Annual Report 2003

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AFig. 3(A and B) Inside of wild-type (A) and phsup1-tm1 (B) petunia flowers. (Cand D) Flower diagrams of wild-type (C) and phsup1-tm1 (D) petunia flowers.Sepals, petals, stamens and a pistil is formed in whorls 1, 2, 3 and 4,respectively. (E and F) Developing flowers of wild-type (E) and phsup1-tm1 (F) petunia. An arrow in (F) indicates ectopic tissue at the base ofphsup1-tm1 flower. (G and H) Transverse sections of anthers in wild-type(G) and phsup1-tm1 (H) petunia flowers. (1-K) PhSUP1 transcript distributionby in situ hybridization. Longitudinal section of young flower (I) andits schematic representation compared with that of SUP in Arabidopsisflower (K). Shown in (J) is a transverse section of developing anther.Hybridization signals are seen between pairs of closed triangles. st, stamen;se, sepal; pe, petal; ov, ovary.compartments (two each of outer and innerlocules), however, the inner locule was notseparated into two in this mutant. In accordancewith this phenotype, PhSUP1 transcriptswere detected in connective tissues ofthe anther (Fig. 3J). These findings indicatethat PhSUP1 plays multifaceted roles in thefloral-organ morphogenesis in the petunia,and the functions are in part common to, butsubstantially different from those ofArabidopsis SUP.Function of photoreceptors in riceIn order to elucidate the function of cryptochrome(blue light receptor: cry) genes inrice, we isolated cDNA clones (OsCRY1a andOsCRY1b) homologous to the ArabidopsisCRY1 (AtCRY1) gene. We also identified asequence homologous to AtCRY2 in the ricegenome, and obtained the correspondingcDNA (OsCRY2). OsCRY1s and OsCRY2show the moderate homology (59%) in aminoacid sequences in the N-half but no homology(16-17%) in the C-half. OsCRY1s weremainly expressed in green tissues, whileOsCRY2 was strongly expressed in etiolatedseedlings and substantially in the flower andcallus.The rice SnRK1 (sucrose non-fermenting-1 related protein kinase 1) family consists ofthree genes, OSK1, OSK24 and OSK35.Promoter::GUS expression analysis showedthat the OSK24P::GUS expression wasdetected in the pericarp at the early stagewith a shift to the endosperm as theendosperm cells were formed, and around15DAF, when cell division of cellularendosperm was almost finished, GUS stainingwas confined to endosperm cells. Theshifting pattern of the OSK24 expressionwas correlated with the appearance of starchgranules in each tissue, which was alsoobserved at another temporal sink organ, thebasal part of the leaf sheath. These findings,together with those obtained by in situmRNA localization experiments, suggestthat OSK24 (rice SnRK1b) most probablyhas a special role in carbohydrate metabolismof the sink organs.Alternative oxidase (AOX), a terminal oxidaseof alternative respiration, is thought tobe closely associated with resistance to environmentalstress. AOX transcript wasinduced by addition of salycilic acid, propionicacid, and sodium vanadidate. At thesame time, cytosolic acidification wasobserved with 31 P-NMR on addition of thosereagents.Mechanism of tolerance to environmentalstressSalt toleranceVacuolar Na + /H +antiporter transportsNa +from the cytoplasm to vacuoles, andplays important roles in salt-tolerance ofplants. Overexpression of three vacuolarNa + /H + antiporter genes, isolated from rice106 Annual Report 2003
(OsNHX1), barley (HvNHX1) and barilla(SkNHX1), suppressed the Na + -, Li + - andhygromycin sensitivity of the yeast nhx1mutant. In addition, the genes suppressedthe sensitivity to high K + concentration. Theexpression of the genes was increased inplants treated with high concentration ofNaCl and KCl. These results suggest thatthe plant Na + /H + antiporters transport severalcations including Na + . The expression ofthe fusion protein of OsNHX1 and sGFP inrice cells showed that OsNHX1-sGFP wasdistributed in the vacuolar membrane.In retrotransposon (Tos17) insertionalmutant population, we isolated two independentOsNHX1 mutant lines, in which neitherOsNHX1 mRNA nor polypeptides were present.The growth of the mutant plants andcells treated with various stresses includingsalt stress was similar to that of wild-type,and there were no significant differences inthe contents of major ions between themutants and wild-type.Analysis of cell death mechanism inplantsEnhanced resistance to salt, cold andwound stress by overproduction of animalcell death suppressors Bcl-xL and Ced-9in tobacco cells – possible contributionthrough improved function of organellaPreviously, we reported that overproductionof animal cell death suppressors Bcl-xLand Ced-9 conferred enhanced resistance toUV-B and paraquat treatment in tobaccoplants. Here, we show that the progenycould germinate in 0.2 M NaCl or at 10˚Cunder light, while control plants could not(Fig. 4). Suspension-cultured Bcl-cells resistedNaCl treatment maintaining an activeSelection of transformed plants using theherbicide resistant geneWe cloned the cDNA for acetolactate synthase(ALS) with two point mutations,W548L and S627I, from the rice, conferringstrong resistance to a pyrimidinylcarboxy(PC) herbicide. The two-point mutated riceALS gene works functionally in rice withoutany adverse effect on rice. Since this gene isexpected to be an effective selection markergene for rice transformation with bispyribacsodium(BS), a selectable marker system consistingof W548L/S627I two-point mutatedrice ALS gene and BS is being developed as acommercial product for plant transformationin general.In addition, several mutations were introducedin an ALS gene artificially. TheP171H/R172S two-point mutated rice ALSgene is also considered to be useful as aselection marker for genetic transformationof rice used with sulfonylurea herbicides.One month at 10˚C at 15,000 lux (16L/8D)Fig. 4Enhanced germination of transgenic plants overproducing animal celldeath suppressor at 10˚C. Seeds were sown on agar medium at 10˚Cunder 15,000 lux for one month. Only overproducers of animal cell deathsuppressors can germinate in this condition.Intracellular pH8.07.26.45.64.8A600NaCl0.750.25CytoplasmVacuole0.5 M NaClWildNaClM65-24(Bcl-xL)-20 0 20 40 60 80 100 -20 0 20 40 60 80 100Time after treatment (min)Time after treatment (min)Fig. 5Change of cytoplasmic and vacuolar pHs caused by 0.5 M NaCl-treatment.Changes of pH in both the cytoplasm and vacuole were determinedby in vivo 31 P-NMR. Inner figure with black background showsquantitative cell death levels at A 600 based on Evans blue staining.Transgenic M65-24 cell line overproducing Bcl-xL resisted the treatment,maintaining the compartment between the cytoplasm and vacuole forlonger period with higher ratio of survived cells.8.07.26.45.64.8A6000.750.25CytoplasmVacuoleAnnual Report 2003 107
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Message from the PresidentIn cooper
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¡X-ray crystallographic studies of
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Topics of Research in This YearComp
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Elucidation of genomic structure ar
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the total library) were mapped onto
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observed among the ribosomal protei
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molecular analyses and morphologica
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with 5 IU equine chorionic gonadotr
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serious problem in rice production
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exogenous genes into a hymenopteran
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Isolation and characterization of B
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oids could be regenerated from endo
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Quantitative trait locus analyses o
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ion, induces hypercholesterolemia,
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is an important source for amino ac
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In this study, 1.4 and 1.6 kb-long
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aqueous solution using cyanuric chl
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Developing a waste selection device
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decreased every five days by 5˚C,
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ticides. If the viral dose that kil
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In addition, the number of crown ro
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Molecular structure of the GARP fam
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X-ray crystallographic studies ofSt
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Rapid and high resolution QTL analy
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Plant regeneration system through m
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Table 1 Transformation Efficiency a
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from the tissues of mutants. The In
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the regenerated plants were transpl
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plants and fungi, and RNA interfere
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Page 63 and 64: efforts are made to obtain single m
Page 65 and 66: was carried out by shotgun sequenci
Page 67 and 68: epetitive sequence specific to Oryz
Page 69 and 70: 75 µM of ABA at 25ºC. More than 8
Page 71 and 72: Change of the molecular weightforms
Page 73 and 74: A unique landrace group recognizedb
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Page 83 and 84: that antigen-specific regulatory T
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Page 89 and 90: Insect Genetics and Evolution Depar
Page 91 and 92: activity, which was found to detoxi
Page 93 and 94: Insect Biomaterial and Technology D
Page 95 and 96: peptides obtained from sericin a re
Page 97 and 98: DNA markers for Nid-1, a resistance
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Page 101 and 102: Plant Science DivisionThe Plant Sci
Page 103 and 104: mutants have been studied in relati
Page 105 and 106: have verified that candidate genes
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Page 109 and 110: mammalian cells, respectively, inhi
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Page 115 and 116: nation rates. However, in our attem
Page 117 and 118: Yeast two-hybrid assay showed inter
Page 119 and 120: and a few NADPH-cytochrome P450 oxi
Page 121 and 122: the R0(53%).Foreign genes (35S + np
Page 123 and 124: gamma ray from 44 TBq 60 Co was use
Page 125 and 126: List of PublicationOriginal Papers1
Page 127 and 128: 35 Fujisaki S, Mizoguchi Y, Takahas
Page 129 and 130: 71 Imai K, Khandoker MY, Yonai M, T
Page 131 and 132: 107 Kiuchi S, Inage Y, Hiraiwa H, U
Page 133 and 134: 147 Miyamoto Y, Sakumoto R, Sakabe
Page 135 and 136: 184 Ochi A, Hossain KS, Magoshi J,
Page 137 and 138: 219 Takahashi M, Nagai T, Okamura N
Page 139 and 140: 258 Watanabe H, Nakashima K, Saito
Page 141 and 142: Author Department Paper.No.Yasushi
Page 143 and 144: Author Department Paper.No.Sadao Wa
Page 145 and 146: Monograph1 Magoshi J, Nakamura S (2
Page 147 and 148: on Lepidopteran Genomics”.NIAS-CO
Page 149 and 150: tion of the high-quality draft sequ
Page 151 and 152: Executive Members andResearch Staff
Page 153 and 154: Insect Growth Regulation Laboratory
Page 155 and 156: Sericultural Science LaboratoryMole
Page 157 and 158: Members of NIAS EvaluationComittee(
Page 159 and 160: Annual Report 2003 153
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