped on soybean chromosome 18 (molecular linkage group G, MLG G) flanked by simple sequence repeat (SSR) markers SSR24 and SSR40 at a distance of 111.9 kb. Among the ten genes marked within this 111.9-kb region between the two markers, three genes (Glyma18g51930, Glyma18g51950, and Glyma18g51960) are nucleotide-binding site and leucine-rich repeat-type genes. These genes may be involved in recognizing the presence of pathogens and ultimately conferring resistance. Based on resistance spectrum analysis and mapping results, we inferred that Rpp6907 is a novel gene different from Rpp1 in PI 200492, PI 561356, PI 587880A, PI 587886, and PI 594538A, or a new Rpp1-b allele.

In our previous study, we reported that luteolin might exert neuroprotective functions by inhibiting the production of proinflammatory mediators, thereby suppressing microglial activation. In this study, we used two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) to study the effect of ubiquitin-specific processing protease 8 (USP8) in luteolin-treated microglia. Western blot analysis verified that USP8 expression is upregulated by luteolin. Researchers have found that USP8 markedly enhanced the stability of neuregulin receptor degradation protein-1 (Nrdp1), which in turn inhibited the production of proinflammatory cytokines in toll-like receptor-triggered macrophages. We next hypothesized that luteolin inhibits microglial inflammation by regulating USP8 gene expression. After transfecting BV2-immortalized murine microglial cells with USP8, a significant reduction in the degradation of Nrdp1 was observed. USP8 overexpression also reduced the production of lipopolysaccharide (LPS)-induced proinflammatory mediators such as inducible nitric oxide synthase (iNOS), nitric oxide (NO), cyclooxygenase-2 (COX-2), and prostaglandin E2 (PGE2). We also found that USP8 siRNA blocked luteolin inhibition of pro-inflammatory gene expression such as iNOS, NO, COX-2, and PGE2. Taken together, our findings suggested that luteolin inhibits microglial inflammation by enhancing USP8 protein production. We concluded that in addition to anti-inflammatory luteolin, USP8 might represent a novel mechanism for the treatment of neuroinflammation and neurodegeneration.

Nitrogen is an important macronutrient required for plant growth, and is a limiting factor for crop productivity. Improving the nitrogen use efficiency (NUE) is therefore crucial. At present, the NUE mechanism is unclear and information on the genes associated with NUE in soybeans is lacking. cystathionine beta synthase (CBS) domain-containing proteins (CDCPs) may be implicated in abiotic stress tolerance in plants. We identified and classified a CBS domain–containing protein superfamily in soybean. A candidate gene for NUE, GmCBS21, was identified. GmCBS21 gene characteristics, the temporal expression pattern of the GmCBS21 gene, and the phenotype of GmCBS21 overexpression in transgenic Arabidopsis thaliana under low nitrogen stress were analyzed. The phenotypes suggested that the transgenic Arabidopsis thaliana seedlings performed better under the nitrogen-deficient condition. GmCBS21-overexpressing transgenic plants exhibit higher low nitrogen stress tolerance than WT plants, and this suggests its role in low nitrogen stress tolerance in plants. We conclude that GmCBS21 may serve as an excellent candidate for breeding crops with enhanced NUE and better yield.

As a vital antioxidant, L-ascorbic acid (AsA) affects diverse biological processes in higher plants. Lack of AsA in cell impairs plant development. In the present study, we manipulated a gene of GDP-mannose pyrophosphorylase which catalyzes the conversion of D-mannose-1-P to GDP-D-mannose in AsA biosynthetic pathway and found out the phenotype alteration of tomato. In the tomato genome, there are four members of GMP gene family and they constitutively expressed in various tissues in distinct expression patterns. As expected, over-expression of SlGMP3 increased total AsA contents and enhanced the tolerance to oxidative stress in tomato. On the contrary, knock-down of SlGMP3 significantly decreased AsA contents below the threshold level and altered the phenotype of tomato plants with lesions and further senescence. Further analysis indicated the causes for this symptom could result from failing to instantly deplete the reactive oxygen species (ROS) as decline of free radical scavenging activity. More ROS accumulated in the leaves and then triggered expressions of defence-related genes and mimic symptom occurred on the leaves similar to hypersensitive responses against pathogens. Consequently, the photosynthesis of leaves was dramatically fallen. These results suggested the vital roles of AsA as an antioxidant in leaf function and defence response of tomato.

Nitrogen (N) is a major limiting factor in crop production, and plant adaptive responses to low N are involved in many post-transcriptional regulation. Recent studies indicate that miRNAs play important roles in adaptive responses. However, miRNAs in soybean adaptive responses to N limitation have been not reported. We constructed sixteen libraries to identify low N-responsive miRNAs on a genome-wide scale using samples from 2 different genotypes (low N sensitive and low N tolerant) subjected to various periods of low nitrogen stress. Using high-throughput sequencing technology (Illumina-Solexa), we identified 362 known miRNAs variants belonging to 158 families and 90 new miRNAs belonging to 55 families. Among these known miRNAs variants, almost 50% were not different from annotated miRNAs in miRBase. Analyses of their expression patterns showed 150 known miRNAs variants as well as 2 novel miRNAs with differential expressions. These differentially expressed miRNAs between the two soybean genotypes were compared and classified into three groups based on their expression patterns. Predicted targets of these miRNAs were involved in various metabolic and regulatory pathways such as protein degradation, carbohydrate metabolism, hormone signaling pathway, and cellular transport. These findings suggest that miRNAs play important roles in soybean response to low N and contribute to the understanding of the genetic basis of differences in adaptive responses to N limitation between the two soybean genotypes. Our study provides basis for expounding the complex gene regulatory network of these miRNAs.

Plants have evolved sophisticated defense mechanisms against herbivores to help them adapt to the environment. Understanding the defense mechanisms in plants can help us control insects in a more effective manner. In this study, we found that compared with Tianlong 2 (a cultivated soybean with insect susceptibility), ED059 (a wild soybean line with insect resistance) contains sharper pubescence tips, as well as lower transcript levels of wound-induced protein kinase ( WIPK ) and salicylic acid-induced protein kinase ( SIPK ), which are important mitogen-activated protein kinases involved in early defense response to herbivores. The observed lower transcript levels of WIPK and SIPK induced higher levels of jasmonic acid (JA), JA biosynthesis enzymes (AOC3) and some secondary metabolites in ED059. Functional analysis of the KTI1 gene via Agrobacterium -mediated transformation in Arabidopsis thaliana indicated that it plays an important role in herbivore defense in ED059. We further investigated the molecular response of third-instar Helicoverpa armigera (Hubner) larvae to Tianlong 2 and ED059. We found apoptotic cells only in the midguts of larvae that fed on ED059. Compared with larvae reared on the susceptible cultivar Tianlong 2, transcript levels of catalase ( CAT ) and glutathione S-transferase ( GST ) were up-regulated, whereas those of CAR , CHSB , and TRY were down-regulated in larvae that fed on the highly resistant variety ED059. We propose that these differences underlie the different herbivore defense responses of ED059 and Tianlong 2.

Soybean is an important oil and protein crop in the world. Cotton bollworm (Helicoverpa armigera) is one of primary defoliating insects which seriously damage the soybean production. The wild soybean, ED059, was reported to be highly resistant to H. armigera. However, the molecular mechanism of resistance to H. armigera remains unknown. In this study, we conducted the transcript and protein profilings for ED059 and a susceptible soybean cultivar (cv.) Tianlong 2. In summary, 2213 genes and 116 proteins were specifically expressed in response to H. armigera in ED059, while 2179 genes and 9 proteins were specifically expressed in cv. Tianlong 2. Four hundred and four genes were significantly up-regulated in response to H. armigera in ED059 while down-regulated or not changed in cv. Tianlong 2. GO analyses showed the specifically expressed genes in ED059 were mainly enriched in metabolism-related pathways. The function of one potential resistant gene involved in the phenylpropanoid pathway was confirmed by use of transgenic Arabidopsis. Our study provided useful resources for the soybean improvement, which could contribute to the elucidation of defense mechanisms against chewing insects in plants.