Cloning of a putative monogalactosyldiacylglycerol synthase gene from rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) plants and its expression in response to submergence and other stresses

Suppression subtractive hybridization was used to construct a subtractive cDNA library from plants of non-submerged and 7-day-submerged rice (Oryza sativa L., FR13A, a submergence-tolerant cultivar). One clone of the subtractive cDNA library, S23, was expressed abundantly during submergence. The full length of S23 was amplified using 5- and 3-rapid amplification of cDNA ends, and found to consist of 1,671 bp with an open reading frame of 1,077 bp (181–1257) encoding 358 amino acids. Its deduced amino acid sequence showed a high homology with monogalactosyldiacylglycerol synthase (UDPgalactose: 1,2-diacylglycerol 3--d-galactosyl transferase; EC 2.4.1.46, MGDG synthase) from Arabidopsis thaliana; therefore, we named the gene OsMGD. Time-course studies showed that the expression of OsMGD in the rice cultivars FR13A and IR42 (submergence-susceptive cultivar) during submergence was gradually increased and that expression in FR13A was higher than in IR42. The expression of OsMGD in FR13A was influenced by benzyladenine and illumination. The accumulation of OsMGD mRNA in both FR13A and IR42 was also increased by ethephon, gibberellin, drought and salt treatment, but cold stress had no effect on the expression of the gene. These results suggest that the expression of OsMGD mRNA requires benzyladenine or illumination, and that the process is also mediated by ethephon and gibberellin. Salt and drought stress have an effect similar to that of submergence. Furthermore, the enhanced expression of OsMGD may relate to photosynthesis, and play an important role during submergence.Abbreviations BA N⁶-Benzyladenine - GA Gibberellin - MGD Monogalactosyldiacylglycerol synthase - RACE Rapid amplification of cDNA ends     

Molecular cloning and characterization of a novel SNAP25-type protein gene <Emphasis Type="Italic">OsSNAP32</Emphasis> in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The SNAP25-type proteins belong to the superfamily of the SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), and function as important components of the vesical trafficking machinery in eukaryotic cells. In this paper, we report the cloning and expression characterization of OsSNAP32 gene, and the subcellular localization of its encoded protein. The OsSNAP32 gene contains five exons and four introns, and is located between RFLP markers C12276S and S1917 on chromosome 2 in rice. The OsSNAP32 has a molecular weight of 31.3 kD, comprises 283 amino acid residues, and contains Qb-SNARE and Qc-SNARE domains in the N- and C-terminal, respectively. Multiple sequence alignment of the SNARE domains indicates that OsSNAP32 protein is homologous to HvSNAP34 and HvSNAP28 (63% and 55% of amino acid identity respectively) from barley. The transient expression method in onion epidermal cells, revealed that OsSNAP32 is located in the plasma membrane, like other SNAP25-type proteins. Semi-quantitative RT-PCR assay showed that the OsSNAP32 is highly expressed in leaves and culms, and low in roots of rice, while hardly detected in immature spikes and flowering spikes. The expression of OsSNAP32 was significantly activated in rice seedlings treated with H2O2, PEG6000, and low temperature or after inoculation with rice blast (Magnaporthe grisea strain Hoku 1). The results suggest that this gene belongs to a novel member of this gene family encoding SNAP25-type proteins, involved in the rice responses to biotic and abiotic stresses.     

A cytosolic NADP-malic enzyme gene from rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) confers salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

NADP-malic enzyme (NADP-ME, EC 1.1.1.40) functions in many different pathways in plant and may be involved in plant defense such as wound and UV-B radiation. Here, expression of the gene encoding cytosolic NADP-ME (cytoNADP-ME, GenBank Accession No. AY444338) in rice (Oryza sativa L.) seedlings was induced by salt stress (NaCl). NADP-ME activities in leaves and roots of rice also increased in response to NaCl. Transgenic Arabidopsis plants over-expressing rice cytoNADP-ME had a greater salt tolerance at the seedling stage than wild-type plants in MS medium-supplemented with different levels of NaCl. Cytosolic NADPH/NADP⁺ concentration ratio of transgenic plants was higher than those of wild-type plants. These results suggest that rice cytoNADP-ME confers salt tolerance in transgenic Arabidopsis seedlings.     

A practical vector for efficient knockdown of gene expression in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

In the last decade, RNA interferences (RNAi) has proven to be an effective strategy to knock out homologous genes in a wide range of species. Based on its principle, a new generation of vectors containing an inverted target sequence separated by an intron as a loop, developing simplifications to the procedure of RNAi construction are required to improve the efficiency of gene inactivation techniques. Here, a novel polymerase chain reaction (PCR)—based RNAi vector pTCK303 with a maize ubiquitin promoter, 2 specific multiple enzyme sites, and a rice intron was constructed for monocot gene silencing. With this vector, only 1 PCR product amplified by a single pair of primers and 2 ligation reactions were needed to create an RNAi construct, which shortened the time span before being transformed into the plant. To test the efficiency of vector pTCK303, a rice geneOsGAS1 was used, and its RNAi construct was introduced into rice calli. Southern blot analysis of the transgenic rice confirmed the presence of theOsGAS1 RNAi structure. The decrease inOsGAS1 level in the transgenic rice was detected by Northern blot probed with anOsGAS1-specific sequence. Moreover, the rate of inhibition of the RNA expression level in RNAi transgenic rice was approximately 85% according to our real-time PCR. Therefore, the RNAi vector pTCK303 based on the homology-dependent gene-silencing mechanisms facilitated the inhibition of endogenous genes in a monocot and was proven to be a practical and efficient platform for silencing a rice gene. These authors contributed equally to this work.     

Expression of a carbonic anhydrase gene is induced by environmental stresses in Rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Expression of the gene (OsCA1) coding for carbonic anhydrase (CA) in leaves and roots of rice was induced by environmental stresses from salts (NaCl, NaHCO₃ and Na₂CO₃), and osmotic stress (10%, w/v, PEG 6000). CA activity of rice seedlings more than doubled under some of these stresses. Transgenic Arabidopsis over-expressing OsCA1 had a greater salt tolerance at the seedling stage than wild-type plants in 1/2 MS medium with 5 mM NaHCO₃, 50 mM NaCl, on 100 mM NaCl. Thus CA expression responds to environmental stresses and is related to stress tolerance in rice.     

Regeneration of transgenic plants from two indica rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) cultivars using shoot apex explants

We have established a reproducible procedure for transformation of shoot apices and regeneration of transgenic plants for two indica rice cultivars, white ponni (WP) and Pusa Basmathi 1 (PB 1). Four-day-old shoot apex explants were transformed by cocultivation with Agrobacterium tumefaciens strain EHA 101 harbouring a binary plasmid pRIT1. The vector contained an improved hygromycin phosphotransferase (hpt) gene for hygromycin resistance driven by actin 1 promoter and the reporter gene beta-glucuronidase intron (INT-GUS) controlled by CaMV 35S promoter. Rice shoots were induced on media containing 0.1 mg/l napthalene acetic acid (NAA), 1.0 mg/l kinetin (kn), 1.0 mg/l N(6)-benzyleaminopurin (BAP), 300 mg/l casaminoacid, 500 mg/l proline, 50 mg/l hygromycin and 500 mg/l cefotaxime. Transgenic plants were raised in pots and seeds were collected. Histochemical and polymerase chain reaction (PCR) analyses of field established transgenic rice plants and their offsprings confirmed the presence of GUS gene. Integration of T-DNA into the genome of putative transgenics was further confirmed by southern analysis. The transformation efficiency of WP was found to be ranging from 5.6 to 6.2% whereas in the case of PB1, it was from 7 to 8%. Progeny analysis of these plants showed a pattern of classical Mendelian inheritance for both hpt and GUS gene.     

Molecular characterization the <Emphasis Type="Italic">YABBY</Emphasis> gene family in <Emphasis Type="Italic">Oryza sativa</Emphasis> and expression analysis of <Emphasis Type="Italic">OsYABBY1</Emphasis>

Members of the YABBY gene family have a general role that promotes abaxial cell fate in a model eudicot, Arabidopsis thaliana. To understand the function of YABBY genes in monocots, we have isolated all YABBY genes in Oryza sativa (rice), and revealed the spatial and temporal expression pattern of one of these genes, OsYABBY1. In rice, eight YABBY genes constitute a small gene family and are classified into four groups according to sequence similarity, exon-intron structure, and organ-specific expression patterns. OsYABBY1 shows unique spatial expression patterns that have not previously been reported for other YABBY genes, so far. OsYABBY1 is expressed in putative precursor cells of both the mestome sheath in the large vascular bundle and the abaxial sclerenchyma in the leaves. In the flower, OsYABBY1 is specifically expressed in the palea and lemma from their inception, and is confined to several cell layers of these organs in the later developmental stages. The OsYABBY1-expressing domains are closely associated with cells that subsequently differentiate into sclerenchymatous cells. These findings suggest that the function of OsYABBY1 is involved in regulating the differentiation of a few specific cell types and is unrelated to polar regulation of lateral organ development.     

Identification and characterization of differentially expressed ESTs of <Emphasis Type="Italic">Gossypium barbadense</Emphasis> infected by <Emphasis Type="Italic">Verticillium dahliae</Emphasis> with suppression subtractive hybridization

The wilt defense reaction of cotton is a complicated continuous process and involves a battery of genes. In this study, we adopted the suppression subtractive hybridization (SSH) technique to isolate differentially expressed ESTs from Gossypium barbadense variety 7124 during the Verticillium wilt defense process. An array of 1165 clones from the subtractive library has been screened with reverse northern blotting, of which 131 ESTs were considered as overexpressed and 16 ESTs were downregulated. Sequence analysis and blast search showed that 83 ESTs were homologous to 45 unique sequences in the databases. Among all these differentially expressed ESTs, at least three kinds of genes were characterized. The majority of ESTs with a deduced identity as aerobic metabolism enzymes were strongly expressed in the infection process. Likewise, ESTs similar to those reported for pathogen-related protein genes were also picked out in this study. These ESTs, in combination with other kinase-like genes and a defensin-like EST, constituted an assembly of genes which responded during pathogenic infection. These results imply that sea-island cotton undergoes strong oxidative stress and results in a series of defense responses when attacked by V. dahliae. To our knowledge, this is the first report on the isolation of global ESTs during the sea-island cotton defense reaction.__________From Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 214–223.Original English Text Copyright © 2005 by Zuo, Wang, Wu, Chai, Sun, Tang.This article was submitted by the authors in English.     

Isolation and characterization of conserved non-coding sequences among rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) paralogous regions

Segmental duplication is particularly frequent within plant genomes and the ability of the original single-copy gene to gain a new function for the change of regulatory elements is one of the prominent consequences of duplication. Thus, it is important to study the pattern of conserved non-coding sequence (CNS) between paralogous genes. We report the result of a survey of CNSs among paralogous regions in rice (Oryza sativa L.), as well as the comparison of CNS dataset between rice and Arabidopsis thaliana. Some common properties, such as the change of A + T content near the CNS boundaries and CNS are enriched in regulatory genes, were observed. However, the content of CNSs differs between rice and Arabidopsis, and it is interesting that the rice metabolic network includes both CNS-poor and CNS-rich genes, which indicated a fine-tuned metabolic network presents in rice.     

Pistil induction by hormones from callus of <Emphasis Type="Italic">Oryza sativa</Emphasis> in vitro

This study describes the successful formation of floral organ pistil from the callus of pistil explants of Oryza sativa L. For induction of floral organs, different explants—including young embryo, lemma, palea and pistil—were used for callus induction with different combinations of N⁶-benzyladenine and 2,4-dichlorophenoxyacetic acid (2,4-D). High frequencies of callus formation from pistil and young embryo explants were achieved. Floral organs were induced after calli from pistils were transferred to medium containing both zeatin and 2,4-D. The morphological characteristics of the pistil-like organs are very similar to those formed in planta though with minor differences. Further histological study revealed that the in vitro pistil contains an ovule within its ovary. Furthermore, a pistil-specific gene, OsMADS3 used as a molecular marker for pistil identity, was expressed in the pistil-like organs as it was in pistils in the flower of the plant.     

Mapping of quantitative trait loci associated with ultraviolet-B resistance in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The detection of quantitative trait loci (QTLs) associated with UV-B resistance in rice should allow their practical application in breeding for such a complex trait, and may lead to the identification of gene characteristics and functions. Considerable variation in UV-B resistance exists within cultivated rice (Oryza sativa L.), but its detailed genetic control mechanism has not been well elucidated. We detected putative QTLs associated with the resistance to enhanced UV-B radiation in rice, using 98 BC₁F₅ (backcross inbred lines; BILs) derived from a cross between Nipponbare (a resistant japonica rice variety) and Kasalath (a sensitive indica rice variety). We used 245 RFLP markers to construct a framework linkage map. BILs and both parents were grown under visible light with or without supplemental UV-B radiation in a growth chamber. In order to evaluate UV-B resistance, we used the relative fresh weight of aerial parts (RFW) and the relative chlorophyll content of leaf blades (RCC). The BIL population exhibited a wide range of variation in RFW and RCC. Using composite interval mapping with a LOD threshold of 2.9, three putative QTLs associated with both RFW and RCC were detected on chromosomes 1, 3 and 10. Nipponbare alleles at the QTLs on chromosome 1 and 10 increased the RFW and RCC, while the Kasalath allele at the QTL on chromosome 3 increased both traits. Furthermore, the existence of both QTLs on chromosomes 1 and 10 for UV-B resistance was confirmed using chromosome segment substitution lines. Plants with Kasalath alleles at the QTL on chromosome 10 were more sensitive to UV-B radiation than plants with them on chromosome 1. These results also provide the information not only for the improvement of UV-B resistance in rice though marker-associated selection, but also for the identification of UV-B resistance mechanisms by using near-isogenic lines.Communicated by D.J. Mackill     

Purification and characterization of two ascorbate peroxidases of rice (<Emphasis Type="Italic">Oryza sativa </Emphasis>L.) expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

To clarify the diversity and function of isozymes of ascorbate peroxidase (APX) in plants, a method of producing large quantities of these proteins is needed. Here, we describe an Escherichia coli expression system for the rapid and economic expression of two rice APX genes, APXa and APXb (GeneBank accession Nos. D45423 and AB053297, respectively). The two genes were cloned into the pGEX-6p-3 vector to allow expression of APX as a glutathione-S-transferase (GST) fusion protein. The GST-APXa and GST-APXb fusion proteins were purified by affinity chromatography using a glutathione-Sepharose 4B column, with final yields of 40 and 73 mg g^–1 dry cells, respectively. Specific activities were 15 and 20 mM ascorbate min^–1 mg^–1 protein, respectively. The K_m values for ascorbate were 4 and 1 mM, respectively, and those for H₂O₂ were 0.3 and 0.7 mM, respectively indicating that the two rice isoenzymes have different properties.Revisions requested 27 September 2004; Revisions received 12 November 2004     

Knockout of a starch synthase gene <Emphasis Type="Italic">OsSSIIIa</Emphasis>/<Emphasis Type="Italic">Flo5</Emphasis> causes white-core floury endosperm in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

To elucidate the role of SSIIIa during starch synthesis in rice (Oryza sativa L.) endosperm, we characterized null mutants of this gene, generated by T-DNA insertions. Scanning electron microscope (SEM) analysis revealed that the starch granules in these mutants are smaller and rounder compared with the wild type controls, and that the mutant endosperm is characterized by a loosely packed central portion exhibiting a floury-like phenotype. Hence, the OsSSIIIa (Oryza sativa SSIIIa) mutations are referred to as white-core floury endosperm 5-1 (flo5-1) and flo5-2. Based upon their X-ray diffraction patterns, the crystallinity of the starch in the flo5 mutant endosperm is decreased compared with wild type. Through determination of the chain-length distribution of the mutant endosperm starch, we found that flo5-1 and flo5-2 mutants have reduced the content of long chains with degree of polymerization (DP) 30 or greater compared with the controls. This suggests that OsSSIIIa/Flo5 plays an important role in generating relatively long chains in rice endosperm. In addition, DP 6 to 8 and DP 16 to 20 appeared to be reduced in endosperm starch of flo5-1 and flo5-2, whereas DP 9 to 15 and DP 22 to 29 were increased in these mutants. By the use of differential scanning calorimetry (DSC), the gelatinization temperatures of endosperm starch were found to be 1–5°C lower than those of the control. We propose a distinct role for OsSSIIIa/Flo5 and the coordinated action of other SS isoforms during starch synthesis in the seed endosperm of rice.     

Characterization of raffinose synthase from rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L. var. Nipponbare)

The putative raffinose synthase gene from rice was cloned and expressed in Escherichia coli. The enzyme displayed an optimum activity at 45°C and pH 7.0, and a sulfhydryl group was required for its activity. The enzyme was specific for galactinol and p-nitrophenyl-α-d-galactoside as galactosyl donors, and sucrose, lactose, 4−β-galactobiose, N-acetyl-d-lactosamine, trehalose and lacto-N-biose were recognized as galactosyl acceptors.     

Conditional genetic effect of allelopathy in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) under different environmental conditions

Five parental rice lines with varied levels of allelopathic potential were employed in a partial diallel crossing program to generate 10 F₁ hybrids. The allelopathic effects of the aqueous leaf extracts of the five rice parents and 10 F₁s grown under two different conditions were assessed at different growth stages using lettuce (Lactuca sativa L.) as a bioassay species. Conditional genetics of rice seedling allelopathy and its genotype × environment effects were analyzed by using additive–dominant developmental genetic models. The results showed that conditional additive and dominant effects expressed alternatively from 3- to 8-leaf stage of rice seedlings. The additive effects were significant in the 5|4, 6|5, and 8|7-leaf phases, whilst the dominance effects appeared to play an important role in the 4|3 and 7|6 leaf phases. The conditional narrow sense heritability was significant in the 5|4, 6|5, and 8|7 leaf phases, the broad sense heritability was pronounced among all the growth periods investigated, suggesting that selection for allelopathic activity should be performed during this three leaf phases in order to improve selection response.     

Identification and fine mapping of a thermo-sensitive chlorophyll deficient mutant in rice (<Emphasis Type="Italic">Oryza sativa </Emphasis>L.)

A thermo-sensitive chlorophyll deficient mutant was isolated from more than 15,000 transgenic rice lines. The mutant displayed normal phenotype at 23°C or lower temperature (permissive temperature). However, when grown at 26°C or higher (nonpermissive temperature) the plant exhibited an abnormal phenotype characterized by yellow green leaves. Genetic analysis revealed that a single nuclear-encoded recessive gene is responsible for the mutation, which is tentatively designed as cde1(t) (chlorophyll deficient 1, temporally). PCR analysis and hygromycin resistance assay indicated the mutation was not caused by T-DNA insertion. To isolate the cde1(t) gene, a map-based cloning strategy was employed and 15 new markers (five SSR and ten InDels markers) were developed. A high-resolution physical map of the chromosomal region around the cde1(t) gene was made using F₂ and F₃ population consisting of 1,858 mutant individuals. Finally, the cde1(t) gene was mapped in 7.5 kb region between marker ID10 and marker ID11 on chromosome 2. Sequence analysis revealed only one candidate gene, OsGluRS, in the 7.5 kb region. Cloning and sequencing of the target region from the cde1(t) mutant showed that a missense mutation occurred in the mutant. So the OsGluRS gene (TIGR locus Os02 g02860) which encode glutamyl-tRNA synthetase was identified as the Cde1(t) gene.     

The Plant Architecture of Rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>)

Plant architecture, a collection of the important agronomic traits that determine grain production in rice, is mainly affected by factors including tillering, plant height and panicle morphology. Recently, significant progress has been made in isolating and collecting of mutants that are defective in rice plant architecture. Although our understanding of the molecular mechanisms that control rice tillering, panicle development and plant height are still limited, new findings have begun to emerge. This review, therefore, summarizes the recent progress in exploring the mechanisms that control rice plant architecture.