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PubMed2000

Mitogen-activated protein kinase and abscisic acid signal transduction.

The phytohormone abscisic acid (ABA) is a classical plant hormone, responsible for regulation of abscission, diverse aspects of plant and seed development, stress responses and germination. It was found that ABA signal transduction in plants can involve the activity of type 2C-phosphatases (PP2C), calcium, potassium, pH and a transient activation of MAP kinase. The ABA signal transduction cascades have been shown to be tissue-specific, the transient activation of MAP kinase has until now only been found in barley aleurone cells. However, type 2C phosphatases are involved in the induction of most ABA responses, as shown by the PP2C-deficient abi-mutants. These phosphatases show high homology with phosphatases that regulate MAP kinase activity in yeast. In addition, the role of farnesyl transferase as a negative regulator of ABA responses also indicates towards involvement of MAP kinase in ABA signal transduction. Farnesyl transferase is known to regulate Ras proteins, Ras proteins in turn are known to regulate MAP kinase activation. Interestingly, Ras-like proteins were detected in barley aleurone cells. Further establishment of the involvement of MAP kinase in ABA signal transduction and its role therein, still awaits more study.

Abscisic Acid↗

PubMed1995

Molecular cloning, characterization and expression analysis of two catalase isozyme genes in barley.

Clones representing two distinct barley catalase genes, Cat1 and Cat2, were found in a cDNA library prepared from seedling polysomal mRNA. Both clones were sequenced, and their deduced amino acid sequences were found to have high homology with maize and rice catalase genes. Cat1 had a 91% deduced amino acid sequence identity to CAT-1 of maize and 92% to CAT B of rice. Cat2 had 72 and 79% amino acid sequence identities to maize CAT-2 and -3 and 89% to CAT A of rice. Barley, maize or rice isozymes could be divided into two distinct groups by amino acid homologies, with one group homologous to the mitochondria-associated CAT-3 of maize and the other homologous to the maize peroxisomal/glyoxysomal CAT-1. Both barley CATs contained possible peroxisomal targeting signals, but neither had favorable mitochondrial targeting sequences. Cat1 mRNA occurred in whole endosperms (aleurones plus starchy endosperm), in isolated aleurones and in developing seeds, but Cat2 mRNA was virtually absent. Both mRNAs displayed different developmental expression patterns in scutella of germinating seeds. Cat2 mRNA predominated in etiolated seedling shoots and leaf blades. Barley genomic DNA contained two genes for Cat1 and one gene for Cat2. The Cat2 gene was mapped to the long arm of chromosome 4, 2.9 cM in telomeric orientation from the mlo locus conferring resistance to the powdery mildew fungus (Erysiphe graminis f.sp. hordei).

Amino Acid Sequence↗

PubMed1991

Characterization of the level, target sites and inheritance of cytosine methylation in tomato nuclear DNA.

The tomato nuclear genome was determined to have a G + C content of 37% which is among the lowest reported for any plant species. Non-coding regions have a G + C content even lower (32% average) whereas coding regions are considerably richer in G + C (46%). 5-methyl cytosine was the only modified base detected and on average 23% of the cytosine residues are methylated. Immature tissues and protoplasts have significantly lower levels of cytosine methylation (average 20%) than mature tissues (average 25%). Mature pollen has an intermediate level of methylation (22%). Seeds gave the highest value (27%), suggesting de novo methylation after pollination and during seed development. Based on isoschizomer studies we estimate 55% of the CpG target sites (detected by Msp I/Hpa II) and 85% of the CpNpG target sites (detected by Bst NI/Eco RI) are methylated. Unmethylated target sites (both CpG and CpNpG) are not randomly distributed throughout the genome, but frequently occur in clusters. These clusters resemble CpG islands recently reported in maize and tobacco. The low G + C content and high levels of cytosine methylation in tomato may be due to previous transitions of 5mC----T. This is supported by the fact that G + C levels are lowest in non-coding portions of the genome in which selection is relaxed and thus transitions are more likely to be tolerated. This hypothesis is also supported by the general deficiency of methylation target sites in the tomato genome, especially in non-coding regions. Using methylation isoschizomers and RFLP analysis we have also determined that polymorphism between plants, for cytosine methylation at allelic sites, is common in tomato. Comparing DNA from two tomato species, 20% of the polymorphisms detected by Bst NI/Eco RII could be attributed to differential methylation at the CpNpG target sites. With Msp I/Hpa II, 50% of the polymorphisms were attributable to methylation (CpG and CpNpG sites). Moreover, these polymorphisms were demonstrated to be inherited in a mendelian fashion and to co-segregate with the methylation target site and thus do not represent variation for transacting factors that might be involved in methylation of DNA. The potential role of heritable methylation polymorphism in evolution of gene regulation and in RFLP studies is discussed.

Base Composition↗

PubMed1991

Analysis of the 5' flanking region responsible for the endosperm-specific expression of a rice glutelin chimeric gene in transgenic tobacco.

The 5' upstream region of the rice storage protein type II glutelin gene was examined for its regulatory function in transgenic tobacco. Chimeric genes containing 5' flanking regions of the glutelin gene transcriptionally fused to the beta-glucuronidase (GUS) reporter gene were introduced into the tobacco genome by Agrobacterium tumefaciens-mediated gene transfer. The chimeric genes were expressed specifically in developing seeds, as opposed to leaves and stems, of the transgenic tobacco. Histochemical analysis revealed that the GUS activity was restricted to the endosperm tissue. A deletion series of the 5' flanking region was created from position -1329 to -74 relative to the transcriptional initiation site and similarly examined in transgenic tobacco. Measurement of GUS activity of the seeds from the transgenic plants bearing the chimeric genes indicated that the region between positions -441 and -237 was required for the temporal and endosperm-specific expression of the GUS activity in tobacco. RNA analysis by northern blotting confirmed the importance of the -441 to -237 region. Addition of up to 888 bp to the -441 deletion resulted in little increase in GUS activity, although all constructs expressing the GUS gene showed a similar tissue and temporal regulation pattern.

Base Sequence↗

PubMed1992

Developmental and organ-specific expression of an ABA- and stress-induced protein in barley.

An mRNA species, HVA1, has been shown to be rapidly induced by abscisic acid (ABA) in barley aleurone layers (Hong, Uknes and Ho, Plant Mol Biol 11: 495-506, 1988). In the current work we have investigated the expression of HVA1 in other organs of barley plants. In developing seeds, HVA1 mRNA is not detected in starchy endosperm cells, yet it accumulates in aleurone layers and embryo starting 25 days after anthesis, and its level remains high in these organs in dry seeds. Although the levels of HVA1 mRNA are equivalent in the dry embryos of dormant and nondormant barley seeds, upon imbibition HVA1 mRNA declines much slower in the dormant than in the nondormant embryos. The HVA1 mRNA and protein levels are highly induced by ABA treatment in all organs of 3-day-old seedlings. However, the induction in the leaf of 7-day-old seedlings is less than one tenth the level observed in the leaf of 3-day-old seedlings. In the leaf, HVA1 mRNA and protein are induced mainly at the base. These observations indicate that the expression of HVA1 is under developmental regulation. Besides the HVA1 protein, a smaller protein (p20) of approximately 20 kDa cross-reacting with anti-HVA1 polyclonal antibodies, is induced by ABA in barley seedlings but not in seeds. HVA1 mRNA is induced by drought, NaCl, cold or heat treatment. Similar to ABA treatment, the drought induction of HVA1 occurs in all the tissues of 3-day-old seedlings, but the induction decreases dramatically in the leaf of 7-day-old plants. The significance of organ-specific, developmentally regulated, and stress-induced expression of HVA1 is discussed.

Abscisic Acid↗

PubMed1996

Molecular cloning and characterization of a gibberellin-inducible, putative alpha-glucosidase gene from barley.

A putative alpha-glucosidase clone has been isolated from a cDNA library constructed from mRNA of barley aleurones treated with gibberellin A 3 (GA). The clone is 2752 bp in length and has an uninterrupted open reading frame encoding a polypeptide of 877 amino acids. A 680 amino acid region is 43% identical to human lysosomal alpha-glucosidase and other glycosyl hydrolases. In isolated aleurones, the levels of the corresponding mRNA increase strongly after the application of GA, similar to the pattern exhibited by low-pI alpha-amylase mRNA. High levels are also observed in the aleurone and scutellum after germination, while low levels are found in developing seeds. The genome contains a single form of this alpha-glucosidase gene and two additional sequences that may be related genes or pseudogenes.

Amino Acid Sequence↗

PubMed1993

A highly conserved alpha-tubulin sequence from Prunus amygdalus.

The sequence of an alpha-tubulin from Prunus amygdalus has been obtained by cDNA cloning. When this sequence is compared to that of the Tub alpha 1 gene from maize it shows a very high degree of similarity, much higher than any of the alpha-tubulin sequences reported so far from plants. The expression of this gene is high in the stages of seed development where a high divisional activity is present. It is preferentially expressed in the radicular tissues as it is gene Tub alpha 1 in maize. Southern analysis indicates that this gene may form a subfamily of alpha-tubulin genes having similar sequence and tissue specificity and existing at least in maize and in Prunus.

Amino Acid Sequence↗

PubMed1995

Cis elements and potential trans-acting factors for the developmental regulation of the Phaseolus vulgaris CHS15 promoter.

A nuclear factor (SBF-1) has previously been identified in Phaseolus vulgaris L. (bean) suspension cell nuclear extracts that binds in vitro to three DNase I-footprinted elements (SBF-1 boxes I, II, and III, 5' to 3') in the 5' region of the bean CHS15 (chalcone synthase) gene promoter. To define the functional role of the three SBF-1 boxes in development, we examined transgenic tobacco plants carrying a series of nested CHS15 promoter-beta-glucuronidase (GUS) fusions for GUS activity by histochemical staining. We show that the CHS15 promoter deleted to position -173 and lacking all three SBF-1 boxes directs the same qualitative pattern of expression in initiating lateral roots and in developing seeds as the full length promoter (-326). Thus, activation of expression in these organs is mediated by sequence elements located downstream of the three SBF-1 boxes. However, specific deletions within the -326 to -173 region modulate expression. Thus, deletion of box II abolishes GUS activity in initiating lateral roots. Further deletion of box III fails to restore expression but subsequent deletion of an additional 43 bp to position -173 re-establishes expression. We show that sequence-specific DNA-binding activities consistent with these results are present in nuclear extracts of bean roots and seeds. These studies reveal cis elements within the CHS15 promoter, and potential trans factors, that permit organ- and tissue-specific developmental patterns of regulation to be combined with a flexible response to environmental cues.

Acyltransferases↗

PubMed1995

The myrosinase gene family in Arabidopsis thaliana: gene organization, expression and evolution.

Myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1.) is in Brassicaceae species such as Brassica napus and Sinapis alba encoded by two differentially expressed gene families, MA and MB, consisting of about 4 and 10 genes, respectively. Southern blot analysis showed that Arabidopsis thaliana contains three myrosinase genes. These genes were isolated from a genomic library and two of them, TGG1 and TGG2, were sequenced. They were found to be located in an inverted mode with their 3' ends 4.4 kb apart. Their organization was highly conserved with 12 exons and 11 short introns. Comparison of nucleotide sequences of TGG1 and TGG2 exons revealed an overall 75% similarity. In contrast, the overall nucleotide sequence similarity in introns was only 42%. In intron 1 the unusual 5' splice border GC was used. Phylogenetic analyses using both distance matrix and parsimony programs suggested that the Arabidopsis genes could not be grouped with either MA or MB genes. Consequently, these two gene families arose only after Arabidopsis had diverged from the other Brassicaceae species. In situ hybridization experiments showed that TGG1 and TGG2 expressing cells are present in leaf, sepal, petal, and gynoecium. In developing seeds, a few cells reacting with the TGG1 probe, but not with the TGG2 probe, were found indicating a partly different expression of these genes.

Amino Acid Sequence↗

PubMed1995

Molecular cloning of two novel rice cDNA sequences encoding putative calcium-dependent protein kinases.

We have isolated, from a cDNA library constructed from rice coleoptiles, two sequences, OSCPK2 and OSCPK11, that encode for putative calcium-dependent protein kinase (CDPK) proteins. OSCPK2 and OSCPK11 cDNAs are related to SPK, another gene encoding a rice CDPK that is specifically expressed in developing seeds [20]. OSCPK2 and OSCPK11-predicted protein sequences are 533 and 542 amino acids (aa) long with a corresponding molecular mass of 59436 and 61079 Da respectively. Within their polypeptide chain, they all contain those conserved features that define a plant CDPK; kinase catalytic sequences are linked to a calmodulin-like regulatory domain through a junction region. The calmodulin-like regulatory domain of the predicted OSCPK2 protein contains 4 EF-hand calcium-binding sites while OSCPK11 has conserved just one canonical EF-hand motif. In addition, OSCPK2- and OSCPK11-predicted proteins contain, at their N-terminal region preceding the catalytic domain, a stretch of 80 or 74 residues highly rich in hydrophilic amino acids. Comparison of the NH2-terminal sequence of all three rice CDPKs so far identified (OSCPK2, OSCPK11 and SPK) indicates the presence of a conserved MGxxC(S/Q)xxT motif that may define a consensus signal for N-myristoylation. OSCPK2 and OSCPK11 proteins are both encoded by a single-copy gene and their polyadenylated transcripts are 2.4 and 3.5 kb long respectively. OSCPK2 and OSCPK11 mRNAs are equally abundant in rice roots and coleoptiles. A 12 h white light treatment of the coleoptiles reduces the amount of OSCPK2 mRNA with only a slight effect on the level of OSCPK11 transcript. With anoxic treatments, OSCPK2 mRNA level declined significantly and promptly while the amount of OSCPK11 transcript remained constant.

Amino Acid Sequence↗

PubMed1994

Repression of the L-asparaginase gene during nodule development in Lupinus angustifolius.

Upon the establishment of an effective nitrogen-fixing symbiosis in amide-transporting plants the enzymatic activity and transcript levels of L-asparaginase are dramatically decreased. This decrease in L-asparaginase activity is essential for the correct functioning of the Rhizobium-legume symbiosis in lupin in which asparagine, synthesized from recently fixed nitrogen, is exported to aerial parts of the plant for use in growth and development. Concomitant with this decrease in L-asparaginase transcript a DNA-binding protein was detected in the nodules. This binding protein was not detectable in ineffective nodules, in nodules treated with nitrate, or in root tips, mature roots, developing flowers or developing seeds. The DNA-binding activity was shown to interact with a 59 bp sequence proximal to the transcription start site. Within this sequence a CTAAAAT direct repeat and a ACTGT/TGTCA incomplete inverted repeat were implicated in the binding of protein to the DNA by DNase I protection experiments. Competitive binding studies with synthesized binding sites were consistent with the CTAAAAT/TGTCA sequence pair proximal to the transcription start site having the highest affinity for the DNA-binding protein. We postulate that this DNA-binding protein is associated with repression of L-asparaginase gene expression in mature lupin root nodules.

Asparaginase↗

PubMed1994

Two ABA-responsive proteins from pea (Pisum sativum L.) are closely related to intracellular pathogenesis-related proteins.

We report here the isolation of cDNAs encoding two abscisic acid-responsive pea (Pisum sativum L.) proteins, ABR17 and ABR18, which are synthesized during late seed development in vivo. Southern blot analyses suggest that ABR17 cDNA corresponds to a single-copy gene, but ABR18 is one member of a family of closely related sequences in the pea genome. The deduced amino acid sequences of ABR17 and ABR18 cDNAs showed similarity to those of the pea disease resistance response proteins, to pathogenesis-related and to stress-induced proteins in other species and to the major birch pollen allergen Betv1.

Abscisic Acid↗

PubMed1991

Auxin-induced expression of the soybean GH3 promoter in transgenic tobacco plants.

The gene encoding the auxin-responsive GH3 mRNA (G. Hagen, A. Kleinschmidt, TJ. Guilfoyle, Planta 162: 147-153 (1984] from soybean was cloned, and its sequence and transcription initiation site were determined. The promoter of the GH3 gene has been fused to the open reading frame of the Escherichia coli uidA gene which encodes beta-glucuronidase (GUS). This fusion gene was introduced into tobacco via Agrobacterium tumefaciens-mediated transformation, and the expression of the gene was examined by fluorometric assay and histochemical staining of young R1 tobacco seedlings and mature plants. In transgenic tobacco plants that have not been exposed to exogenous auxin, expression of the fusion gene is largely restricted to roots of young green plants and developing floral organs, including ovules, developing seeds, and pollen, of mature plants. Application of exogenous auxin to tobacco seedlings or plant organs results in a greater than 50-fold increase in expression of GUS. Auxin-induced GUS expression is greatest in vascular tissue, but not restricted to this tissue. The auxin-deduced GUS expression was characterized for kinetics, auxin specificity and dose response.

Amino Acid Sequence↗

PubMed1995

Identification of a cDNA that encodes a 1-acyl-sn-glycerol-3-phosphate acyltransferase from Limnanthes douglasii.

Two different techniques were used to isolate potential cDNAs for acyl-CoA: 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPA-AT) enzymes from Limnanthes douglasii. Both heterologous screening with the maize pMAT1 clone and in vivo complementation of the Escherichia coli mutant JC201 which is deficient in LPA-AT activity, were carried out. Clones identified by these procedures were different. Homology searches demonstrated that the clone isolated by heterologous probing, pLAT1, encodes a protein which is most similar to the maize (open reading frame in pMAT1) and yeast SLC1 proteins, which are putative LPA-AT sequences. This L. douglasii sequence shows much lower homology to the E. coli LPA-AT protein PlsC, which is the only LPA-AT sequence confirmed by over-expression studies. The clone isolated by complementation, pLAT2, encodes a protein with homology to both SLC1 and PlsC. It was not possible to over-express the complementing protein encoded by pLAT2 but further experimentation on membranes from complemented JC201 demonstrated that they possess a substrate specificity distinctly different from PlsC and similar to Limnanthes sp. microsome specificity. This data strongly supports the contention that pLAT2 is an LPA-AT clone. Northern blot analysis revealed different expression patterns for the two genes in pLAT1 and pLAT2. Transcription of the gene encoding the insert of pLAT2 occurred almost exclusively in developing seed tissue, whilst the cDNA of pLAT1 hybridised to poly(A)+ mRNA from seed, stem and leaf, demonstrating more widespread expression throughout the plant. Southern blot analysis indicated that the cDNA of pLAT2 was transcribed from a single-copy gene while that for pLAT1 was a member of a small gene family.

1-Acylglycerol-3-Phosphate O-Acyltransferase↗

PubMed1995

Expression and in vitro targeting of a sunflower oleosin.

Two distinct cDNAs encoding oleosins (oil body proteins) have been identified by degenerate PCR as transcripts present in the developing seeds of sunflower (Helianthus annuus L. cv. Dwarf Sunbred). One (pSOM) of these is closely related to a reported sunflower oleosin, whilst the other (pSO5) has not been previously described. Different expression patterns were observed for the two cDNAs, pSO5 being expressed earlier than pSOM in seed maturation and oil deposition. The results support the contention that oleosin proteins are synthesised either during or closely after the formation of the oil body. Translation in vitro of synthetic oleosin transcripts was enhanced by the addition of microsomes but suppressed by the addition of purified signal recognition particle (SRP) complex. Deletion of 62 amino acid residues at the C-terminus of the oleosin did not alter the in vitro targeting of the protein to the microsomal membrane. Taken together these data support the idea that oleosins are targeted to the ER membrane as part of oil body biogenesis.

Amino Acid Sequence↗

PubMed1994

Tissue-specific expression and temporal regulation of the rice glutelin Gt3 gene are conferred by at least two spatially separated cis-regulatory elements.

The rice glutelin Gt3 promoter was fused to a beta-glucuronidase (GUS) reporter gene and its expression evaluated in transgenic tobacco plants. Histochemical analysis revealed that the expression of the introduced Gt3 promoter/GUS (beta-glucuronidase) chimeric gene was confined to endosperm tissue of developing seeds. 5'-promoter deletion analysis revealed that two domains of the Gt3 promoter, -346 to -263 bp (domain I) and -945 to -726 bp (domain II) from the transcriptional start site, were essential for optimum expression of the GUS reporter gene. Removal of 5' sequences upstream of -726 resulted in a reduction in overall promoter activity and a shift in temporal expression from a maximum of 16-20 days after flowering to 24 days. Removal of DNA sequences from the 5' end to -346 yielded a promoter fragment that was still able to confer endosperm-specific expression, although a further deletion to -263 abolished promoter activity. These data suggest that at least two cis-regulatory elements are required for endosperm specificity and temporal regulation of glutelin Gt3 gene expression.

Base Sequence↗

PubMed1990

The extensin gene family in oilseed rape (Brassica napus L.): characterisation of sequences of representative members of the family.

A family of cross-hybridising cDNA clones has been isolated from a cDNA library produced with poly(A)+ RNA from the roots of oilseed-rape (Brassica napus L.). The clones were selected as abundantly expressed in root by differential screening of the root cDNA library with cDNA probes prepared from root, green leaf, etiolated leaf and developing seed. mRNA species corresponding to the selected abundant clones were expressed in roots at levels of at least 400 times those in other organs, as shown by Northern blot analysis and RNase protection assays. Complete nucleotide sequence determination of the cDNA clones showed that they encoded proteins homologous to carrot extensin and were the products of at least three different genes. An extensin gene, designated extA, was obtained from an oilseed rape (B. napus L.) genomic library screened with a cDNA species encoding a protein expressed abundantly in roots. The gene is a member of a multigene family, consisting of about 3 members per haploid genome with strong homology to the probe, and a further 20 or so members with weaker homology. The isolated gene, although not identical to the cDNA probe, was also found to be specifically expressed in roots, and was transcribed into a mRNA species approximately 1,300 nucleotides in size. A single transcription start was identified by S1 mapping. The complete nucleotide sequence of the extA gene and its flanking regions has been determined and shown to encode a protein homologous to carrot and tomato extensins.

Amino Acid Sequence↗

PubMed1995

Tissue-specific and developmental regulation of a gene encoding a low molecular weight sulfur-rich protein in soybean seeds.

A gene corresponding to a cDNA clone, SE60, encoding a low molecular weight sulfur-rich protein in soybean seeds was isolated from a soybean genomic library and characterized at the nucleotide level. The SE60 gene is interrupted by an intervening sequence of 694 bp in size. The 5' flanking region of the gene contained various regulatory sequences such as the RY repeat and CACA elements found in other seed protein genes of legumes. The SE60 gene encoded a preprotein of 75 amino acids, having a signal sequence of 28 amino acids at the N-terminus. The mature protein of 47 amino acids was basic and cysteine-rich. Northern blot analysis suggested that the SE60 gene is expressed in a tissue-specific and developmentally regulated manner during soybean seed development. The SE60 genes form a small multigene family composed of about four members in the soybean genome.

Amino Acid Sequence↗