Arsenic biotransformation by Streptomyces sp. isolated from rice rhizosphere

Isolation and functional analysis of microbes mediating the methylation of arsenic (As) in paddy soils is important for understanding the origin of dimethylarsinic acid (DMA) in rice grains. Here, we isolated from the rice rhizosphere a unique bacterium responsible for As methylation. Strain GSRB54, which was isolated from the roots of rice plants grown in As‐contaminated paddy soil under anaerobic conditions, was classified into the genus Streptomyces by 16S ribosomal RNA sequencing. Sequence analysis of the arsenite S‐adenosylmethionine methyltransferase (arsM) gene revealed that GSRB54 arsM was phylogenetically different from known arsM genes in other bacteria. This strain produced DMA and monomethylarsonic acid when cultured in liquid medium containing arsenite [As(III)]. Heterologous expression of GSRB54 arsM in Escherichia coli promoted methylation of As(III) by converting it into DMA and trimethylarsine oxide. These results demonstrate that strain GSRB54 has a strong ability to methylate As. In addition, DMA was detected in the shoots of rice grown in liquid medium inoculated with GSRB54 and containing As(III). Since Streptomyces are generally aerobic bacteria, we speculate that strain GSRB54 inhabits the oxidative zone around roots of paddy rice and is associated with DMA accumulation in rice grains through As methylation in the rice rhizosphere.     

Arsenic mitigates cadmium toxicity in rice seedlings

Two contrasting rice (Oryza sativa L.) cultivars, i.e. Wuyujing 3 (WYJ3, Cd-tolerant) and Shanyou 63 (SY63, Cd-sensitive), were grown on a red soil (Ultisol) to study both individual and combined phytotoxicity of arsenic (As) and cadmium (Cd) in terms of Cd and As availability, their uptake and accumulation, antioxidant defense activity and oxidative damage. The antioxidant defense system examined in this study included enzymatic and non-enzymatic molecular antioxidants such as superoxide dismutase (SOD), peroxidase (POD), glutathione (GSH) and ascorbic acid (AsA). Results showed that As or Cd treatment decreased root and shoot biomass in both cultivars compared with their corresponding control (no Cd or As treatment), although less severe inhibition of plant growth was observed in WYJ3 than in SY63. Moreover, rice growth was inhibited more severely by Cd treatment than by As treatment, which could be explained by the higher amount of available Cd (60%) (0.1 M HCl-extractable Cd) compared to the lower amount of available As (15%) (0.5 M NaH₂PO₄-extractable As) in their postharvest soils. However, shoot biomass in cultivar SY63, and root and shoot biomass in cultivar WYJ3 were significantly higher in the As plus Cd treatment than in the Cd treatment alone, showing that the combined toxicity of these two heavy metals was not additive and on the contrary, As mitigated Cd-induced growth inhibition. The As plus Cd treatment also significantly decreased As or Cd concentrations both in roots and in shoots of the two rice cultivars compared with the As or Cd treatment alone, respectively. On the other hand, treatment with As or Cd alone significantly decreased the SOD and POD activities, and GSH and AsA concentrations, while the activities of these enzymes and the concentrations of GSH and AsA were significantly higher in the As plus Cd treatment than in the Cd treatment alone, resulting in less severe oxidative damage as indicated by the lower concentration of MDA in the As plus Cd treatment (P < 0.05). However, no significant difference was observed in the antioxidant defense activity between the As plus Cd treatment and the As treatment alone. These results suggest that the combined toxicity of As and Cd in rice is lower than that of individual Cd or As, which might be attributed to the decreased uptake and accumulation of Cd and As, and the less oxidative stress caused by the interactive effects of As with Cd both in rhizosphere and in plants.     

Recent advances in rice biotechnology--towards genetically superior transgenic rice

Rice biotechnology has made rapid advances since the first transgenic rice plants were produced 15 years ago. Over the past decade, this progress has resulted in the development of high frequency, routine and reproducible genetic transformation protocols for rice. This technology has been applied to produce rice plants that withstand several abiotic stresses, as well as to gain tolerance against various pests and diseases. In addition, quality improving and increased nutritional value traits have also been introduced into rice. Most of these gains were not possible through conventional breeding technologies. Transgenic rice system has been used to understand the process of transformation itself, the integration pattern of transgene as well as to modulate gene expression. Field trials of transgenic rice, especially insect-resistant rice, have recently been performed and several other studies that are prerequisite for safe release of transgenic crops have been initiated. New molecular improvisations such as inducible expression of transgene and selectable marker-free technology will help in producing superior transgenic product. It is also a step towards alleviating public concerns relating to issues of transgenic technology and to gain regulatory approval. Knowledge gained from rice can also be applied to improve other cereals. The completion of the rice genome sequencing together with a rich collection of full-length cDNA resources has opened up a plethora of opportunities, paving the way to integrate data from the large-scale projects to solve specific biological problems.     

Arsenic mitigates cadmium toxicity in rice seedlings

Two contrasting rice (Oryza sativa L.) cultivars, i.e. Wuyujing 3 (WYJ3, Cd-tolerant) and Shanyou 63 (SY63, Cd-sensitive), were grown on a red soil (Ultisol) to study both individual and combined phytotoxicity of arsenic (As) and cadmium (Cd) in terms of Cd and As availability, their uptake and accumulation, antioxidant defense activity and oxidative damage. The antioxidant defense system examined in this study included enzymatic and non-enzymatic molecular antioxidants such as superoxide dismutase (SOD), peroxidase (POD), glutathione (GSH) and ascorbic acid (AsA). Results showed that As or Cd treatment decreased root and shoot biomass in both cultivars compared with their corresponding control (no Cd or As treatment), although less severe inhibition of plant growth was observed in WYJ3 than in SY63. Moreover, rice growth was inhibited more severely by Cd treatment than by As treatment, which could be explained by the higher amount of available Cd (60%) (0.1 M HCl-extractable Cd) compared to the lower amount of available As (15%) (0.5 M NaH₂PO₄-extractable As) in their postharvest soils. However, shoot biomass in cultivar SY63, and root and shoot biomass in cultivar WYJ3 were significantly higher in the As plus Cd treatment than in the Cd treatment alone, showing that the combined toxicity of these two heavy metals was not additive and on the contrary, As mitigated Cd-induced growth inhibition. The As plus Cd treatment also significantly decreased As or Cd concentrations both in roots and in shoots of the two rice cultivars compared with the As or Cd treatment alone, respectively. On the other hand, treatment with As or Cd alone significantly decreased the SOD and POD activities, and GSH and AsA concentrations, while the activities of these enzymes and the concentrations of GSH and AsA were significantly higher in the As plus Cd treatment than in the Cd treatment alone, resulting in less severe oxidative damage as indicated by the lower concentration of MDA in the As plus Cd treatment (P < 0.05). However, no significant difference was observed in the antioxidant defense activity between the As plus Cd treatment and the As treatment alone. These results suggest that the combined toxicity of As and Cd in rice is lower than that of individual Cd or As, which might be attributed to the decreased uptake and accumulation of Cd and As, and the less oxidative stress caused by the interactive effects of As with Cd both in rhizosphere and in plants.     

Rearrangements of large-insert T-DNAs in transgenic rice

Introduction of large-DNA fragments into cereals by Agrobacterium-mediated transformation is a useful technique for map-based cloning and molecular breeding. However, little is known about the organization and stability of large fragments of foreign DNA introduced into plant genomes. In this study, we produced transgenic rice plants by Agrobacterium-mediated transformation with a large-insert T-DNA containing a 92-kb region of the wheat genome. The structures of the T-DNA in four independent transgenic lines were visualized by fluorescence in situ hybridization on extended DNA fibers (fiber FISH). By using this cytogenetic technique, we showed that rearrangements of the large-insert T-DNA, involving duplication, deletion and insertion, had occurred in all four lines. Deletion of long stretches of the large-insert DNA was also observed in Agrobacterium.     

稻田氮肥的氨挥发损失与稻季大气氮的湿沉降

通过田间小区与大田试验,对稻季期间氮肥的氨挥发损失和大气氮湿沉降状况进行了收集和监测。结果表明,每次施肥后的1～3日内氨挥发损失达到最大值,氨挥发损失受当地气候条件(如光照、温度、湿度、风速、降雨量)、施肥时期以及田面水的NH4^+-N浓度等因素的影响,大气氮湿沉降与施肥量和降雨量有关,稻季内由湿沉降带入土壤或地表水中的氮为7．51kg·hm^-2。其中,NH4^+-N的比例为39．8％～73.2％,平均为55．5％;稻季中总氨挥发量与湿沉降的NH4^+-N平均浓度和总沉降量的相关系数分别达到0．988和0．996,呈显著相关性。     

Inheritance of gusA and neo genes in transgenic rice

Inheritance of foreign genes neo and gusA in rice (Oryza sativa L. cv. IR54 and Radon) has been investigated in three different primary (T₀) transformants and their progeny plants. T₀ plants were obtained by co-transforming protoplasts from two different rice suspension cultures with the neomycin phosphotransferase II gene [neo or aph (3) II] and the -glucuronidase gene (uidA or gusA) residing on separate chimeric plasmid constructs. The suspension cultures were derived from callus of immature embryos of indica variety IR54 and japonica variety Radon. One transgenic line of Radon (AR2) contained neo driven by the CaMV 35S promoter and gusA driven by the rice actin promoter. A second Radon line (R3) contained neo driven by the CaMV 35S promoter and gusA driven by a promoter of the rice tungro bacilliform virus. The third transgenic line, IR54-1, contained neo driven by the CaMV 35S promoter and gusA driven by the CaMV 35S.Inheritance of the transgenes in progeny of the transgenic rice was investigated by Southern blot analysis and enzyme assays. Southern blot analysis of genomic DNA showed that, regardless of copy numbers of the transgenes in the plant genome and the fact that the two transgenes resided on two different plasmids before transformation, the introduced gusA and neo genes were stably transmitted from one generation to another and co-inherited together in transgenic rice progeny plants derived from self-pollination. Analysis of GUS and NPT II activities in T₁ to T₂ plants provided evidence that inheritance of the gusA and neo genes was in a Mendelian fashion in one plant line (AR2), and in an irregular fashion in the two other plant lines (R3 and IR54-1). Homozygous progeny plants expressing the gusA and neo genes were obtained in the T₂ generation of AR2, but the homozygous state was not found in the other two lines of transgenic rice.     

Arsenic in the human food chain,biotransformation and toxicology – Review focusing on seafood arsenic

Fish and seafood are main contributors of arsenic (As) in the diet. The dominating arsenical is the organoarsenical arsenobetaine (AB), found particularly in finfish. Algae, blue mussels and other filter feeders contain less AB, but more arsenosugars and relatively more inorganic arsenic (iAs), whereas fatty fish contain more arsenolipids. Other compounds present in smaller amounts in seafood include trimethylarsine oxide (TMAO), trimethylarsoniopropionate (TMAP), dimethylarsenate (DMA), methylarsenate (MA) and sulfur-containing arsenicals. The toxic and carcinogenic arsenical iAs is biotransformed in humans and excreted in urine as the carcinogens dimethylarsinate (DMA) and methylarsonate (MA), producing reactive intermediates in the process. Less is known about the biotransformation of organoarsenicals, but new insight indicates that bioconversion of arsenosugars and arsenolipids in seafood results in urinary excretion of DMA, possibly also producing reactive trivalent arsenic intermediates. Recent findings also indicate that the pre-systematic metabolism by colon microbiota play an important role for human metabolism of arsenicals. Processing of seafood may also result in transformation of arsenicals.     

根癌农杆菌介导的水稻转基因技术体系的优化

对根癌农杆菌介导水稻遗传转化的各重要因素逐一分析,比较不同基因型、培养基成份、光照、继代次数、菌液浓度、侵染时间、干燥情况等因素对遗传转化的影响。通过培养条件的优化,建立了一个高效的水稻转化技术体系。研究结果表明,相比较于暗培养,光照培养进行愈伤诱导可以使诱导天数缩短4~5d;诱导培养基中加入适量的激素可以使籼稻愈伤诱导提高25%~30%;分化培养基中加入适量的氨基酸和甘露醇可以使植株再生频率提高15%~20%。     

Heterologous expression of artificial miRNAs from rice dwarf virus in transgenic rice

In contrast to hairpin RNAs, in which heterogeneous small RNAs are processed from double-stranded RNA to have potential off-target effects on endogenous other genes, artificial miRNAs (amiRNAs) have advantages of exquisite specificity and non-transitivity to thus target individual genes and groups of endogenous genes. Earlier studies showed that amiRNA engineering based on osa-miRNA528 precursor could efficiently trigger endogenous gene silencing and modulate agronomic traits in rice. However, both the expression efficiency of heterologous amiRNAs based on osa-miRNA528 precursor and the correlation of copy number with the relative expression level of amiRNAs remain unknown. In the present study, five amiRNAs (S9-1174, S9-1192, S11-864, S11-868 and S11-869) targeting different sites of S9 and S11 negative strands in rice dwarf virus (RDV) genome were constructed using endogenous osa-miRNA528 precursor as backbone. After identification by Northern blot, two amiRNAs (S9-1174 and S9-1192) targeting S9 negative strand in RDV genome were highly expressed, whereas in three tested amiRNAs targeting S11 negative strand in RDV genome, only two amiRNAs (S11-868 and S11-869) were processed efficiently. T0 generation transgenic rice containing amiRNAs (S9-1174, S9-1192, S11-868 and S11-869) exhibited different expression ratios of amiRNAs, accounting for 90.0, 90.0, 66.7 and 77.8 %, respectively. In addition, combination analysis with the relative amiRNA expression levels and its copy number revealed that the relative expression levels of amiRNAs had no relation to the copy number of T-DNA insert in transgenic rice.     

Characterization of rice tryptophan decarboxylases and their direct involvement in serotonin biosynthesis in transgenic rice

l-Tryptophan decarboxylase (TDC) and l-tyrosine decarboxylase (TYDC) belong to a family of aromatic l-amino acid decarboxylases and catalyze the conversion of tryptophan and tyrosine into tryptamine and tyramine, respectively. The rice genome has been shown to contain seven TDC or TYDC-like genes. Three of these genes for which cDNA clones were available were characterized to assign their functions using heterologous expression in Escherichia coli and rice (Oryza sativa cv. Dongjin). The purified products of two of the genes were expressed in E. coli and exhibited TDC activity, whereas the remaining gene could not be expressed in E. coli. The recombinant TDC protein with the greatest TDC activity showed a K _m of 0.69 mM for tryptophan, and its activity was not inhibited by phenylalanine or tyrosine, indicating a high level of substrate specificity toward tryptophan. The ectopic expression of the three cDNA clones in rice led to the abundant production of the products of the encoded enzymes, tyramine and tryptamine. The overproduction of TYDC resulted in stunted growth and a lack of seed production due to tyramine accumulation, which increased as the plant aged. In contrast, transgenic plants that produced TDC showed a normal phenotype and contained 25-fold and 11-fold higher serotonin in the leaves and seeds, respectively, than the wild-type plants. The overproduction of either tyramine or serotonin was not strongly related to the enhanced synthesis of tyramine or serotonin derivatives, such as feruloyltyramine and feruloylserotonin, which are secondary metabolites that act as phytoalexins in plants.     

The structures of integration sites in transgenic rice

Extensive genomic sequencing and sequence motif analysis have been conducted over the integration sites of two transgenic rice plants, #478 and #559, carrying the luciferase gene and/or hygromycin phosphotransferase gene. The transgenes reside in a region with inverted structure and a large duplication of rice genome over 2 kb. Integration was found at the AT-rich region and/or at the repetitive sequence region, including a SAR-like structure, retrotransposon and telomere repeats. The presence of a patch of sequence homology between plasmid and target DNA, and a small region of duplication involving the target DNA around the recombination site, implicated illegitimate recombination in the process of gene integration. Massive rearrangement of genomic DNA including deletion or translocation was also observed at the integration site and the flanking region of the transgene. The recognition sites of DNA topoisomerases I or II were observed in the rearranged sequences. Since only three junctions of transgenic rice were implicated in the illegitimate recombination and extensive rearrangement of the rice genome, rice protoplasts may be active in this process.     

Genetic manipulation of gibberellin metabolism in transgenic rice

The 'green revolution' was fueled by the introduction of the semi-dwarf trait into cereal crop cultivars. The semi-dwarf cultivars--which respond abnormally to the plant growth hormone gibberellin (GA)--are more resistant to wind and rain damage and thus yield more grain when fertilized. To generate dwarf rice plants using a biotechnological approach, we modified the level of GA by overproduction of a GA catabolic enzyme, GA 2-oxidase. When the gene encoding GA 2-oxidase, OsGA2ox1, was constitutively expressed by the actin promoter, transgenic rice showed severe dwarfism but failed to set grain because GA is involved in both shoot elongation and reproductive development. In contrast, OsGA2ox1 ectopic expression at the site of bioactive GA synthesis in shoots under the control of the promoter of a GA biosynthesis gene, OsGA3ox2 (D18), resulted in a semi-dwarf phenotype that is normal in flowering and grain development. The stability and inheritance of these traits shows the feasibility of genetic improvement of cereal crops by modulation of GA catabolism and bioactive GA content.     

Expression and characterization of a thermostable l-aminoacylase in transgenic rice

Journal of Plant Biochemistry and Biotechnology - The gene encoding a thermostable l-aminoacylase (LAA) from Deinococcus radiodurans BCRC12827 was isolated and expressed in transgenic rice under...     

辽河下游平原不同水分条件下稻田氨挥发

应用通气密闭室法,研究了辽河下游平原不同水分条件下潮棕壤稻田生态系统施用氮肥后的NH3挥发.结果表明稻田施用氮肥后有明显NH3挥发损失,整个生长期间总挥发量为11.64～34.01 kg N·hm-2,占施氮量的4.66%～11.66%;不同施肥时期的损失量为分蘖期＞孕穗期＞移栽前,挥发高峰出现在施氮肥后的2～4 d内.稻田水分状况对NH3挥发损失具有重要影响,田面积水条件下NH3挥发总量和肥料氮损失率都较大,且不同施氮水平间差异显著(P＜0.05),挥发量随施氮量的增加而增加;田面不积水条件下NH3挥发量相对较小.氮肥用量、田面水NH4 浓度和pH是影响NH3挥发的重要因素;氮肥用量为180 kg N·hm-2时,不同磷水平对NH3挥发的影响不显著.