Analysis of sialyltransferase-like proteins from Oryza sativa

Sialic acids are widely distributed among living creatures, from bacteria to mammals, but it has been commonly accepted that they do not exist in plants. However, with the progress of genome analyses, putative gene homologs of animal sialyltransferases have been detected in the genome of some plants. In this study, we cloned three genes from Oryza sativa (Japanese rice) that encode sialyltransferase-like proteins, designated OsSTLP1, 2, and 3, and analyzed the enzymatic activity of the proteins. OsSTLP1, 2, and 3 consist of 393, 396, and 384 amino acids, respectively, and each contains sequences similar to the sialyl motifs that are highly conserved among animal sialyltransferases. The recombinant soluble forms of OsSTLPs produced by COS-7 cells were analyzed for sialyltransferase-like activity. OsSTLP1 exhibited such activity toward the oligosaccharide Galbeta1,4GlcNAc and such glycoproteins as asialofetuin, alpha1-acid glycoprotein, and asialo-alpha1-acid glycoprotein; OsSTLP3 exhibited similar activity toward asialofetuin; and OsSTLP2 exhibited no sialyltransferase-like activity. The sialic acid transferred by OsSTLP1 or 3 was linked to galactose of Galbeta1,4GlcNAc through alpha2,6-linkage. This is the first report of plant proteins having sialyltransferase-like activity.     

DIPOS: database of interacting proteins in Oryza sativa

Rice is an important crop throughout the world and is the staple food for about half the world's population. For better breeding and improved production, we need to know the function of rice molecules which facilitate their function through interactions with each other. The database of interacting proteins in Oryza sativa (DIPOS) provides comprehensive information of interacting proteins in rice, where the interactions are predicted using two computational methods, i.e., interologs and domain based methods. DIPOS contains 14?614?067 pairwise interactions among 27?746 proteins, covering about 41% of the whole Oryaza sativa proteome. Furthermore, each interaction is assigned a confidence score which further enables biologists to sort out the important proteins. Biological explanations of pathways and interactions are also provided based on the database. Public access to the DIPOS is available at and .     

Genome Characterization of a Breeding Line Derived from a Cross Between Oryza sativa and Oryza rufipogon

A preliminary screening was conducted on BC3F1 and BC4F1 backcross families developed from crossing Oryza sativa (MR219) and O. rufipogon (IRGC105491). Despite earlier results showing that O. rufipogon alleles (wild introgression) contributed to both number of panicles (qPPL-2) and tillers (qTPL-2) at loci RM250, RM208, and RM48 in line A20 of the BC2F2 population, we observed that wild introgression was lost at loci RM250 and RM208 but retained at locus RM48 in BC3F1 and BC4F1. Progeny tests conducted utilizing genotype and phenotype data on both BC4F1 and a reference population, BC2F7 (A20 line), did not show significant differences between groups having the MR219 allele and wild introgression at locus RM48. This suggests that there is no additive and transgressive effect of wild introgression in the BC3F1 and BC4F1 generated. The presence of wild introgression was largely due to gene contamination by cross-pollination during field breeding practices.     

Identification of a set of RFLP probes for subspecies differentiation in Oryza sativa L.

Sixty-eight indica-japonica tester-differentiating RFLP probes were tested in seven indica and seven japonica varieties of rice (Oryza sativa L.) with four enzyme digestions (EcoRI, EcoRV, HindIII and DraI). Twenty-one DNA clones were isolated as indica-japonica subspecies-differentiating probes. A set of 13 probes was established as core probes for subspecies differentiation and a pooled blotting analysis was carried out to facilitate the application of RFLP in rice genetics and breeding practice. A dendrogram of 12 wide-compatibility varieties was constructed based on RFLPs detected by 13 core probes with single enzyme digestions. It was speculated that most RFLPs of indica-japonica differentiating probes were generated by insertions/deletions, which may be of great significance for the origin and differentiation of subspecies in Oryza sativa L.     

Characterization of interspecific hybrids and backcross progenies from a cross between Oryza minuta and Oryza sativa

Oryza minuta, a tetraploid wild relative of cultivated rice, is an important source for the genetic improvement. Interspecific hybrids were obtained from the cross of O. sativa L. (IR24) and O. minuta (Acc. No. 101133) with 5.58% crossability, which ranged from 0.11% to 1.62% in the backcross generations. The chromosome numbers of the backcross progenies were 24 to 48. Seven yield-related traits of the parents, hybrid F₁, and backcross progenies were evaluated. Simple sequence repeat markers analysis showed that the polymorphism ratio of SSR bands between IR24 and Acc. No. 101133 was 93.2%. The average donor segment number, length, donor genome size, and percentage of donor genome of 92 BC₃F₁ plants (2n=24) were 24.1, 17.8 cM, 438.4 cM and 26.2%, respectively. They were complex variation and uneven among the chromosomes. These introgression lines could be used to identify the favorable genes of O. minuta and provide a new platform for the genetic improvement of cultivated rice.     

Characterization of uridine-diphosphate dependent flavonoid glucosyltransferase from Oryza sativa

We cloned a uridine-diphosphate dependent glycosyltransferase RUGT-10 from Oryza sativa. The recombinant enzyme was expressed by glutathione-S transferase gene fusion system in Escherichia coli. RUGT10 showed different regioselectivity depending on the structures of substrates (e.g. flavanone, flavonol, and flavone). Apparently, flavanone such as naringenin and eriodictyol gave one 7-O-glucoside while flavone and flavonol gave more than two products with preferential glucosylation position of hydroxyl group at C-3 position.     

Characterization and mapping of a spotted leaf mutant in rice (Oryza sativa)

Spotted leaf mutant belongs to a class of mutants that can produce necrotic lesions spontaneously in plants without any attack by pathogens. These mutants have no beneficial effect on plant productivity but provide a unique opportunity to study programmed cell death in plant defense responses. A novel rice spotted leaf mutant (spl30) was isolated through low-energy heavy ion irradiation. Lesion expression was sensitive to light and humidity. The spl30 mutant caused a decrease in chlorophyll and soluble protein content, with marked accumulation of reactive oxygen species (ROS) around the lesions. In addition, the spl30 mutant significantly enhanced resistance to rice bacterial blight (X. oryzae pv. oryzae) from China (C1–C7). The use of SSR markers showed that the spl30 gene was located between markers XSN2 and XSN4. The genetic distance between the spl30 gene and XSN2 and between spl30 and XSN4 was 1.7 cM and 0.2 cM, respectively. The spl30 gene is a new gene involved in lesion production and may be related to programmed cell death in rice. The ability of this mutant to confer broad resistance to bacterial blight provides a model for studying the interaction between plants and pathogenic bacteria.     

PIF4, a phytochrome-interacting bHLH factor,functions as a negative regulator of phytochrome B signaling in Arabidopsis

Plants sense and respond to red and far-red light using the phytochrome (phy) family of photoreceptors. However, the mechanism of light signal transduction is not well defined. Here, we report the identification of a new mutant Arabidopsis locus, srl2 (short under red-light 2), which confers selective hypersensitivity to continuous red, but not far-red, light. This hypersensitivity is eliminated in srl2phyB, but not srl2phyA, double mutants, indicating that this locus functions selectively and negatively in phyB signaling. The SRL2 gene encodes a bHLH factor, designated PIF4 (phytochrome-interacting factor 4), which binds selectively to the biologically active Pfr form of phyB, but has little affinity for phyA. Despite its hypersensitive morphological phenotype, the srl2 mutant displays no perturbation of light-induced expression of marker genes for chloroplast development. These data suggest that PIF4 may function specifically in a branch of the phyB signaling network that regulates a subset of genes involved in cell expansion. Consistent with this proposal, PIF4 localizes to the nucleus and can bind to a G-box DNA sequence motif found in various light-regulated promoters.     

Characterization of a vacuolar zinc transporter OZT1 in rice (Oryza sativa L.)

The CDF family is a ubiquitous family that has been identified in prokaryotes, eukaryotes, and archaea. Members of this family are important heavy metal transporters that transport metal ions out of the cytoplasm. In this research, a full length cDNA named Oryza sativa Zn Transporter 1 (OZT1) that closely related to rat ZnT-2 (Zn Transporter 2) gene was isolated from rice. The OZT1 encoding a CDF family protein shares 28.2 % ~ 84.3 % of identities and 49.3 % ~ 90.9 % of similarities with other zinc transporters such as RnZnT-2, HsZnT-8, RnZnT-8 and AtMTP1. OZT1 was constitutively expressed in various rice tissues. The OZT1 expression was significantly induced both in the seedlings of japonica rice Nipponbare and indica rice IR26 in response to Zn²⁺ and Cd²⁺ treatments. Besides, OZT1 expression was also increased when exposed to other excess metals, such as Cu²⁺, Fe²⁺ and Mg²⁺. Subcellular localization analysis indicated that OZT1 localized to vacuole. Heterologous expression of OZT1 in yeast increased tolerance to Zn²⁺ and Cd²⁺ stress but not the Mg²⁺ stress. Together, OZT1 is a CDF family vacuolar zinc transporter conferring tolerance to Zn²⁺ and Cd²⁺ stress, which is important to transporting and homeostasis of Zn, Cd or other heavy metals in plants.     

High-throughput functional affinity purification of mannose binding proteins from Oryza sativa

We have used affinity chromatography in combination with mass spectrometry to isolate, identify, and assign a preliminary functional annotation to a large number of both known and novel proteins from rice. Rice (Oryza sativa) leaf, root, and seed tissue extracts were fractionated by column affinity chromatography using alpha-D-mannose as the ligand. Bound fractions were eluted and subjected to one-dimensional electrophoresis, followed by high-performance liquid chromatography-tandem mass spectrometric analysis of separated proteins. This multiplexed technology resulted in the isolation and identification of 136 distinct mannose binding proteins from rice. A comparative analysis demonstrates very little overlap of identified proteins between the respective tissues, and confirms the correctly compartmentalized presence of a significant number of proteins from largely tissue-specific biochemical pathways. Over 30% of the identified proteins with a previously annotated function are directly involved in sugar metabolism, including several highly expressed known rice lectins. Direct comparison of the peptide sequences identified in this study to those peptides identified in the most comprehensive survey of the rice proteome to date indicates that our current data represents a significant enrichment of proteins unique to this dataset. Nearly 15% of the identified proteins, identified on the basis of exact peptide matching to sequences in the rice genomic database, represent proteins without a previously known functional annotation, indicating the potential of this combined chromatographic approach to assign a preliminary function to novel proteins in a high-throughput fashion.     

水稻叶片对镉胁迫响应的蛋白质差异表达

为揭示水稻镉抗性的分子机理,以抗镉水稻品种P1312777和镉敏感水稻品种IR24为材料,在镉离子浓度为0(对照)、50和100 μmol·L-1条件下水培处理7 d,应用蛋白质组学方法分析了2种水稻叶片对镉胁迫响应的蛋白质差异表达.结果表明:镉胁迫下水稻PI312777叶片中共检测到差异表达蛋白质点31个,通过MALDI-TOF/MS分析,鉴定了其中的24个蛋白质(包括20个不同蛋白质,4个重复检出蛋白质);IR24叶片中共检测到差异表达蛋白质点19个,其中15个蛋白质得到鉴定.PI312777叶片鉴定出的20个蛋白质覆盖了IR24叶片鉴定的15个蛋白质,前者有4个与光合作用相关,11个与细胞防御代谢相关,3个与其他代谢相关,2个为功能未知蛋白.与对照相比,不同浓度镉胁迫下,抗镉水稻PI312777叶片中热激蛋白、谷胱甘肽还原酶、蛋白酶体α亚基6型、果糖1,6-二磷酸醛缩酶、硫氧还蛋白和DNA重组修复蛋白均上调表达;镉敏感水稻IR24叶片中热激蛋白、谷胱甘肽还原酶、蛋白酶体α亚基6型的表达无显著差异,果糖1,6-二磷酸醛缩酶和硫氧还蛋白则下调表达.此外,DNA重组修复蛋白仅在镉胁迫的PI312777叶片中表达.水稻PI312777比IR24具有更强的镉抗性与这些差异表达的蛋白质密切相关.     

Production and characterization of a complete set of individual chromosome additions from Oryza officinalis to Oryza sativa using RFLP and GISH analyses

Monosomic alien addition lines (MAALs) are valuable materials for comparative analyses of two distinct genomes, for elucidating introgression mechanisms, and for dissecting genes controlling complex traits. In the study reported here, MAALs of rice containing the complete genome of Oryza sativa and individual chromosomes of Oryza officinalis were produced. Interspecific hybridizations were made between O. sativa L. ssp. Japonica (CV, Hejiang 19, 2 n=24, AA) and O. officinalis (Acc. HY018, 2 n=24, CC). Two backcrosses were made to the cultivated rice to obtain BC₂F₁ plants. Through RFLP and GISH analyses, 25 MAALs (2 n=25, AA+1C) were identified and divided into 12 syntenic groups, designated MAALs 1–12. MAALs 1, 2, 3, 5, 7 and 10 were each represented by one plant, MAALs 8, 11 and 12 by two plants, MAALs 6 and 9 by four plants, and MAAL 4 by five plants. An ideogram of the C-genome of O. officinalis was constructed, based on GISH analysis of the interspecific hybrid and the MAALs. Comparative RFLP maps showed strong syntenic associations between the A-genomes and C-genomes. Chromosomal arrangements such as translocations and duplications were detected in different alien chromosomes of the MAALs. The complete set of O. officinalis MAALs generated here provides a novel manipulation platform for exploiting and utilizing the O. officinalis genome and carrying out genetic studies.     

Proteomic identification of small, copper-responsive proteins in germinating embryos of Oryza sativa

Background and Aims

Although copper (Cu) is an essential micronutrient for plants and algae, excess Cu is toxic to most plants and can cause a wide range of deleterious effects. To investigate the response of rice (Oryza sativa) to Cu stress, a proteomic approach was used to analyse Cu stress-induced changes in the expression of low molecular-weight proteins in germinating rice seed embryos.

Methods

Rice seeds were germinated in the presence or absence of 200 µm Cu for 6 d, and embryos, including newly formed shoots and radicles, were isolated. After proteins were extracted from the germinating embryos and separated by two-dimensional PAGE, 16 proteins in the 6- to 25-kDa range were identified using MALDI-TOF mass spectrometry.

Key Results and Conclusions

Thirteen of the proteins identified, including metallothionein-like protein, membrane-associated protein-like protein, putative wall-associated protein kinase, pathogenesis-related proteins and the putative small GTP-binding protein Rab2, were up-regulated by Cu stress. Three proteins, a putative small cytochrome P450 (CYP90D2), a putative thioredoxin and a putative GTPase, were down-regulated by Cu stress. As far as is known, this study provides the first proteomic evidence that metallothionein and CYP90D2 are Cu-responsive proteins in plants. These findings may lead to a better understanding of plant molecular responses to toxic metal exposure.Key words: Copper, metallothionein, rice, Oryza sativa, proteomics, seed germination     

Intra and inter subspecific variation in soluble proteins of Oryza sativa L.

Summary Types representing three subspecies of Oryza sativa, namely, indica, japonica and javanica, and a group of intermediate types collected from North East India, were studied for variation in soluble proteins using acrylamide gel electrophoresis. The study revealed that there was a marked variation within and between varietal groups. Variability for number and intensity of protein bands in indica was wider than in japonica and javanica. Protein pattern in the group comprising N.E. Indian types transgressed that of all three established groups by displaying a wide spectrum. Relative homology, as measured from percentage similarities of N.E. Indian types to the three subspecies, suggested the existence of six different groups. Comparison of varietal groups for the protein mobility pattern showed that japonica and javanica varieties tended to show higher percentages of slow mobility proteins than indica. It appears, from the narrow variability and relatively low percentage of slow mobility proteins, that the japonica and javanica races are of later origin compared to indica. However, the study with a limited number of types suggested the monophyletic origin of varietal groups from an indica-like base predominantly found in the secondary centres of origin of O. sativa.