Activity of a maize ubiquitin promoter in transgenic rice

We have used the maize ubiquitin 1 promoter, first exon and first intron (UBI) for rice (Oryza sativa L. cv. Taipei 309) transformation experiments and studied its expression in transgenic calli and plants. UBI directed significantly higher levels of transient gene expression than other promoter/intron combinations used for rice transformation. We exploited these high levels of expression to identify stable transformants obtained from callus-derived protoplasts co-transfected with two chimeric genes. The genes consisted of UBI fused to the coding regions of the uidA and bar marker genes (UBI:GUS and UBI:BAR). UBI:GUS expression increased in response to thermal stress in both transfected protoplasts and transgenic rice calli. Histochemical localization of GUS activity revealed that UBI was most active in rapidly dividing cells. This promoter is expressed in many, but not all, rice tissues and undergoes important changes in activity during the development of transgenic rice plants.     

Evaluation of the stress-inducible production of choline oxidase in transgenic rice as a strategy for producing the stress-protectant glycine betaine

Glycine betaine (GB) is a compatible solute that is also capable of stabilizing the structure and function of macromolecules. Several GB-producing transgenic rice lines were generated in which the Arthrobacter pascens choline oxidase (COX) gene, fused to a chloroplast targeting sequence (TP) was expressed under the control of an ABA-inducible promoter (SIP; stress-inducible promoter) or a ubiquitin (UBI) gene promoter that is considered to be constitutive. This comparison led to interesting observations that suggest complex regulation with respect to GB synthesis and plant growth response under stress. In spite of the use of the well-studied stress-inducible promoter, the highest level of GB accumulation (up to 2.60 micromol g(-1) DW) in the SIP lines grown under saline conditions was not as high as in the UBI lines (up to 3.12 micromol g(-1) DW). Therefore, the use of an ABA-inducible promoter was not more beneficial for de novo production of GB. Interestingly, saline growth conditions enhanced GB accumulation by up to 89% in the SIP lines, whereas up to 44% increase was seen in a UBI line. In all these cases the GB levels were many-fold below the range reported for plant species that produce GB naturally. In spite of lower GB concentrations, statistically greater levels of stress tolerance were found in SIP lines than in UBI lines, suggesting that the stress protection observed in SIP plants cannot be totally explained by the increase in the GB content.     

<Emphasis Type="Italic">Ds</Emphasis> insertion mutagenesis as an efficient tool to produce diverse variations for rice breeding

The availability of diversified germplasm resources is the most important for developing improved rice varieties with higher seed yield or tolerance to various biotic or abiotic stresses. Here we report an efficient tool to create increased variations in rice by maize Ac/Ds transposon (a gene trap system) insertion mutagenesis. We have generated around 20,000 Ds insertion rice lines of which majority are homozygous for Ds element. We subjected these lines to phenotypic and abiotic stress screens and evaluated these lines with respect to their seed yields and other agronomic traits as well as their tolerance to drought, salinity and cold. Based on this evaluation, we observed that random Ds insertions into rice genome have led to diverse variations including a range of morphological and conditional phenotypes. Such differences in phenotype among these lines were accompanied by differential gene expression revealed by GUS histochemical staining of gene trapped lines. Among the various phenotypes identified, some Ds lines showed significantly higher grain yield compared to wild-type plants under normal growth conditions indicating that rice could be improved in grain yield by disrupting certain endogenous genes. In addition, several 1,000s of Ds lines were subjected to abiotic stresses to identify conditional mutants. Subsequent to these screens, over 800 lines responsive to drought, salinity or cold stress were obtained, suggesting that rice has the genetic potential to survive under abiotic stresses when appropriate endogenous genes were suppressed. The mutant lines that have higher seed yielding potential or display higher tolerance to abiotic stresses may be used for rice breeding by conventional backcrossing combining with molecular marker-assisted selection. In addition, by exploiting the behavior of Ds to leave footprints upon remobilization, we have shown an alternative strategy to develop new rice varieties without foreign DNA sequences in their genome. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Constitutive expression of a meiotic recombination protein gene homolog, OsTOP6A1, from rice confers abiotic stress tolerance in transgenic Arabidopsis plants

Plant productivity is greatly influenced by various environmental stresses, such as high salinity and drought. Earlier, we reported the isolation of topoisomerase 6 homologs from rice and showed that over expression of OsTOP6A3 and OsTOP6B confers abiotic stress tolerance in transgenic Arabidopsis plants. In this study, we have assessed the function of nuclear-localized topoisomerase 6 subunit A homolog, OsTOP6A1, in transgenic Arabidopsis plants. The over expression of OsTOP6A1 in transgenic Arabidopsis plants driven by cauliflower mosaic virus-35S promoter resulted in pleiotropic effects on plant growth and development. The transgenic Arabidopsis plants showed reduced sensitivity to stress hormone, abscisic acid (ABA), and tolerance to high salinity and dehydration at the seed germination; seedling and adult stages as reflected by the percentage of germination, fresh weight of seedlings and leaf senescence assay, respectively. Concomitantly, the expression of many stress-responsive genes was enhanced under various stress conditions in transgenic Arabidopsis plants. Moreover, microarray analysis revealed that the expression of a large number of genes involved in various processes of plant growth and development and stress responses was altered in transgenic plants. Although AtSPO11-1, the homolog of OsTOP6A1 in Arabidopsis, has been implicated in meiotic recombination; the present study demonstrates possible additional role of OsTOP6A1 and provides an effective tool for engineering crop plants for tolerance to different environmental stresses. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A relationship between tolerance to dehydration of rice cell lines and ability for ABA synthesis under stress.

Plant dehydration is commonly caused by some adverse environmental conditions such as salinity, drought and freezing. As the plant hormone abscisic acid (ABA) is involved in responses to water stress, we studied its putative relationship with the degree of tolerance to these abiotic stresses. For this purpose we used cell lines that had been established from mature embryos of rice (Oryza sativa L, cvs. Bahia and Bomba), and selected by their high (L-T) or low (L-S) levels of tolerance to each type of stress. Tolerance of rice calli to either osmotic, saline, or freezing stress was generally improved by a previous treatment with ABA. This ABA effect was evident in those callus lines with low tolerance (L-S), as their ability to recover from stress increased up to three fold. Independent of the cultivar used, there were no significant differences in the endogenous ABA contents between untreated L-T and L-S lines. However, upon stress, the increase in endogenous ABA was higher in L-T than in L-S lines. These results, together with those obtained by using Fluridone, an inhibitor of ABA synthesis, show that differences in the level of cell tolerance to osmotic, saline and freezing stress are related to their different capacity of ABA synthesis under stress conditions.     

Overexpression of the mitogen-activated protein kinase gene OsMAPK33 enhances sensitivity to salt stress in rice (Oryza sativa L.)

Mitogen-activated protein kinases (MAPK) signalling cascades are activated by extracellular stimuli such as environmental stresses and pathogens in higher eukaryotic plants. To know more about MAPK signalling in plants, aMAPK cDNA clone, OsMAPK33, was isolated from rice. The gene is mainly induced by drought stress. In phylogenetic analysis, OsMAPK33 (Os02g0148100) showed approximately 47-93% identity at the amino acid level with other plant MAPKs. It was found to exhibit organ-specific expression with relatively higher expression in leaves as compared with roots or stems, and to exist as a single copy in the rice genome. To investigate the biological functions of OsMAPK33 in rice MAPK signalling, transgenic rice plants that either overexpressed or suppressed OsMAPK33 were made. Under dehydration conditions, the suppressed lines showed lower osmotic potential compared with that of wild-type plants, suggesting a role of OsMAPK33 in osmotic homeostasis. Nonetheless, the suppressed lines did not display any significant difference in drought tolerance compared with their wild-type plants. With increased salinity, there was still no difference in salt tolerance between OsMAPK33-suppressed lines and their wild-type plants. However, the overexpressing lines showed greater reduction in biomass accumulation and higher sodium uptake into cells, resulting in a lower K+/Na+ ratio inside the cell than that in the wild-type plants and OsMAPK33-suppressed lines. These results suggest that OsMAPK33 could play a negative role in salt tolerance through unfavourable ion homeostasis. Gene expression profiling of OsMAPK33 transgenic lines through rice DNA chip analysis showed that OsMAPK33 altered expression of genes involved in ion transport. Further characterization of downstream components will elucidate various biological functions of this novel rice MAPK.     

UBI4, the polyubiquitin gene of Saccharomyces cerevisiae , is a heat shock gene that is also subject to catabolite derepression control

 Carbon and nitrogen regulation of UBI4, the stress-inducible polyubiquitin gene of Saccharomyces cerevisiae, was investigated using a UBI4 promoter-LacZ fusion gene (UBI4-LacZ). Expression of this gene in cells grown on different media indicated that the UBI4 promoter is more active during growth on respiratory than on fermentable carbon sources but is not subject to appreciable control by nitrogen catabolite repression. UBI4-LacZ expression was virtually identical in cells having constitutively high (ras2, sra1-13) or constitutively low (ras2) levels of cyclic AMP-dependent protein kinase activity, indicating that this kinase does not exert a major influence on UBI4 expression. Catabolite derepression control of the UBI4 promoter was confirmed by measurements of UBI4-LacZ expression in hap mutant and wild-type strains before and after transfer from glucose to lactate. Mutagenesis of the perfect consensus for HAP2/3/4 complex binding at position −542 resulted in considerable reduction of UBI4 promoter derepression with respiratory adaptation in HAP wild-type cells and abolished the reduced UBI4-LacZ derepression normally seen when aerobic cultures of the hap1 mutant are transferred from glucose to lactate. This HAP2/3/4 binding site is therefore a major element contributing to catabolite derepression of the UBI4 promoter, although data obtained with hap1 mutant cells indicated that HAP1 also contributes to this derepression. The HAP2/3/4 and HAP1 systems are normally found to activate genes for mitochondrial (respiratory) functions. Their involvement in mediating higher activity of the UBI4 promoter during respiratory growth may reflect the contribution of UBI4 expression to tolerance of oxidative stress. Received: 3 June 1996 / Accepted: 20 August 1996     

UBI4, the polyubiquitin gene of Saccharomyces cerevisiae, is a heat shock gene that is also subject to catabolite derepression control

Carbon and nitrogen regulation of UBI4, the stress-inducible polyubiquitin gene of Saccharomyces cerevisiae, was investigated using a UBI4 promoter-LacZ fusion gene (UBI4-LacZ). Expression of this gene in cells grown on different media indicated that the UBI4 promoter is more active during growth on respiratory than on fermentable carbon sources but is not subject to appreciable control by nitrogen catabolite repression. UBI4-LacZ expression was virtually identical in cells having constitutively high (ras2, sra1-13) or constitutively low (ras2) levels of cyclic AMP-dependent protein kinase activity, indicating that this kinase does not exert a major influence on UBI4 expression. Catabolite derepression control of the UBI4 promoter was confirmed by measurements of UBI4-LacZ expression in hap mutant and wild-type strains before and after transfer from glucose to lactate. Mutagenesis of the perfect consensus for HAP2/3/4 complex binding at position ?542 resulted in considerable reduction of UBI4 promoter derepression with respiratory adaptation in HAP wild-type cells and abolished the reduced UBI4-LacZ derepression normally seen when aerobic cultures of the hap1 mutant are transferred from glucose to lactate. This HAP2/3/4 binding site is therefore a major element contributing to catabolite derepression of the UBI4 promoter, although data obtained with hap1 mutant cells indicated that HAP1 also contributes to this derepression. The HAP2/3/4 and HAP1 systems are normally found to activate genes for mitochondrial (respiratory) functions. Their involvement in mediating higher activity of the UBI4 promoter during respiratory growth may reflect the contribution of UBI4 expression to tolerance of oxidative stress.     

Overexpression of a ItICE1 gene from Isatis tinctoria enhances cold tolerance in rice

ItICE1, a ICE1-like gene, was isolated from a cDNA library from cold-treated woad (Isatis tinctoria L.) tissues. Expression analysis revealed that the ItICE1 gene was expressed constitutively and was predominant in the leaves of woad seedlings and that its mRNA accumulation was altered by salt stress and abscisic acid application, but not by dehydration and cold stresses. The transgenic rice lines overexpressing ItICE1 showed no growth retardation under normal growth conditions as well as enhanced tolerance to cold stress. Physiological assays showed that ItICE1 not only increased the accumulation of free proline and chlorophyll in transgenic rice lines under cold stress, but also reduced malondialdehyde content and electrolyte leakage. The analysis of gene expression in transgenic rice lines indicated that the maize ubiquitin promoter could respond to cold stress and upregulate ItICE1 gene expression level under its control. Under cold stress conditions, transgenic lines had a remarkably increased expression of OsDREB1A, J013078A14, 001-125-G03, 001-023-B08 and J023042N13 compared to wild-type plants (P < 0.05), implying that ItICE1 functions in the CBF/DREB1 cold-response pathway. These results demonstrate that ItICE1 plays an important regulatory role in the improvement of tolerance to cold stress in rice and is potentially useful for improving the cold tolerance of other plants.     

Putrescine accumulation in Arabidopsis thaliana transgenic lines enhances tolerance to dehydration and freezing stress

Polyamines have been globally associated to plant responses to abiotic stress. Particularly, putrescine has been related to a better response to cold and dehydration stresses. It is known that this polyamine is involved in cold tolerance, since Arabidopsis thaliana plants mutated in the key enzyme responsible for putrescine synthesis (arginine decarboxilase, ADC; EC 4.1.1.19) are more sensitive than the wild type to this stress. Although it is speculated that the overexpression of ADC genes may confer tolerance, this is hampered by pleiotropic effects arising from the constitutive expression of enzymes from the polyamine metabolism. Here, we present our work using A. thaliana transgenic plants harboring the ADC gene from oat under the control of a stress-inducible promoter (pRD29A) instead of a constitutive promoter. The transgenic lines presented in this work were more resistant to both cold and dehydration stresses, associated with a concomitant increment in endogenous putrescine levels under stress. Furthermore, the increment in putrescine upon cold treatment correlates with the induction of known stress-responsive genes, and suggests that putrescine may be directly or indirectly involved in ABA metabolism and gene expression.Key words: cold acclimation, dehydration, putrescine, polyamines, stress     

异源表达CkLEA1基因增强了拟南芥对非生物胁迫的耐受性

植物在生长过程中会受到各种非生物胁迫的伤害,导致生长发育和产量受到严重影响,胚胎晚期丰富蛋白（late embryogenesis abundant proteins,LEA蛋白）在植物抵抗非生物胁迫过程中起着重要的保护作用。在前期的研究基础上,将受多种胁迫诱导的柠条锦鸡儿CkLEA1（GenBank登录号KC309408）基因转入野生型拟南芥,通过实时荧光定量PCR从7株T₃代纯合体中筛选出3个转基因株系做进一步研究。种子萌发率实验发现,在200 mmol/L NaCl和400 mmol/L甘露醇处理下,转基因株系萌发率均高于野生型拟南芥。干旱处理2周大的幼苗后,转基因株系明显比野生型更抗旱,存活率高于野生型,并且失水率低于野生型。同时,转基因株系积累了较少的丙二醛（MDA）,超氧化物歧化酶（SOD）活性和谷胱甘肽（GSH）含量也高于野生型。这些结果表明,柠条锦鸡儿CkLEA1基因在种子萌发阶段提高了拟南芥对盐和渗透胁迫的耐受性,并且提高了转基因拟南芥幼苗生长阶段对干旱胁迫的抵抗能力。     

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.     

Overexpression of OsHsp17.0 and OsHsp23.7 enhances drought and salt tolerance in rice

Heat shock proteins (Hsps) play an important role in plant stress tolerance. We previously reported that expression of OsHsp17.0 and OsHsp23.7 could be enhanced by heat shock treatment and/or other abiotic stresses. In this paper, stress tolerance assays of transgenic rice plants overexpressing OsHsp17.0 and OsHsp23.7 have been carried out. Both OsHsp17.0-OE and OsHsp23.7-OE transgenic lines demonstrated higher germination ability compared to wild-type (WT) plants when subjected to mannitol and NaCl. Phenotypic analysis showed that transgenic rice lines displayed a higher tolerance to drought and salt stress compared to WT plants. In addition, transgenic rice lines showed significantly lower REC, lower MDA content and higher free proline content than WT under drought and salt stresses. These results suggest that OsHsp17.0 and OsHsp23.7 play an important role in rice acclimation to salt and drought stresses and are useful for engineering drought and salt tolerance rice.