A single limit dextrinase gene is expressed both in the developing endosperm and in germinated grains of barley.
The single gene encoding limit dextrinase (pullulan 6-glucanohydrolase; EC 184.108.40.206) in barley (Hordeum vulgare) has 26 introns that range in size from 93 to 822 base pairs. The mature polypeptide encoded by the gene has 884 amino acid residues and a calculated molecular mass of 97,417 D. Limit dextrinase mRNA is abundant in gibberellic acid-treated aleurone layers and in germinated grain. Gibberellic acid response elements were found in the promoter region of the gene. These observations suggest that the enzyme participates in starch hydrolysis during endosperm mobilization in germinated grain. The mRNA encoding the enzyme is present at lower levels in the developing endosperm of immature grain, a location consistent with a role for limit dextrinase in starch synthesis. Enzyme activity was also detected in developing grain. The limit dextrinase has a presequence typical of transit peptides that target nascent polypeptides to amyloplasts, but this would not be expected to direct secretion of the mature enzyme from aleurone cells in germinated grain. It remains to be discovered how the enzyme is released from the aleurone and whether another enzyme, possibly of the isoamylase group, might be equally important for starch hydrolysis in germinated grain. (+info)
Mannose inhibits Arabidopsis germination via a hexokinase-mediated step.
Low concentrations of the glucose (Glc) analog mannose (Man) inhibit germination of Arabidopsis seeds. Man is phosphorylated by hexokinase (HXK), but the absence of germination was not due to ATP or phosphate depletion. The addition of metabolizable sugars reversed the Man-mediated inhibition of germination. Carbohydrate-mediated regulation of gene expression involving a HXK-mediated pathway is known to be activated by Glc, Man, and other monosaccharides. Therefore, we investigated whether Man blocks germination through this system. By testing other Glc analogs, we found that 2-deoxyglucose, which, like Man, is phosphorylated by HXK, also blocked germination; no inhibition was observed with 6-deoxyglucose or 3-O-methylglucose, which are not substrates for HXK. Since these latter two sugars are taken up at a rate similar to that of Man, uptake is unlikely to be involved in the inhibition of germination. Furthermore, we show that mannoheptulose, a specific HXK inhibitor, restores germination of seeds grown in the presence of Man. We conclude that HXK is involved in the Man-mediated repression of germination of Arabidopsis seeds, possibly via energy depletion. (+info)
Cucumber cotyledon lipoxygenase during postgerminative growth. Its expression and action on lipid bodies.
In cucumber (Cucumis sativus), high lipoxygenase-1 (LOX-1) activity has been detected in the soluble fraction prepared from cotyledons of germinating seeds, and the involvement of this enzyme in lipid turnover has been suggested (K. Matsui, M. Irie, T. Kajiwara, A. Hatanaka  Plant Sci 85: 23-32; I. Fuessner, C. Wasternack, H. Kindl, H. Kuhn  Proc Natl Acad Sci USA 92: 11849-11853). In this study we have investigated the expression of the gene lox-1, corresponding to the LOX-1 enzyme. LOX-1 expression is highly coordinated with that of a typical glyoxysomal enzyme, isocitrate lyase, during the postgerminative stage of cotyledon development. In contrast, although icl transcripts accumulated in tissue during in vitro senescence, no accumulation of lox-1 mRNA could be observed, suggesting that lox-1 plays a specialized role in fat mobilization. LOX-1 is also known to be a major lipid body protein. The partial peptide sequences of purified LOX-1 and lipid body LOX-1 entirely coincided with that deduced from the lox-1 cDNA sequence. The data strongly suggest that LOX-1 and lipid body LOX-1 are derived from a single gene and that LOX-1 can exist both in the cytosol and on the lipid bodies. We constructed an in vitro oxygenation system to address the mechanism of this dual localization and to investigate the action of LOX-1 on lipids in the lipid bodies. LOX-1 cannot act on the lipids in intact lipid bodies, although degradation of lipid body proteins, either during seedling growth or by treatment with trypsin, allows lipid bodies to become susceptible to LOX-1. We discuss the role of LOX-1 in fat mobilization and its mechanism of action. (+info)
Cloning and characterization of TPE4A, a thiol-protease gene induced during ovary senescence and seed germination in pea.
A cDNA clone encoding a thiol-protease (TPE4A) was isolated from senescent ovaries of pea (Pisum sativum) by reverse transcriptase-polymerase chain reaction. The deduced amino acid sequence of TPE4A has the conserved catalytic amino acids of papain. It is very similar to VSCYSPROA, a thiol-protease induced during seed germination in common vetch. TPE4A mRNA levels increase during the senescence of unpollinated pea ovaries and are totally suppressed by treatment with gibberellic acid. In situ hybridization indicated that TPE4A mRNA distribution in senescent pea ovaries is different from that of previously reported thiol-proteases induced during senescence, suggesting the involvement of different proteases in the mobilization of proteins from senescent pea ovaries. TPE4A is also induced during the germination of pea seeds, indicating that a single protease gene can be induced during two different physiological processes, senescence and germination, both of which require protein mobilization. (+info)
Extragenic suppressors of the arabidopsis zwi-3 mutation identify new genes that function in trichome branch formation and pollen tube growth.
The plant cytoskeleton plays a pivotal role in determining the direction of cell wall expansion, and ultimately the cell's final shape. However, the mechanisms by which localized expansion events are initiated remain obscure. Mutational analysis of the trichome (plant hair) morphogenic pathway in Arabidopsis has identified at least eight genes that determine trichome branch number. One of these genes, ZWICHEL (ZWI), encodes a novel member of the kinesin superfamily of motor proteins. Mutations in the ZWI gene cause a reduction in the number of trichome branches. To identify additional genes involved in trichome branch initiation, we screened for extragenic suppressors of the zwi-3 mutation and isolated three suppressors that rescued the branch number defect of zwi-3. These suppressors define three genes, named suz, for suppressor of zwichel-3. All of the suppressors were shown to be allele specific. One of the suppressors, suz2, also rescued the trichome branch number defect of another branch mutant, furca1-2. Plants homozygous for suz2 have more than the wild-type number of trichome branches. This suggests that SUZ2 is a negative regulator of trichome branching and may interact with ZWI and FURCA1. The suz1 and suz3 mutants display no obvious phenotype in the absence of the zwi-3 mutation. The suz1 zwi-3 double mutants also exhibited a male-sterile phenotype due to a defect in pollen tube germination and growth, whereas both the suz1 and the zwi-3 single mutants are fertile. The synthetic male sterility of the suz1 zwi-3 double mutants suggests a role for SUZ1 and ZWI in pollen germination and pollen tube growth. DNA sequence analysis of the zwi-3 mutation indicated that only the tail domain of the zwi-3 protein would be expressed. Thus, the suz mutations show allele-specific suppression of a kinesin mutant that lacks the motor domain. (+info)
Molecular and biochemical properties and physiological roles of plant phospholipase D.
Recent advances have thrust the study of plant phospholipase D (PLD) into the molecular era. This review will highlight some of the recent progress made in elucidating the molecular and biochemical nature of plant PLDs as well as their roles in plant physiology. (+info)
Root formation in ethylene-insensitive plants.
Experiments with ethylene-insensitive tomato (Lycopersicon esculentum) and petunia (Petunia x hybrida) plants were conducted to determine if normal or adventitious root formation is affected by ethylene insensitivity. Ethylene-insensitive Never ripe (NR) tomato plants produced more below-ground root mass but fewer above-ground adventitious roots than wild-type Pearson plants. Applied auxin (indole-3-butyric acid) increased adventitious root formation on vegetative stem cuttings of wild-type plants but had little or no effect on rooting of NR plants. Reduced adventitious root formation was also observed in ethylene-insensitive transgenic petunia plants. Applied 1-aminocyclopropane-1-carboxylic acid increased adventitious root formation on vegetative stem cuttings from NR and wild-type plants, but NR cuttings produced fewer adventitious roots than wild-type cuttings. These data suggest that the promotive effect of auxin on adventitious rooting is influenced by ethylene responsiveness. Seedling root growth of tomato in response to mechanical impedance was also influenced by ethylene sensitivity. Ninety-six percent of wild-type seedlings germinated and grown on sand for 7 d grew normal roots into the medium, whereas 47% of NR seedlings displayed elongated tap-roots, shortened hypocotyls, and did not penetrate the medium. These data indicate that ethylene has a critical role in various responses of roots to environmental stimuli. (+info)
Differences in spatial expression between 14-3-3 isoforms in germinating barley embryos.
The family of 14-3-3 proteins is ubiquitous in eukaryotes and has been shown to exert an array of functions. We were interested in the possible role of 14-3-3 proteins in seed germination. Therefore, we studied the expression of 14-3-3 mRNA and protein in barley (Hordeum distichum L.) embryos during germination. With the use of specific cDNA probes and antibodies, we could detect individual expression of three 14-3-3 isoforms, 14-3-3A, 14-3-3B, and 14-3-3C. Each homolog was found to be expressed in barley embryos. Whereas protein levels of all three isoforms were constant during germination, mRNA expression was found to be induced upon imbibition of the grains. The induction of 14-3-3A gene expression during germination was different from that of 14-3-3B and 14-3-3C. In situ immunolocalization analysis showed similar spatial expression for 14-3-3A and 14-3-3B, while 14-3-3C expression was markedly different. Whereas 14-3-3A and 14-3-3B were expressed throughout the embryo, 14-3-3C expression was tissue specific, with the strongest expression observed in the scutellum and the L2 layer of the shoot apical meristem. These results show that 14-3-3 homologs are differently regulated in barley embryos, and provide a first step in acquiring more knowledge about the role of 14-3-3 proteins in the germination process. (+info)