Nucleotide Sequence of a 28-kbp Portion of Rice Mitochondrial DNA: the Existence of Many Sequences That Correspond to Parts of Mitochondrial Genes in Intergenic Regions

The nucleotide sequence of a 27,588-bp region of rice mitochondrialDNA was determined. This sequence contains putative genes thatencode initiator methionine tRNA (trnfM), subunits III (nad3)and IV (nad4) of the NADH dehydrogenase complex, and ribosomalproteins S3 (rps3), S12 (rps12) and L16 (rp116). An open readingframe that contains sequences homologous to parts of rps2 andatpA is also present. In addition to these regions, there aremany short sequences with homology to fragments of mitochondrialDNAs from rice or other plants. These sequences may be remnantsof multiple rearrangements of the genome and their presenceseems to explain, in part, the large sizes of the mitochondrialgenomes of higher plants. (Received July 15, 1994; Accepted September 26, 1994)     

Cloning and Characterization of a Probenazole-Inducible Gene for an Intracellular Pathogenesis-Related Protein in Rice

Probenazole (3-allyloxy-1,2-benzisothiazole-1,1-dioxide) inducesdisease resistance in rice against rice blast fungus. To investigatethe molecular mechanism of probenazole-induced resistance, weisolated and characterized a cDNA clone of a probenazole-induciblegene in rice, which encoded a protein designated PBZ1. Sequenceanalysis revealed that significant homology at the amino acidlevel exists between the predicted PBZ1 protein and intracellularpathogenesis-related (IPR) proteins. Accumulation of PBZ1 mRNAwas not induced by wounding, but markedly induced by inoculationwith rice blast fungus. In addition, it was induced sooner byinoculation with rice blast fungus. In addition, it was inducedsooner by inoculation with an incompatible race than that witha compatible race. On the other hand, when the accumulationof the PBZ1 mRNA was examined after treatment with probenazole-relatedcompounds, it was not fully correlated with anti-rice blastactivity. However, it was induced after treatement with N-cyanomethyl-2-chloro-isonicotinamide(NCI), which belongs to another group of compounds known toinduce disease resistance. Thus, although the accumulation ofthe PBZ1 mRNA was not fully correlated with anti-rice blastactivity, our findings suggest that the PBZ1 gene has an importantfunction during the disease resistance response in rice. (Received June 19, 1995; Accepted October 13, 1995)     

Molecular Cloning and Characterization of a cDNA for the r{beta} Subunit of a G Protein from Rice

We isolated a cDNA for the rß subunit of a heterotrimericG protein from rice (Oryza sativa L. cv. Nipponbare). The aminoacid sequence deduced from the cDNA was 76% and 94% homologusto the sequences of the rß subunits from Arabidopsisand maize (AGrß1 and ZGrß1), respectively. (Received July 28, 1995; Accepted December 25, 1995)     

A Far-Upstream Sequence of the Wheat Histone H3 Promoter Functions Differently in Rice and Tobacco Cultured Cells

The cis-regulatory function of a far-upstream sequence (–1,711to –186) of the promoter of the wheat gene for histoneH3 (TH012) was analyzed in cultured rice and tobacco cells ina transient expression system with the gene for rß-D-glucuronidaseas a reporter gene. The far-upstream sequence was necessaryfor full activity of the H3 promoter in rice cells but did notenhance the activity of the proximal promoter in tobacco cells.Dissection analysis of the far-upstream sequence revealed theexistence of several positive and negative cis-acting sequencesin this region, some of which functioned differently in riceand tobacco cells. In gain-of-function experiments with ricecells, the sequence from –848 to –704, containingthe CCAAT and octamer (CaCGGATC) motifs, functioned in an orientation-independentmanner, whereas the sequence from –703 to –486 functionedin an orientation-dependent manner. By contrast, both sequencesexhibited an orientation-dependent cis-function in tobacco cells.These findings suggest that some cis-regulatory sequences inthe far-upstream region of the H3 promoter function differentlyin rice and tobacco cells. (Received May 10, 1995; Accepted August 8, 1995)     

Distribution of Endogenous Gibberellins in Vegetative Shoots of Rice

Levels of endogenous gibberellins in rice seedlings (Oryza sativaL., cv. Nipponbare) were compared between young and old leavesat the 4- and 5-leaf stages. The levels of GA₁, GA₁₉ and GA₅₃were higher in the youngest leaf than in older leaves at the5-leaf stage, but they did not differ significantly betweenthe leaf sheath and the leaf blade. At the 4-leaf stage, thelevel of GA₁, was highest in the third leaf sheaths which containedyoung elongating tissues. These results indicate that gibberellinsare synthesized in young vegetative tissues to promote theirelongation growth. The levels of GA₁ in the youngest leaf sheathsof two cultivars of dwarf rice, Tan-ginbozu and Waito-C, wereapproximately 10% of that in the normal rice at the 5-leaf stage.This result could explain the retardation of shoot elongationin these dwarf cultivars. (Received February 15, 1995; Accepted June 1, 1995)     

Tobacco Nuclear Genes for Photosystem I Subunits, psaD, psaE and psaH Share an Octamer Motif Bound with Nuclear Proteins

In Nicotiana sylvestris, nuclear-encoded photosystem I (PSI)genes, psaD, psaE and psaH, share an octamer motif bound withthree phosphoproteins. This motif is not found in the chloroplastgenome. From the view point of endosymbiont hypothesis, theseresults suggest that a set of ancient PSI genes acquired a commoncis-element in the nucleus after they were transferred fromthe ancestral organelle. (Received March 20, 1995; Accepted August 9, 1995)     

A Step in the Biosynthesis of Gibberellins that Is Controlled by the Mutation in the Semi-Dwarf Rice Cultivar Tan-Ginbozu

Genetic analysis and a comparison of endogenous levels of gibberellinsbetween the semi-dwarf rice cultivar Tan-ginbozu and the correspondingnormal cultivar Ginbozu have confirmed that Tan-ginbozu is agibberellin deficient mutant and that the semi-dwarfism of Tan-ginbozuis controlled by a single recessive gene. A step in the biosynthesisof gibberellins that is blocked by the mutation in Tan-ginbozuhad been considered to be the synthesis of ent-kaurene or anearlier step. However, the rate of production of ent-kaureneby Tan-ginbozu was almost the same as that by Ginbozu. By contrast,accumulation of only a small amount of ent-kaurene was detectedin Tan-ginbozu, and the amount that accumulated was similarto that in Ginbozu that had been treated with 6.9 x 10^-8 M uniconazole-P(an effective inhibitor of three oxidative steps in the pathwayfrom ent-kaurene to ent-kaurenoic acid via entkaurenol and ent-kaurenal).The height of the treated Ginbozu plants was reduced to thesame as that of Tanginbozu plants. Resembling Tan-ginbozu plants,Ginbozu plants that had been treated with uniconazole-P respondedwell to ent-kaurenoic acid and slightly to ent-kaurene and ent-kaurenol.Since the growth-promoting activity of enf-kaurenal in Tan-ginbozuwas similar to that of ent-kaurene, our results suggest thatthe mutation in Tan-ginbozu blocks the three oxidative stepswhereby ent-kaurene is converted to ent-kaurenoic acid. (Received June 9, 1995; Accepted February 15, 1996)     

Changes in the Chlorophyll Contents of Leaves and in Levels of Cytokinins in Root Exudates during Ripening of Rice Cultivars Nipponbare and Akenohoshi

Changes in fluxes of cytokinins in exudates transported viathe xylem from roots of rice plants cvs. Nipponbare (a standardJapanese cultivar) and Akenohoshi (a slowly senescing cultivar)were measured by mass spectrometry with deuterium-labeled standards.The fluxes of zeatin (Z), trans-ribosylzeatin (trans-RZ), N⁶-isopentenyladenine(iP), and "conjugated Z" (Z in the hydrolysates of highly polarfractions) decreased from heading to the late ripening stagein both cultivars. At the late ripening stage, iP and Z couldno longer be detected, while the flux of N⁶-isopentenyladenosine(iPA) increased slightly. In Akenohoshi, conjugated Z was thepredominant cytokinin from heading to the middle of the ripeningstage. The flux of each of the cytokinins in Akenohoshi washigher than that in Nipponbare at every time point, with theexception of the flux of iPA just after heading. The total concentrationof cytokinins in the xylem exudate of Akenohoshi was higherthan that of Nipponbare after the middle of ripening stage.The chlorophyll content of the third leaves, which were senescingrapidly, was significantly correlated with the flux of totalcytokinins per plant or per unit leaf area. These results suggestthat the larger amounts of cytokinins, in particular conjugatedZ, transported from the roots to the shoots caused the slowsenescence of leaves in Akenohoshi during the ripening stage. (Received May 9, 1994; Accepted July 1, 1995)     

Molecular Cloning and Characterization of cDNA for a Rice Protein that Contains Seven Repetitive Segments of the Trp-Asp Forty-Amino-Acid Repeat (WD-40 Repeat)

A cDNA clone for a polypeptide that contained seven repetitivesegments of the Trp-Asp forty-amino-acid repeat (WD-40 repeat)was isolated from a cDNA library prepared from the greeningleaves of rice. The cDNA was 1,285 bp long and contained anopen reading frame that encoded a protein of 334 amino acidresidues, which was designated it RWD (rice protein containingthe WD-40 repeat). RWD exhibited greater homology to a groupof receptor for activated C-kinase (RACK), a product of auxin-regulatedgene from cultured cells (arcA) and a Chlamydomonas ßsubunit-like polypeptide (Cblp) rather than to the ßsubunits of heterotrimeric G protein complexes. The mRNA forRWD (1.3 kb) was found in all organs of rice plants, in particular,in roots. Therefore, RWD is suggested to be a protein that isexpressed constitutively. (Received September 27, 1994; Accepted February 8, 1995)     

Partial Male Sterility in Transgenic Tobacco Carrying Antisense and Sense PAL cDNA under the Control of a Tapetum-Specific Promoter

Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5 [EC] ) catalyzes thefirst step in the synthesis of phenylpropanoids. An antisenseor sense PAL cDNA of sweet potato under the control of a tapetum-specificpromoter of rice was introduced into tobacco. A reduction inpollen fertility was observed in two out of seventeen antisensePAL transformants and in six out of nineteen sense PAL transformants.The pollen fertility of these plants ranged from 8% to 60%.The distorted pollen grains that did not germinate lacked starchand flavonols. PAL activity in anthers at the microspore stagewas also reduced to some extent and the level of PAL activitywas positively correlated with the number of fertile pollengrains at the flowering stage. Although it was unclear how theantisense or sense transgene affected the PAL activity in anthers,our results clearly demonstrate that the PAL activity in theanther tapetum has a significant effect on the development ofmicrospores. (Received October 9, 1995; Accepted March 10, 1996)     

CYP9A17v2组成型过量表达参与棉铃虫对拟除虫菊酯的抗性

微粒体细胞色素P450氧化酶介导的解毒代谢增强是棉铃虫Helicoverpa armigera对拟除虫菊酯类杀虫剂产生抗性的主要原因。作者前期的研究表明, CYP9A12和CYP9A14组成型过量表达与棉铃虫YGF品系对拟除虫菊酯的高水平抗性相关, CYP9A12和CYP9A14的功能表达研究结果为其参与对拟除虫菊酯抗性提供了直接证据。本研究通过对棉铃虫CYP9A17v2的克隆、mRNA表达水平和功能表达的研究, 以期明确该基因是否参与棉铃虫对拟除虫菊酯的抗性。结果表明: CYP9A17v2与CYP9A12的氨基酸序列具有很高的相似性（94%）。与棉铃虫对照品系（YG）相比, CYP9A17v2在YGF抗性品系末龄幼虫脂肪体中具有10.9倍的组成型过量表达, 而在中肠中未发现过量表达。用酿酒酵母Saccharomyces cerevisiae异源表达的CYP9A17v2能够代谢多种拟除虫菊酯（顺式氰戊菊酯、溴氰菊酯和氟氯氰菊酯）。据此认为CYP9A17v2组成型过量表达参与了棉铃虫对拟除虫菊酯的抗性。至此, CYP9A亚家族中已有3个P450基因（CYP9A12, CYP9A14 和 CYP9A17v2）被证实参与了棉铃虫对拟除虫菊酯的氧化解毒代谢。     

Stoichiometries of Photosystem I and Photosystem II in Rice Mutants Differently Deficient in Chlorophyll b

Stoichiometries of photosystem I (PSI) and photosystem II (PSII)reaction centers in a cultivar of rice, Norin No. 8, and threechlorophyll b-deficient mutants derived from the cultivar wereinvestigated. Quantitation of PSI by photooxidation of P-700and chromatographic assay of vitamin K₁ showed that, on thebasis of chlorophyll, the mutants have higher concentrationsof PSI than the wildtype rice. Greater increases were observedin the PSII contents measured by photoreduction of Q_A, bindingof a radioactive herbicide and atomic absorption spectroscopyof Mn. Consequently, the PSII to PSI ratio increased from 1.1–1.3in the wild-type rice to 1.8 in chlorina 2, which contains noChl b, and to 2.0–3.3 in chlorina 11 and chlorina 14,which have chlorophyll a/b ratios of 9 and 13, respectively.Measurement of oxygen evolution with saturating single-turnoverflashes revealed that, whereas at most 20% of PSII centers areinactive in oxygen evolution in the wildtype rice, the non-functionalPSII centers amount to about 50% in the three mutant strains.The fluorescence induction kinetics was also analyzed to estimateproportions of the inactive PSII in the mutants. The data obtainedsuggest that plants have an ability to adjust the stoichiometryof the two photosystems and the functional organization of PSIIin response to the genetically induced deficiency of chlorophyllb. (Received July 29, 1994; Accepted February 7, 1996)     

Metabolism of Gibberellins in Cell-Free Extracts of Anthers from Normal and Dwarf Rice

Cell-free extracts were prepared from anthers of normal anddwarf rice (Oryza sativa L.), and the metabolism of radioisotope-labeledgibberellins in the extracts was analyzed by HPLC and gas chromatography-massspectrometry (GC/MS). GA₁₂ was converted to GA₁₅ and GA₃₄ inthe extracts. GA₂₀ was converted to GA¹, GA₈ and GA₂₉, but GA₉was converted only to GA₃₄. The extracts of the dwarf cultivar,Waito-C (dy mutant), showed the same 3ß-hydroxylationactivity as did those of the normal cultivar, Nihonbare, indicatingthat the dy gene is not expressed in the anthers. These resultssuggest that the regulation of the biosynthesis of gibberellinsin rice is organ-specific. (Received November 9, 1989; Accepted January 10, 1990)