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  • 1
    ISSN: 1573-5028
    Keywords: development ; glucuronidase reporter ; heat shock genes ; in situ hybridization ; tissue specificity ; transgenic plants
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The heat shock (hs) response during plant growth and development was analyzed in tobacco and Arabidopsis using chimaeric β-glucuronidase reporter genes (hs-Gus) driven by a soybean hs promoter. Fluorimetric measurements and histochemical staining revealed high Gus activities in leaves, roots, and flowers exclusively after heat stress. The highest levels of heat-inducible expression were found in the vascular tissues. Without heat stress, a developmental induction of hs-Gus was indicated by the accumulation of high levels of Gus in transgenic tobacco seeds. There was no developmental induction of hs-Gus in Arabidopsis seeds. In situ hybridization to the RNA of the small heat shock protein gene Athsp17.6 in tissue sections revealed an expression in heat-shocked leaves but no expression in control leaves of Arabidopsis. However, a high level of constitutive expression of hs gene was detected in meristematic and provascular tissues of the Arabidopsis embryo. The developmental and tissue-specific regulation of the hs response is discussed.
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  • 2
    ISSN: 1573-5028
    Keywords: osmotic-stress ; osmotin-like proteins ; pathogenesis-related proteins ; phytophthora infestans ; salicylic acid ; stress-inducible genes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have characterized three cDNAs encoding osmotin-like proteins from potato (Solanum commersonii) cell cultures. These cDNAs (pA13, pA35, and pA81) have extensive nucleotide identity in the coding regions but low homology in the 3′ non-coding sequences, and may encode three isoforms of potato pathogenesis-related (PR) type 5 proteins. Using gene-specific probes, RNA gel blot analyses showed constitutive accumulation of osmotin-like protein mRNAs in cell cultures, leaves, stems, roots and flowers, with high abundance in the roots and mature flowers. Treatments with abscisic acid (ABA), low temperature, and NaCl increased the accumulation of all three mRNAs in S. commersonii cell cultures and plants grown in vitro. Salicylic acid (SA), and wounding resulted in a moderate increase in the levels of pA13 and pA81 but not pA35 mRNAs. Infection with the fungus Phytophthora infestans activated strong and non-systemic expression of all three osmotin-like protein genes. The accumulation of osmotin-like proteins, however, was detected only in P. infestans-infected tissues but not in plants treated with ABA, SA, NaCl, low temperature, or wounding.
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  • 3
    ISSN: 1573-5028
    Keywords: 3-hydroxy-3-methylglutaryl coenzyme A reductase ; gene family ; Solanum tuberosum ; isoprenoid metabolism ; cis-acting elements ; pollen expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) catalyzes a key step in isoprenoid metabolism leading to a range of compounds that are important for the growth, development and health of the plant. We have isolated 7 classes of genomic clones encoding HMGR from a potato genomic library. Comparison of nucleic acid sequences reveals a high degree of identity between all seven classes of clones and the potato hmg 1 gene described by Choi et al. (Plant Cell 4: 1333, 1992), indicating that all are members of the same subfamily in potato. A representative member (hmg 1.2) of the most abundant class of genomic clones was selected for further characterization. Transgenic tobacco and potato containing the β-glucuronidase (GUS) reporter gene under the control of the hmg 1.2 promoter expressed GUS activity constitutively at a low level in many plant tissues. High levels of GUS activity were observed only in the pollen. GUS assays of isolated pollen, correlations of GUS activity with the HMGR activity of anthers, hmg 1.2 promoter deletion studies, and segregation analysis of the expression of hmg 1.2::GUS among the R2 pollen of R1 progeny plants demonstrated that the hmg 1.2 promoter controls pollen expression.
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  • 4
    ISSN: 1573-5028
    Keywords: BY-2 cells ; mRNA stability ; NT-1 ; post-transcriptional control
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Plants and other higher eukaryotes have the ability to recognize and target specific transcripts for rapid decay from among the majority of relatively stable mRNAs present within cells. However, little is known about the nature of unstable transcripts in plants, or the mechanisms that facilitate their rapid degradation. As a first step toward understanding how plants distinguish between unstable and stable transcripts, a novel differential screen was used to identify cDNAs for genes with unstable transcripts (GUTs), solely on the basis of the instability of their mRNAs. cDNA probes were prepared from tobacco cells that had been depleted of highly unstable mRNAs by treatment for 90 min with a transcriptional inhibitor, and from control, untreated cells. GUT clones were selected on the basis of weak hybridization to the former probe relative to the latter probe. Half-life measurements performed on the mRNAs hybridizing to eight GUT clones indicated that each was unstable, with a half-life on the order of about an hour or less. All eight of the cDNAs corresponded to new tobacco genes, and four showed sequence similarity with genes from other species, including the eukaryotic family of DNAJ homologs, a tomato wound-inducible protein, and histone H3. In addition to providing information about the types of transcripts that are inherently unstable in plants, the GUT clones should provide excellent tools for the identification of cis- and trans-acting determinants of mRNA instability.
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  • 5
    ISSN: 1573-5028
    Keywords: glycine-rich proteins (GRPs) ; subtractive hybridization ; somatic embryogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We report the first successful isolation by subtractive hybridization of a gene expressed specifically during somatic embryogenesis. Embryogenic cell clusters, 32–50 μm in diameter, were isolated by sieving and density-gradient centrifugation. The cDNA library was constructed from proglobulars which were formed from embryogenic cell clusters 3 days after transfer to auxin-free modified Lin and Staba's medium. For use as probe in screening, the same cDNA used for library construction was enriched for specific sequences using subtractive hybridization. The cDNA used for subtraction was prepared from suspension cultures 5 days after subculturing in auxin-containing medium. Nine independent differentially expressed cDNA clones were obtained from a screen of 150 000 recombinant phages. Northern analysis indicated one of these, CFM6, to be expressed specifically during somatic embryogenesis. In addition, one hybridizing transcript was detected in plantlet cotyledons, and two transcripts were detected in hypocotyls. Two separate and distinct hybridizing transcripts are expressed specifically in hypocotyl tissue. The amino acid sequence deduced from the nucleotide sequence of the CEM6 cDNA indicates that it encodes a glycine-rich protein containing a hydrophobic signal-sequence like domain. Its early embryo-specific expression and sequence characteristics suggest an important role as a cell wall protein in embryogenesis.
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  • 6
    ISSN: 1573-5028
    Keywords: chalcone synthase ; flavonoid ; flower development ; organ-specific gene expression ; phenylpropanoid
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Recent studies on chalcone synthase (CHS) and the related stilbene synthase (STS) suggest that the structure of chs-like genes in plants has evolved into different forms, whose members have both different regulation and capacity to code for different but related enzymatic activities. We have studied the diversity of chs-like genes by analysing the structure, expression patterns and catalytic properties of the corresponding enzymes of three genes that are active during corolla development in Gerbera hybrida. The expression patterns demonstrate that chs-like genes are representatives of three distinct genetic programmes that are active during organ differentiation in gerbera. Gchs1 and gchs3 code for typical CHS enzymes, and their gene expression pattern temporally correlates with flavonol (gchs1, gchs3) and anthocyanin (gchs1) synthesis during corolla development. Gchs2 is different. The expression pattern does not correlate with the pigmentation pattern, the amino acid sequence deviates considerably from the consensus of typical CHSs, and the catalytic properties are different. The data indicate that it represents a new member in the large superfamily of chs and chs-related genes.
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  • 7
    ISSN: 1573-5028
    Keywords: biosynthesis ; gene structure ; narbonin ; recombinant protein ; 2S globulin ; seed storage protein ; Victa narbonensis L.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract cDNA and genomic clones encoding narbonin, a 2S globulin from the seed of narbon bean (Vicia narbonensis L.), were obtained using the polymerase chain reaction (PCR) and sequenced. The full-length cDNA as well as genomic clones contain a single open reading frame (ORF) of 873 bp that encodes a protein with 291 amino acids comprising the mature narbonin polypeptide (M r ca. 33 100) and an initiation methionine. The deduced amino acid sequence lacks a transient N-terminal signal peptide. The genomic clones do not contain any intron. No homology was found to nucleic acid and protein sequences so far registered in sequence data libraries. The biosynthesis of narbonin during embryogenesis is developmentally-regulated and its pattern of synthesis closely resembles that of typical seed storage globulins. However, during seed germination narbonin was degraded very slowly, indicating that it may have other function than storage protein. Southern analysis suggests the existence of a small narbonin gene family. Narbonin genes were also found in four different species of the genus Vicia as well as in other legumes such as Canavalia ensiformis and Glycine max. In Escherichia coli a recombinant narbonin was produced which yielded crystals like those prepared from narbonin purified from seeds.
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  • 8
    ISSN: 1573-5028
    Keywords: 1-aminocyclopropane-1-carboxylic acid (ACC) ; ACC synthase ; ethylene ; potato ; ozone ; Solanum tuberosum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Acute or chronic exposure of potato plants to ozone (O3) induces ethylene production. We isolated a 1586 bp cDNA (pOIP-1) encoding 1-aminocyclopropane-1-carboxylate (ACC) synthase from a cDNA library constructed with mRNA extracted from O3-treated leaves. The clone has a 1365 bp open reading frame and a 221 bp trailing sequence. The active site found in all ACC synthases and 11 of the 12 amino acid residues conserved in aminotransferases are found in pOIP-1. Northern analysis showed that the mRNA encoding ACC synthase was detectable 1 h after the onset of O3 exposure, and the message increased over time as did ethylene production. Concurrent with the increased ACC synthase mRNA was a decrease in the message for the Rubisco small subunit (rbcS) with no change in the large subunit (rbcL). When the plants were treated with aminooxyacetic acid (AOA), both ethylene production and level of ACC synthase transcript were inhibited. The decline in rbcS was also inhibited by AOA suggesting a correlation between ethylene production and loss of rbcS. Based on nuclear run-on studies it appears that the increase in ACC synthase mRNA may result from O3-induced transcriptional activity.
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  • 9
    ISSN: 1573-5028
    Keywords: electron transfer ; light-harvesting complex I ; membrane localization ; photosynthesis ; processing site ; transit peptide
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We report here the isolation and nucleotide sequence of a cDNA clone encoding a phtosystem I polypeptide that is recognized by a polyclonal antibody prepared against subunit II of the photosystem I reaction center. The transit peptide processing site was determined to occur after Met50 by N terminal sequencing. The decuced sequence of this protein predicts that the polypeptide has a net positive charge (pI=9.6) and no membrane spanning regions are evident from the hydropathy plot. Based on these considerations and the fact that subunit II is solubilized by alkali treatment of thylakoids, we concluded that subunit II is an extrinsic membrane protein. The absence of hydrophobic regions characteristic of thylakoid transfer domains furthermore implies that subunit II is localized on the stromal side of the membrane.
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  • 10
    ISSN: 1573-5028
    Keywords: Spinacia oleracea ; chloroplast DNA ; inverted repeat transcription terminator
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The expression of the psbA, trnH-GUG and rps19′ genes from spinach chloroplasts, coding respectively for the 32 kDa protein, the tRNAHis (GUG), and the putative ribosomal protein CS19′, has been studied by cloning, Northern hybridization and 3′ and 5′ S1 mapping experiments. It is demonstrated that the putative transcription termination signal of the psbA gene does not function as a rho-independent terminator of transcription in E. coli, whatever its orientation. Evidence is presented suggesting that, in spinach, the psbA and trnH-GUG genes are probably cotranscribed. The 3′-OH extremities of transcripts observed downstream from the putative psbA terminator are interpreted as resulting from processing of the psbA precursor. Using different approaches, it is shown that the rps19′ gene, located on the other strand and overlapping the trnH-GUG gene, is not expressed.
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