大豆异黄酮浸种对盐胁迫大豆幼苗的生理效应

大豆异黄酮( Soybean isoflavones)是在大豆生长过程中形成并在成熟种子和叶片中积累较多的一类具有生物活性的次生代谢物,通常可作为人们日常生活中的一类营养保健品.研究了外源大豆苷或染料木苷溶液(0.01 mg/L)浸种处理对盐胁迫栽培大豆(N23674品种)和滩涂野大豆(BB52种群)及其经逐代耐盐性筛选的杂交后代(4076株系,F5)幼苗叶片伤害率、光合作用、Na+含量和Na+/K+值、活性氧清除酶活性及内源大豆异黄酮含量等生理指标的影响.结果表明:盐胁迫下,两种外源大豆异黄酮浸种处理均可显著抑制叶片相对电解质渗透率和硫代巴比妥酸反应物(TBARS)含量的上升及净光合速率(Pn)的下降,降低Na+含量和Na+/K+值,增强超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性,提高内源大豆异黄酮含量,从而表现对盐害的缓解效应,其中对耐盐性较弱的栽培大豆N23674品种效应更明显.这为大豆异黄酮在大豆耐盐育种、化学调控和盐碱地种植利用等提供了理论依据.     

Mapping QTLs for seed yield and drought susceptibility index in soy-bean（Glycine max L.） across different environments

Drought stress has long been a major constraint in maintaining yield stability of soybean （Glycine max （L.） Merr.） in rainfed ecosystems. The identification of consistent quantitative trait loci （QTL） involving seed yield per plant （YP） and drought susceptibility index （DSI） in a population across different environments would therefore be important in molecular marker-assisted breeding of soybean cultivars suitable for rainfed regions. The YP of a recombinant line population of 184 F2：7：11 lines from a cross of Kefengl and Nannong1138-2 was studied under water-stressed （WS） and well-watered （WW） conditions in field （F） and greenhouse （G） trials, and DSI for yield was calculated in two trials. Nineteen QTLs associated with YP-WS and YP-WW, and 10 QTLs associated with DSI, were identi- fied. Comparison of these QTL locations with previous findings showed that the majority of these regions control one or more traits re- lated to yield and other agronomic traits. One QTL on molecular linkage group （MLG） K for YP-F, and two QTLs on MLG C2 for YP-G, remained constant across different water regimes. The regions on MLG C2 for YP-WW-F and MLG H for YP-WS-F had a pleiotropic effect on DSI-F, and MLG A1 for YP-WS-G had a pleiotropic effect on DSI-G. The identification of consistent QTLs for YP and DSI across different environments will significantly improve the efficiency of selecting for drought tolerance in soybean.     

NaCl胁迫下野生和栽培大豆幼苗体内离子的再转运

采用NaCl根际处理和叶面饲喂^22Na方法,研究了野生大豆(Glycine soja)——耐盐的BB52、盐敏感的N23232和栽培大豆(Glycine max)——较耐盐的Lee68幼苗在盐胁迫及解除过程中对Na^ 、Cl^-的吸收和再转运。结果表明,在NaCl根际处理12h过程中,BB52和Lee68幼苗根对Na^ 、Cl^-吸收和向茎、叶的运输逐渐增加,10h时趋于稳定,Na^ 、Cl^-含量高低顺序是根＞茎＞叶。但N23232的Na^ 、Cl^-含量则是茎＞根＞叶。在用NaCl对根处理10h后再解除NaCl处理的0～36h内,BB52吸收的Na^ 、Cl^-较多地留于根部或转运至根茎过渡区,叶中较少。N23232吸收的Na^ 较多地转运至茎部,而Cl^-含量在幼苗各部分无差异。叶片饲喂^22Na 10h后,BB52吸收^22Na较N23232多,并较多地向根部运输。从离子再转运角度讨论了BB52的耐盐性。     

Production of free and glycosylated isoflavones in in vitro soybean (Glycine max L.) hypocotyl cell suspensions and comparison with industrial seed extracts

Glycine max is one of the major sources of phytochemicals, in particular of isoflavones, a class of phytoestrogens with ascertained beneficial effects on human health. In the present study, in vitro callus production from soybean hypocotyl seedling explants and cell suspensions were optimized. Time-courses having 20, 40 and 60 g/L of initial cell inoculum were performed to determine the concentration most suitable for isoflavone production. The amount of total polyphenols and total flavonoids as well as the antioxidant capacity of both cell and culture media fractions were measured by means of spectrophotometric methods. The levels of aglycone and glycosylated isoflavones (didzein, genistein, glycitein, didzin, genistin, glycitin), as well as of ferulic acid, vanillic acid and vanillin, were determined by HPLC–DAD. On average, 93.5 % of the total (cells plus media) isoflavones in soybean cell suspensions were detected as aglycones. Concentrated cell cultures as well as industrial soybean seed extracts were enzymatically hydrolyzed to release the aglycones and their metabolic profiles were analysed by HPLC–DAD. In contrast to cell suspensions, in undigested seed extract the aglycon form represented only 16.8 % of the total isoflavones amount. After enzymatic treatment, the antioxidant capacity increased by 30 and 33 %, respectively, in concentrated cell and seed extracts, demonstrating the presence of a larger amount of bioactive metabolites after digestion. At the present extraction conditions, soybean concentrated cell suspensions yielded 5.8-fold more total isoflavones (mostly in the free form) than seed extracts, leading to hypothesise their possible use as ingredients for cosmetic and nutraceutical applications.     

Mechanical impedance increases aluminium tolerance of soybean (Glycine max) roots

The effect of aluminium (Al) on root elongation was studied in solution culture and sand culture. Compared to solution culture, in sand culture a ten times higher Al supply was necessary to inhibit root elongation to a comparable degree. This was due to a much lower Al uptake into the 5 mm root tips in sand culture. Fe concentrations in root tips were also lower in sand culture. Ca concentrations were higher and less depressed by Al, whereas Mg and K concentrations were not affected by the culture substrate. Regressions of Al concentrations in root tips versus inhibition of root elongation by Al revealed root damage at lower Al concentrations in sand culture. The effect of culture substrate on Al tolerance was independent of N source and could also be shown in flowing solution culture with and without sand. The results indicate that mechanical impedance in sand culture decreased Al uptake. This may be due to enhanced exudation of organic complexors thus reducing activites of monomeric Al species.     

RNA sequencing analysis of salt tolerance in soybean (Glycine max)

Salt stress causes foliar chlorosis and scorch, plant stunting, and eventually yield reduction in soybean. There are differential responses, namely tolerance (excluder) and intolerance (includer), among soybean germplasm. However, the genetic and physiological mechanisms for salt tolerance is complex and not clear yet. Based on the results from the screening of the RA-452 x Osage mapping population, two F_4:6 lines with extreme responses, most tolerant and most sensitive, were selected for a time-course gene expression study in which the 250 mM NaCl treatment was initially imposed at the V1 stage and continued for 24 h (hrs). Total RNA was isolated from the leaves harvested at 0, 6, 12, 24 h after the initiation of salt treatment, respectively. The RNA-Seq analysis was conducted to compare the salt tolerant genotype with salt sensitive genotype at each time point using RNA-Seq pipeline method. A total of 2374, 998, 1746, and 630 differentially expressed genes (DEGs) between salt-tolerant line and salt-sensitive line, were found at 0, 6, 12, and 24 h, respectively. The expression patterns of 154 common DEGs among all the time points were investigated, of which, six common DEGs were upregulated and seven common DEGs were downregulated in salt-tolerant line. Moreover, 13 common DEGs were dramatically expressed at all the time points. Based on Log2 (fold change) of expression level of salt-tolerant line to salt-sensitive line and gene annotation, Glyma.02G228100, Glyma.03G226000, Glyma.03G031000, Glyma.03G031400, Glyma.04G180300, Glyma.04G180400, Glyma.05 g204600, Glyma.08G189600, Glyma.13G042200, and Glyma.17G173200, were considered to be the key potential genes involving in the salt-tolerance mechanism in the soybean salt-tolerant line.     

氯化钠胁迫下野生和栽培大豆幼苗体内的多胺水平变化

以通用的较耐盐的栽培大豆Lee68品种和对盐敏感的野生大豆N23232种群为参照,研究了盐胁迫下耐盐野生大豆BB52种群幼苗体内多胺(PAs)组分、含量及多胺氧化酶(PAO)活性的变化。结果表明,盐胁迫下BB52幼苗根PAs中Put和Spm含量下降较Lee68和N23232显著,但Spd含量下降较少．BB52叶片PAs中Put含量下降,Spd上升,(Spd+Spin)／Put值增加和Put／PAs值降低幅度与耐盐性呈正相关趋势．盐胁迫下,各材料根和叶中PAO活性上升,N23232上升最明显．探讨了多胺水平与BB52耐盐性的关系。     

Nucleotide sequences encoding glycinin B4 polypeptide of the cultivated soybean Glycine max and its presumed wild-type ancestor Glycine soja in connection with the origin of cultivated soybean

Nucleotide sequences of cDNAs encoding soybean glycinin B4 polypeptide were compared in three soybean cultivars and two plant introductions of wild soybean Glycine soja. Only two nucleotide substitutions were found in three cultivars G. max, as compared with G. max and G. soja having nucleotide sequences which contain four nucleotide substitutions. These data serve as additional evidence, at molecular level, indicating the origin of G. max from G. soja. On the other hand, the time period required for four nucleotide substitutions' accumulation, as calculated from parameters of molecular evolution of 11S globulins, is much longer than the term having passed after soybean domestication.     

Effect of ammonium- and nitrate-nitrogen nutrition on aluminium tolerance of soybean (Glycine max L.)

Ammonium-nitrogen supply increased Al tolerance (parameter root elongation rate) of soybean (Glycine max L.) plants compared to nitrate-nitrogen supply when grown at constant pH in solution culture. This protective effect of ammonium against Al could only partially be attributed to lowered activity of monomeric aluminium species in the ammonium solution. For ammonium and nitrate-grown plants the relationship between Al concentration in the root tips and total length could be described by the same regression equation. The higher Al tolerance of soybean plants grown in the presence of ammonium was due to restricted ad/absorption of Al which resulted from competition with positively charged Al species for binding sites in the apoplast. Induction of higher symplastic Al tolerance is unlikely because preculture with ammonium decreased rather than increased aluminium tolerance of the plants.     

Ectopic phytocystatin expression leads to enhanced drought stress tolerance in soybean (Glycine max) and Arabidopsis thaliana through effects on strigolactone pathways and can also result in improved seed traits

Ectopic cystatin expression has long been used in plant pest management, but the cysteine protease, targets of these inhibitors, might also have important functions in the control of plant lifespan and stress tolerance that remain poorly characterized. We therefore characterized the effects of expression of the rice cystatin, oryzacystatin‐I (OCI), on the growth, development and stress tolerance of crop (soybean) and model (Arabidopsis thaliana) plants. Ectopic OCI expression in soybean enhanced shoot branching and leaf chlorophyll accumulation at later stages of vegetative development and enhanced seed protein contents and decreased the abundance of mRNAs encoding strigolactone synthesis enzymes. The OCI‐expressing A. thaliana showed a slow‐growth phenotype, with increased leaf numbers and enhanced shoot branching at flowering. The OCI‐dependent inhibition of cysteine proteases enhanced drought tolerance in soybean and A. thaliana, photosynthetic CO₂ assimilation being much less sensitive to drought‐induced inhibition in the OCI‐expressing soybean lines. Ectopic OCI expression or treatment with the cysteine protease inhibitor E64 increased lateral root densities in A. thaliana. E64 treatment also increased lateral root densities in the max2‐1 mutants that are defective in strigolactone signalling, but not in the max3‐9 mutants that are defective in strigolactone synthesis. Taken together, these data provide evidence that OCI‐inhibited cysteine proteases participate in the control of growth and stress tolerance through effects on strigolactones. We conclude that cysteine proteases are important targets for manipulation of plant growth, development and stress tolerance, and also seed quality traits.     

Proteomics-Based Investigation of Salt-Responsive Mechanisms in Roots of Bradyrhizobium japonicum-Inoculated Glycine max and Glycine soja Seedlings

Journal of Plant Growth Regulation - Salt stress is one of the environmental factors most limiting crop productivity worldwide. Plant roots are the primary site for salt sensing; therefore, a...     

Microscopy of a cytoplasmic male-sterile soybean from an interspecific cross between Glycine max and G. soja (Leguminosae)

Cytoplasmic male sterility has been found independently in soybean three times since 1995, but no microscopic investigation has been published. The purpose of this microscopic study was to establish the developmental sequence leading to sterility in a cytoplasmic male-sterile soybean line that has been found to be stable under all environmental conditions tested and to demarcate the temporal and spatial parameters that result in degeneration of the microspores and pollen grains. Light microscopy showed an abnormal development and/or premature degeneration of the tapetum after meiosis II, but some pollen grains persisted until after microspore mitosis. The pollen grains never completely filled with reserves. Premature formation of the endothecium also was evident. Histochemical staining for water-insoluble carbohydrates revealed an abnormal pattern of starch deposition in anther walls that coincided with lack of pollen filling. Electron microscopy showed degeneration of the inner mitochondrial membrane in the tapetal cells as the first detectable change leading to cell degeneration. Subsequently, the tapetal endoplasmic reticulum exhibited atypical concentric rings. Pollen grains displayed mitochondria with unusually enlarged inner mitochondrial spaces, degraded plastids, a rudimentary intine, and no starch or lipid reserves. Results link mitochondrial degeneration, premature formation of the endothecium, and energy deprivation to male sterility.     

Screening of Glycine max and Glycine soja genotypes for multiple shoot formation at the cotyledonary node

Summary To identify genotypes which may give better plant regeneration responses in vitro, multiple shoots were induced from 155 Glycine max and 13 Glycine soja genotypes from maturity groups 000 to VII on B5 medium supplemented with 1 or 5 mol benzylaminopurine (BAP). The average number of shoots formed show genotype specific and hormone concentration specific responses, with number of shoots ranging from 1 to 12 for different genotypes. The results were reproducible with different seed lots of the same genotype and genotypes with similar genetic backgrounds responded in a similar fashion. No hybrid vigor was observed, except in one instance of F₁ hybrids between low shoot producers where the number of shoots obtained were higher than either parent. The root forming ability of cuttings of soybean plants grown in vivo showed general agreement with shoot forming ability in vitro. The ability to form multiple shoots appears to be genetically controlled.This research was supported by funds from the Illinois Agricultural Experiment Station and Agrigenetics Research Associates     

Mapping of the genomic regions controlling seed storability in soybean (Glycine max L.)

Seed storability is especially important in the tropics due to high temperature and relative humidity of storage environment that cause rapid deterioration of seeds in storage. The objective of this study was to use SSR markers to identify genomic regions associated with quantitative trait loci (QTLs) controlling seed storability based on relative germination rate in the F_2:3 population derived from a cross between vegetable soybean line (MJ0004-6) with poor longevity and landrace cultivar from Myanmar (R18500) with good longevity. The F_2:4 seeds harvested in 2011 and 2012 were used to investigate seed storability. The F₂ population was genotyped with 148 markers and the genetic map consisted of 128 SSR loci which converged into 38 linkage groups covering 1664.3 cM of soybean genome. Single marker analysis revealed that 13 markers from six linkage groups (C1, D2, E, F, J and L) were associated with seed storability. Composite interval mapping identified a total of three QTLs on linkage groups C1, F and L with phenotypic variance explained ranging from 8.79 to 13.43%. The R18500 alleles increased seed storability at all of the detected QTLs. No common QTLs were found for storability of seeds harvested in 2011 and 2012. This study agreed with previous reports in other crops that genotype by environment interaction plays an important role in expression of seed storability.     

Effect of chaotropic agents on reversible unfolding of a soybean (Glycine max) seed acid phosphatase

In this work we examined the effect of urea and guanidinium chloride on the structural stability of a single isoform of soybean seed acid phosphatase, based on the intensity of tryptophan fluorescence as a function of denaturant concentration. The free energy of unfolding, DeltaGu, was calculated at 25 degrees C as a function of the concentrations of both chaotropic agents; the conformational stability, DeltaG (H2O), was determined to be 2.48 kcal mol(-1). Center of mass, determined from analysis of fluorescence data, was used as a parameter to assess conformational changes. Our results indicate that complete enzyme inactivation occurred before full enzyme unfolding in both cases, and suggest that there are differences between the conformational flexibility of the active-site and that of the macromolecule as a whole.     

Evaluation of the geographical pattern of genetic diversity of Glycine soja and Glycine max based on four single copy nuclear gene loci: For conservation of soybean germplasm

Genetic diversity and its geographical patterns play a very important role in species conservation and exploitation. Here, nucleotide polymorphism patterns of four single copy nuclear gene loci in wild (Glycine soja) and cultivated soybean (Glycine max) populations from different geographical regions as well as their demographic history were analyzed. The results showed that: (1) Southern subpopulation has the highest, while central subpopulation revealed the lowest genetic diversity among three Chinese G. soja subpopulations. (2) Northern Chinese G. max subpopulation depicted higher genetic diversity than other two Chinese, Korean, Japanese and American G. max subpopulations. (3) Significant genetic differentiation (P < 0.001) was observed among Chinese G. soja subpopulations from three ecological zones. There was also a significant genetic differentiation（P < 0.01）between three Chinese and Japanese subpopulations of G. max. (4) The demographic dynamics revealed that effective population size of G. soja is expanding, while it was constant in G. max. G. soja is a useful germplasm resource to widen the genetic diversity of G. max. This study suggests that native populations of G. soja from different geo-ecological regions should be protected to conserve the genetic diversity.