Assessing genetic diversity and geographical differentiation in a global collection of wild soybean (Glycine soja Sieb. et Zucc.) and assigning a mini-core collection.

Wild soybean (Glycine soja), the ancestor of the cultivated soybean (G. max), is a crucial resource for capturing the genetic diversity of soybean species. In this study, we used a set of 78 genome-wide microsatellite markers to analyse the genetic diversity and geographic differentiation patterns in a global collection of 2,050 G. soja accessions and a mini-core collection of G. max stored in two public seed banks. We observed a notable reduction in the genetic diversity of G. max compared with G. soja and identified a close phylogenetic relationship between G. max and a G. soja subpopulation located in central China. Furthermore, we revealed substantial genetic divergence between northern and southern subpopulations, accompanied by diminished genetic diversity in the northern subpopulations. Two clusters were discovered among the accessions from north-eastern China-one genetically close to those from South Korea and Southern Japan, and another close to those from Amur Oblast, Russia. Finally, 192 accessions were assigned to a mini-core collection of G. soja, retaining 73.8% of the alleles detected in the entire collection. This mini-core collection is accessible to those who need it, facilitating efficient evaluation and utilization of G. soja genetic resources in soybean breeding initiatives.

Molecular Basis Underlying Common Cutworm Resistance of the Primitive Soybean Landrace Peking.

The common cutworm (CCW; Spodoptera litura) is one of the major insect pests of soybean in Asia and Oceania. Although quantitative trail loci related to CCW resistance have been introduced into leading soybean cultivars, these do not exhibit sufficient resistance against CCW. Thus, understanding the genetic and metabolic resistance mechanisms of CCW as well as integrating other new resistance genes are required. In this study, we focused on a primitive soybean landrace, Peking, which has retained resistances to various pests. We found a resistance to CCW in Peking by the detached-leaf feeding assay, and subsequently determined the genetic and metabolic basis of the resistance mechanism using chromosome segment substitution lines (CSSLs) of Peking. Several characteristic metabolites for Peking were identified by the metabolomic approach using liquid chromatography/mass spectrometry combined with a principle component analysis. The structure of seven metabolites were determined by nuclear magnetic resonance (NMR) analysis. The genomic segments of Peking on chromosome 06 (Chr06) and Chr20 had a clear association with these metabolites. Moreover, a line possessing a Peking genomic segment on Chr20 inhibited growth of the CCW. The genetic factors and the metabolites on Chr20 in Peking will be useful for understanding mechanisms underlying CCW resistance and breeding resistant soybean cultivars.

A cellulose synthase-derived enzyme catalyses 3-O-glucuronosylation in saponin biosynthesis.

Triterpenoid saponins are specialised metabolites distributed widely in the plant kingdom that consist of one or more sugar moieties attached to triterpenoid aglycones. Despite the widely accepted view that glycosylation is catalysed by UDP-dependent glycosyltransferase (UGT), the UGT which catalyses the transfer of the conserved glucuronic acid moiety at the C-3 position of glycyrrhizin and various soyasaponins has not been determined. Here, we report that a cellulose synthase superfamily-derived glycosyltransferase (CSyGT) catalyses 3-O-glucuronosylation of triterpenoid aglycones. Gene co-expression analyses of three legume species (Glycyrrhiza uralensis, Glycine max, and Lotus japonicus) reveal the involvement of CSyGTs in saponin biosynthesis, and we characterise CSyGTs in vivo using Saccharomyces cerevisiae. CSyGT mutants of L. japonicus do not accumulate soyasaponin, but the ectopic expression of endoplasmic reticulum membrane-localised CSyGTs in a L. japonicus mutant background successfully complement soyasaponin biosynthesis. Finally, we produced glycyrrhizin de novo in yeast, paving the way for sustainable production of high-value saponins.

High-Throughput Screening and Characterization of a High-Density Soybean Mutant Library Elucidate the Biosynthesis Pathway of Triterpenoid Saponins.

Triterpenes (C30) constitute one of the diverse class of natural products with potential applications in food, cosmetic and pharmaceutical industries. Soyasaponins are oleanane-type triterpenoids widespread among legumes and particularly abundant in soybean seeds. They have associated with various pharmacological implications and undesirable taste properties of soybean-based food products. Uncovering the biosynthetic genes of soyasaponins will provide new opportunities to control the pathway for human benefits. However, the pathway of soyasaponin biosynthesis has not been fully elucidated in part because of a paucity of natural mutants. Here, we applied a structured high-density soybean mutant library for the forward genetic screening of triterpenoid biosynthesis. The seed soyasaponin polymorphism in the mutant library was evaluated using a high-throughput thin-layer chromatography and liquid chromatography tandem mass spectrometry analysis. This screening identified 35 mutants (3.85% of 909 mutant lines) with seven unusual soyasaponin phenotypes (Categories 1-7), which was greater than the number of natural mutants reported previously (22 mutants, 0.18% of ∼12,428 accessions). Nine unique intermediates of soyasaponin biosynthesis were identified and their chemical structures were estimated based on their MS/MS fragment patterns. Based on published information, 19 mutants could be associated with loss of function of four individual soyasaponin biosynthesis genes identified through expressed sequence tag mining or positional cloning, whereas the remaining 16 mutants were novel and may facilitate discovery of the unknown biosynthetic genes of soyasaponins. Our approach and library may help to identify new phenotype materials and causative genes associated with specialized metabolite production and other traits.

Sucrose supply from leaves is required for aerenchymatous phellem formation in hypocotyl of soybean under waterlogged conditions.

Background and Aims: Soil waterlogging often causes oxygen deficiency in the root systems of plants and severely inhibits plant growth. Formation of aerenchyma - interconnected spaces that facilitate the movement of gases between and within the aerial and submerged parts of plants - is an adaptive trait for coping with waterlogged conditions. Soybean (Glycine max) forms porous secondary tissues known as aerenchymatous phellem (AP), which are derived from the outermost cell layer of phellogen. To understand what factors other than waterlogging are involved in phellogen and AP formation, we examined how their formation in soybean seedlings was affected by darkness, CO2 deficiency and blockage of phloem transport. Methods: Aerenchymatous phellem and phellogen formation were expressed as area ratios in cross-sections of hypocotyl. CO2 was depleted by use of calcium oxide and sodium hydroxide. Phloem transport was blocked by heat-girdling of hypocotyls. Sucrose levels were measured by spectrophotometry. Key Results: Under light conditions, waterlogging induced the accumulation of high concentrations of sucrose in hypocotyls, followed by phellogen and AP formation in hypocotyls. Phellogen formation and AP formation were inhibited by darkness, CO2 deficiency and blockage of phloem transport. Phellogen formation and AP formation were also inhibited by excision of shoots above the epicotyl, but they recovered following application of sucrose (but not glucose or fructose application) to the cut surface. Conclusions: The results demonstrate that sucrose derived from leaves is essential for AP and phellogen formation in soybean hypocotyls under waterlogged soil conditions. Maintenance of a high sucrose concentration is thus essential for the development of phellogen and AP and the differentiation of phellogen to AP.

Transcriptomic analysis reveals the flooding tolerant mechanism in flooding tolerant line and abscisic acid treated soybean.

Soybean is highly sensitive to flooding stress and exhibits markedly reduced plant growth and grain yield under flooding conditions. To explore the mechanisms underlying initial flooding tolerance in soybean, RNA sequencing-based transcriptomic analysis was performed using a flooding-tolerant line and ABA-treated soybean. A total of 31 genes included 12 genes that exhibited similar temporal patterns were commonly changed in these plant groups in response to flooding and they were mainly involved in RNA regulation and protein metabolism. The mRNA expression of matrix metalloproteinase, glucose-6-phosphate isomerase, ATPase family AAA domain-containing protein 1, and cytochrome P450 77A1 was up-regulated in wild-type soybean under flooding conditions; however, no changes were detected in the flooding-tolerant line or ABA-treated soybean. The mRNA expression of cytochrome P450 77A1 was specifically up-regulated in root tips by flooding stress, but returned to the level found in control plants following treatment with the P450 inhibitor uniconazole. The survival ratio and root fresh weight of plants were markedly improved by 3-h uniconazole treatment under flooding stress. Taken together, these results suggest that cytochrome P450 77A1 is suppressed by uniconazole treatment and that this inhibition may enhance soybean tolerance to flooding stress.

Quantitative proteomics reveals that peroxidases play key roles in post-flooding recovery in soybean roots.

Soybean is an important legume crop that exhibits markedly reduced growth and yields under flooding conditions. To unravel the mechanisms involved in recovery after flooding in soybean root, gel-free proteomic analysis was performed. Morphological analysis revealed that growth suppression was more severe with increased flooding duration. Out of a total of 1645 and 1707 identified proteins, 73 and 21 proteins were changed significantly during the recovery stage following 2 and 4 days flooding, respectively. Based on the proteomic, clustering, and in silico protein-protein interaction analyses, six key enzymes were analyzed at the mRNA level. Lipoxygenase 1, which was increased at the protein level during the recovery period, was steadily down-regulated at the mRNA level. The peroxidase superfamily protein continuously increased in abundance during the course of recovery and was up-regulated at the mRNA level. HAD acid phosphatase was decreased at the protein level and down-regulated at the transcript level, while isoflavone reductase and an unknown protein were increased at both the protein and mRNA levels. Consistent with these findings, the enzymatic activity of peroxidase was decreased under flooding stress but increased significantly during the recovery sage. These results suggest that peroxidases might play key roles in post-flooding recovery in soybean roots through the scavenging of toxic radicals.

Analyses of flooding tolerance of soybean varieties at emergence and varietal differences in their proteomes.

Flooding of fields due to heavy and/or continuous rainfall influences soybean production. To identify soybean varieties with flooding tolerance at the seedling emergence stage, 128 soybean varieties were evaluated using a flooding tolerance index, which is based on plant survival rates, the lack of apparent damage and lateral root development, and post-flooding radicle elongation rate. The soybean varieties were ranked according to their flooding tolerance index, and it was found that the tolerance levels of soybean varieties exhibit a continuum of differences between varieties. Subsequently, tolerant, moderately tolerant and sensitive varieties were selected and subjected to comparative proteomic analysis to clarify the tolerance mechanism. Proteomic analysis of the radicles, combined with correlation analysis, showed that the ratios of RNA binding/processing related proteins and flooding stress indicator proteins were significantly correlated with flooding tolerance index. The RNA binding/processing related proteins were positively correlated in untreated soybeans, whereas flooding stress indicator proteins were negatively correlated in flooded soybeans. These results suggest that flooding tolerance is regulated by mechanisms through multiple factors and is associated with abundance levels of the identified proteins.

Analysis of flooding-responsive proteins localized in the nucleus of soybean root tips.

Flooding stress has negative impact on soybean cultivation as it severely impairs plant growth and development. To examine whether nuclear function is affected in soybean under flooding stress, abundance of nuclear proteins and their mRNA expression were analyzed. Two-day-old soybean seedlings were treated with flooding for 2 days, and nuclear proteins were purified from root tips. Gel-free proteomics analysis identified a total of 39 flooding-responsive proteins, of which abundance of 8 and 31 was increased and decreased, respectively, in soybean root tips. Among these differentially regulated proteins, the mRNA expression levels of five nuclear-localized proteins were further analyzed. The mRNA levels of four proteins, which are splicing factor PWI domain-containing protein, epsilon2-COP, beta-catenin, and clathrin heavy chain decreased under flooding stress, were also down-regulated. In addition, mRNA level of a receptor for activated protein kinase C1(RACK1) was down-regulated, though its protein was accumulated in the soybean nucleus in response to flooding stress. These results suggest that several nuclear-related proteins are decreased at both the protein and mRNA level in the root tips of soybean under flooding stress. Furthermore, RACK1 might have an important role with accumulation in the soybean nucleus under flooding-stress conditions.

Regulatory modules controlling maize inflorescence architecture.

Genetic control of branching is a primary determinant of yield, regulating seed number and harvesting ability, yet little is known about the molecular networks that shape grain-bearing inflorescences of cereal crops. Here, we used the maize (Zea mays) inflorescence to investigate gene networks that modulate determinacy, specifically the decision to allow branch growth. We characterized developmental transitions by associating spatiotemporal expression profiles with morphological changes resulting from genetic perturbations that disrupt steps in a pathway controlling branching. Developmental dynamics of genes targeted in vivo by the transcription factor RAMOSA1, a key regulator of determinacy, revealed potential mechanisms for repressing branches in distinct stem cell populations, including interactions with KNOTTED1, a master regulator of stem cell maintenance. Our results uncover discrete developmental modules that function in determining grass-specific morphology and provide a basis for targeted crop improvement and translation to other cereal crops with comparable inflorescence architectures.

The synthesis of cytosolic ascorbate peroxidases in germinating seeds and seedlings of soybean and their behavior under flooding stress.

Cytosolic ascorbate peroxidases (cAPXs) of soybean have been found by proteome analysis to be downregulated in submerged seedlings. To elucidate the physiological meaning of this downregulation, soybean cAPXs were characterized in this study. Vigorous synthesis was detected in germinating seeds and seedlings. Expression of the corresponding genes was detected clearly in tissues that actively underwent cell division. The gene expression was suppressed by flooding stress, but not by salinity, cold or drought stress. The expression recovered 1 d after release from flooding stress, accompanied by growth resurgence.

Comprehensive analysis of endoplasmic reticulum-enriched fraction in root tips of soybean under flooding stress using proteomics techniques.

Flooding is a serious problem for soybean cultivation because it markedly reduces growth and grain yields. Here, 2 proteomics techniques were used to evaluate whether endoplasmic reticulum (ER)-enriched fraction is altered in soybean under flooding stress. Two-day-old soybeans were treated with flooding for 2 days, and rough ER-enriched fraction was then purified from root tips. Flooding-responsive protein of ER-enriched fraction was identified using gel-free and 1D-gel based proteomics techniques, and 117 proteins were increased and 212 proteins were decreased in soybean root tips in response to flooding stress. Among the identified proteins, 111 were functionally categorized as being involved in protein synthesis, post-translational modification, protein folding, protein degradation, and protein activation. Among differentially regulated proteins, the mRNA expression levels of 14 proteins that were predicted to be localized in the ER were analyzed. Notably, 3-ketoacyl-CoA reductase 1 was up-regulated and eight genes related to stress, hormone metabolism, cell wall and DNA repair were down-regulated within 1 day under flooding conditions. In addition, the expression of luminal-binding protein 5 was specifically induced in flood-stressed roots, whereas arabinogalactan protein 2 and methyltransferase PMT2 were down-regulated. Taken together, these results suggest that flooding mainly affects the function of protein synthesis and glycosylation in the ER in root tips of soybean.

Organ-specific proteomics analysis for identification of response mechanism in soybean seedlings under flooding stress.

Flooding is one of the severe environmental factors which impair growth and yield in soybean plant. To investigate the organ specific response mechanism of soybean under flooding stress, changes in protein species were analyzed using a proteomics approach. Two-day-old soybeans were subjected to flooding for 5 days. Proteins were extracted from root, hypocotyl and leaf, and separated by two-dimensional polyacrylamide gel electrophoresis. In root, hypocotyl and leaf, 51, 66 and 51 protein species were significantly changed, respectively, under flooding stress. In root, metabolism related proteins were increased; however these proteins were decreased in hypocotyl and leaf. In all 3 organs, cytoplasm localized proteins were decreased, and leaf chloroplastic proteins were also decreased. Isoflavone reductase was commonly decreased at protein level in all 3 organs; however, mRNA of isoflavone reductase gene was up-regulated in leaf under flooding stress. Biophoton emission was increased in all 3 organs under flooding stress. The up-regulation of isoflavone reductase gene at transcript level; while decreased abundance at protein level indicated that flooding stress affected the mRNA translation to proteins. These results suggest that concurrence in expression of isoflavone reductase gene at mRNA and protein level along with imbalance in other disease/defense and metabolism related proteins might lead to impaired growth of root, hypocotyl and leaf of soybean seedlings under flooding stress.

Organ-specific proteomic analysis of drought-stressed soybean seedlings.

Changes in protein levels in drought-stressed soybean seedlings were analyzed using a proteomics approach. Three-day-old soybean seedlings were subjected to drought stress or treated with 10% polyethylene glycol (PEG) as osmotic stress. After treatment, the proteins were extracted from the leaf, hypocotyl, and root and separated using two-dimensional polyacrylamide gel electrophoresis. The root was the most drought-responsive organ, with the levels of 32, 13, and 12 proteins changing in response to drought stress, PEG treatment, and both, respectively. In the leaves of PEG-treated and drought-stressed seedlings, metabolism-related proteins increased and energy production- and protein synthesis-related proteins decreased. For 3 proteins present in all organs in drought-stressed plants, mRNA was differentially regulated: heat shock protein 70 and actin isoform B were upregulated, and methionine synthase was downregulated. mRNA expression patterns reflected those of protein levels, suggesting transcriptional regulation of these proteins. Western blot analysis confirmed the increase in ascorbate peroxidase in drought-stressed plants. The downregulation of mRNA and decreased protein levels of methionine synthase in the leaves, hypocotyl, and roots of drought-stressed plants, but not in other treatments, indicated that methionine synthase is a drought response protein. These results also suggest that the decreased methionine synthase in response to drought stress can impair the soybean seedling growth.

Characterization of calnexin in soybean roots and hypocotyls under osmotic stress.

Calnexin is an endoplasmic reticulum-localized molecular chaperone protein which is involved in folding and quality control of proteins. To evaluate the expression of calnexin in soybean seedlings under osmotic stress, immunoblot analysis was performed using a total membrane protein fraction. Calnexin constantly accumulated at an early growth stage of soybean under normal growth conditions. Expression of this protein decreased in 14-day-old soybean roots when treated with 10% polyethylene glycol for 2 days. Other abiotic stresses such as drought, salinity, cold as well as abscisic acid treatment, similarly reduced accumulation of calnexin and this reduction was correlated with reduction in root length in soybean seedlings under abiotic stresses. When compared between soybean and rice, calnexin expression was not changed in rice under abiotic stresses. Using Flag-tagged calnexin, a 70 kDa heat shock cognate protein was identified as an interacting protein. These results suggest that osmotic or other abiotic stresses highly reduce accumulation of the calnexin protein in developing soybean roots. It is also suggested that calnexin interacts with a 70 kDa heat shock cognate protein and probably functions as molecular chaperone in soybean.

Proteomics techniques for the development of flood tolerant crops.

Proteomics is a useful analytical approach for investigating crop responses to stress. Recent remarkable advances in proteomic techniques allow for the identification of a wider range of proteins than was previously possible. The application of proteomic techniques to clarify the molecular mechanisms underlying crop responses to flooding stress may facilitate the development of flood tolerant crops. Flooding is an environmental stress found worldwide and may increase in frequency due to changes in global climate. Waterlogging resulting from flooding causes significant reductions in the growth and yield of several crops. Transient flooding displaces gases in soil pores and often causes hypoxia in plants grown on land with poor drainage. Changes in protein expression and post-translational modification of proteins occur as plants activate their defense system in response to flooding stress. In this review, we discuss the contributions that proteomic studies have made toward increasing our understanding of the well-organized cellular response to flooding in soybean and other crops. The biological relevance of the proteins identified using proteomic techniques in regard to crop stress tolerance will be discussed as well.

A comparative proteomic analysis of the early response to compatible symbiotic bacteria in the roots of a supernodulating soybean variety.

To reveal the processes involved in the early stages of symbiosis between soybean plants and root nodule bacteria, we conducted a proteomic analysis of the response to bacterial inoculation in the roots of supernodulating (En-b0-1) and non-nodulating (En1282) varieties, and their parental normal-nodulating variety (Enrei). A total of 56 proteins were identified from 48 differentially expressed protein spots in normal-nodulating variety after bacterial inoculation. Among 56 proteins, metabolism- and energy production-related proteins were upregulated in supernodulating and downregulated in non-nodulating varieties compared to normal-nodulating variety. The supernodulating and non-nodulating varieties responded oppositely to bacterial inoculation with respect to the expression of 11 proteins. Seven proteins of these proteins was downregulated in supernodulating varieties compared to non-nodulating variety, but expression of proteasome subunit alpha type 6, gamma glutamyl hydrolase, glucan endo-1,3-beta glucosidase, and nodulin 35 was upregulated. The expression of seven proteins mirrored the degree of nodule formation. At the transcript level, expression of stem 31kDa glycoprotein, leucine aminopeptidase, phosphoglucomutase, and peroxidase was downregulated in the supernodulating variety compared to the non-nodulating variety, and their expression in the normal-nodulating variety was intermediate. These results suggest that suppression of the autoregulatory mechanism in the supernodulating variety might be due to negative regulation of defense and signal transduction-related processes.

Characterization of a novel flooding stress-responsive alcohol dehydrogenase expressed in soybean roots.

Alcohol dehydrogenase (Adh) is the key enzyme in alcohol fermentation. We analyzed Adh expression in order to clarify the role of Adh of soybeans (Glycine max) to flooding stress. Proteome analysis confirmed that expression of Adh is significantly upregulated in 4-day-old soybean seedlings subjected to 2 days of flooding. Southern hybridization analysis and soybean genome database search revealed that soybean has at least 6 Adh genes. The GmAdh2 gene that responded to flooding was isolated from soybean cultivar Enrei. Adh2 expression was markedly increased 6 h after flooding and decreased 24 h after floodwater drainage. In situ hybridization and Western blot indicated that flooding strongly induces Adh2 expression in RNA and protein levels in the root apical meristem. Osmotic, cold, or drought stress did not induce expression of Adh2. These results indicate that Adh2 is a flooding-response specific soybean gene expressed in root tissue.

Involvement of two rice ETHYLENE INSENSITIVE3-LIKE genes in wound signaling.

Ethylene and jasmonic acid (JA) have been proposed as key compounds for wound signaling in plants. In Arabidopsis, ETHYLENE INSENSITIVE3 (EIN3), which is an essential transcription factor for ethylene signaling, is regulated at the post-transcriptional level, while transcriptional regulation of EIN3 or EIN3-LIKE (EIL) genes has not been well documented. The expression of 6 rice EIL genes (OsEIL1-6) was analyzed and only OsEIL1 and 2 were found to be wound-inducible EIL. OsEIL2 was also induced by JA. Electrophoretic mobility shift assays showed that recombinant OsEIL1 and 2 proteins bound to specific DNA sequences that are recognized by a wound-inducible tobacco EIL. Accumulation of OsEIL1 and 2 transcripts reached a maximum at 1 and 0.5 h after wounding, respectively, and the corresponding DNA-binding activity in nuclear extracts of rice leaves was increased at 1 h after wounding. Candidates for OsEIL-target genes were selected by microarray analysis of wounded rice and by promoter sequence analyses of wound-inducible genes identified by microarray analysis. In OsEIL1- and/or 2-suppressed rice plants, the expression of at least four of 18 candidate genes analyzed was down-regulated. These results indicate the importance of inducible OsEILs in wound signaling in rice.

Cytosolic ascorbate peroxidase 2 (cAPX 2) is involved in the soybean response to flooding.

Proteomic analyses of soybean seedlings responding to flooding were conducted to identify key proteins involved. The seeds were germinated on a spongy matrix for two days, and then subjected to flooding for three days. After flooding, the total number of roots, the length of the main root, the lengths of the lateral and adventitious roots, and the fresh weight of the underground tissues of flooded soybean seedlings were significantly suppressed compared with nontreated plants. To identify the early flooding-responsive proteins, the seedling roots were used for preparing cytosolic and membrane fractions. After two-dimensional polyacrylamide gel electrophoresis and silver staining, 208 proteins were detected, and the levels of 44 were different from those of the control. The expression pattern of 10 proteins among the 44 from six different soybean cultivars confirmed that the 10 were flooding-responsive proteins. One of the 10 proteins was dominantly down-regulated under flooding conditions and was identified as cytosolic ascorbate peroxidase 2 (cAPX 2). Northern-hybridization showed that the abundance of cAPX 2 transcript decreased significantly after flooding, as did the enzymatic activity of APX. These results suggest that cAPX 2 is involved in flooding stress responses in young soybean seedlings.