Showing papers in "Plant and Cell Physiology in 1999"

Compilation and Characterization of Arabiopsis thaliana Response Regulators Implicated in His-Asp Phosphorelay Signal Transduction

Abstract: His-Asp phosphorelays are evolutionary-conserved powerful biological tactics for intracellular signal transduction. Such a phosphorelay is generally made up of "sensor histidine (His)-kinases", "response regulators", and "histidine-containing (HPt) phosphotransmitters". In the higher plant, Arabidopsis thaliana, results from recent intensive studies suggested that His-Asp phosphorelays may be widely used for propagating environmental stimuli, such as phytohormones (e.g., ethylene and cytokinin). In this study, we first inspected extensively the occurrence of Arabidopsis response regulators in order to compile and characterize them. The results showed that this higher plant has, at least, 14 members of the family of response regulators that can be classified into two distinct subtypes (type-A and type-B), as judged from their structural designs, biochemical properties, and expression profiles. Comparative studies were conducted for each representative (ARR3 and ARR4 for type-A, and ARR10 for type-B). It was suggested that expression of the type-A response regulator is cytokinin-inducible, while that of the type-B response regulator appears to be not. Results from yeast two-hybrid analyses suggested that the type-B response regulator may have an ability to stably interact with a set of HPt phosphotransmitters (AHPs). These and other results will be discussed with special reference to the His-Asp phosphorelay signaling network in Arabidopsis thaliana.

Overexpression of an Arabidopsis peroxisomal ascorbate peroxidase gene in tobacco increases protection against oxidative stress.

Abstract: The Arabidopsis gene APX3 that encodes a putative peroxisomal membrane-bound ascorbate peroxidase was expressed in transgenic tobacco plants. /l.PA'J-expressing lines had substantial levels of APX3 mRNA and protein. The H2O2 can be converted to more reactive toxic molecules, e.g. 'OH, if it is not quickly removed from plant cells. The expression of APX3 in tobacco could protect leaves from oxidative stress damage caused by aminotriazole which inhibits catalase activity that is found mainly in glyoxysomes and peroxisomes and leads to accumulation of H2O2 in those organelles. However, these plants did not show increased protection from oxidative damage caused by paraquat which leads to the production of reactive oxygen species in chloroplasts. Therefore, protection provided by the expression of APX3 seems to be specific against oxidative stress originated from peroxisomes, not from chloroplasts, which is consistent with the hypothesis that APX3 is a peroxisomal membrane-bound antioxidant enzyme.

Photosynthesis and Plant Growth at Elevated Levels of CO2

Abstract: In this review, we discuss the effects of elevated CO2 levels on photosynthesi s in relation to the whole plant growth in terrestrial higher C3 plants. Short-term CO2 enrichment stimulates the rate of photosynthesi s. Plant mass is also enhanced by CO2 enrichment. However, the effects of long-term CO2 enrichment on photosynthesis are variable. Generally, the prolonged exposure to CO2 enrichment reduces the initial stimulation of photosynthesi s in many species, and frequently suppresses photosynthesis. These responses are attributed to secondary responses related to either excess carbohydrate accumulation or decreased N content rather than direct responses to CO2. Accumulation of carbohydrates in leaves may lead to the repression of photosynthetic gene expression and excess starch seems to hinder CO2 diffusion. Therefore, the species which have the sink organs for carbohydrate accumulation do not show the suppression of photosynthesi s. The suppression of photosynthesis by CO 2 enrichment is always associated with decreases in leaf N and Rubisco contents. These decreases are not due to dilution of N caused by a relative increase in the plant mass but are the result of a decrease in N allocation to leaves at the level of the whole plant, and the decrease in Rubisco content is not selective. Leaf senescence and plant development are also accelerated by CO2 enrichment. However, they are independent of each other in some species. Thus, various responses to CO 2 observed at the level of a single leaf result from manifold responses at the level of the whole plant grown under conditions of CO2 enrichment.

Induction of Rice Cytosolic Ascorbate Peroxidase mRNA by Oxidative Stress; the Involvement of Hydrogen Peroxide in Oxidative Stress Signalling

Abstract: The oxidative stress response of rice cytosolic ascorbate peroxidase (APX) was examined. The transcript level of cytosolic APX was significantly increased when suspension cultures of germinating rice embryos were treated with paraquat (7.9-fold) or H 2O: (6.1-fold). Induction by paraquat reached a maximum at 8 h. Induction by H2O2 peaked earlier at 4 h of treatment. This result suggests that the induction by paraquat might be caused by the H2O2 generated from superoxide. Treatment with a superoxide dismutase inhibitor, diethyldithiocarbamate, which is supposed to decrease the cellular H2O2 level, reduced the paraquat induction of cytosolic APX. In contrast, when APX and catalase were inhibited by hydroxyurea or aminotriazole, cellular H2O2 content was elevated and cytosolic APX mRNA was markedly increased without paraquat or H2O2 treatment. This suggests that cytosolic APX is regulated by the H2O2 level within cells. An increase in H 2O2 content was observed in the paraquat treated embryos, suggesting that paraquat induction of cytosolic APX was caused by H 2O2 generated through superoxide dismutation. These results indicate that H2O2 is involved in oxidative stress signalling, leading to the induction of cytosolic APX.

Cloning and secondary structure analysis of caleosin, a unique calcium-binding protein in oil bodies of plant seeds.

Abstract: Plant seed oil bodies comprise a matrix of triacylglycerols surrounded by a monolayer of phospholipids embedded with abundant oleosins and some minor proteins. Three minor proteins, temporarily termed Sops 1-3, have been identified in sesame oil bodies. A cDNA sequence of Sop1 was obtained by PCR cloning using degenerate primers derived from two partial amino acid sequences, and subsequently confirmed via immunological recognition of its over-expressed protein in Escherichia coli. Alignment with four published homologous sequences suggests Sop1 as a putative calcium-binding protein. Immunological cross-recognition implies that this protein, tentatively named caleosin, exists in diverse seed oil bodies. Caleosin migrated faster in SDS-PAGE when incubated with Ca2+. A single copy of caleosin gene was found in sesame genome based on Southern hybridization. Northern hybridization revealed that both caleosin and oleosin genes were concurrently transcribed in maturing seeds where oil bodies are actively assembled. Hydropathy plot and secondary structure analysis suggest that caleosin comprises three structural domains, i.e., an N-terminal hydrophilic calcium-binding domain, a central hydrophobic anchoring domain, and a C-terminal hydrophilic phosphorylation domain. Compared with oleosin, a conserved proline knot-like motif is located in the central hydrophobic domain of caleosin and assumed to involve in protein assembly onto oil bodies.

Glutamine Synthetase in Higher Plants: Regulation of Gene and Protein Expression from the Organ to the Cell

Abstract: Compared to other enzymatic systems, the regulation of GS isoenzyme expression shows a unique diversity. Considering that GS is one of the oldest existing and functioning genes found in all extant life forms, we can hypothesise that the evolution of metabolic pathways from primitive pre-procaryotes to lower and then higher plants might have gradually refined the function of GS to provide reduced nitrogen forms for the rest of the metabolism (Kumada et al. 1993). This refinement might explain the genetic and biological diversity encountered in the various modes of expression and regulation of higher plant GS isoenzymes both at the cellular and intracellular levels (Fig. 1). Although model plants are valuable sources of information helping to decipher fine regulatory control mechanisms (Lam et al. 1996), the study of this genetic diversity appears to be one of the most promising areas of research, necessary to better understand ammonia assimilation in plants and more generally improve nitrogen use efficiency.

Highly purified thermo-stable oxygen-evolving photosystem II core complex from the thermophilic cyanobacterium Synechococcus elongatus having His-tagged CP43.

Abstract: The carboxyl terminus of the CP43 subunit of photosystem II (PSII) in the thermophilic cyanobacterium, Synechococcus elongatus, was genetically tagged with six consecutive histidine residues to create a metal binding site on the PSII supramolecular complex. The histidine-tagging enabled rapid isolation of an intact cyanobacterial PSII core complex from dodecyl maltoside-solubilized thylakoids by a simple one-step Ni(2+)-affinity column chromatography. The isolated core complex was in a dimeric form with a molecular mass of about 580 kDa, consisting of five major intrinsic membrane proteins (CP47, CP43, D1, D2 and cytochrome b-559), three extrinsic proteins (33 kDa, 12 kDa, and cytochrome c-550), and a few low molecular mass membrane proteins, and evolved oxygen at a rate as high as 3,400 mumol (mg Chl)-1 h-1 at 45 degrees C with ferricyanide as an electron acceptor. The core complex emitted thermoluminescence B2-, B1- and Q-bands arising from S2QB-, S3QB- and S2QA- charge recombinations at respective emission temperatures of 45, 38 and 20 degrees C, all of which were higher by about 15 degrees C as compared with those in mesophilic spinach BBY membranes. These results indicated that the isolated core complex well retained the intact properties of thermoluminescence of thermophilic cyanobacterial cells, the deeper stabilization of PSII charge pairs. The isolated complex was extremely stable in terms of both protein composition and function, exhibiting no release of extrinsic proteins, no proteolytic degradation in any of its subunits, accompanied by only a slight (less than 10%) loss in oxygen evolution, after dark-incubation at 20 degrees C for 8 d. These properties of the thermophilic PSII core complex are highly useful for various types of studies on PSII.

Overproduction of Arabidopsis thaliana FeSOD confers oxidative stress tolerance to transgenic maize.

Abstract: Transgenic maize (Zea mays L.) plants have been generated by particle gun bombardment that overproduce an Arabidopsis thaliana iron superoxide dismutase (FeSOD). To target this enzyme into chloroplasts, the mature Fesod coding sequence was fused to a chloroplast transit peptide from a pea ribulose-1,5-bisphosphate carboxylase gene. Expression of the chimeric gene was driven by the CaMV 35S promoter. Growth characteristics and in vitro oxidative stress tolerance of transgenic lines grown in control and chilling temperatures were evaluated. The transgenic line with the highest transgenic FeSOD activities had enhanced tolerance toward methyl viologen and had increased growth rates.

Expression of Atropa belladonna putrescine N-methyltransferase gene in root pericycle.

Abstract: The cDNAs encoding putrescine N-methyltransferase (PMT), which catalyzes the S-adenosylmethionine-dependent N-methylation of putrescine at the first committed step in the biosynthetic pathways of tropane alkaloids, were isolated from Atropa belladonna and Hyoscyamus niger. These PMTs, however, lacked the N-terminal tandem repeat arrays previously found in Nicotiana PMTs. AbPMT1 RNA was much more abundant in the root of A. belladonna than was AbPMT2 RNA. The 5'-flanking region of the AbPMT1 gene was fused to the beta-glucuronidase (GUS) reporter gene and transferred to A. belladonna. Histochemical analysis showed that GUS is expressed specifically in root pericycle cells and that the 0.3-kb 5'-upstream region was sufficient for pericycle-specific expression. Treatment of A. belladonna roots with methyl jasmonate did not up-regulate the expression of GUS or endogenous AbPMT genes. The regulation of tropane alkaloid biosynthesis is discussed and compared with that of nicotine biosynthesis.

Iron Deficiency Induced Changes in Ascorbate Content and Enzyme Activities Related to Ascorbate Metabolism in Cucumber Roots

Abstract: Ascorbate content and the activities of ascorbate free-radical reductase (AFR-R) and ascorbate peroxidase (APX) were investigated in order to determine whether they are affected under Fe deficiency. Plasma membrane vesicles, cell wall and cytosolic fractions were isolated from the roots of cucumber (Cucumis sativus L.) plants grown in the absence or in the presence of Fe. Plasma membrane vesicles showed NADH-dependent reducing activities with Fe3+-citrate, ferricyanide and AFR as electron acceptors. Only AFR-R activity was stimulated ca. 3-fold in Fe deficient plasma membranes. No significant change in cytosolic AFR-R activity was induced by Fe depletion, while the activity of cytosolic APX was more than twice that of the non-deficient control. Furthermore, the content of ascorbate (AA) was enhanced ca. 1.7-fold in Fe deficient roots. These results indicate that metabolic changes resulting in enhanced AA levels and activities of AFR-R and APX in the roots could be related to plant responses to Fe deficiency stress.

Differential expression of genes for response regulators in response to cytokinins and nitrate in Arabidopsis thaliana.

Abstract: In Arabidopsis thaliana, a number of response regulators are presumably involved in His-Asp phosphorelay signal transduction in response to environmental stimuli, such as phytohormones. Previously, it was shown that expression of a certain set of genes for response regulators are cytokinin- and nitrate-responsive in their mRNA accumulation, under certain growth conditions [Taniguchi et al. (1998) FEBS Lett. 429: 259, Brandstatter and Kieber (1998) Plant Cell 10: 1009]. To answer the critical question of whether or not other response regulator genes, so far identified in Arabidopsis thaliana, are also cytokinin-inducible, here an extended comparative examination was carried out. It was demonstrated that not all of response regulator genes are necessarily cytokinin-responsive in their transcription. Rather, the members of a certain subfamily (type-A) are cytokinin-responsive, but those belonging to the other (type-B) are not. The presumed nitrate-responsiveness was also assessed for the same set of response regulators, and the analogous view was supported. These results suggest that the two subtypes of response regulators differ from each other, as judged from not only their structural designs, but also the expression profiles of their transcripts in response to plant stimuli.

Molecular Cloning and Expression Analysis of a Gene for a Sucrose Transporter in Maize (Zea mays L.)

Abstract: Here we report the cloning of a sucrose transporter cDNA from maize (Zea mays L.) and an analysis of the expression of the gene. A cDNA clone (ZmSUT1) was identified as a sucrose transporter cDNA from its sequence homology at the amino acid level to sucrose transporters that have been cloned from other higher plant species. Based on the results of genomic Southern hybridization, ZmSUT1 appears to be a single copy gene. A Northern blot analysis of seedlings and leaf blades suggests that the sucrose transporter is involved in the export of accumulated carbohydrates from source leaf blades. From the measurements of transcript levels and carbohydrate contents in mature leaf blades, we propose that the expression of the gene for the maize sucrose transporter is positively regulated by the amounts of soluble sugars such as sucrose and glucose in source leaves of maize. In addition, based on the tissue specificity of the expression of the gene in maize plants at the reproductive stage, it is possible that the sucrose transporter acts in sink tissues such as pedicles as well as in source tissues such as leaf blades.

Identification and characterization of Arabidopsis mutants with reduced quenching of chlorophyll fluorescence.

Abstract: Regulation of nonradiative dissipation of absorbed light energy in PSII is an indispensable process to avoid photoinhibition in plants. To dissect molecular mechanisms of the regulation, we identified Arabidopsis mutants with reduced quenching of Chl fluorescence using a fluorescence imaging system. By analyses of Chl fluorescence induction pattern in the light and quantum yield of both photosystems, 37 mutants were classified into three groups. The first group was characterized by an extremely high level of minimum Chl fluorescence at the open PSII center possibly due to a defect in PSII. Mutants with significant reduction in the nonphotochemical quenching formation but not in quantum yield of both photosystems were classified into the second group. Mutants in the third group showed reduction in quantum yield of both photosystems possibly due to a defect in the electron transport activity. Mutants in the second and third groups were further characterized by light intensity dependence of Chl fluorescence parameters and steady state redox level of P700.

Developmental transitions and dynamics of the cortical ER of Arabidopsis cells seen with green fluorescent protein.

Abstract: Arabidopsis thaliana plants were stably transformed with DNA encoding green fluorescent protein and with sequences ensuring retention in the endoplasmic reticulum (ER). Confocal laser scanning microscopy shows fluorescent ER in many cells of seedlings so allowing developmental changes to be documented. The arrangement of the cortical ER changes as cells mature in the hypocotyl and root epidermis. In the root, cells that have completed expansion have reticulate cortical ER resembling the ER described in many previous studies. Expanding cells, however, show extensive perforated sheets of cortical ER which transform quite abruptly into a loose reticulum at the basipetal end of the elongation zone. The reticulum compacts in trichoblasts beginning at sites where root hairs are about to emerge. The compacted form is maintained throughout the hair until growth ceases and the open reticulate form returns. All forms of cortical ER are dynamic and we use a color overlay method to distinguish stable and moving structures in a single composite image. Reticulate ER continuously rearranges its polygonal layout and perforations move and change their shape in the ER sheets of younger cells. ER deeper in the cell (i.e. not close to the plasma membrane) moves more actively so that almost no tubules remain stable even over short periods of less than one minute. The function of the perforated sheets of cortical ER present in growing cells is unknown.

Sequence analysis of Expressed Sequence Tags from an ABA-Treated cDNA Library Identifies Stress Response Genes in the Moss Physcomitrella patens

Abstract: Partial cDNA sequencing was used to obtain 169 expressed sequence tags (ESTs) in the moss, Physcomitrella patens. The source of ESTs was a random cDNA library constructed from 7 day-old protonemata following treatment with 10(-4) M abscisic acid (ABA). Analysis of the ESTs identified 69% with homology to known sequences, 61% of which had significant homology to sequences of plant origin. More importantly, at least 11 ESTs had significant similarities to genes which are implicated in plant stress-responses, including responses which may involve ABA. These included a cDNA associated with desiccation tolerance, two heat shock protein genes, one cold acclimation protein cDNA and five others that may be involved in either oxidative or chemical stress or both, i.e., Zn/Cu-superoxide dismutase, NADPH protochlorophyllide oxidoreductase (PorB), selenium binding protein, glutathione peroxidase and glutathione S transferase. Analysis of codon usage between P. patens and seed plants indicated that although mosses and higher plants are to a large extent similar, minor variations also exists that may represent the distinctiveness of each group.

Over expression of mitochondrial citrate synthase gene improves the growth of carrot cells in Al-phosphate medium.

Abstract: A mitochondrial citrate synthase (CS) of Arabidopsis thaliana was introduced into carrot (Daucus carota L. cv. MS Yonsun) cells by Agrobacterium tumefaciens-mediated transformation. Transgenic cell lines had high CS activity, the highest value observed was 0.24 mumol (mg protein)-1 min-1 which was 1.9-fold of that in wild-type cells. Transcript levels of DcCS were similar between transgenic lines, but those of AtCS were increased as the CS activity of cells was increased. Isoelectric focussing revealed that the CS polypeptide of the transgenic lines had a pI value different from that of the wild-type cells, although the molecular mass was the same. These results indicate that the CS polypeptides of A. thaliana were expressed and processed to the mature form in carrot cells. The growth rate and excretion was 2.2-2.8 and 2.8-4.0 fold greater in the transgenic cells than in the wild type cells, respectively. Phosphate uptake from Al-phosphate also increased in transgenic cells. It appears, the overexpression of mitochondrial citrate synthase in carrot cells improves the growth rate in Al-phosphate medium possibly as a result of increased citrate excretion.

Features of Photosynthesis in Haloxylon species of Chenopodiaceae that are Dominant Plants in Central Asian Deserts

Abstract: Vladimir I. Pyankov, Clanton C. Black Jr., Elena G. Artyusheva, Elena V. Voznesenskaya, Maurice S.B. Ku and Gerald E. Edwards 1 Department of Plant Physiology, Urals State University, Lenin Avenue 51, 620083 Ekaterinburg, Russia 2 Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, U.S.A. 3 Morphology and Anatomy Department, Komarov Botanical Institute RAS, Prof. Popov Street 2, 197376 St. Petersburg, Russia 4 Botany Department, Washington State University, Pullman, WA 99164-4238, U.S.A.

Identification of clp genes expressed in senescing Arabidopsis leaves.

Abstract: Clp protease is a highly selective protease in E. coli, which consists of two types of subunits, the regulatory subunit with ATPase activity, ClpA, and the catalytic subunit, ClpP. In order to examine the possible association of plant Clp protease with the degradation of protein in senescing chloroplasts, we isolated a cDNA clone for ClpC which is a plant homologue of ClpA from Arabidopsis thaliana in addition to ERD1 which we had isolated earlier [Kiyosue et al. (1993) Biochem. Biophys. Res. Commun. 196: 1214]. We also isolated a clone for the plastidic gene, clpP (pclpP) and cDNA clones for putative nuclear clpP genes (nclpP1-6). We analyzed the expression of these clp genes in Arabidopsis leaves after various dark periods and during natural senescence. The expression of erd1 was increased by dark-induced and by natural senescence, as reported earlier [Nakashima et al. (1997) Plant J. 12: 851], while that of AtclpC was decreased. Two catalytic subunits nclpPs (nclpP3 and nclpP5) showed high expression in naturally senescing leaves, but the expression of pclpP and the other nclpPs was not changed. Immunoblot analysis of chloroplast protein and in vitro import analysis demonstrated that both nucleus-encoded regulatory subunits as well as nClpP5 were localized in the chloroplast stroma. These observations suggest that chloroplast Clp protease is composed of very complicated combinations of subunits, and that ERD1, nClpP5 and pClpP have a role in the concerted degradation of protein in senescing chloroplasts.

Multiple functional proteins are produced by cleaving asn-gln bonds of a single precursor by vacuolar processing enzyme

Abstract: Precursor-accumulating vesicles mediate transport of the precursors of seed proteins to protein storage vacuoles in maturing pumpkin seeds. We isolated the precursor-accumulating vesicles and characterized a 100-kDa component (PV100) of the vesicles. Isolated cDNA for PV100 encoded a 97,310-Da protein that was composed of a hydrophobic signal peptide and the following three domains: an 11-kDa Cys-rich domain with four CXXXC motifs, a 34-kDa Arg/Glu-rich domain composed of six homologous repeats, and a 50-kDa vicilin-like domain. Both immunocytochemistry and immunoblots with anti-PV100 antibodies showed that <10-kDa proteins and the 50-kDa vicilin-like protein were accumulated in the vacuoles. To identify the mature proteins derived from PV100, soluble proteins of the vacuoles were separated, and their molecular structures were determined. Mass spectrometry and peptide sequencing showed that two Cys-rich peptides, three Arg/Glu-rich peptides, and the vicilin-like protein were produced by cleaving Asn-Gln bonds of PV100 and that all of these proteins had a pyroglutamate at their NH2 termini. To clarify the cleavage mechanism, in vitro processing of PV100 was performed with purified vacuolar processing enzyme (VPE). Taken together, these results suggested that VPE was responsible for cleaving Asn-Gln bonds of a single precursor, PV100, to produce multiple seed proteins. It is likely that the Asn-Gln stretches not only provide cleavage sites for VPE but also produce aminopeptidase-resistant proteins. We also found that the Cys-rich peptide functions as a trypsin inhibitor. Our findings suggested that PV100 is converted into different functional proteins, such as a proteinase inhibitor and a storage protein, in the vacuoles of seed cells.

Hypergravity Increases the Molecular Mass of Xyloglucans by Decreasing Xyloglucan-Degrading Activity in Azuki Bean Epicotyls

Abstract: Elongation growth of dark-grown azuki bean (Vigna angularis Ohwi et Ohashi cv. Takara) epicotyls was suppressed by hypergravity at 30 x g and above. Acceleration at 300 x g significantly decreased the mechanical extensibility of cell walls. The amounts of cell wall polysaccharides (pectin, hemicellulose-II and cellulose) per unit length of epicotyls increased under the hypergravity condition. Hypergravity also increased the amounts and the weight-average molecular mass of xyloglucans in the hemicellulose-II fraction, while decreasing the activity of xyloglucan-degrading enzymes extracted from epicotyl cell walls. These results suggest that hypergravity increases the amounts and the molecular mass of xyloglucans by decreasing xyloglucan-degrading activity. Modification of xyloglucan metabolism as well as the thickening of cell walls under hypergravity conditions seems to be involved in making the cell wall mechanically rigid, thereby inhibiting elongation growth of azuki bean epicotyls.

Two tropinone reductases, that catalyze opposite stereospecific reductions in tropane alkaloid biosynthesis, are localized in plant root with different cell-specific patterns.

Abstract: In the plant species that produce tropane alkaloids, two tropinone reductases (TRs) catalyze the stereospecific reductions of the 3-carbonyl group of tropinone. This reduction is a key branch point that determines the metabolite flow into the separate alkaloid groups, each with different stereospecific configurations. In this study, a specific antibody was prepared for each of the TRs by immunizing mice with recombinant TR protein and subsequent immuno-affinity purification of the antiserum. Immunoblot analyses revealed that accumulation of both TRs was highest in the lateral roots of Hyoscyamus niger throughout its development. In cultured roots, TR proteins were accumulated in a basal region but not in root apex. These patterns were similar to that of hyoscyamine 6 beta-hydroxylase (H6H), an enzyme that catalyzes a downstream step in the same biosynthetic pathway. However, an immunohistochemical analysis revealed that the two TRs and H6H were accumulated with different cell-specific patterns in the cultured root, suggesting transportation of the alkaloid intermediate(s) across the different cell layers.

Analysis of Lipoxygenase mRNA Accumulation in the Common Bean (Phaseolus vulgaris L.) during Development and under Stress Conditions

Abstract: Plant lipoxygenases (LOX, EC 1.13.11.12) have been involved in processes such as stress responses and development. The levels of these enzymes and their corresponding mRNAs are modulated during these processes as well as by different effectors such as jasmonic acid (JA), its methyl ester (MeJA) or abscisic acid (ABA). A new lipoxygenase (LOX) cDNA clone, PvLOX2, was isolated from a Phaseolus vulgaris nodule library and used to study the LOX mRNA accumulation pattern in some developmental stages and in plants subjected to hormone and stress treatments. In nodules, LOX mRNA reaches a maximum level around day 14 to 16 after Rhizobium tropici inoculation, as compared with LOX mRNA present in uninoculated and inoculated roots at the same days. LOX antigen is detected in the nodule parenchyma and in the uninfected cells. During germination, bean LOX transcripts start to accumulate 48 h after imbibition, remains at the same level until 72 h after imbibition and then declines. In hypocotyl, LOX mRNA is abundant in the growing region and almost absent in the mature region. After water stress or ABA treatment, this mRNA increases in the mature region and decreases in the growing region. In bean seedlings, LOX mRNA is accumulated in response to some types of stresses such as cold and desiccation. Wounding, MeJA or ABA treatment of mature leaves also induces LOX mRNA accumulation. These results indicate that in common bean plants LOX is required during development and stress conditions.

Molecular cloning and biochemical characterization of a novel cytochrome P450, flavone synthase II, that catalyzes direct conversion of flavanones to flavones.

Abstract: Cytochrome P450 cDNAs, AFNS2 and TFNS5, were isolated from snapdragon and torenia petal cDNA libraries, respectively, based on the sequence homology with licorice CYP93B1 cDNA encoding (2S)-flavanone 2-hydroxylase. They were expressed in yeast and identified to encode flavone synthase II catalyzing direct conversion of flavanones to flavones probably via 2-hydroxyflavanones.

The Requirement of Selenium for the Growth of Marine Coccolithophorids, Emiliania huxleyi, Gephyrocapsa oceanica and Helladosphaera sp. (Prymnesiophyceae)

Abstract: Marine coccolithophorids, Emiliania huxleyi, Gephyrocapsa oceanica and Helladosphaera sp. were found to require selenium for their growth. The optimum concentrations were 1-10 nM for SeC>2 and SeO3~ and above 1 fJM. for SeO4~. During the depletion of selenium algal growth was strongly suppressed accompanied with the decrease in net photosynthesis.

Plastidic RNA polymerase σ factors in Arabidopsis

Abstract: In plant cells, plastid DNA is transcribed by at least two types of RNA polymerase, plastid-encoded RNA polymerase (PEP) and nuclear-encoded RNA polymerase (NEP). PEP is homologous to eubacterial transcription machinery, but its regulatory subunit, sigma (sigma) factor, is not encoded on the plastid DNA. We previously cloned the three nuclear-encoded sigma factor genes from Arabidopsis thaliana and designated them as sigA, sigB, and sigC. By means of RFLP mapping, sigA and sigB were mapped on chromosome I and sigC on the chromosome III. Based on comparison of the genomic structure of the three sig genes, intron sites in the 3' half of the genes were shown to be identical between sigB and sigC but divergent in sigA, consistent with the phylogenetic relevance of the three gene products. A transient expression assay of GFP fusions in Arabidopsis protoplasts showed that the N-termini of all three sig gene products functioned as chloroplast-targeting signals. We also constructed transgenic Arabidopsis lines harboring the sigA-promoter or the sigB-promoter uidA fusion. Both the sigA- and sigB-promoters were similarly activated at cotyledons, hypocotyls, rosette leaves, cauline leaves, sepals, and siliques but not at roots, seeds, or other flower organs. In addition, the two promoters were repeatedly activated in young seedlings under continuous light, possibly in an oscillated fashion.

Phosphate as a limiting factor for the cell division of tobacco BY-2 cells.

Abstract: The re-addition of phosphate to tobacco BY-2 cells deprived of phosphate for 3 d induced cells to semi-synchronously re-enter the cell cycle from a static state. Though the addition of auxin to auxin-starved tobacco BY-2 cells also induced cell division (Ishida et al. 1993), some major differences were observed between these two systems. BY-2 cells lost the ability to re-enter the cell cycle after prolonged periods of auxin deprivation, but in contrast retained this ability after longer periods in the absence of phosphate. By differential cDNA screening we identified a phosphate-induced gene phi-1. phi-1 was rapidly induced by the addition of phosphate with transcript levels starting to decrease by the start of DNA synthesis. phi-1 does not share any significant homology with any gene with known functions over its full length. However, the N-terminus shared some homology with plasma membrane ATPases suggesting that it may be involved in some process of phosphorylation. Immunolocalization of the phi-1 gene product revealed that it rapidly accumulated in the cytoplasm prior to the start of plastid and nuclear DNA synthesis. These results are discussed in relation to the role of phosphate in inducing plant cell division.