We screened a rice (Oryza sativa L. 'Nipponbare') full-length cDNA expression library through functional complementation in yeast (Saccharomyces cerevisiae) to find novel cation transporters involved ...in salt tolerance. We found that expression of a cDNA clone, encoding the rice homolog of Shaker family K⁺ channel KAT1 (OsKAT1), suppressed the salt-sensitive phenotype of yeast strain G19 (Δena1-4), which lacks a major component of Na⁺ efflux. It also suppressed a K⁺-transport-defective phenotype of yeast strain CY162 (Δtrk1Δtrk2), suggesting the enhancement of K⁺ uptake by OsKAT1. By the expression of OsKAT1, the K⁺ contents of salt-stressed G19 cells increased during the exponential growth phase. At the linear phase, however, OsKAT1-expressing G19 cells accumulated less Na⁺ than nonexpressing cells, but almost the same K⁺. The cellular Na⁺ to K⁺ ratio of OsKAT1-expressing G19 cells remained lower than nonexpressing cells under saline conditions. Rice cells overexpressing OsKAT1 also showed enhanced salt tolerance and increased cellular K⁺ content. These functions of OsKAT1 are likely to be common among Shaker K⁺ channels because OsAKT1 and Arabidopsis (Arabidopsis thaliana) KAT1 were able to complement the salt-sensitive phenotype of G19 as well as OsKAT1. The expression of OsKAT1 was restricted to internodes and rachides of wild-type rice, whereas other Shaker family genes were expressed in various organs. These results suggest that OsKAT1 is involved in salt tolerance of rice in cooperation with other K⁺ channels by participating in maintenance of cytosolic cation homeostasis during salt stress and thus protects cells from Na⁺.
We examined the function and intracellular localization of the product of the Na+/H+ antiporter gene (OsNHX1) cloned from rice (Oryza sativa). OsNHX1 has the ability to suppress Na+, Li+ and ...hygromycin sensitivity of yeast nhx1 mutants and sensitivity to a high K+ concentration, a novel phenotype of the nhx1 mutants. Analysis using rice cells expressing a fusion protein of OsNHX1 and green fluorescent protein and Western blot analysis using antibodies specific for OsNHX1 confirmed the localization of OsNHX1 on the tonoplasts. These results indicate that the OsNHX1 gene encodes a vacuolar (Na+, K+)/H+ antiporter. Treatment with high concentrations of NaCl and KCl increased the transcript levels of OsNHX1 in rice roots and shoots. In addition, overexpression of OsNHX1 improved the salt tolerance of transgenic rice cells and plants. These results suggest that OsNHX1 on the tonoplasts plays important roles in the compartmentation of Na+ and K+ highly accumulated in the cytoplasm into the vacuoles, and the amount of the antiporter is one of the most important factors determining salt tolerance in rice.
Freeze-drying (FD) medium containing ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) is reported to be beneficial for maintenance of sperm DNA integrity after FD. Recently, ...trehalose has also been reported to have notable ability to stabilize the protein structure and biomembranes of sperm in a dry state. In this study, we examined the effect of a combination of EGTA and different concentrations of trehalose in FD medium on sperm DNA integrity and the in vitro development of IVM porcine oocytes after intracytoplasmic sperm injection (ICSI) using freeze-dried boar sperm. Ejaculated sperm from a boar were suspended in basic FD medium supplemented with 0, 3.75, 7.5, 15, 30, 60, or 90 mM trehalose and freeze-dried. After rehydration, the sperm in all groups were subjected to DNA damage detection using a Halomax kit. It was found that the level of DNA damage in 15-mM group was significantly lower than that in 0-mM group, and no difference was observed between the 15-, 7.5-, and 3.75-mM groups. Moreover, there were no significant differences in the DNA damage level among 0, 3.75 mM, and other groups treated with trehalose. When freeze-dried sperm were used for ICSI, the fertilization rates and blastocyst formation rates (observed at 10 hours and 6 days of IVC after ICSI, respectively) in the 7.5- and 15-mM groups were not different from those in 0-mM group. These results suggest that FD medium supplemented with trehalose at appropriate concentrations improves sperm DNA integrity, but does not improve fertilization and preimplantation embryo development of IVM oocytes following ICSI.
Canopy temperature can be a good indicator of stomatal conductance. To understand the genetic basis of phenotypic differences in stomatal conductance between average and high-yielding rice (Oryza ...sativa L.) cultivars, we conducted a quantitative trait locus (QTL) analysis of canopy temperature. We developed reciprocal series of backcross inbred lines (BC1F6) derived from a cross between the average-yielding japonica cultivar ‘Koshihikari’ and the high-yielding indica cultivar ‘Takanari’. A stable QTL, qCTd11 (QTL for canopy temperature difference on chromosome 11) on the short arm of chromosome 11, accounted for 10.4 and 19.8% of the total phenotypic variance in the two lines; the ‘Takanari’ allele decreased the canopy temperature difference value. A chromosome segment substitution line carrying the Takanari qCTd11 showed a greater reduction in canopy temperature than ‘Koshihikari’, and had higher stomatal conductance and photosynthetic rate. These results suggest that qCTd11 is not only involved in canopy temperature, but is also involved in both stomatal conductance and photosynthetic rate.
Many agronomic traits that are important in rice breeding are controlled by multiple genes. The extensive time and effort devoted so far to identifying and selecting such genes are still not enough ...to target multiple agronomic traits in practical breeding in Japan because of a lack of suitable plant materials in which to efficiently detect and validate beneficial alleles from diverse genetic resources. To facilitate the comprehensive analysis of genetic variation in agronomic traits among Asian cultivated rice, we developed 12 sets of chromosome segment substitution lines (CSSLs) with the japonica background, 11 of them in the same genetic background, using donors representing the genetic diversity of Asian cultivated rice. Using these materials, we overviewed the chromosomal locations of 1079 putative QTLs for seven agronomic traits and their allelic distribution in Asian cultivated rice through multiple linear regression analysis. The CSSLs will allow the effects of putative QTLs in the highly homogeneous japonica background to be validated.
Heading date, a crucial factor determining regional and seasonal adaptation in rice (Oryza sativa L.), has been a major selection target in breeding programs. Although considerable progress has been ...made in our understanding of the molecular regulation of heading date in rice during last two decades, the previously isolated genes and identified quantitative trait loci (QTLs) cannot fully explain the natural variation for heading date in diverse rice accessions.
To genetically dissect naturally occurring variation in rice heading date, we collected QTLs in advanced-backcross populations derived from multiple crosses of the japonica rice accession Koshihikari (as a common parental line) with 11 diverse rice accessions (5 indica, 3 aus, and 3 japonica) that originate from various regions of Asia. QTL analyses of over 14,000 backcrossed individuals revealed 255 QTLs distributed widely across the rice genome. Among the detected QTLs, 128 QTLs corresponded to genomic positions of heading date genes identified by previous studies, such as Hd1, Hd6, Hd3a, Ghd7, DTH8, and RFT1. The other 127 QTLs were detected in different chromosomal regions than heading date genes.
Our results indicate that advanced-backcross progeny allowed us to detect and confirm QTLs with relatively small additive effects, and the natural variation in rice heading date could result from combinations of large- and small-effect QTLs. We also found differences in the genetic architecture of heading date (flowering time) among maize, Arabidopsis, and rice.
Na
+/H
+ exchanger catalyzes the countertransport of Na
+ and H
+ across membranes. We isolated a rice cDNA clone the deduced amino acid sequence of which had homology with a putative Na
+/H
+ ...exchanger in
Saccharomyces cerevisiae, NHX1. The sequence contains 2330 bp with an open reading frame of 1608 bp. The deduced amino acid sequence is similar to that of NHX1 and NHE isoforms in mammals, and shares high similarity with the sequences within predicted transmembrane segments and an amiloride-binding domain. The expression of the gene was increased by salt stress. These results suggest that the product of the novel gene,
OsNHX1, functions as a Na
+/H
+ exchanger, and plays important roles in salt tolerance of rice.
We isolated two cDNA clones (OsCLC-1 and OsCLC-2) homologous to tobacco CLC-Nt1, which encodes a voltage-gated chloride channel, from rice (Oryza sativa L. ssp. japonica, cv. Nipponbare). The deduced ...amino acid sequences were highly conserved (87.9% identity with each other). Southern blot analysis of the rice genomic DNA revealed that OsCLC-1 and OsCLt-2 were single-copy genes on chromosomes 4 and 2, respectively. OsCLC-1 was expressed in most tissues, whereas OsCLC-2 was expressed only in the roots, nodes, internodes and leaf sheaths. The level of expression of OsCLC-1, but not of OsCLC-2, was increased by treatment with NaCl. Both genes could partly substitute for GEF1, which encodes the sole chloride channel in yeast, by restoring growth, under ionic stress. These results indicate that both genes are chloride channel genes. The proteins from both genes were immunochemically detected in the tonoplast fraction. Tagged synthetic green fluorescent protein which was fused to OsCLC-1 or OsCLC-2 localized in the vacuolar membranes. These results indicate that the proteins may play a role in the transport of chloride ions across the vacuolar membrane. We isolated loss-of-function mutants of both genes from a panel of rice mutants produced by the insertion of a retrotransposon, Tos17, in the exon region, and found inhibition of growth at all life stages.
Boar sperm freeze‐dried with trehalose showed a protective effect against sperm DNA fragmentation. However, normal fertilization and embryonic development were not improved. Damaged sperm may ...activate maternal DNA repair genes when injected into oocytes. Therefore, we investigated the expression profile of some DNA repair genes in porcine oocytes after intra‐cytoplasmic sperm injection. First, the expression levels of MGMT, UDG, XPC, MSH2, XRCC6 and RAD51 genes that are concerned with different types of DNA repair were examined in in vitro mature (IVM) oocytes injected with ejaculated sperm, or freeze‐dried sperm with or without trehalose. Quantitative reverse transcription polymerase chain reaction revealed that expression of six DNA repair genes in the oocytes at 4 h after injection did not differ among the four groups. Next, we investigated the gene expression levels of these genes at different stages of maturation. The relative expression levels of UDG and XPC were significantly up‐regulated in mature oocytes compared with earlier stages. Furthermore, there was an increased tendency in relative expression of MSH2 and RAD51. These results suggested two possible mechanisms that messenger RNA of DNA repair genes are either accumulated during IVM to be ready for fertilization or increased expression levels of DNA repair genes in oocytes caused by suboptimal IVM conditions.
A two-point mutated gene of acetolactate synthase (ALS) was isolated from rice cells, which was cultured together with an ALS-inhibiting herbicide, bispyribac-sodium (BS). The mutations involved ...residues of tryptophan at position 548 to leucine (W548L) and serine at position 627 to isoleucine (S627I). The ALS expressed in Escherichia coli from this gene showed resistance to multiple herbicides including pyrimidinylcarboxylate (PC), sulfonylurea and imidazolinone herbicides, and showed stronger resistance to PC herbicides than to other herbicides. BS, a PC herbicide, had almost no effect on the enzyme even at 100 μM, which is an approximately 10,000-fold higher concentration than the concentration required for 50% inhibition of the wild-type. The resistance level of W548L/S627I mutating ALS to BS was stronger than the additive effect predicted from the degree of resistance of each single amino acid mutated ALS. Transformed rice cells carrying this gene and a regenerated rice plant expressed resistance to BS, suggesting that this gene is useful as a selectable marker for introducing foreign traits into rice when used with PC herbicides.
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