Polyamine synthesis in plants. Purification and properties of amidinotransferase from soybean (Glycine max) axes

Three-day-old soybean (Glycine max) seedlings were exposed to 0.4 M sorbitol solution for 4 h to induce amidinotransferase activity, with the corresponding enzyme being purified to homogeneity by chromatographic separation on DEAE-Sephacel, Sephacryl S-300 and L-arginine Sepharose 4B. The purified enzyme used L-arginine and L-glycine as the major donor/acceptor of the amidino group, respectively, with formation of guanidinoacetic acid and ornithine products being confirmed by ESI-MS. The enzyme is a tetrameric protein having a molecular mass of 240,000 Da, whose thiol group is needed for enzymatic activity. The K(M)s for arginine and glycine were 3.8 and 0.89 mM, respectively, with optimal temperature and pH being 37 degrees C and 9.5, respectively. The soybean amidinotransferase could be indirectly involved in nitrogen metabolism, as suggested by the observation that arginine:glycine amidinotransferase in soybean axes is indirectly involved in putrescine biosynthesis and displays feedback control at high levels of an endogenous regulator, putrescine.     

Interval mapping of quantitative trait loci for reproductive,morphological, and seed traits of soybean (Glycine max L.)

Quantitative trait loci (QTL) were mapped in segregating progeny from a cross between two soybean (Glycine max (L.) Merr.) cultivars: Minsoy (PI 27.890) and Noir 1 (PI 290.136). The 15 traits analyzed included reproductive, morphological, and seed traits, seed yield and carbon isotope discrimination ratios (¹³C/¹²C). Genetic variation was detected for all of the traits, and transgressive segregation was a common phenomenon. One hundred and thirty-two linked genetic markers and 24 additional unlinked markers were used to locate QTL by interval mapping and one-way analysis of variance, respectively. Quantitative trait loci controlling 11 of the 15 traits studied were localized to intervals in 6 linkage groups. Quantitative trait loci for developmental and morphological traits (R1, R5, R8, plant height, canopy height, leaf area, etc.) tended to be clustered in three intervals, two of which were also associated with seed yield. Quantitative trait loci for seed oil were separated from all the other QTL. Major QTL for maturity and plant height were linked to RFLP markers R79 (31% variation) and G173 (53% variation). Quantitative trait loci associated with unlinked markers included possible loci for seed protein and weight. Linkage between QTL is discussed in relation to the heritability and genetic correlation of the traits.     

Genetic analysis of field germination in soybean (Glycine max. (L) merill.)

Summary Genetic analysis for germination percentage was carried out in the F₃ and F₄ generations of a diallel cross involving six promising genotypes of soybean. Results indicated a high amount of genetic variability and a moderately high heritability together with genetic advance, suggesting a possible improvement for this character through hybridization and selection. Correlations at different levels revealed a strong negative association of germination with only one seed character: seed weight. This observation was further confirmed from path coefficient analysis. These findings strongly suggest that to base selection on seed weight which may not influence the seed quality of soybean.     

Epistatic expression of quantitative trait loci (QTL) in soybean [Glycine max (L.) Merr.] determined by QTL association with RFLP alleles

Quantitative trait values for seed oil and protein content or for maturity were measured in recombinant inbred lines (RIL) of soybean derived from a cross between two soybean cultivars: Minsoy PI 27890 and Noir 1 PI 290136. Seed oil was found to be inversely correlated to protein content. By analyzing DNA from plants with extreme phenotypes, we were able to identify quantitative trait loci (QTL) for these traits as being linked to several restriction fragment length polymorphism (RFLP) loci, including R183 for oil and protein content and R79 for maturity. Cumulative distributions of trait values were graphed for those RIL with Minsoy alleles and for those with Noir 1 alleles. As already suggested by the alleles found associated with extreme phenotypes, the distributions were consistent with an independent and additive expression of the maturity QTL linked to R79. That is, the cumulative distributions for plants with Minsoy alleles and for plants with Noir 1 alleles were similar in shape, but the entire Noir 1 curve had been shifted to later maturity dates. In contrast, the trait distributions for a locus affecting oil and protein content linked to R183 were not compatible with an additive model. These results suggest that this approach can be used for rapid identification of QTLs with epistatic expression.     

GTP-specific pyrophosphorylation of thiamin in dark-grown soybean (Glycine max) seedling axes

ATP:thiamin pyrophosphotransferase (TPT: EC 2.7.6.2) was purified 5 900-fold from 48 h dark-grown soybean [ Glycine max (L.), Merr. cv. Ransom II] seedling axes. TPT activity was monitored during purification by measuring the formation of thiamin pyrophosphate (TPP) from [2-¹⁴C]-thiamin at optimal pH (7.3). Although other nucleoside triophosphates were active as pyrophosphate donors (apparent K_ms from 21 to 138 m M ), GTP was the preferred nucleotide with an apparent K_m of 0.021 m M . TPT activity was extremely sensitive to TPP formation, suggesting product feedback inhibition of TPT activity in vivo. Sulfhydryl, H⁺ and Mg²⁺ concentrations, either independently or in concert, were found to affect TPT activity.     

Megagametophyte abnormalities of near-isogenic female partial-sterile soybean mutants (Glycine max; Leguminosae)

Megagametogenesis of soybean, Glycine max (L.) Merr., cultivars Clark and Clark k2, and F1 hybrid of Clark (female parent) crossed with Clark k2 (male parent) were studied using stereo light microscopy and confocal scanning laser microscopy. Reproductive development in Clark and Clark k2 plants was compared to F1 hybrid plants. In mature pods, 6.4% of the ovules of Clark, 8.1% of the ovules of Clark k2, and 41.4% of the ovules of F1 hybrid plants were aborted. This female partial sterility was due to incomplete megagametophyte development: undeveloped polar nuclei—or developed but not in a position for fertilization; increased megagametophyte wall thickness; abnormal shape and/or premature degeneration of synergids and intact synergids throughout the life of the ovule; egg cell not well-developed or absent; and megagametophyte remaining uninucleate. Each of these abnormalities contributed to either lack of double fertilization or early megagametophyte abortion. Electronic Publication     

Identification of a new major QTL associated with resistance to soybean cyst nematode (Heterodera glycines)

Resistance of soybean [Glycine max (L.) Merr.] to cyst nematode (SCN) (Heterodera glycines Ichinohe), one of the most destructive pathogens affecting soybean, involves a complex genetic system. The identification of QTLs associated with SCN resistance may contribute to the understanding of such system. The objective of this work was to identify and map QTLs for resistance to SCN Race 14 with the aid of molecular markers. BC₃F_2:3 and F_2:3 populations, both derived from an original cross between resistant cv. Hartwig and the susceptible line BR-92–31983 were screened for resistance to SCN Race 14. Four microsatellite (Satt082, Sat_001, Satt574 and Satt301) and four RAPD markers (OPAA-11₇₉₅, OPAE-08₈₃₇, OPR-07₅₄₈ and OPY-07₂₀₃₀) were identified in the BC₃F_2:3 population using the bulked segregant analysis (BSA) technique. These markers were amplified in 183 F_2:3 families and mapped to a locus that accounts for more than 40% of the resistance to SCN Race 14. Selection efficiency based on these markers was similar to that obtained with the conventional method. In the case of the microsalellite markers, which identify homozygous resistant genotypes, the efficiency was even higher. This new QTL has been mapped to the soybean linkage group D2 and, in conjunction with other QTLs already identified for SCN resistance, will certainly contribute to our understanding of the genetic basis of resistance of this important disease in soybean. Received: 12 October 1999 / Accepted: 14 April 2000     

Unusual regulatory properties of sucrose-phosphate synthase purified from soybean (Glycine max) leaves

Sucrose-phosphate (SPS) from source leaves of soybean ( Glycine max (L.) Merr. cv. Ransom II) was purified 74-fold to a final specific activity of 1.8 U (mg protein)¹. The partially purified preparation was free from phosphoglucoseisomerase (EC 5.3.1.9), pyrophosphatase (EC 3.6.1.1), phosphoenolpyruvate-phosphatase (EC 3.1.3.-), phosphofructokinase (EC 2.7.1.11), and uridine diphosphatase (EC 3.6.1.6), and was used for characterization of the kinetic and regulatory properties of the enzyme. The enzyme showed hyperbolic saturation kinetics for both fructose-6-phosphate (K_m=0.57 m M ) and UDPGlucose (UDPG) (K_m=4.8 m M ). The activity of SPS was inhibited by the product UDP. In vitro this inhibition could be partially overcome by the presence of Mg²⁺. Inorganic orthophosphate was only slightly inhibitory (35% inhibition at 25 m M phosphate). Glucose-6-phosphate (up to 20 m M ) had no effect on activity, and did not show any significant interaction with phosphate inhibition. A range of potential effectors was tested and had no effect on SPS activity: Glucose-1-phosphate, fructose-1, 6-bisphosphate, α-glycero-phosphate, dihydroxyacetone-phosphate, 3-phosphoglyceric acid, (all at 5 m M ), sucrose at 100 m M and pyrophosphate at 0.1 m M . The apparent lack of allosteric regulation of soybean SPS makes this enzyme markedly different from SPS previously characterized from spinach and maize.     

Occurrence of ferredoxin-dependent glutamate synthase in plant cell fraction of soybean root nodules (Glycine max)

Ferredoxin-dependent glutamate synthase (EC 1.4.7.1) and NADH-dependent glutamate synthase (EC 1.4.1.14) have been identified in the plant cells of soybean nodules. Ferredoxin-dependent glutamate synthase is 2-fold more active than NADH-dependent enzyme in vitro. Ferredoxin-dependent glutamate synthase cross-reacts with IgG against ferredoxin-dependent glutamate synthase of rice green leaves, whereas NADH-dependent glutamate synthase does not recognize the IgG, indicating that there are two distinct enzyme proteins. Ferredoxin-dependent glutamate synthase is composed of polypeptide chain(s) of 165 kDa and has a high affinity to spinach leaf ferredoxin as an electron carrier.     

Effects of temperature on germination and mitochondrial dehydrogenases in two soybean (Glycine max) cultivars

The effects of eight germination temperatures from 10°C to 35°C on germination and dehydrogenase activities of two soybean (Glycine max [L.] Merr.) cultivars were investigated after 48 h of seedling growth. Axis fresh weights of cv. Chippewa increased as germination temperature increased from 10°C to 35°C. In contrast, axis fresh weights for the cv. Wells increased more slowly with increasing temperature and reached a maximum at c. 25°C. In general, in vitro activities of glutamate dehydrogenase (GDH), NADP-isocitrate dehydrogenase (NADP-ICDH), and malate dehydrogenase (MDH) from the axes of cv. Chippewa correlated well with increases in axis fresh weights. GDH and MDH activities from axes of the cv. Wells also reflected increases in axis fresh weights although the correlation was not as evident as for the cv. Chippewa. NADP-ICDH activity from ‘Wells’ axes was highest at 35°C even though germination was poor at this temperature. GDH and MDH activities from cotyledons of both cultivars were not correlated with axis weight increases. No GDH activity was detected in ‘Wells’ cotyledons from seeds germinated at 35°C.     

Identification of genomic regions determining flower and pod numbers development in soybean （Glycine max L.）

Flower and pod numbers per plant are important agronomic traits underlying soybean yield.So far quantitative trait loci (QTL) detected for flower and pod-related traits have mainly focused on the final stage,and might therefore have ignored genetic effects expressed during a specific developmental stage.Here,dynamic expressions of QTL for flower and pod numbers were identified using 152 recombinant inbred lines (RILs) and a linkage map of 306 markers.Wide genetic variation was found among RILs;17 unconditional and 18 conditional QTL were detected for the two traits at different developmental stages over two years.Some QTL were detected only at one stage and others across two or more stages,indicating that soybean flower and pod numbers development may be governed by time-dependent gene expression.Three main QTL (qfn-Chr18-2,qfn-Chr20-1,and qfn-Chr19) were detected for flower number,and two main QTL (qpn-Chr11 and qpn-Chr20) were detected for pod number.The phenotypic variation explained by them ranged from 6.1% to 34.7%.The markers linked to these QTL could be used in marker-assisted selection for increasing soybean flower and pod numbers,with the ultimate aim of increasing soybean yield.Comparison of the QTL regions for flower and pod numbers traits with the related genes reported previously showed that seven and four related genes were located in the QTL regions of qfn-Chr11 and qfn-Chr19,respectively.Tbese results provide a basis for fine mapping and cloning of flower and pod development-related genes.     

Effects of quercetin and enhanced UV-B radiation on the soybean (Glycine max) leaves

The possible ameliorative effects of quercetin on soybean [Glycine max (L.) Merr.] leaves exposed to UV-B radiation were conducted in greenhouse. The symmetrical leaves supplied with quercetin solution (0.2%, 1%) were exposed to UV-B radiation (0, 3.5, 6.5 kJ m⁻² d⁻¹). 0.2% quercetin ameliorated leaf photosynthesis, improved leaf water content (LWC), and decreased lipid oxidation. The unfavorable effect on photosynthetic parameter was displayed in 1% quercetin treatment. The effect of quercetin on phenylalanine ammonia lyase (PAL) activity varied with the quercetin concentration, UV-B radiation intensity and leaf development. In the later development polyphenol oxidase (PPO) activity was increased significantly by quercetin treatments. We suggested that quercetin with suitable concentration could serve as UV-B protective agent partly due to its antioxidant capacity.     

Histopathology of roots of Glycine max (L.) Merrill induced by root-knot nematode (Meloidogyne incognita)

In Glycine max, the second-stage juveniles of Meloidogyne incognita entered the roots through the apical meristem or elongation zone. The juveniles induced giant cells in the zone of vascular strands. Near the head of the nematode and adjacent to the giant cells, the vascular strands exhibited abnormalities in their shapes and structures; both xylem and phloem were found to be affected. The giant cells had dense and granular cytoplasm, and large nuclei with large nucleoli. Some parenchyma cells exhibited hypertrophy, while others exhibited hyperplasia. The distinctive feature of the study is reporting the occurrence of abnormal xylem, abnormal phloem and abnormal parenchyma.     

Genetic and cytological analyses of a partial-female-sterile mutant (PS-1) in soybean (Glycine max; Leguminosae)

Soybean partial-female-sterile mutant 1 (PS-1) was recovered from a gene-tagging study. The objectives were to study the inheritance, linkage, allelism, and certain aspects of the reproductive biology of the PS-1 mutant. For inheritance and linkage tests, PS-1 was crossed to flower color mutant Harosoy-w4 and to chlorophyll-deficient mutant CD-1, also recovered from the gene-tagging study. For allelism tests, reciprocal crosses were made with PS-1 and three other partial-sterile mutants (PS-2, PS-3, and PS-4) recovered from the same gene-tagging study. The PS-1 mutant is inherited in a 3:1 ratio and is a single recessive gene. Linkage results indicated that the gene for partial sterility in PS-1 is not linked either to the w4 locus or to the CD-1 locus. Allelism tests showed that the gene in PS-1 is nonallelic to the gene in PS-2, PS-3, and PS-4. Investigations of developing and mature pollen indicated no differences in morphology, stainability, or fluorescence between normal and partial-sterile genotypes. The PS-1 mutant is completely male fertile. Confocal scanning laser microscopy was used to determine that early embryo abortion in PS-1 is due indirectly to abnormal migration of the fused polar nucleus, which prevented it from being fertilized. Subsequent absence of endosperm development leads directly to abortion of the proembryo.     

大豆耐旱种质鉴定和相关根系性状的遗传与QTL定位

从301份黄淮海和长江中下游地区代表性大豆地方品种和育成品种(系)中按根系类型选取59份,在苗期干旱胁迫和非胁迫条件下对地上部和地下部性状进行2年重复鉴定,发现材料间性状隶属函数值具有丰富遗传变异,以株高、叶龄、根干重和茎叶干重隶属函数的算术平均数为抗旱综合指标从中筛选出汉中八月黄、晋豆14,科丰1号,圆黑豆等强耐旱型(1级)和临河大粉青、宁海晚黄豆等干旱敏感型(5级)材料。比根干重、比总根长、比根体积与耐旱隶属函数平均值均呈极显著正相关,可作为耐旱性的根系性状指标。利用“科丰1号×南农1138 2”(1级×4级)衍生的RIL群体为材料,对耐旱相关根系性状采用主基因 多基因混合遗传模型分离分析法进行遗传分析并进行QTL定位。结果表明,该两亲本间比根干重、比总根长、比根体积的遗传均为两对主基因加多基因模型,后两者主基因间有连锁(重组率分别为4.30%和1.93%);主基因遗传率为62.26%～91.81%,多基因遗传率为2.99%～24.75%;耐旱相关根系性状各主要由1对主基因控制,另1对效应较小。QTL分析检测到5、3、5个QTLs分别控制比根重、比根总长、比根体积,位于N6 C2、N8 D1b W、N11 E、N18 K连锁群上。3性状各有1个贡献率大的QTL(Dw1,Rl1,Rv1),而且均位在N6 C2的STAS8_3T STAS8_6T相同距离的区段上,其他QTLs效应均较小。分离分析与QTL定位的结果相对一致。     

RFLP tagging of QTLs conditioning specific leaf weight and leaf size in soybean

 Selection for high specific leaf weight (SLW) in soybean [Glycine max (L) Merr.] may increase apparent photosynthetic rate per unit leaf area (AP), which in turn may improve seed yield. In general, the SLW and leaf size are negatively correlated in soybean. To maximize total photosynthetic performance, and perhaps the seed yield, of a soybean cultivar, it would be necessary to establish a large leaf area rapidly while maintaining a high SLW. The objective of the present study was to identify quantitative trait loci (QTLs) conditioning SLW and leaf size in soybean. One hundred and twenty F₄-derived lines from a ‘Young’×PI416937 population were evaluated using restriction fragment length polymorphism (RFLP) markers. The genetic map consisted of 155 loci on 33 linkage groups (LGs) covering 973 cM of map distance. The phenotypic data were collected from two different environments – a greenhouse at Athens, Ga. and a field site at Windblow, N.C. The SLW and leaf-size measurements were made on leaves from the 8th and 9th node of soybean plants at the V12 stage of development. Combined over environments, six putative independent RFLP markers were associated with SLW, and four of these loci were consistent across environments. Individually, the six markers each explained between 8 and 18% of the phenotypic variation among lines for SLW. The Young alleles contributed to a greater SLW at four of the six independent marker loci, and transgressive segregation occurred among the progeny for SLW. Three putative independent RFLP markers were associated with leaf size, each explaining between 6 to 11% of the phenotypic variation in the trait, and one of these markers was identified in both environments. There was no correlation between SLW and leaf size in this population. Similarly, none of the six QTLs conditioning SLW were linked to any of the three QTLs for leaf size. In this soybean population, it is possible to select for progeny lines with greater SLW than either parent perhaps without affecting the leaf size. It is feasible to pyramid all of the desirable alleles for greater SLW and large leaf size in a single genetic background. Received: 16 August 1997 / Accepted: 20 October 1997