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.     

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.     

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     

Two rice cytosolic ascorbate peroxidases differentially improve salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

In order to determine the different roles of rice (Oryza sativa L.) cytosolic ascorbate peroxidases (OsAPXa and OsAPXb, GenBank accession nos. D45423 and AB053297, respectively) under salt stress, transgenic Arabidopsis plants over-expressing OsAPXa or OsAPXb were generated, and they all exhibited increased tolerance to salt stress compared to wild-type plants. Moreover, transgenic lines over-expressing OsAPXb showed higher salt tolerance than OsAPXa transgenic lines as indicated by root length and total chlorophyll content. In addition to ascorbate peroxidase (APX) activity, antioxidant enzyme activities of catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GR), which are also involved in the salt tolerance process, and the content of H₂O₂ were also assayed in both transgenic and wild-type plants. The results showed that the overproduction of OsAPXb enhanced and maintained APX activity to a much higher degree than OsAPXa in transgenic Arabidopsis during treatment with different concentrations of NaCl, enhanced the active oxygen scavenging system, and protected plants from salt stress by equilibrating H₂O₂ metabolism. Our findings suggest that the rice cytosolic OsAPXb gene has a more functional role than OsAPXa in the improvement of salt tolerance in transgenic plants. Zhenqiang Lu and Dali Liu contributed equally.     

The Effect of Salinity Pretreatment on Cd Accumulation and Cd-Induced Stress in <Emphasis Type="Italic">BADH</Emphasis><Emphasis Type="Italic">-</Emphasis>Transgenic and Nontransgenic Rice Seedlings

The influence of betaine aldehyde dehydrogenase (BADH) and salinity pretreatment on oxidative stress under cadmium (Cd) toxicity was investigated in rice cv. Xiushui 11 and its BADH-transgenic line Bxiushui 11. The results showed that plants previously treated with 4.25 and 8.5 mM NaCl, respectively, for 5 days each had higher Cd concentrations in both roots and shoots of the two rice genotypes compared with the controls. Malondialdehyde (MDA) content in both leaves and roots was increased by salinity pretreatment and was significantly lower in the salinity-pretreatment plants than in the controls when the plants were consequently exposed to Cd stress. Salinity pretreatment also increased proline content and the activities of superoxide dismutase (SOD) and peroxidase (POD) in both leaves and roots. It can be assumed that salinity pretreatment enhances the defensive ability of plants against oxidative stress through increasing activities of antioxidative enzymes. The BADH-transgenic line (Bxiushui 11) had lower Cd and MDA content, higher SOD and POD activities, and higher proline content than its wild type (Xiushui 11). The current results suggest that betaine, a product of BADH expression, improves the tolerance of rice plants to Cd stress through increasing the activities of antioxidative enzymes and osmoprotectant content.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of Indica rice genotypes: an assessment of factors affecting the transformation efficiency

An efficient Agrobacterium-mediated method for transformation of popular Bangladeshi Indica rice genotypes has been developed. Mature embryo-derived calluses as well as immature embryos were used as the target material. Transgenic plant production frequency was higher using the immature embryos than mature embryo-derived calluses. However, 3-week-old mature embryo-derived calluses served as an excellent starting material. The super-binary vector (pTOK233) was generally more effective than the binary vector (pC1301-Xa21mSS) particularly with recalcitrant Bangladeshi genotypes such as BR22. However, transformation of the Japonica cultivar Taipei-309 was equally effective with either plasmid. Inclusion of acetosyringone (200M) in co-cultivation media proved essential for successful transformation and the optimum co-cultivation period found was to be 3days. A large number of morphologically normal, fertile transgenic plants were obtained which expressed gus as determined by histochemical staining. Integration of the hpt gene into the genome of transgenic plants was confirmed by molecular analysis. Mendelian inheritance of transgenes (hpt and gus gene) was observed in T1 progeny.     

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.     

Transgenic rice as a novel production system for <Emphasis Type="Italic">Melanocarpus</Emphasis> and <Emphasis Type="Italic">Pycnoporus</Emphasis> laccases

Laccases have numerous biotechnological applications, among them food processing. The widespread use of laccases has increased the demand for an inexpensive and safe source of recombinant enzyme. We explored the use of a rice-based system for the production of two fungal laccases derived from the ascomycete Melanocarpus albomyces and the basidiomycete Pycnoporus cinnabarinus. High-expression levels of active recombinant laccases were achieved by targeting expression to the endosperm of rice seeds. The laccase cDNAs were fused to a plant-derived signal sequence for targeting to the secretory pathway, and placed under the control of a constitutive seed-specific promoter fused to an intron for enhanced expression. This construct enabled the recovery of on average 0.1-1% of soluble laccase in total soluble proteins (TSP). The highest yields of recombinant laccases obtained in rice seeds were 13 and 39 ppm for riceMaL and ricePycL, respectively. The rice-produced laccases were purified and characterized. The wild-type and the recombinant proteins showed similar biochemical features in terms of molecular mass, pI, temperature and optimal pH and the N-terminus was correctly processed. Although presenting lower kinetic parameters, the rice-produced laccases were also suitable for the oxidative cross-linking of a food model substrate [maize-bran feruloylated arabinoxylans (AX)].     

Cloning,characterization, and transformation of the phosphoethanolamine <Emphasis Type="Italic">N</Emphasis>-methyltransferase gene (<Emphasis Type="Italic">ZmPEAMT1</Emphasis>) in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

N-methylation of phosphoethanolamine, the committing step in choline (Cho) biosynthesis in plants, is catalyzed by S-adenosyl-l-methionine: phosphoethanolamine N-methyltransferase (PEAMT, EC 2.1.1.103). Herein we report the cloning and characterization of the novel maize phosphoethanolamine N-methyltransferase gene (ZmPEAMT1) using a combination of bioinformatics and a PCR-based allele mining strategy. The cDNA sequence of ZmPEAMT1 gene is 1,806 bp in length and translates a 495 amino acids peptide. The upstream promoter sequence of ZmPEAMT1 were obtained by TAIL-PCR, and contained four kinds of putative cis-acting regulatory elements, including stress-responsive elements, phytohormone-responsive elements, pollen developmental special activation elements, and light-induced signal transduction elements, as well as several other structural features in common with the promoter of rice and Arabidopsis homologies. RT-PCR analysis showed that expression of ZmPEAMT1 was induced by salt stress and suppressed by high temperature. Over-expression of ZmPEAMT1 enhanced the salt tolerance, root length, and silique number in transgenic Arabidopsis. These data indicated that ZmPEAMT1 maybe involved in maize root development and stress resistance, and maybe having a potential application in maize genetic engineering. Note: Nucleotide sequence data are available in GenBank under the following accession numbers: maize (Zea mays, ZmPEAMT1, AY626156; ZmPEAMT2, AY103779); rice (Oryza sativa, OsPEAMT1/Os01g50030, NM_192178; OsPEAMT2/Os05g47540, XM_475841); wheat (Triticum aestivum, TaPEAMT, AY065971); Arabidopsis (Arabidopsis thaliana, AtNMT1/At3g18000, AY091683; AtNMT2/At1g48600, NM_202264; AtNMT3/At1g73600, NM_106018); oilseed rape (Brassica napus, BnPEAMT, AY319479), tomato (Lycopersicon esculentum, AF328858), spinach (Spinacia oleracea, AF237633).     

Gene cloning and heterologous expression of pyranose 2-oxidase from the brown-rot fungus, <Emphasis Type="Italic">Gloeophyllum</Emphasis><Emphasis Type="Italic">trabeum</Emphasis>

A pyranose 2-oxidase gene from the brown-rot basidiomycete Gloeophyllum trabeum was isolated using homology-based degenerate PCR. The gene structure was determined and compared to that of several pyranose 2-oxidases cloned from white-rot fungi. The G. trabeum pyranose 2-oxidase gene consists of 16 coding exons with canonical promoter CAAT and TATA elements in the 5′UTR. The corresponding G. trabeum cDNA was cloned and contains an ORF of 1,962 base pairs encoding a 653 amino acid polypeptide with a predicted molecular weight of 72 kDa. A Hisx6 tagged recombinant G. trabeum pyranose 2-oxidase was generated and expressed heterologously in Escherichia coli yielding 15 U enzyme activity per ml of induced culture. Structural alignment and phylogenetic analysis were performed and are discussed.     

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.     

Protoplasts from <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-transformed cell line of <Emphasis Type="Italic">Medicago sativa</Emphasis> L. regenerated to hairy roots

Summary Protoplasts were isolated from Agrobacterium rhizogenes A4-transformed cell line of Medicago sativa L. The highest yield of protoplasts (4.2×10⁶ per g fresh weight) was obtained from 12-d-old calluses after being subeultured on fresh medium. The viability of protoplasts reached over 80%. Protoplasts were induced to undergo sustained divisions when cultured in Durand et al. (DPD) medium supplemented with 2 mgl⁻¹ (9.05 μM) 2,4-dichlorophenoxyacetic acid, 0,2mgl⁻¹ (0.93 μM) kinetin, 0.3 M mannitol, 2% (w/v) sucrose, and 500 mgl⁻¹ casein hydrolyzate at a plating density of 1.0×10⁵ per ml. An agarose-beads culture method was appropriate for protoplast division of transformed alfalfa. The division frequency was about 30%. Numerous hairy roots were induced from protocalluses on Murashige and Skoog medium without growth regulators. Paper electrophoresis revealed that all of the regenerated hairy roots tested synthesized the corresponding opines. This protoplast culture system would be valuable for further somatic hybridization in forage legumes.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Authentic seed-specific activity of the <Emphasis Type="Italic">Perilla</Emphasis> oleosin 19 gene promoter in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

The Perilla (Perilla frutescens L. cv. Okdong) oleosin gene, PfOle19, produces a 19-kDa protein that is highly expressed only in seeds. The activity of the −2,015 bp 5′-upstream promoter region of this gene was investigated in transgenic Arabidopsis plants using the fusion reporter constructs of enhanced green fluorescent protein (EGFP) and β-glucuronidase (GUS). The PfOle19 promoter directs Egfp expression in developing siliques, but not in leaves, stems or roots. In the transgenic Arabidopsis, EGFP fluorescence and histochemical GUS staining were restricted to early seedlings, indehiscent siliques and mature seeds. Progressive 5′-deletions up to the −963 bp position of the PfOle19 promoter increases the spatial control of the gene expression in seeds, but reduces its quantitative levels of expression. Moreover, the activity of the PfOle19 promoter in mature seeds is 4- and 5-fold greater than that of the cauliflower mosaic virus 35S promoter in terms of both EGFP intensity and fluorometric GUS activity, respectively.