Nitrate concentration and nitrate reductase activity in the leaves of three legumes and three cereals*

Nitrate concentration and nitrate reductase activity (NRA) were studied in the leaves of soybean (Glycine max), groundnut (Arachis hypogaea and cowpea (Vigna unguiculata) and sorghum (Sorghum bicolor), pearl millet (Pennisetum americanum) and maize (Zea mays) at three nitrogen fertiliser levels in two field experiments. Higher nitrate concentrations were detected in the leaves of groundnut, cowpea and pearl millet than in sorghum and maize. Nitrate content in the leaves and leaf NRA were not related across crop species, nor was a generalised pattern of leaf NRA and leaf nitrate observed within legumes or within cereals. Nitrogen application resulted in higher nitrate availability in the leaves, with varied leaf NRA.     

Comparison of the starch synthesis genes between maize and rice: copies, chromosome location and expression divergence

Gene duplication and divergence are important evolutionary processes. It has been suggested that a whole genome duplication (WGD) event occurred in the Gramineae, predating its divergence, and a second WGD occurred in maize during its evolution. In this study we compared the fate of the genes involved in the core pathway of starch biosynthesis following the ancient and second WGDs in maize and rice. In total, thirty starch synthesis genes were detected in the maize genome, which covered all the starch synthesis gene families encoded by 27 genes in rice. All of these genes, except ZmGBSSIIb and ZmBEIII, are anchored within large-scale synteny blocks of rice and maize chromosomes. Previous findings and our results indicate that two of the current copies of many starch synthesis genes (including AGPL, AGPS, GBSS, SSII, SSIII, and BEII) probably arose from the ancient WGD in the Gramineae and are still present in the maize and rice genome. Furthermore, two copies of at least six genes (AGPS1, SSIIb, SSIIIb, GBSSII, BEI, and ISA3) appear to have been retained in the maize genome after its second WGD, although complete coding regions were only detected among the duplicate sets of AGPS1, SSIIb, and SSIIIb. The expression patterns of the remaining duplicate sets of starch synthesis genes (AGPL1/2, AGPS1/2, SSIIa/b, SSIIIa/b, GBSSI/II, and BEIIa/b) differ in their expression and could be classified into two groups in maize. The first group is mainly expressed in the endosperm, whereas the second is expressed in other organs and the early endosperm development. The four duplicate sets of ZmGBSSII, ZmSSIIb, ZmSSIIIb and AGPS1, which arose from the second WGD diverged in gene structure and/or expression patterns in maize. These results indicated that some duplicated starch synthesis genes were remained, whereas others diverged in gene structure and/or expression pattern in maize. For most of the duplicated genes, one of the copies has disappeared in the maize genome after the WGD and the subsequent “diploidization”. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genomic structure and expression of alternative oxidase genes in legumes

Mitochondria isolated from chickpea (Cicer arietinum) possess substantial alternative oxidase (AOX) activity, even in non‐stressed plants, and one or two AOX protein bands were detected immunologically, depending on the organ. Four different AOX isoforms were identified in the chickpea genome: CaAOX1 and CaAOX2A, B and D. CaAOX2A was the most highly expressed form and was strongly expressed in photosynthetic tissues, whereas CaAOX2D was found in all organs examined. These results are very similar to those of previous studies with soybean and siratro. Searches of available databases showed that this pattern of AOX genes and their expression was common to at least 16 different legume species. The evolution of the legume AOX gene family is discussed, as is the in vivo impact of an inherently high AOX capacity in legumes on growth and responses to environmental stresses.     

Molecular cloning and expression analysis of three genes encoding starch synthase II in rice

Three starch synthase (SS) genes, OsSSII-1, OsSSII-2 and OsSSII-3, were identified in rice (Oryza sativa L.) and localized to chromosomes 10, 2 and 6, respectively. The three OsSSII full-length cDNAs were cloned, and the predicted amino acid sequences were found to share 52–73% similarity with other members of the plant SSII family. The SS activity of each OsSSII was confirmed by expression and enzyme activity assay in Escherichia coli. Expression profile analysis revealed that OsSSII-1 was expressed in endosperms, leaves and roots; OsSSII-2 was mainly expressed in leaves, while OsSSII-3 was mainly expressed in endosperms. Similar to the OsSSI proteins, the OsSSII-2 and OsSSII-3 proteins were found in the soluble as well as the starch-granule-bound fractions in rice. The roles of the OsSSII proteins in starch biosynthesis in rice and the evolutionary relationships of the genes encoding monocotyledonous and dicotyledonous class-II SS enzymes are discussed.Abbreviations CDS Coding domain sequence - EST Expressed sequence tag - GB Granule-bound - Glc Glucose - SS Starch synthase     

Expression of a soybean nodule‐enhanced phosphoenolpyruvate carboxylase gene that shows striking similarity to another gene for a house‐keeping isoform

Three different cDNAs for phosphoenolpyruvate carboxylase (PEPC) were isolated from soybean root nodules. The full-length cDNA of the most abundant isoform (GmPEPC7) was very similar to another one (GmPEPC15), the nucleotide sequence of which is identical to that of a reported clone (gmppc1) (Vazquez-Tello, A., Whittier, R.F., Kawasaki, T., Sugimoto, T., Kawamura, Y., Shibata, D. (1993) Plant Physiol. 103, 1025–1026). In the coding region, the newly isolated GmPEPC7 and the previously reported were gmppc1 99% and 98% identical at the amino acid and nucleotide levels, respectively. In contrast, they exhibited only 39% identity in the 3′ non-coding region, indicating that they are encoded by distinct genes. Northern blot analysis with 3′ non-coding regions as isoform-specific probes showed that GmPEPC7 is nodule-enhanced whereas GmPEPC15 (gmppc1) is expressed in most soybean tissues. The third clone (GmPEPC4) was much less homologous to the above two clones and thus was not further characterized. It was also shown by in situ hybridization that the nodule-enhanced isoform is expressed in all cell types in nodules, including in Bradyrhizobium-infected and uninfected cells and cortical cells. A relatively strong hybridization signal was detected in the vascular bundle pericycle. Southern blot analysis indicated that there are only two PEPC genes exhibiting a high degree of similarity in the soybean genome, one for the nodule-enhanced GmPEPC7 and the other for the constitutively expressed gmppc1. A phylogenetic tree based on the amino acid sequences of soybean PEPCs and nodule-enhanced PEPCs of alfalfa and pea suggested that the soybean nodule-enhanced isoform evolved from the housekeeping PEPC gene after the ureid-translocating and amide-translocating legumes diverged from each other.     

The structural organisation of the gene encoding class II starch synthase of wheat and barley and the evolution of the genes encoding starch synthases in plants

Wheat and barley contain at least four classes of starch synthases in the endosperm, granule bound starch synthase I (GBSSI) and starch synthases I, II and III (SSI, SSII, SSIII). In this work, SSII in barley is shown to be associated with the starch granule by using antibodies. A cDNA from barley encoding SSII and the genes for SSII from barley and Aegilops tauschii (A. tauschii, the D genome donor to wheat) are characterised. Fluorescent in situ hybridisation (FISH) and PCR were used to localise the wheat SSII gene to the short arm of chromosome 7, showing synteny with the location of the rice SSII gene to the short arm of chromosome 6. Comparison of the genes encoding SSII of A. tauschii, barley and Arabidopsis showed a conserved exon-intron structure although the size of the introns varied considerably. Extending such comparison between the genes encoding starch synthases (GBSSI, SSI, SSII and SSIII) from A. tauschii and Arabidopsis showed that the exon-intron structures are essentially conserved. Separate and distinct genes for the individual starch synthases therefore existed before the separation of monocotyledons and dicotyledons. Electronic Publication     

A comprehensive expression analysis of the starch synthase gene family in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

To elucidate the roles of the isogenes encoding starch synthase (EC 2.4.1.21) in rice (Oryza sativa L.), a comprehensive expression analysis of the gene family was conducted. Extensive searches for starch synthase genes were done in the databases of both the whole genome and full-length cDNAs of rice, and ten genes were revealed to comprise the starch synthase gene family. Multi-sequence alignment analysis of the starch synthase proteins from rice and other plant species suggested that they were grouped into five classes, soluble starch synthase I (SSI), SSII, SSIII, SSIV and granule-bound starch synthase (GBSS). In rice, there was one gene for SSI, three for SSII and two each for SSIII, IV and GBSS. The expression pattern of the ten genes in the developing caryopsis was examined by semi-quantitative RT–PCR analysis. Based on the temporal expression patterns, the ten genes could be divided into three groups: (i) early expressers (SSII-2, III-1, GBSSII), which are expressed in the early stage of grain filling; (ii) late expressers (SSII-3, III-2, GBSSI), which are expressed in the mid to later stage of grain filling; and (iii) steady expressers (SSI, II-1, IV-1, IV-2), which are expressed relatively constantly during grain filling. Within a caryopsis, the three gene groups spatially share their expression, i.e. early expressers in the pericarp, the late expressers in the endosperm and the steady expressers in both tissues. In addition, this grouping was reflected in the expression pattern of various rice tissues: expression in non-endosperm, endosperm or all tissues examined. The implications in this spatio-temporal work sharing of starch synthesis isogenes are discussed.Abbreviations DAF Days after flowering - GBSS Granule-bound starch synthase - SS Soluble starch synthase     

Differences in uptake kinetics of ammonium and nitrate in legumes and cereals

Influx isotherms were obtained for nitrate and ammonium from three legumes, Cajanus cajan (L.) Millsp., Cicer arietinum L. and Arachis hypogaea L. and three cereals, Sorghum bicolor (L.) Moench., Pennisetum glaucum L. and Zea mays L. The transition in influx isotherms for both nitrogen sources was found to be within the concentration range (0.05–2.5 mM) tested. There were significant differences in Km and Vmax for ammonium between legumes and cereals. The difference in the kinetic properties for nitrate uptake between the two groups of plants only became apparent at the higher concentration tested. Legumes translocated absorbed nitrate and ammonium to shoots more rapidly than cereals. Results show that there are significant differences in uptake and translocation of ammonium and nitrate between legumes and cereals.     

Diversification of genes encoding granule-bound starch synthase in monocots and dicots is marked by multiple genome-wide duplication events

Starch is one of the major components of cereals, tubers, and fruits. Genes encoding granule-bound starch synthase (GBSS), which is responsible for amylose synthesis, have been extensively studied in cereals but little is known about them in fruits. Due to their low copy gene number, GBSS genes have been used to study plant phylogenetic and evolutionary relationships. In this study, GBSS genes have been isolated and characterized in three fruit trees, including apple, peach, and orange. Moreover, a comprehensive evolutionary study of GBSS genes has also been conducted between both monocots and eudicots. Results have revealed that genomic structures of GBSS genes in plants are conserved, suggesting they all have evolved from a common ancestor. In addition, the GBSS gene in an ancestral angiosperm must have undergone genome duplication ～251 million years ago (MYA) to generate two families, GBSSI and GBSSII. Both GBSSI and GBSSII are found in monocots; however, GBSSI is absent in eudicots. The ancestral GBSSII must have undergone further divergence when monocots and eudicots split ～165 MYA. This is consistent with expression profiles of GBSS genes, wherein these profiles are more similar to those of GBSSII in eudicots than to those of GBSSI genes in monocots. In dicots, GBSSII must have undergone further divergence when rosids and asterids split from each other ～126 MYA. Taken together, these findings suggest that it is GBSSII rather than GBSSI of monocots that have orthologous relationships with GBSS genes of eudicots. Moreover, diversification of GBSS genes is mainly associated with genome-wide duplication events throughout the evolutionary course of history of monocots and eudicots.     

Diverse origins of waxy foxtail millet crops in East and Southeast Asia mediated by multiple transposable element insertions

The naturally occurring waxy and low-amylose variants of foxtail millet and other cereals, like rice and barley, originated in East and Southeast Asia under human selection for sticky foods. Mutations in the GBSS1 gene for granule-bound starch synthase 1 are known to be associated with these traits. We have analyzed the gene in foxtail millet, and found that, in this species, these traits were originated by multiple independent insertions of transposable elements and by subsequent secondary insertions into these elements or deletion of parts of the elements. The structural analysis of transposable elements inserted in the GBSS1 gene revealed that the non-waxy was converted to the low-amylose phenotype once, while shifts from non-waxy to waxy occurred three times, from low amylose to waxy once and from waxy to low amylose once. The present results, and the geographical distribution of different waxy molecular types, strongly suggest that these types originated independently and were dispersed into their current distribution areas. The patterns of GBSS1 variation revealed here suggest that foxtail millet may serve as a key to solving the mystery of the origin of waxy-type cereals in Asia. The GBSS1 gene in foxtail millet provides a new example of the evolution of a gene involved in the processes of domestication and its post-domestication fate under the influence of human selection. Electronic Supplementary Material Supplementary material is available for this article at     

Soybean 14-3-3 gene family: identification and molecular characterization

The 14-3-3s are a group of proteins that are ubiquitously found in eukaryotes. Plant 14-3-3 proteins are encoded by a large multigene family and are involved in signaling pathways to regulate plant development and protection from stress. Recent studies in Arabidopsis and rice have demonstrated the isoform specificity in 14-3-3s and their client protein interactions. However, detailed characterization of 14-3-3 gene family in legumes has not been reported. In this study, soybean 14-3-3 proteins were identified and their molecular characterization performed. Data mining of soybean genome and expressed sequence tag databases identified 18 14-3-3 genes, of them 16 are transcribed. All 16 SGF14s have higher expression in embryo tissues suggesting their potential role in seed development. Subcellular localization of all transcribed SGF14s demonstrated that 14-3-3 proteins in soybean have isoform specificity, however, some overlaps were also observed between closely related isoforms. A comparative analysis of SGF14s with Arabidopsis and rice 14-3-3s indicated that SGF14s also group into epsilon and non-epsilon classes. However, unlike Arabidopsis and rice 14-3-3s, SGF14s contained only one kind of gene structure belonging to each class. Overall, soybean consists of the largest family of 14-3-3 proteins characterized to date. Our results provide a solid framework for further investigations into the role of SGF14s and their involvement in legume-specific functions.     

Functional conservation and divergence of GmCHLI genes in polyploid soybean

Polyploidy is prevalent in nature. As the fate of duplicated genes becomes more complicated when the encoded proteins function as oligomers, functional investigations into duplicated oligomer‐encoding genes in polyploid genomes will facilitate our understanding of how traits are expressed. In this study, we identified GmCHLI1, a gene encoding the I subunit of magnesium (Mg)‐chelatase, which functions in hexamers as responsible for the semi‐dominant etiolation phenotype in soybean. Four GmCHLI copies derived from two polyploidy events were identified in the soybean genome. Further investigation with regard to expression patterns indicated that these four copies have diverged into two pairs; mutation in the other copy of the pair that includes GmCHLI1 also resulted in a chlorophyll‐deficient phenotype. Protein interaction assays showed that these four GmCHLIs can interact with each other, but stronger interactions were found with mutated subunits. The results indicate that, in polyploidy, deficiency in each copy of duplicated oligomer‐encoding genes could result in a mutant phenotype due to hetero‐oligomer formation, which is different from the model of allelic dosage or functional redundancy. In addition, we interestingly found an increase in isoflavonoids in the heterozygous etiolated plants, which might be useful for improving soybean seed quality.     

A combined reduction in activity of starch synthases II and III of potato has novel effects on the starch of tubers

A chimeric antisense construct has been used to generate transgenic potatoes ( Solanum tuberosum L.) in which activities of both of the main starch synthases responsible for amylopectin synthesis in the tuber (SSII and SSIII) are reduced. The properties of starch from tubers of these plants have been compared with those of starches from transgenic plants in which activity of either SSII or SSIII has been reduced. Starches from the three types of transgenic plant are qualitatively different from each other and from the starch of control plants with unaltered starch synthase activities, with respect to granule morphology, the branch lengths of amylopectin, and the gelatinisation behaviour analysed by viscometry. The effects of reducing SSII and SSIII together cannot be predicted from consideration of the effects of reducing these two isoforms individually. These results indicate that different isoforms of starch synthase make distinct contributions to the synthesis of amylopectin, and that they act in a synergistic manner, rather than independently, during amylopectin synthesis.     

Microevolution of symbiotic Bradyrhizobium populations associated with soybeans in east North America

Microevolution and origins of Bradyrhizobium populations associated with soybeans at two field sites (A and B, 280 km apart in Canada) with contrasting histories of inoculation was investigated using probabilistic analyses of six core (housekeeping) gene sequences. These analyses supported division of 220 isolates in five lineages corresponding either to B. japonicum groups 1 and 1a or to one of three novel lineages within the genus Bradyrhizobium. None of the isolates from site A and about 20% from site B (the only site with a recent inoculation history) were attributed to inoculation sources. The data suggest that most isolates were of indigenous origin based on sequence analysis of 148 isolates of soybean‐nodulating bacteria from native legumes (Amphicarpaea bracteata and Desmodium canadense). Isolates from D. canadense clustered with B. japonicum group 1, whereas those from A. bracteata were placed in two novel lineages encountered at soybean field sites. One of these novel lineages predominated at soybean sites and exhibited a significant clonal expansion likely reflecting selection by the plant host. Homologous recombination events detected in the 35 sequence types from soybean sites had an effect on genetic diversification that was approximately equal to mutation. Interlineage transfer of core genes was infrequent and mostly attributable to gyrB that had a history of frequent recombination. Symbiotic gene sequences (nodC and nifH) of isolates from soybean sites and native legumes clustered in two lineages corresponding to B. japonicum and B. elkani with the inheritance of these genes appearing predominantly by vertical transmission. The data suggest that soybean‐nodulating bacteria associated with native legumes represent a novel source of ecologically adapted bacteria for soybean inoculation.     

大豆类黄酮生物合成关键酶CHS基因的克隆及表达分析

根据查尔酮合成酶(CHS)基因DNA序列的保守区域设计了PCR引物,通过RT-PCR扩增从大豆叶片中克隆出3个参与类黄酮合成的CHS基因,分别命名为GmCHS1、GmCHS2和GmCHS3。在大豆基因组数据库进行同源比对,发现这3个基因分别与大豆基因组上Gm08g11610、Gm05g28610和Gm08g11520相对应,DNA序列一致性达95%～98%,推导氨基酸序列一致性达98%以上。进化分析显示,大豆中3个CHS蛋白与决明、菜豆CHS蛋白亲缘关系较近。表达分析显示,这3个基因在不同品种间有表达水平的差异,这可能是不同大豆品种中类黄酮含量不同的重要原因之一。     

Structure and chromosomal arrangement of leghemoglobin genes in kidney bean suggest divergence in soybean leghemoglobin gene loci following tetraploidization

We have determined the structure of one of the leghemoglobin (Lb) genes of Phaseolus vulgaris (kidney bean) and deduced the chromosomal arrangement of leghemoglobin genes by genomic hybridizations with Lb and two other sequences, each specific to the 5' or 3' region of the soybean leghemoglobin loci. By comparing this organization with two other species of legumes, Glycine max (soybean) and G. soja (wild soybean), a phylogeny of leghemoglobin gene loci was traced. The intragenic structure of the kidney bean leghemoglobin gene shows the same intron/exon arrangement as that of soybean leghemoglobin genes and extensive sequence homologies in both coding as well as 5' and 3' non-coding regions. The presence in the kidney bean genome of four leghemoglobin genes suggests that tandem duplications of a single primordial plant globin gene had occurred to generate four leghemoglobin genes in an `Lb-locus' before Glycine and Phaseolus species diverged. Chromosome duplication by tetraploidization in Glycine generated two loci containing four genes each. A large deletion in one of the two four-gene loci in soybean resulted in the generation of the Lbc₂ locus containing two leghemoglobin genes, one functional and another pseudo (LbΨ₂). This pseudogene, unlike that present on the main locus, is represented by only two and a half exons and appears to be truncated. The two other truncated genes (LbT₁ and LbT₂) were probably generated similarly in the genome of Glycine spp. following tetraploidization before the divergence of G. max and G. soja.