Gene flow from transgenic rice to red rice (Oryza sativa L.) in the field

In this study, we simulate a transgenic rice crop highly infested with red rice to examine transgene transfer from a transgenic line (A2504) resistant to glufosinate ammonium to cohabitant red rice. The red rice was sown along with the transgenic line at the highest density found in naturally infested crops in the region. Agricultural practices similar to those used to control red rice infestation in northern Italy rice fields were used to reproduce the local rice production system. During the first 2 years, the field was treated with herbicide at the appropriate time; in the first year the dosage of herbicide was three times the recommended amount. In this first year, detectable red rice plants that escaped herbicide treatment were manually removed. Nevertheless, two herbicide‐resistant hybrid plants (named 101 and 104) were identified in the experimental field during the second year of cultivation. Phenotypic and molecular characterisation suggests the hybrid nature of these two plants, deriving from crossing events involving A2504, respectively, with red rice (plant 101) and the buffer cultivar Gladio (plant 104). The progeny of two subsequent generations of the two plants were examined and the presence of the transgene detected, indicating stable transfer of the transgene across generations. In conclusion, despite control methods, red rice progeny tolerant to the herbicide can be expected following use of transgenic rice and, consequently, difficulties in controlling this weed with chemicals will emerge in a relatively short time.     

A rice (Oryza sativa L.) MAP kinase gene, OsMAPK44, is involved in response to abiotic stresses

We have isolated and characterized a putative rice MAPK gene (designated OsMAPK44) encoding for a protein of 593 amino acids that has the MAPK family signature and phosphorylation activation motif, TDY. Alignment of the predicted amino acid sequences of OsMAPK44 showed high homology with other rice MAPKs. Under normal conditions, the OsMAPK44 gene is highly expressed in root tissues, but relatively less in leaf and stem tissues of the japonica type rice plant (O. sativa L. Donggin). mRNA expression of the gene is highly inducible by salt and drought treatment, but not by cold treatment. Moreover, the mRNA level of the OsMAPK44 is up-regulated by exogenously applied Abscisic acid (ABA) and H₂O₂. When we compared the OsMAPK44 gene expression level between a salt sensitive indica cultivar (IR64) and a salt resistant indica cultivar (Pokkali), they showed some difference in expression kinetics with the salt treatment. OsMAPK44 gene expression in Pokkali was slightly up-regulated within 30 min and then disappeared rapidly, while IR64 maintained its expression for 1 h following down-regulation. Under the salinity stress, OsMAPK44 overexpression transgenic rice plants showed less damage and greater ratio of potassium and sodium than OsMAPK44 suppressed transgenic lines did, suggesting that OsMAPK44 may have a role to prevent damages due to working for favorable ion balance in the presence of salinity.     

Comparative expression analysis of genes encoding metallothioneins in response to heavy metals and abiotic stresses in rice (Oryza sativa) and Arabidopsis thaliana

To get insights into the functions of metallothionein (MT) in plant response to multiple stresses, expressions of 10 rice MT genes (OsMTs) and 7 Arabidopsis MT genes (AtMTs) were comprehensively analyzed under combined heavy metal and salt stress. OsMT1a, OsMT1b, OsMT1c, OsMT1g, and OsMT2a were increased by different heavy metals. Notably, ABA remarkably increased OsMT4 up to 80-fold. Combined salt and heavy metals (Cd, Pb, Cu) synergistically increased OsMT1a, OsMT1c, and OsMT1g, whereas combined salt and H₂O₂ or ABA synergistically increased OsMT1a and OsMT4. Heavy metals decreased AtMT1c, AtMT2b, and AtMT3 but cold or ABA increased AtMT1a, AtMT1c, and AtMT2a. AtMT4a was markedly increased by salt stress. Combined salt and other stresses (Pb, Cd, H₂O₂) synergistically increased AtMT4a. Taken together, these findings suggest that MTs in monocot and dicot respond differently to combined stresses, which provides a valuable basis to further determine the roles of MTs in broad stress tolerance.     

Identification of rice (Oryza sativa L.) signal factors capable of inducing Agrobacterium vir gene expression

Two kinds of signal factors capable of inducing Agrobaorerium vir gene expression were purified and identified from leaf extracts of panicle-differentiating to flowering stage of rice (Oryza saliva L. cv. IR 72) detected by Agrobacterium vir(?) lacZ. fusion genes. The induction was similar to that observed with 5 μm actosyringone (AS). Based on the comprehensive analysis of the data by UV, IR, NMR, MS, HMQC and HMBC, the structures of these two signal factors are identified as 5, 7, 4'-trihydroxy-3', 5'-dimethoxy-flavone (named tricin) and 5, 4' -dihydroxy-3', 5' -dimethoxy-7- (β-D-glucosyloxy) -flavone, respectively. These results demonstrate that monocotyledonous plants do contain highly efficient vir gene inducing factors of Agrobacterium, and the reason why monocotyledonous plants are difficult to transform by Ayrobacterium is not due to absence of vir gene inducing factors, but due to the signal factors only produced in specific stage and tissue of monocotyledonous plants     

Wheat LEA genes, PMA80 and PMA1959, enhance dehydration tolerance of transgenic rice (Oryza sativa L.)

Drought and salt stresses are two major factors that lower plant productivity. Transgenic approaches offer powerful means to better understand and then minimize loss of yield due to these abiotic stresses. In this study, we have generated transgenic rice plants expressing a wheat LEA group 2 protein (PMA80) gene, and separately the wheat LEA group 1 protein (PMA1959) gene. Molecular analysis of the transgenic plants revealed the stable integration of the transgenes. Immunoblot analysis showed the presence of the LEA group 2 protein (39 kDa) and the LEA group 1 protein (25 kDa) in most of the plant lines. Second-generation transgenic plants were subjected to dehydration or salt stress. The results showed that accumulation of either PMA80 or PMA1959 correlates with increased tolerance of transgenic rice plants to these stresses.     

受非生物胁迫和稻瘟病胁迫双重诱导的OsWRKY转录因子基因表达特征分析

OsWRKY 转录因子在水稻非生物胁迫和抗病反应中具有相当重要的调节作用。为阐明其调节作用提供依据,研究了疑似功能广泛的 OsWRKY 转录因子表达谱,采用五个 OsWRKY 转录因子基因,即 Os-WRKY7、OsWRKY11、OsWRKY30、OsWRKY70和 OsWRKY89,利用 real-time PCR 研究各种非生物胁迫和稻瘟菌胁迫诱导表达特征,以及各种激素对 OsWRKY 表达量的影响。所采用的五个基因均受到稻瘟菌胁迫的诱导,而且各种非生物胁迫也能不同程度地诱导其表达。在各个激素处理下,有些被诱导或被抑制,也有未受影响。五个 OsWRKY 基因均有可能参与稻瘟病胁迫响应。其中 OsWRKY7和 OsWRKY70可能是在JA 和 SA 相互拮抗调控下参与,OsWRKY89可能是通过非本研究涉及的其他激素途径参与。在非生物胁迫方面,OsWRKY7可能通过 ABA 途径参与干旱、高盐和极端温度胁迫;OsWRKY11有可能参与高盐胁迫;OsWRKY30有可能参与高盐和高温胁迫;OsWRKY70可能参与高盐、干旱和极端温度胁迫;OsWRKY89可能参与高温胁迫,但并不是通过本研究所涉及的四种激素途径。     

Identification of salt-stress responsive genes in rice (Oryza sativa L.) by cDNA array

To identify salt stress-responsive genes, we constructed a cDNA library with the salt-tolerant rice cultivar, Lansheng. About 15000 plasmids were extracted and dotted on filters with Biomeck 2000 HDRT system or by hand. Thirty genes were identified to display altered expression levels responding to 150 mmol/L NaCl. Among them eighteen genes were up-regulated and the remainders down-regulated. Twenty-seven genes have their homologous genes in Gen-Bank Databases. The expression of twelve genes was studied by Northern analysis. Based on the functions, these genes can be classified into five categories, including photosynthesis-related gene, transport-related gene, metabolism-related gene, stress- or resistance-related gene and the others with various functions. The results showed that salt stress influenced many aspects of rice growth. Some of these genes may play important roles in plant salt tolerance.     

Receptor‐like kinase OsSIK1 improves drought and salt stress tolerance in rice (Oryza sativa) plants

Receptor‐like kinases (RLKs) play essential roles in plant growth, development and responses to environmental stresses. A putative RLK gene, OsSIK1, with extracellular leucine‐rich repeats was cloned and characterized in rice (Oryza sativa). OsSIK1 exhibits kinase activity in the presence of Mn²⁺, and the OsSIK1 kinase domain has the ability to autophosphorylate and phosphorylate myelin basic protein (MBP). OsSIK1 promoter‐GUS analysis revealed that OsSIK1 is expressed mainly in the stem and spikelet in rice. The expression of OsSIK1 is mainly induced by salt, drought and H₂O₂ treatments. Transgenic rice plants with overexpression of OsSIK1 show higher tolerance to salt and drought stresses than control plants. On the contrary, the knock‐out mutants sik1‐1 and sik1‐2, as well as RNA interference (RNAi) plants, are sensitive to drought and salt stresses. The activities of peroxidase, superoxide dismutase and catalase are enhanced significantly in OsSIK1‐overexpressing plants. Also, the accumulation of H₂O₂ in leaves of OsSIK1‐overexpressing plants is much less than that of the mutants, RNAi plants and control plants, as measured by 3,3′‐diamino benzidine (DAB) staining. We also show that OsSIK1 affects stomatal density in the abaxial and adaxial leaf epidermis of rice. These results indicate that OsSIK1 plays important roles in salt and drought stress tolerance in rice, through the activation of the antioxidative system.     

Accumulation of glycinebetaine in rice plants that overexpress choline monooxygenase from spinach and evaluation of their tolerance to abiotic stress

BACKGROUND AND AIMS: Glycinebetaine (GB), a quaternary ammonium compound, is a very effective compatible solute. In higher plants, GB is synthesized from choline (Cho) via betaine aldehyde (BA). The first and second steps in the biosynthesis of GB are catalysed by choline monooxygenase (CMO) and by betaine aldehyde dehydrogenase (BADH), respectively. Rice (Oryza sativa), which has two genes for BADH, does not accumulate GB because it lacks a functional gene for CMO. Rice plants accumulate GB in the presence of exogenously applied BA, which leads to the development of a significant tolerance to salt, cold and heat stress. The goal in this study was to evaluate and to discuss the effects of endogenously accumulated GB in rice. METHODS: Transgenic rice plants that overexpressed a gene for CMO from spinach (Spinacia oleracea) were produced by Agrobacterium-mediated transformation. After Southern and western blotting analysis, GB in rice leaves was quantified by (1)H-NMR spectroscopy and the tolerance of GB-accumulating plants to abiotic stress was investigated. KEY RESULTS: Transgenic plants that had a single copy of the transgene and expressed spinach CMO accumulated GB at the level of 0.29-0.43 micromol g(-1) d. wt and had enhanced tolerance to salt stress and temperature stress in the seedling stage. CONCLUSIONS: In the CMO-expressing rice plants, the localization of spinach CMO and of endogenous BADHs might be different and/or the catalytic activity of spinach CMO in rice plants might be lower than it is in spinach. These possibilities might explain the low levels of GB in the transgenic rice plants. It was concluded that CMO-expressing rice plants were not effective for accumulation of GB and improvement of productivity.     

Overexpression of the rice Osmyb4 gene increases chilling and freezing tolerance of Arabidopsis thaliana plants

The expression of the gene Osmyb4, detected at low level in rice (Oryza sativa) coleoptiles grown for 3 days at 29 degrees C, is strongly induced by treatments at 4 degrees C. At sublethal temperatures of 10 and 15 degrees C, its expression in rice seedlings is already evident, but this effect cannot be vicariated by other stresses or ABA treatment. We demonstrate by transient expression that Myb4 transactivates the PAL2, ScD9 SAD and COR15a cold-inducible promoters. The Osmyb4 function in vivo is demonstrated overexpressing its cDNA in Arabidopsis thaliana plants (ecotype Wassilewskija) under the control of the constitutive CaMV 35S promoter. Myb4 overexpressing plants show a significant increased cold and freezing tolerance, measured as membrane or Photosystem II (PSII) stability and as whole plant tolerance. Finally, in Osmyb4 transgenic plants, the expression of genes participating in different cold-induced pathways is affected, suggesting that Myb4 represents a master switch in cold tolerance.     

Molecular mapping of genes conferring aluminum tolerance in rice (Oryza sativa L.)

Crop productivity on acid soil is restricted by multiple abiotic stress factors. Aluminum (Al) tolerance seems to be a key to productivity on soil with a pH below 5.0, but other factors such as Mn toxicity and the deficiency of P, Ca and Mg also play a role. The development of Al-tolerant genotypes of rice is an urgent necessity for improving crop productivity in developing countries. Inhibition of root growth is a primary and early symptom of Al toxicity. The present study was conducted to identify genetic factors controlling the aluminum tolerance of rice. Several parameters related to Al tolerance, most importantly the relative root growth under Al stress versus non-stress conditions, were scored in 188 F₃ selfed families from a cross between an Al-tolerant Vietnamese local variety, Chiembau, and an Al-susceptible improved variety, Omon269–65. The two varieties are both Oryza sativa ssp. indica, but showed a relatively high level of DNA polymorphism, permitting the assembly of an RFLP map consisting of 164 loci spanning 1,715.8 cM, and covering most of the rice genome. A total of nine different genomic regions on eight chromosomes have been implicated in the genetic control of root and shoot growth under aluminum stress. By far the greatest effects on aluminum tolerance were associated with the region near WG110 on chromosome 1. This region does not seem to correspond to most of the genes that have been mapped for aluminum tolerance in other species, nor do they correspond closely to one another. Most results, both from physiological studies and from molecular mapping studies, tend to suggest that aluminum tolerance is a complex multi-genic trait. The identification of DNA markers (such as WG110) that are diagnostic for aluminum tolerance in particular gene pools provides an important starting point for transferring and pyramiding genes that may contribute to the sustainable improvement of crop productivity in aluminum-rich soils. The isolation of genes responsible for aluminum tolerance is likely to be necessary to gain a comprehensive understanding of this complex trait. Received: 29 March 2000 / Accepted: 16 August 2000