Functional analysis of soybean genes involved in flavonoid biosynthesis by virus-induced gene silencing

Virus-induced gene silencing (VIGS) is a powerful tool for functional analysis of genes in plants. A wide-host-range VIGS vector, which was developed based on the Cucumber mosaic virus (CMV), was tested for its ability to silence endogenous genes involved in flavonoid biosynthesis in soybean. Symptomless infection was established using a pseudorecombinant virus, which enabled detection of specific changes in metabolite content by VIGS. It has been demonstrated that the yellow seed coat phenotype of various cultivated soybean lines that lack anthocyanin pigmentation is induced by natural degradation of chalcone synthase ( CHS ) mRNA. When soybean plants with brown seed coats were infected with a virus that contains the CHS gene sequence, the colour of the seed coats changed to yellow, which indicates that the naturally occurring RNA silencing is reproduced by VIGS. In addition, CHS VIGS consequently led to a decrease in isoflavone content in seeds. VIGS was also tested on the putative flavonoid 3'-hydroxylase ( F3'H ) gene in the pathway. This experiment resulted in a decrease in the content of quercetin relative to kaempferol in the upper leaves after viral infection, which suggests that the putative gene actually encodes the F3'H protein. In both experiments, a marked decrease in the target mRNA and accumulation of short interfering RNAs were detected, indicating that sequence-specific mRNA degradation was induced. The present report is a successful demonstration of the application of VIGS for genes involved in flavonoid biosynthesis in plants; the CMV-based VIGS system provides an efficient tool for functional analysis of soybean genes.     

Biotic elicitors as a means of increasing isoflavone concentration of soybean seeds

Soybean (Glycine max) seeds contain isoflavones that have positive impacts on human health. Four greenhouse experiments were conducted to determine if isoflavone concentration of mature soybean seeds could be increased using elicitor compounds. The effects on soybean seed isoflavone concentrations following foliar applications of two lipo‐chitooligosaccharides (LCO) [Bj V (C_18:1 MeFuc) and Bj V (Ac, C¹⁶, MeFuc)], chitosan, actinomycetes spores (Streptomyces melanosporofaciens strain EF‐76) and yeast extract at different concentrations and growth stages were evaluated. Combined chitosan seed treatment and foliar applications were also evaluated. Concentrations of daidzein, genistein, glycitein, and total isoflavones were determined by HPLC. Foliar applications of LCOs, chitosan, and actinomycetes caused a marked increase in individual and total isoflavone concentration (ranging between 21% and 84%) of mature seeds when compared to untreated control plants. There were limited differences between the different concentrations and stages of application tested for chitosan and actinomycetes; however, response to LCOs was greatest at higher concentrations (i.e. 10^‐6 M) when applied at the early podding stage. Compared to untreated plants, combined seed treatment and foliar applications of chitosan increased individual and total isoflavone concentration of mature soybean seeds by 16% to 93%. Trends were similar for different cultivars, however, the magnitude of the response varied. Finally, response to foliar applications of yeast extract was highly concentration dependent with increases of up to 56% in total isoflavone observed with 2 mg mL^‐1. Results indicate that elicitors hold promise as a way of increasing isoflavone concentration of mature soybean seeds.     

Modification of isoflavones in soybean seeds via expression of multiple phenolic biosynthetic genes

To modify the level and composition of isoflavones, the important bioactive constituents of soybean seeds, soybean was transformed via co-bombardment of embryogenic cultures with three DNA cassettes containing the CHS6-chalcone synthase and IFS2-isoflavone synthase genes, and a fragment of PAL5-phenylalanine ammonia-lyase gene, all in sense orientation under the lectin promoter mixed with the selectable marker gene, HPT (hygromycin phosphotransferase) under the 35S promoter. Four of six fertile lines produced integrated all four genes.Isoflavone levels were lower in T1 mature seeds of 5 of the 6 lines compared to the control. Transgene segregation was found in one selected line, with formation of additional sublines with different transgene composition found also in the homozygous plants. Decreased isoflavone concentrations (by about 70%) were found in T4 homozygous seeds of the two lines studied in detail here. The embryo axes accumulated most of the glycitein and contained a higher isoflavone concentration than the cotyledons. Expression of transgenes driven by the lectin promoter reduced the isoflavone concentration only in the cotyledons and not in embryo axes, indicating that this promoter is preferably active in cotyledons.     

Salt stress induced soybean <Emphasis Type="Italic">GmIFS1</Emphasis> expression and isoflavone accumulation and salt tolerance in transgenic soybean cotyledon hairy roots and tobacco

Effects of isoflavones on plant salt tolerance were investigated in soybean (Glycine max L. Merr. cultivar N23674) and tobacco (Nicotiana tabacum L.). Leaf area, fresh weight, net photosynthetic rate (Pn), and transpiration rate (Tr) of soybean N23674 plants treated with 80 mM NaCl were significantly reduced, while a gene (GmIFS1) encoding for 2-hydroxyisoflavone synthase was highly induced, and isoflavone contents significantly increased in leaves and seeds. To test the impact of isoflavones to salt tolerance, transgenic soybean cotyledon hairy roots expressing GmIFS1 (hrGmIFS1) were produced. Salt stress slightly increased isoflavone content in hairy roots of the transgenic control harboring the empty vector but substantially reduced the maximum root length, root fresh weight, and relative water content (RWC). The isoflavone content in hrGmIFS1 roots, however, was significantly higher, and the above-mentioned root growth parameters decreased much less. The GmIFS1 gene was also transformed into tobacco plants; plant height and leaf fresh weight of transgenic GmIFS1 tobacco plants were much greater than control plants after being treated with 85 mM NaCl. Leaf antioxidant capacity of transgenic tobacco was significantly higher than the control plants. Our results suggest that salt stress-induced GmIFS1 expression increased isoflavone accumulation in soybean and improved salt tolerance in transgenic soybean hairy roots and tobacco plants.     

Improvement of protein quality in transgenic soybean plants

Glycinin is one of the abundant storage proteins in soybean seeds. A modified Gy1 (A1aB1b) proglycinin gene with a synthetic DNA encoding four continuous methionines (V3-1) was connected between the hpt gene and the modified green fluorescent protein sGFP(S65T) gene, and a resultant plasmid was introduced into soybean by particle bombardment in order to improve nutritional value of its seeds. After the selection with hygromycin, the efficiency of gene introduction was evaluated. More than 60 % of the regenerated plants tolerant to hygromycin yielded the hpt and V3-1 fragment by polymerase chain reaction (PCR) analysis, and the expression of sGFP was detected in about 50 % of putative transgenic soybeans. Southern hybridization confirmed the presence of transgenes in T0 plants and the transgenic soybeans hybridized with the hpt and V3-1 genes were analyzed showed different banding patterns. Most of the transgenic plants were growing, flowering normally and produced seeds. Analysis of seed obtained from transgenic soybean plants expressing hpt and V3-1 genes showed higher accumulation of glycinin compared with non-transgenic plants. In addition, protein expression in transgenic soybean plants was observed by using 2D-electrophoresis.     

Virus-induced gene silencing as a tool for functional genomics in a legume species

Virus-induced gene silencing (VIGS) is an attractive reverse-genetics tool for studies of gene function. However, efficient VIGS has only been accomplished in a few plant species. In order to extend the application of VIGS, we examined whether a VIGS vector based on Pea early browning virus (PEBV) would produce recognizable phenotypes in Pisum sativum. A plasmid vector of PEBV was modified to allow agro-inoculation and insertion of heterologous sequences. cDNA fragments of the P. sativum phytoene desaturase (PDS), LEAFY (LFY) and KORRIGAN1 (KOR1) homologues were inserted into the PEBV RNA2 vector, replacing the genes required for nematode transmission. Pisum sativum inoculated with PEBV carrying a fragment of PsPDS developed characteristic photo-bleached leaves and this phenotype was associated with a significant reduction in PsPDS mRNA. The P. sativum homologue of LFY is known as UNIFOLIATA (UNI). Plants inoculated with PEBV carrying a fragment of UNI developed distorted flowers and leaves with modified architecture, which are also observed in UNI-mutants. In Arabidopsis thaliana, the KOR1-mutant is characterized by an extreme dwarf phenotype. Pisum sativum plants inoculated with PEBV carrying a fragment of PsKOR1 displayed a significant reduction in height and inhibition of root growth. The PEBV VIGS vector did not affect the ability of P. sativum to flower, set seeds, and form nodules characteristic of symbiosis with rhizobium. These results suggest that the PEBV vector can be applied to functional genomics in a legume species to study genes involved in a wide range of biological processes.     

Heterologous gene silencing induced by tobacco rattle virus (TRV) is efficient for pursuing functional genomics studies in woody plants

Virus-induced gene silencing (VIGS) is an effective tool for studying the functions of plant genes, but only a few VIGS vectors available for woody plants were reported so far. Here we present an effective heterologous VIGS system in woody plants based on tobacco rattle virus (TRV) vectors. We first tested whether the TRV-vector can be directly applied to infect woody plant species, such as Vernicia fordii, Populus tomentosa Carr. and Camellia oleifera. The results revealed that TRV-mediated VIGS could be effectively elicited in V. fordii, weakly in P. tomentosa Carr., but not in C. oleifera. TRV-based VIGS vectors with heterologous phytoene desaturase (PDS) sequences from various woody plant species silenced successfully the endogenous PDS gene in Nicotina benthamiana and V. fordii. The photobleached leaf phenotype of silenced plants significantly correlated with the down-regulation of endogenous PDS as compared with controls. To further confirm the reliability of VIGS in V. fordii, we also isolated the cloroplastos alterados 1 gene from P. tomentosa Carr., and the silencing pheotypes of albino leaves were observed in V. fordii 2 weeks after inoculation using a heterologous TRV-based VIGS system. Taken together, we have successfully developed an Agrobacterium-mediated VIGS assay in V. fordii and demonstrated that V. fordii as a heterologous VIGS system provides a valuable tool for functional genomic analysis in woody plant species.     

Increase of isoflavones in soybean callus by Agrobacterium-mediated transformation

Plant secondary metabolites have always been a focus of study due to their important roles in human medicine and nutrition. We transferred the isoflavone synthase (IFS) gene into soybean [Glycine max (L.) Merr.] using the Agrobacterium-mediated transformation method in an attempt to produce transformed soybean plants which produced increased levels of the secondary metabolite, isoflavone. Although the trial to produce transgenic plant failed due to unestablished hygromycin selection, transformed callus cell lines were obtained. The induction rate and degree of callus were similar among the three cultivars tested, but light illumination positively influenced the frequency of callus formation, resulting in a callus induction rate of 74% for Kwangan, 67% for Sojin, and 73% for Duyou. Following seven to eight subcultures on selection media, the isoflavone content of the transformed callus lines were analyzed by high-performance liquid chromatography. The total amount of isoflavone in the transformed callus cell lines was three- to sixfold higher than that in control callus or seeds. Given the many positive effects of isoflavone on human health, it may be possible to adapt our transformed callus lines for industrialization through an alternative cell culture system to produce high concentrations of isoflavones.     

Relationships between soybean shoot nitrogen components and soybean aphid populations

Defining the relationships between soybean (Glycine max [L.] merr.) shoot nitrogen (N) components and soybean aphid (Aphis glycines Matsumura) populations will increase understanding of the biology of this important insect pest. In this 2-year field study, caged soybean plants were infested with soybean aphids (initial infestation of 0, 10, 50, or 100 aphids plant^?1) at the fifth node developmental stage. Soybean aphid populations, soybean shoot dry weight, and shoot concentrations of nitrate-N, ureide-N, and total N were measured starting at full bloom through full seed soybean development stages. Soybean aphid population as well as shoot concentration of ureide-N increased rapidly starting at full bloom, peaked at beginning seed, and dramatically decreased by full seed soybean reproductive stages. Regression analysis indicated significant relationships (P = 0.01; r = 0.71) between soybean aphid populations and shoot ureide-N concentration. Thus, soybean aphid population levels appear to coincide with shoot ureide-N concentrations in the soybean plant.     

Novel major quantitative trait loci regulating the content of isoflavone in soybean seeds

Despite their medicinal, pharmaceutical, and nutritional importance of isoflavones, the genetic basis controlling the amounts of isoflavones in soybean seeds is still not well understood. The main obstacle is the great variability in the content of isoflavone in seeds harvested from different environments. In this study, quantitative trait loci (QTL) for the content of different isoflavones including daidzein, genistein, and glycitein were investigated in a population of recombinant inbred lines derived from the cross of “Hwangkeum” (Glycine max) by “IT182932” (Glycine soja). Seeds analyzed were harvested in three different experimental environments. QTL analyses for isoflavone content were conducted by composite interval mapping across a genomewide genetic map. Two major QTL were mapped to soybean chromosomes 5 and 8, which were designated QDZGT1 and QDZGT2, respectively. Both loci have not been previously reported in other isoflavone sources. The results from this study will be useful in cloning genes that can control the contents of isoflavones in soybean and for the development of soybean lines containing a high or low isoflavone content.     

Expression of <Emphasis Type="Italic">FLOWERING LOCUS T</Emphasis> from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> induces precocious flowering in soybean irrespective of maturity group and stem growth habit

The flowering integrator gene FLOWERING LOCUS T (FT) in Arabidopsis thaliana is conserved between diverse plant species and is thought to be the flowering signal ‘‘florigen’’, a universal long-distance mobile signal. In soybean, two FT homologs having a function to induce flowering in Arabidopsis have been identified. In this study, we showed that the expression of FT from Arabidopsis by the Apple latent spherical virus (ALSV) vector promoted precocious flowering and terminated vegetative growth in a wide range of genotypes of soybean, without using a short-day treatment. Four determinate and two indeterminate cultivars, infected with ALSV expressing FT (FT-ALSV), set flower buds on shoot apices and terminated vegetative growth at the fourth- to seventh-node stages under long-day conditions. In contrast, non-infected, healthy plants did not set flower buds on shoot apices at the same stage under the same conditions. After flowering, soybean cultivars infected with FT-ALSV, belonging to different maturity groups and stem growth habits, matured and produced seeds. The results suggest that the basic flowering pathway controlled by FT in A. thaliana might also be conserved in soybean. A system for precocious flowering and shortening of generation time using FT-ALSV would be a useful and novel technology for efficient soybean breeding.     

Coating Soybean Seed with Oxamyl for Control of Heterodera glycines

Oxamyl coated on soybean (Glycine max (L.) Merr. cv. Elgin) seeds in solutions of 20, 40, 80, and 160 mg/ml had no serious deleterious effects on seedling emergence and growth when planted in sterile soil. Seedling emergence on day 3 was less than that of the uncoated control, but by day 7 emergence was equal to, or greater than, the control. Shoot and root growth from seed coated with oxamyl in 40 and 80 mg/ml solutions was greater than that of the control. In soil infested with soybean cyst nematode, Heterodera glycines, shoot weight of soybean plants from seeds coated with oxamyl in 80 mg/ml solution was 11 and 9% greater at weeks 3 and 7, respectively, than from uncoated seeds. Numbers of juveniles (J3 and J4) and adults of H. glycines observed on the roots of plants from oxamyl-coated seeds were 83, 42, and 49% less at weeks 3, 5, and 7, respectively, than numbers on the roots of the untreated control. Numbers of J2 extracted from the roots of plants from oxamyl-coated seeds were 75% less at weeks 5 and 7 than those extracted from roots of uncoated seeds. The numbers of J2 extracted from the soil planted to oxamyl-coated seeds were 51 and 33% less at weeks 5 and 7, respectively, than from soil planted to uncoated seed.     

大豆出苗期和苗期对盐胁迫的响应及耐盐指标评价

比较了4个大豆品种出苗期和苗期的耐盐性,测定150 mmol/L NaCl胁迫下的株高、下胚轴长、侧根数、地上干/鲜重、根干/鲜重、MDA含量、SOD活性、游离Pro含量,并将幼苗移栽到田间生长至成熟。结果表明:出苗期和苗期盐胁迫下4个品种的株高都显著降低、地上干/鲜重和根干/鲜重降低;出苗期胁迫侧根数减少,下胚轴长降低;而苗期胁迫侧根数增加,下胚轴长升高。未胁迫条件下,出苗期和苗期耐盐性强的品种22021-1的MDA含量和SOD活性高于耐盐性弱的品种22293-1。胁迫后,22021-1的MDA含量降低、SOD活性升高,其MDA含量分别比对照低51.03%和21.45%,SOD活性比对照高5.85%和45.77%;22293-1的MDA含量出苗期比对照高58.97%,苗期基本无变化,SOD活性出苗期和苗期升高都不显著。MDA和SOD可以作为大豆耐盐性筛选指标。早期的短时胁迫对不同耐盐性大豆品种的经济产量影响不同。     

Control of seed coat thickness and permeability in soybean : a possible adaptation to stress

Although the seed coat, through its thickness and permeability, often regulates seed germination, very little is known about the control of its development. Using soybean (Glycine max [L.] Merrill) explants, podbearing cuttings in which defined solutions can be substituted for the roots, we have demonstrated that cytokinin and mineral nutrients moving through the xylem can control soybean seed coat development. Lack of cytokinin and minerals in the culture solution, causes a thicker, less permeable seed coat to develop. The seeds with thickened coats will imbibe water rapidly if scarified; furthermore, these scratched seeds also germinate and produce normal plants. Inasmuch as stress (e.g. drought) decreases mineral assimilation and cytokinin production by the roots, the resulting delay in germination could be an adaptive response to stress.     

Application of gibberellic acid to the surface of soybean seed (t Glycine max (L.) Merr.) and symbiotic nodulation,plant development,final grain and protein yield under short season conditions

In short-season soybean production areas, low soil temperature is the major factor limiting soybean establishment, nodulation and nitrogen fixation. Gibberellic acid (GA) pretreatment of crop seeds can overcome low soil temperature inhibition of seed germination and seedling development. However, previous studies have found that the application of GAs decreased legume nodulation and nitrogen fixation under optimal growth conditions. A field experiment was conducted under short season conditions in eastern Canada to determine whether the application of GA₃ to soybean seed could accelerate germination, and increase plant nodulation and nitrogen fixation. The results indicated that GA₃ application accelerated seedling emergence but decreased plant nodulation and nitrogen accumulation at early plant growth stages. However, these initial negative effects were overcome as the plants developed. Gibberellic acid applied to soybean seed at the time of planting did not influence final grain and protein yield.     

Molecular sequence variations of the lipoxygenase-2 gene in soybean

Soybean lipoxygenase genes comprise a multi-gene family, with the seed lipoxygenase isozymes LOX1, LOX2, and LOX3 present in soybean seeds. Among these, the LOX2 isozyme is primarily responsible for the “beany” flavor of most soybean seeds. The variety, Jinpumkong 2, having null alleles (lx1, lx2, and lx3) lacks the three seed lipoxygenases; so, sequence variations between the lipoxygenase-2 genes of Pureunkong (Lx2) and Jinpumkong 2 (lx2) cultivars were examined. One indel, four single nucleotide polymorphisms (SNPs), a 175-bp fragment in the 5′-flanking sequence, and a missense mutation within the coding region were found in Jinpumkong 2. The distribution of the sequence variations was investigated among 90 recombinant inbred lines (RILs) derived from a cross of Pureunkong × Jinpumkong 2 and in 480 germplasm accessions with various origins and maturity groups. Evidence for a genetic bottleneck was observed: the 175-bp fragment was rare in Glycine max, but present in the majority of the G. soja accessions. Furthermore, the 175-bp fragment was not detected in the 5′ upstream region of the Lx2 gene on chromosome (Chr) 13 in Williams 82; instead, a similar 175-bp fragment was positioned in the homeologous region on Chr 15. The findings indicated that the novel fragment identified was originally present in the Lx2 region prior to the recent genome duplication in soybean, but became rare in the G. max gene pool. The missense mutation of the conserved histidine residue of the lx2 allele was developed into a single nucleotide-amplified polymorphism (SNAP) marker. The missense mutation showed a perfect correlation with the LOX2-lacking phenotype, so the SNAP marker is expected to facilitate breeding of soybean cultivars which lack the LOX2 isozyme.     

Infestation of Riptortus pedestris (Fabricius) decreases the nutritional quality and germination potential of soybean seeds

The bean bug, Riptortus pedestris (Fabricius), is one of the major pests of soybean; this pest mainly feeds on soybean pods. Its population is highest when the soybean plants bear mature seeds. We aimed to determine the effect of bean bug infestation on soybean according to changes in nutrient levels and seed germination potential. Newly emerged adult males of R. pedestris (n = 5 per plant) were released into a meshed cage containing soybean plants at the R5 stage. Another meshed cage with soybean plants was treated as control. Healthy seeds harvested from control plants were selected and accounted as undamaged seeds and seeds from bug-released cages were visually categorized into 7 different classes according to the intensity of damage incurred due to bean bug infestation. The seeds were then compared in terms of changes in weight, protein, lipid, and carbohydrate content, and germination potential. Elevated protein levels were observed in seeds that showed a higher intensity of damage. On the other hand, the lipid and carbohydrate contents and germination potential were reduced in seeds showing a higher degree of damage. Germination potential in terms of lipid and carbohydrate loss caused by bug infestation is also discussed.