An insight into the molecular basis of salt tolerance of L-myo-inositol 1-P synthase (PcINO1) from Porteresia coarctata (Roxb.) Tateoka, a halophytic wild rice

The molecular basis of salt tolerance of L-myo-inositol 1-P synthase (MIPS; EC 5.5.1.4) from Porteresia coarctata (Roxb.) Tateoka (PcINO1, AF412340) earlier reported from this laboratory, has been analyzed by in vitro mutant and hybrid generation and subsequent biochemical and biophysical studies of the recombinant proteins. A 37-amino acid stretch between Trp-174 and Ser-210 has been confirmed as the salt-tolerance determinant domain in PcINO1 both by loss or gain of salt tolerance by either deletion or by addition to salt-sensitive MIPS(s) of Oryza (OsINO1) and Brassica juncea (BjINO1). This was further verified by growth analysis under salt environment of Schizosaccharomyces pombe transformed with the various gene constructs and studies on the differential behavior of mutant and wild proteins by Trp fluorescence, aggregation, and circular dichroism spectra in the presence of salt. 4,4'-Dianilino-1,1'-binaphthyl-5,5-disulfonic acid binding experiments revealed a lower hydrophobic surface on PcINO1 than OsINO1, contributed by this 37-amino acid stretch explaining the differential behavior of OsINO1 and PcINO1 both with respect to their enzymatic functions and thermodynamic stability in high salt environment. Detailed amino acid sequence comparison and modeling studies revealed the interposition of polar and charged residues and a well-connected hydrogen-bonding network formed by Ser and Thr in this stretch of PcINO1. On the contrary, hydrophobic residues clustered in two continuous stretches in the corresponding region of OsINO1 form a strong hydrophobic patch on the surface. It is conceivable that salt-tolerant MIPS proteins may be designed out of the salt-sensitive plant MIPS proteins by replacement of the corresponding amino acid stretch by the designated 37-amino acid stretch of PcINO1.     

A novel salt-tolerant L-myo-inositol-1-phosphate synthase from Porteresia coarctata (Roxb.) Tateoka, a halophytic wild rice: molecular cloning, bacterial overexpression, characterization, and functional introgression into tobacco-conferring salt tolerance phenotype

l-myo-Inositol-1-phosphate synthase (EC 5.5.1.4, MIPS), an evolutionarily conserved enzyme protein, catalyzes the synthesis of inositol, which is implicated in a number of metabolic reactions in the biological kingdom. Here we report on the isolation of the gene (PINO1) for a novel salt-tolerant MIPS from the wild halophytic rice, Porteresia coarctata (Roxb.) Tateoka. Identity of the PINO1 gene was confirmed by functional complementation in a yeast inositol auxotrophic strain. Comparison of the nucleotide and deduced amino acid sequences of PINO1 with that of the homologous gene from Oryza sativa L. (RINO1) revealed distinct differences in a stretch of 37 amino acids, between amino acids 174 and 210. Purified bacterially expressed PINO1 protein demonstrated a salt-tolerant character in vitro compared with the salt-sensitive RINO1 protein as with those purified from the native source or an expressed salt-sensitive mutant PINO1 protein wherein amino acids 174-210 have been deleted. Analysis of the salt effect on oligomerization and tryptophan fluorescence of the RINO1 and PINO1 proteins revealed that the structure of PINO1 protein is stable toward salt environment. Furthermore, introgression of PINO1 rendered transgenic tobacco plants capable of growth in 200-300 mm NaCl with retention of approximately 40-80% of the photosynthetic competence with concomitant increased inositol production compared with unstressed control. MIPS protein isolated from PINO1 transgenics showed salt-tolerant property in vitro confirming functional expression in planta of the PINO1 gene. To our knowledge, this is the first report of a salt-tolerant MIPS from any source.     

Salinity-induced enhancement of L-myo-inositol 1-phosphate synthase in rice (Oryza sativa L.)

The salt-tolerant varieties of rice (Oryza sativa L.) exhibit enhanced activity of the chloroplast form of L-myo-inositol 1-phosphate synthase (EC 5.5.4.1) under NaCl treatment either during the seedling stage or in fully grown plants during field growth. The salt-induced enhancement was noticeable only in chloroplasts from light-grown plants. The effects of these treatments on the cytosolic inositol synthase activity were less pronounced. While the effect of salt on the activity of the two forms was marginal in the salt-sensitive varieties during seedling growth, salinity affected the chloroplast inositol synthase activity adversely in these varieties during growth of the plants under field conditions. The salt-enhanced activities of inositol synthase(s) in the highly salt-tolerant varieties studied were found to be comparable to that observed in Porteresia coarctata, a halophytic wild rice species. The implications of these findings, which suggest a role of the inositol pathway in osmoregulation, are discussed.     

Mapping of QTLs associated with cytosolic glutamine synthetase and NADH-glutamate synthase in rice (Oryza sativa L.).

Ninety-eight backcross inbred lines (BC1F6) developed between Nipponbare, a japonica rice, and Kasalath, an indica rice were employed to detect putative quantitative trait loci (QTLs) associated with the contents of cytosolic glutamine synthetase (GS1; EC 6.3.1.2) and NADH-glutamate synthase (NADH-GOGAT; EC 1.4.1.14) in leaves. Immunoblotting analyses showed transgressive segregations toward lower or greater contents of these enzyme proteins in these backcross inbred lines. Seven chromosomal QTL regions for GS1 protein content and six for NADH-GOGAT protein content were detected. Some of these QTLs were located in QTL regions for various biochemical and physiological traits affected by nitrogen recycling. These findings suggested that the variation in GS1 and NADH-GOGAT protein contents in this population is related to the changes in the rate of nitrogen recycling from senescing organs to developing organs, leading to changes in these physiological traits. Furthermore, a structural gene for GS1 was mapped between two RFLP markers, C560 and C1408, on chromosome 2 and co-located in the QTL region for one-spikelet weight. A QTL region for NADH-GOGAT protein content was detected at the position mapped for the NADH-GOGAT structural gene on chromosome 1. A QTL region for soluble protein content in developing leaves was also detected in this region. Although fine mapping is required to identify individual genes in the future, QTL analysis could be a useful post-genomic tool to study the gene functions for regulation of nitrogen recycling in rice.     

Introgression of a novel salt-tolerant L-myo-inositol 1-phosphate synthase from Porteresia coarctata (Roxb.) Tateoka (PcINO1) confers salt tolerance to evolutionary diverse organisms

We have previously demonstrated that introgression of PcINO1 gene from Porteresia coarctata (Roxb.) Tateoka, coding for a novel salt-tolerant L-myo-inositol 1-phosphate synthase (MIPS) protein, confers salt tolerance to transgenic tobacco plants (Majee, M., Maitra, S., Dastidar, K.G., Pattnaik, S., Chatterjee, A., Hait, N.C., Das, K.P. and Majumder, A.L. (2004) A novel salt-tolerant L-myo-inositol-1-phosphate synthase from Porteresia coarctata (Roxb.) Tateoka, a halophytic wild rice: molecular cloning, bacterial overexpression, characterization, and functional introgression into tobacco-conferring salt-tolerance phenotype. J. Biol. Chem. 279, 28539-28552). In this communication we have shown that functional introgression of the PcINO1 gene confers salt-tolerance to evolutionary diverse organisms from prokaryotes to eukaryotes including crop plants albeit to a variable extent. A direct correlation between unabated increased synthesis of inositol under salinity stress by the PcINO1 gene product and salt tolerance has been demonstrated for all the systems pointing towards the universality of the application across evolutionary divergent taxa.     

Identification, cDNA cloning, and gene expression of soluble starch synthase in rice (Oryza sativa L.) immature seeds.

Three forms of soluble starch synthase were resolved by anion-exchange chromatography of soluble extracts from immature rice (Oryza sativa L.) seeds, and each of these forms was further purified by affinity chromatograph. The 55-, 57-, and 57-kD proteins in the three preparations were identified as candidates for soluble starch synthase by western blot analysis using an antiserum against rice granule-bound starch synthase. It is interesting that the amino-terminal amino acid sequence was identical among the three proteins, except that the 55-kD protein lacked eight amino acids at the amino terminus. Thus, these three proteins are products of the same gene. The cDNA clones coding for this protein have been isolated from an immature rice seed library in lambda gt11 using synthetic oligonucleotides as probes. The deduced amino acid sequence of this protein contains a lysine-X-glycine-glycine consensus sequence for the ADP-glucose-binding site of starch and glycogen synthases. Therefore, we conclude that this protein corresponds to a form of soluble starch synthase in immature rice seeds. The precursor of the enzyme contains 626 amino acids, including a 113-residue transit peptide at the amino terminus. The mature form of soluble starch synthase shares a significant but low sequence identity with rice granule-bound starch synthase and Escherichia coli glycogen synthase. However, several regions, including the substrate-binding site, are highly conserved among these three enzymes. Blot hybridization analysis demonstrates that the gene encoding soluble starch synthase is a single-copy gene in the rice genome and is expressed in both leaves and immature seeds. These results suggest that soluble and granule-bound starch synthases play distinct roles in starch biosynthesis of plant.     

水稻黄叶少分蘖突变体yllt1的鉴定及基因定位

采用⁶⁰Co-γ射线诱变籼稻（Oryza sativa subsp.indica）保持系‘T98B’获得一份兼具黄叶和少分蘖表型的突变体yllt1（yellow leaf and less tillering 1）,利用色素含量测定、构建显隐性混池和基因表达量测定等方法从表型和遗传层面对其遗传特征进行分析。结果显示：yllt1苗期叶绿素a和叶绿素b含量为野生型水稻品种‘T98B’的77.78%和60.00%,叶绿体发育异常,缺乏功能性叶绿体类囊体片层;其分蘖盛期的单株分蘖数为野生型的21.43%。遗传分析发现,在突变体yllt1与‘T98B’的杂交F₂群体中,黄叶与少分蘖性状的重组率为0.00%,表明yllt1同时控制叶色与分蘖表型;yllt1呈隐性遗传,受一个细胞核基因独立控制。该研究进一步采用连锁分析法将yllt1精细定位到第11染色体上,经测序分析推断发生了突变的登录号为LOC_Os11g05552的基因是yllt1的目的基因;该基因编码叶绿体前体信号识别颗粒54 kD（cpSRP54）蛋白,其第1外显子的第29位碱基C发生了缺失,将造成其蛋白产物从N-端至C-端氨基酸组成的严重破坏。RT-qPCR分析结果显示,yllt1叶中叶绿素合成基因OsCAO1、OsCAO2与OsNOL等的表达量明显下调;茎中分蘖正向调控基因OsTAC1受到显著抑制,而负调控基因OsTB1与OsDLT的表达量明显增强。研究结果表明cpSRP54同时参与了水稻叶色和分蘖的调控。     

Identification of neutral genes at pollen sterility loci Sd and Se of cultivated rice (Oryza sativa) with wild rice (O. rufipogon) origin

Pollen sterility is one of the main hindrances against the utilization of strong intersubspecific (indica-japonica) heterosis in rice. We looked for neutral alleles at known pollen sterility loci Sd and Se that could overcome this pollen sterility characteristic. Taichung 65, a typical japonica cultivar, and its near isogenic lines E7 and E8 for pollen sterility loci Sd and Se were employed as tester lines for crossing with 13 accessions of wild rice (O. rufipogon). Pollen fertility and genotypic segregations of the molecular markers tightly linked with Sd and Se loci were analyzed in the paired F(1)s and F(2) populations. One accession of wild rice (GZW054) had high pollen fertility in the paired F(1)s between Taichung 65 and E7 or E8. Genotypic segregations of the molecular markers tightly linked with Sd and Se loci fit the expected Mendelian ratio (1:2:1), and non-significances were shown among the mean pollen fertilities with the maternal, parental, and heterozygous genotypes of each molecular markers tightly linked with Sd and Se loci. Evidentially, it indicated that the alleles of Sd and Se loci for GZW054 did not interact with those of Taichung 65 and its near isogenic lines, and, thus were identified as neutral alleles Sd(n) and Se(n). These neutral genes could become important germplasm resources for overcoming pollen sterility in indica-japonica hybrids, making utilization of strong heterosis in such hybrids viable.     

Distinguishing rice (Oryza sativa Poaceae) from wild Oryza species through Phytolith analysis: Results of preliminary research

Asian rice is an important grain, not only in its homeland but in many areas of the world. Identifying rice in the archaeological record is a challenge, especially in the moist tropics, where organic materials preserve only when charred. Phytolith analysis, the identification of opaline silica bodies, provides an alternative method for identifying this important crop. Results of our research suggest thatOryza contributes phytoliths that are genus-specific, that bulliform characteristics alone do not permit separation of wild and domesticatedOryza in regions where species overlap, and that a number of phytolith types, especially silicified glumes, show promise for separating wild from domesticated forms. With further research it should be possible to identify rice through its phytolith assemblage in archaeological soils in the heartland of its domestication and use.     

Gene flow from cultivated rice (Oryza sativa) to its weedy and wild relatives

BACKGROUND AND AIMS: Transgene escape through gene flow from genetically modified (GM) crops to their wild relative species may potentially cause environmental biosafety problems. The aim of this study was to assess the extent of gene flow between cultivated rice and two of its close relatives under field conditions. METHODS: Experiments were conducted at two sites in Korea and China to determine gene flow from cultivated rice (Oryza sativa L.) to weedy rice (O. sativa f. spontanea) and common wild rice (O. rufipogon Griff.), respectively, under special field conditions mimicking the natural occurrence of the wild relatives in Asia. Herbicide resistance (bar) and SSR molecular finger printing were used as markers to accurately determine gene flow frequencies from cultivated rice varieties to their wild relatives. KEY RESULTS: Gene flow frequency from cultivated rice was detected as between approx. 0.011 and 0.046 % to weedy rice and between approx. 1.21 and 2.19 % to wild rice under the field conditions. CONCLUSIONS: Gene flow occurs with a noticeable frequency from cultivated rice to its weedy and wild relatives, and this might cause potential ecological consequences. It is recommended that isolation zones should be established with sufficient distances between GM rice varieties and wild rice populations to avoid potential outcrosses. Also, GM rice should not be released when it has inserted genes that can significantly enhance the ecological fitness of weedy rice in regions where weedy rice is already abundant and causing great problems.     

NAD+ mediated differential thermotolerance between chloroplastic and cytosolic L-myo-inositol-1-phosphate synthase from Diplopterygium glaucum (Thunb.) Nakai

Relative thermotolerance of the enzyme, L-myo-inositol-1-phosphate synthase (MIPS; EC: 5.5.1.4), from the chloroplastic and cytosolic sources of Diplopterygium glaucum was studied. The purification involved streptomycin sulphate precipitation, ammonium sulphate fractionation, ion-exchange chromatography, and molecular sieve chromatography. After the final chromatography, 16.62% of chloroplastic and 13.47% of cytosolic MIPS could be recovered. Between 15 degrees C and 55 degrees C, the two forms of MIPS exhibited differential thermal stability, which is related to the presence of the MIPS co-factor, NAD+. Added NAD+ increased the lower thermotolerance of the chloroplastic MIPS and the removal of 'built-in' NAD+ decreased the higher thermal stability of the cytosolic MIPS.     

Transfer of bacterial blight and blast resistance from the tetraploid wild rice Oryza minuta to cultivated rice,Oryza sativa

Summary Oryza minuta J. S. Presl ex C. B. Presl is a tetraploid wild rice with resistance to several insects and diseases, including blast (caused by Pyricularia grisea) and bacterial blight (caused by Xanthomonas oryzae pv. oryzae). To transfer resistance from the wild species into the genome of cultivated rice (Oryza sativa L.), backcross progeny (BC₁, BC₂, and BC₃) were produced from interspecific hybrids of O. sativa cv IR31917-45-3-2 (2n=24, AA genome) and O. minuta Acc. 101141 (2n=48, BBCC genomes) by backcrossing to the O. sativa parent followed by embryo rescue. The chromosome numbers ranged from 44 to 47 in the BC₁ progeny and from 24 to 37 in the BC₂ progeny. All F₁ hybrids were resistant to both blast and bacterial blight. One BC₁ plant was moderately susceptible to blast while the rest were resistant. Thirteen of the 16 BC₂ progeny tested were resistant to blast; 1 blast-resistant BC₂, plant 75-1, had 24 chromosomes. A 3 resistant: 1 susceptible segregation ratio, consistent with the action of a major, dominant gene, was observed in the BC₂F₂ and BC₂F₃ generations. Five of the BC₁ plants tested were resistant to bacterial blight. Ten of the 21 BC₂ progeny tested were resistant to Philippine races 2, 3, and 6 of the bacterial blight pathogen. One resistant BC₂, plant 78-1, had 24 chromosomes. The segregation of reactions of the BC₂F₂, BC₂F₃, and BC₂F₄ progenies of plant 78-1 suggested that the same or closely linked gene(s) conferred resistance to races 2, 3, 5, and 6 of the bacterial blight pathogen from the Philippines.     

Comparative RFLP mapping of an allotetraploid wild rice species (Oryza latifolia) and cultivated rice (O. sativa)

The purpose of this study was to construct a comparative RFLP map of an allotetraploid wild rice species, Oryza latifolia, and to study the relationship between the CCDD genome of O. latifolia and the AA genome of O. sativa. A set of RFLP markers, which had been previously mapped to the AA genome of cultivated rice, were used to construct the comparative map. Fifty-eight F₂ progeny, which were derived from a single F₁ plant, were used for segregation analysis. The comparative RFLP map contains 149 DNA markers, including 145 genomic DNA markers from cultivated rice, 3 cDNA markers from oat, and one known gene (waxy, from maize). Segregation patterns reflected the allotetraploid ancestry of O. latifolia, and the CC and DD genomes were readily distinguished by most probes tested. There is a high degree of conservation between the CCDD genome of O. latifolia and the AA genome of O. sativa based on our data, but some inversions and translocations were noted.     

Genetic differentiation among Sri Lankan traditional rice (Oryza sativa) varieties and wild rice species by AFLP markers

Traditional rice varieties are one important component of the biodiversity of Sri Lanka. However, no proper studies have been performed on genetic diversity of the Sri Lankan traditional rice varieties used in our breeding programs. In the present study, the genetic diversity of 46 traditional rice (Oryza sativa) varieties and 5 wild rice species is assesed using Amplified Fragment Length Polymorphism (AFLP) markers. Ten primer combinations generated a total of 784 fragments. Of these, 772 fragments were polymorphic (98.4%). UPGMA analysis based on Jaccard's similarity separated the accessions into four major clusters. A cophenetic correlation with r=0.786 strongly supported the clustering pattern of UPGMA dendrogram. A principal coordinate analysis (PCoA) also confirmed the UPGMA clusters. Accessions referred to the same cluster showed similar morphological characteristics (e.g. height, grain colour etc.) while accessions which are known to be morphologically distinct appeared genetically separated. In addition, the clustering pattern distinctly separated lowland and upland rice varieties. This genetic diversity assessment at the molecular level provides reliable information for selection of germplasm in the development of new rice varieties and in conservation of traditional rice genetic resources.