RESUMO
The tropical intertidal ecosystem is defined by trees - mangroves - which are adapted to an extreme and extremely variable environment. The genetic basis underlying these adaptations is, however, virtually unknown. Based on advances in pyrosequencing, we present here the first transcriptome analysis for plants for which no prior genomic information was available. We selected the mangroves Rhizophora mangle (Rhizophoraceae) and Heritiera littoralis (Malvaceae) as ecologically important extremophiles employing markedly different physiological and life-history strategies for survival and dominance in this extreme environment. For maximal representation of conditional transcripts, mRNA was obtained from a variety of developmental stages, tissues types, and habitats. For each species, a normalized cDNA library of pooled mRNAs was analysed using GSFLX pyrosequencing. A total of 537,635 sequences were assembled de novo and annotated as > 13,000 distinct gene models for each species. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology annotations highlighted remarkable similarities in the mangrove transcriptome profiles, which differed substantially from the model plants Arabidopsis and Populus. Similarities in the two species suggest a unique mangrove lifestyle overarching the effects of transcriptome size, habitat, tissue type, developmental stage, and biogeographic and phylogenetic differences between them.
Assuntos
Perfilação da Expressão Gênica , Rhizophoraceae/genética , Mapeamento de Sequências Contíguas , Regulação da Expressão Gênica de Plantas , Genes de Plantas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de DNARESUMO
The glaucocystophyte Cyanophora paradoxa contains cyanelles, plastids with prokaroytic features such as a peptidoglycan wall and a central proteinaceous inclusion body. While this central body includes the majority of the enzyme ribulose 1,5-bisphosphate carboxylase/oxgenase Rubisco), the presence of a carbon-concentrating mechanism (CCM) in C. paradoxa has only been hypothesized. Here, we present physiological data in support of a CCM: CO(2) exchange activity as well as apparent affinity against inorganic carbon were found to increase under CO(2)-limiting stress. Further, expressed sequence tags (ESTs) of C. paradoxa were obtained from two cDNA libraries, one from cells grown in high [CO(2)] conditions and one from cells grown under low [CO(2)] conditions. A cDNA microarray platform assembled from 2378 cDNA sequences revealed that 142 genes significantly responded to a shift from high to low [CO(2)]. Trends in gene expression were comparable to those reported for Chlamydomonas reinhardtii and the cyanobacterium Synechocystis 6803, both possessing a CCM. Among genes regulated by [CO(2)], transcripts were identified encoding carbonic anhydrases (CAs), Rubisco activase and a putative bicarbonate transporter in C. paradoxa, likely functionally involved in the CCM. These results and the polyhedric appearance of the central body further support the hypothesis of a unique 'eukaryotic carboxysome' in Cyanophora.
Assuntos
Aclimatação/fisiologia , Dióxido de Carbono/metabolismo , Carbono/metabolismo , Cyanophora/metabolismo , Células Cultivadas , Cyanophora/citologia , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/fisiologia , Biblioteca GênicaRESUMO
Regionally distinct elongation responses to water stress in the maize primary root tip have been observed in the past. A genetic basis for such differential responses has been demonstrated. Normalized bar-coded cDNA libraries were generated for four regions of the root tip, 0-3 mm (R1), 3-7 mm (R2), 7-12 mm (R3), and 12-20 mm (R4) from the root apex, and transcript profiles for these regions were sampled. This permitted a correlation between transcript nature and regional location for 15 726 expressed sequence tags (ESTs) that, in approximately equal numbers, derived from three conditions of the root: water stress (water potential: -1.6 MPa) for 5 h and for 48 h, respectively, and well watered (5 h and 48 h combined). These normalized cDNA libraries provided 6553 unigenes. An analysis of the regional representation of transcripts showed that populations were largely unaffected by water stress in R1, correlating with the maintenance of elongation rates under water stress known for R1. In contrast, transcript profiles in regions 2 and 3 diverged in well-watered and water-stressed roots. In R1, transcripts for translation and cell cycle control were prevalent. R2 was characterized by transcripts for cell wall biogenesis and cytoskeleton formation. R3 and R4 shared prevalent groups of transcripts responsible for defence mechanisms, ion transport, and biogenesis of secondary metabolites. Transcripts which were followed for 1, 6, and 48 h of water stress showed distinct region-specific changes in absolute expression and changes in regulated functions.
Assuntos
Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Transcrição Gênica , Água/metabolismo , Zea mays/genética , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Zea mays/metabolismoRESUMO
Under salt stress conditions, the level of CpNpG-methylation (N is any nucleoside) of the nuclear genome of the facultative halophyte Mesembryanthemum crystallinum in the CCWGG sequences (W = A or T) increases two-fold and is coupled with hypermethylation of satellite DNA on switching-over of C3-photosynthesis to the crassulacean acid metabolism (CAM) pathway of carbon dioxide assimilation. The methylation pattern of the CCWGG sequences is not changed in both the 5'-promoter region of the gene of phosphoenolpyruvate carboxylase, the key enzyme of C4-photosynthesis and CAM, and in the nuclear ribosomal DNA. Thus, a specific CpNpG-hypermethylation of satellite DNA has been found under conditions of expression of a new metabolic program. The functional role of the CpNpG-hypermethylation of satellite DNA is probably associated with formation of a specialized chromatin structure simultaneously regulating expression of a large number of genes in the cells of M. crystallinum plants on their adaptation to salt stress and switching-over to CAM metabolism.
Assuntos
Adaptação Biológica , Metilação de DNA , DNA de Plantas/química , Regulação da Expressão Gênica de Plantas , Mesembryanthemum/genética , Cloreto de Sódio/farmacologia , Adaptação Biológica/efeitos dos fármacos , Adaptação Biológica/fisiologia , Sequência de Bases , DNA de Plantas/metabolismo , Genoma de Planta , Mesembryanthemum/efeitos dos fármacos , Mesembryanthemum/metabolismo , Dados de Sequência Molecular , Sementes/citologia , Sementes/metabolismo , Cloreto de Sódio/metabolismoRESUMO
Serial Analysis of Gene Expression was used to define number and relative abundance of transcripts in the root tip of well-watered maize seedlings (Zea mays cv FR697). In total, 161,320 tags represented a minimum of 14,850 genes, based on at least two tags detected per transcript. The root transcriptome has been sampled to an estimated copy number of approximately five transcripts per cell. An extrapolation from the data and testing of single-tag identifiers by reverse transcription-PCR indicated that the maize root transcriptome should amount to at least 22,000 expressed genes. Frequency ranged from low copy number (2-5, 68.8%) to highly abundant transcripts (100-->1,200; 1%). Quantitative reverse transcription-PCR for selected transcripts indicated high correlation with tag frequency. Computational analysis compared this set with known maize transcripts and other root transcriptome models. Among the 14,850 tags, 7,010 (47%) were found for which no maize cDNA or gene model existed. Comparing the maize root transcriptome with that in other plants indicated that highly expressed transcripts differed substantially; less than 5% of the most abundant transcripts were shared between maize and Arabidopsis (Arabidopsis thaliana). Transcript categories highlight functions of the maize root tip. Significant variation in abundance characterizes transcripts derived from isoforms of individual enzymes in biochemical pathways.
Assuntos
Regulação da Expressão Gênica de Plantas , Raízes de Plantas/genética , RNA de Plantas/genética , Transcrição Gênica , Zea mays/genética , Sequência de Bases , DNA de Plantas/genética , Enzimas/genética , Biblioteca Gênica , Proteínas de Plantas/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Transcript regulation in response to high salinity was investigated for salt-tolerant rice (var Pokkali) with microarrays including 1728 cDNAs from libraries of salt-stressed roots. NaCl at 150 mM reduced photosynthesis to one tenth of the prestress value within minutes. Hybridizations of RNA to microarray slides probed for changes in transcripts from 15 min to 1 week after salt shock. Beginning 15 min after the shock, Pokkali showed upregulation of transcripts. Approximately 10% of the transcripts in Pokkali were significantly upregulated or downregulated within 1 hr of salt stress. The initial differences between control and stressed plants continued for hours but became less pronounced as the plants adapted over time. The interpretation of an adaptive process was supported by the similar analysis of salinity-sensitive rice (var IR29), in which the immediate response exhibited by Pokkali was delayed and later resulted in downregulation of transcription and death. The upregulated functions observed with Pokkali at different time points during stress adaptation changed over time. Increased protein synthesis and protein turnover were observed at early time points, followed by the induction of known stress-responsive transcripts within hours, and the induction of transcripts for defense-related functions later. After 1 week, the nature of upregulated transcripts (e.g., aquaporins) indicated recovery.
Assuntos
Perfilação da Expressão Gênica , Genes de Plantas , Oryza/genética , Cloreto de Sódio/metabolismo , Adaptação Fisiológica , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Oryza/metabolismo , Oryza/fisiologia , Fotossíntese , Raízes de Plantas/metabolismo , Raízes de Plantas/fisiologia , Transcrição GênicaRESUMO
Transcript expression of Saccharomyces cerevisiae at high salinity was determined by microarray analysis of 6144 open reading frames (ORFs). From cells grown in 1 m NaCl for 10, 30, and 90 min, changes in transcript abundance >2-fold were classified. Salinity-induced ORFs increased over time: 107 (10 min), 243 (30 min), and 354 (90 min). Up-regulated, functionally unknown ORFs increased from 17 to 149 over this period. Expression patterns were similar early, with 67% of up-regulated transcripts after 10 min identical to those at 30 min. The expression profile after 90 min revealed different up-regulated transcripts (identities of 13% and 22%, respectively). Nucleotide and amino acid metabolism exemplified the earliest responses to salinity, followed by ORFs related to intracellular transport, protein synthesis, and destination. Transcripts related to energy production were up-regulated throughout the time course with respiration-associated transcripts strongly induced at 30 min. Highly expressed at 90 min were known salinity stress-induced genes, detoxification-related responses, transporters of the major facilitator superfamily, metabolism of energy reserves, nitrogen and sulfur compounds, and lipid, fatty acid/isoprenoid biosynthesis. We chose severe stress conditions to monitor responses in essential biochemical mechanisms. In the mutant, Deltagpd1/gpd2, lacking glycerol biosynthesis, the stress response was magnified with a partially different set of up-regulated ORFs.
Assuntos
Regulação Fúngica da Expressão Gênica , Fases de Leitura Aberta , Saccharomyces cerevisiae/genética , Solução Salina Hipertônica/farmacologia , Transcrição Gênica , Divisão Celular/efeitos dos fármacos , Replicação do DNA , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Cinética , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Transcrição Gênica/efeitos dos fármacosRESUMO
We have characterized transcripts for three potassium channel homologs in the AKT/KAT subfamily (Shaker type) from the common ice plant (Mesembryanthemum crystallinum), with a focus on their expression during salt stress (up to 500 mM NaCl). Mkt1 and 2, Arabidopsis AKT homologs, and Kmt1, a KAT homolog, are members of small gene families with two to three isoforms each. Mkt1 is root specific; Mkt2 is found in leaves, flowers, and seed capsules; and Kmt1 is expressed in leaves and seed capsules. Mkt1 is present in all cells of the root, and in leaves a highly conserved isoform is detected present in all cells with highest abundance in the vasculature. MKT1 for which antibodies were made is localized to the plasma membrane. Following salt stress, MKT1 (transcripts and protein) is drastically down-regulated, Mkt2 transcripts do not change significantly, and Kmt1 is strongly and transiently (maximum at 6 h) up-regulated in leaves and stems. The detection and stress-dependent behavior of abundant transcripts representing subfamilies of potassium channels provides information about tissue specificity and the complex regulation of genes encoding potassium uptake systems in a halophytic plant.
Assuntos
Regulação da Expressão Gênica de Plantas , Mesembryanthemum/classificação , Mesembryanthemum/fisiologia , Canais de Potássio/genética , Transcrição Gênica , Sequência de Aminoácidos , Arabidopsis/química , Arabidopsis/genética , Sequência de Bases , Hibridização In Situ , Dados de Sequência Molecular , Filogenia , Canais de Potássio/química , Canais de Potássio/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Seeds of the common ice plant (Mesembryanthemum crystallinum) germinate in distinct sub-populations over a time period of more than 4 weeks following imbibition. Distinguishing early (E)- and late (L)-germinating seeds is the expression of a homologue of the transcriptional activator VP1. The deduced amino acid sequence of ice plant VP1 (MVP1) is 39% identical (50% similar) to the sequence of the Arabidopsis VP1 homologue, ABI3. The amount of Mvp1 mRNA, transcribed from a single gene, is different in E and L seeds after water uptake. The levels of the Mvp1 transcripts are very low in immature and mature seeds and they increased during 6 days of imbibition. This expression profile of Mvp1 is different from known Vp1/ABI3-like genes in other plants. Cycloheximide (at 35 microM) abolishes the increase of Mvp1, and L seeds are turned into E seeds, which develop normally when the inhibitor is applied for a short time during imbibition. E seeds treated for the same time period are developmentally impaired and show no radicle elongation. We suggest that the presence and late disappearance of Mvp1 in L seeds is responsible for dormancy and after-ripening of late-germinating ice plant seeds.
Assuntos
Proteínas de Ligação a DNA/genética , Genes de Plantas , Desenvolvimento Vegetal , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/genética , Sementes/crescimento & desenvolvimento , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Sequência de Aminoácidos , Cicloeximida/farmacologia , Germinação , Dados de Sequência Molecular , Plantas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/biossíntese , Sementes/genética , Homologia de Sequência de AminoácidosRESUMO
Mitr1 and Mitr2 from Mesembryanthemum crystallinum (common ice plant) are members of a family of genes homologous to H+[or Na+]/myo-inositol symporters (ITRs), not previously studied in plants. MITR1 complemented an Itr1-deficient yeast strain. Mitr1 is strongly expressed in roots, moderately in stems, and weakly in leaves. Its transcripts increased in all organs, most dramatically in roots, under salinity stress. Mitr2 constitutes a rare transcript, slightly upregulated by salt stress in leaves only. Mitr1 transcripts are present in all cells in the root tip, but become restricted to phloem-associated cells in mature roots. Peptide antibodies against the two proteins indicated the presence of MITR1 in all organs and of MITR2 in leaves. Both are located in the tonoplast. MITR1 acts in removing sodium from root vacuoles, correlated with findings of low root sodium, while leaf vacuoles accumulate sodium in the ice plant. Up-regulation in leaves and stems is also found for Na+/H+-antiporter (Nhx-type) transcripts. Under comparable stress conditions, Nhx-and Itr-like transcripts in Arabidopsis were regulated differently. In the ice plant, co-ordinate induction of Na+/H+-antiporters and Na+/myo-inositol symporters transfers sodium from vacuoles in root cells into the leaf mesophyll as a halophytic strategy that lowers the osmotic potential. The tissue-specific differential expression of Itr- and Nhx-type transcripts suggests that the vacuolar sodium/inositol symporters function to reduce sodium amounts in cells of the root and vascular tissue, while sodium/proton antiporters in leaf tissues function to partition sodium into vacuoles for storage.
Assuntos
Proteínas de Transporte/genética , Inositol/metabolismo , Proteínas de Plantas , Plantas/genética , Sódio/metabolismo , Simportadores , Sequência de Aminoácidos , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Transporte Biológico , Proteínas de Transporte/metabolismo , DNA Complementar/química , DNA Complementar/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Teste de Complementação Genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mutação , Plantas/efeitos dos fármacos , Plantas/metabolismo , RNA de Plantas/efeitos dos fármacos , RNA de Plantas/genética , RNA de Plantas/metabolismo , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Cloreto de Sódio/farmacologia , Distribuição Tecidual , Transcrição Gênica/efeitos dos fármacos , Vacúolos/metabolismoRESUMO
Transgenic tobacco (Nicotiana tabacum L, cv. SR-1) expressing mannitol 1-phosphate dehydrogenase, MTLD, in chloroplasts and myo-inositol O-methyltransferase, IMT1, in the cytosol after crossing of lines which expressed these foreign genes separately has been analysed. Plants expressing both enzymes accumulated mannitol and D-ononitol in amounts comparable to those following single gene transfer and showed phenotypically normal growth during the vegetative stage. Induction of flowering for transgenovar and wild-type occurred at the same time, but during flowering the phenotype of the transformed plants changed. Compared to wild-type, transgenic plants were characterized by curled, smaller upper leaves and elongated stems during flowering; incomplete development of flower buds with shorter sepals and pedicels resulted in increased abortion. Flowers completing development were normal. The vegetative biomass of the transformed plants was slightly higher than that of wild-type. Concentrations of soluble sugars and potassium were lower than in wild-type only in the apical parts of the transgenic plants. Both enzymes, under control of the CaMV 35S promoter, promoted accumulation of mannitol and D-ononitol in the youngest leaves close to the vegetative meristem and in flowers, suggesting that their presence could signal lower sink demand leading to a decrease in carbon import to flowers and developing seed capsules. The interpretation here is that increases of inert carbohydrates in developing sinks interfere with metabolism, such as respiration or glycolysis. This interference may be less significant in source tissues during vegetative growth than in sink tissues during seed development.
Assuntos
Metabolismo dos Carboidratos , Nicotiana/metabolismo , Plantas Tóxicas , Íons , Fenótipo , Plantas Geneticamente Modificadas/metabolismoRESUMO
The reasons for the presence of a multitude of plasma membrane-localized water channels (PIP aquaporins) in plants may be functional differences in water (or other solute) transport, or in developmental, environmental or tissue-specific regulation of expression. We compared tissue- and cell-specific expression of McMipA, an abundantly expressed PIP from the common ice plant (Mesembryanthemum crystallinum), with that of the previously characterized McMipB [Yamada et al. (1997) Plant Cell Physiol. 38: 1326]. The activity of a 2.2 kb DNA fragment containing the promoter region of McMipA in a fusion with the GUS coding region was studied in transgenic tobacco. The McMipA promoter was active in pericycle and cortex cells in roots and in phloem-associated cells and cells surrounding the pericycle in shoots. In green leaves, mesophyll cells and the minor veins showed GUS activity, but the major veins did not. In floral tissues, GUS activity was observed in the pistil and anthers of immature buds and the tip of the mature pistil and pollen. Neither the apical meristem nor root tip showed any GUS activity. The differences in tissue specificity between the McMipA and McMipB promoters indicated that the two PIPs, MC-MIPA and MC-MIPB, serve different functions in plants.
Assuntos
Canais Iônicos/genética , Canais Iônicos/metabolismo , Nicotiana/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/fisiologia , Plantas Tóxicas , Plantas/genética , Regiões Promotoras Genéticas , Aquaporinas , Sequência de Bases , Glucuronidase/genética , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/biossíntese , Mapeamento por RestriçãoRESUMO
We have characterized transcripts for nine major intrinsic proteins (MIPs), some of which function as water channels (aquaporins), from the ice plant Mesembryanthemum crystallinum. To determine the cellular distribution and expression of these MIPs, oligopeptide-based antibodies were generated against MIP-A, MIP-B, MIP-C, or MIP-F, which, according to sequence and functional characteristics, are located in the plasma membrane (PM) and tonoplast, respectively. MIPs were most abundant in cells involved in bulk water flow and solute flux. The tonoplast MIP-F was found in all cells, while signature cell types identified different PM-MIPs: MIP-A predominantly in phloem-associated cells, MIP-B in xylem parenchyma, and MIP-C in the epidermis and endodermis of immature roots. Membrane protein analysis confirmed MIP-F as tonoplast located. MIP-A and MIP-B were found in tonoplast fractions and also in fractions distinct from either the tonoplast or PM. MIP-C was most abundant but not exclusive to PM fractions, where it is expected based on its sequence signature. We suggest that within the cell, MIPs are mobile, which is similar to aquaporins cycling through animal endosomes. MIP cycling and the differential regulation of these proteins observed under conditions of salt stress may be fundamental for the control of tissue water flux.
Assuntos
Aquaporinas/metabolismo , Plantas/metabolismo , Sequência de Aminoácidos , Aquaporinas/química , Sequência de Bases , Primers do DNA , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Cloreto de SódioRESUMO
Past efforts to improve plant tolerance to drought, high salinity and low-temperature through breeding and genetic engineering have had limited success owing to the genetic complexity of stress responses. Progress is now anticipated through comparative genomics studies of an evolutionarily diverse set of model organisms, and through the use of techniques such as high-throughput analysis of expressed sequence tags, large-scale parallel analysis of gene expression, targeted or random mutagenesis, and gain-of-function or mutant complementation. The discovery of novel genes, determination of their expression patterns in response to abiotic stress, and an improved understanding of their roles in stress adaptation (obtained by the use of functional genomics) will provide the basis of effective engineering strategies leading to greater stress tolerance.
Assuntos
Engenharia Genética/métodos , Fenômenos Fisiológicos Vegetais , Plantas/genética , Desastres , Concentração OsmolarRESUMO
A comparison of drought tolerance in plants at extreme ends of the evolutionary spectrum is beginning to show the mechanisms involved.
Assuntos
Dessecação , Fenômenos Fisiológicos Vegetais , Evolução Biológica , Desastres , Doenças das Plantas , Água/metabolismoRESUMO
Methylenetetrahydrofolate reductase (MTHFR) is the least understood enzyme of folate-mediated one-carbon metabolism in plants. Genomics-based approaches were used to identify one maize and two Arabidopsis cDNAs specifying proteins homologous to MTHFRs from other organisms. These cDNAs encode functional MTHFRs, as evidenced by their ability to complement a yeast met12 met13 mutant, and by the presence of MTHFR activity in extracts of complemented yeast cells. Deduced sequence analysis shows that the plant MTHFR polypeptides are of similar size (66 kDa) and domain structure to other eukaryotic MTHFRs, and lack obvious targeting sequences. Southern analyses and genomic evidence indicate that Arabidopsis has two MTHFR genes and that maize has at least two. A carboxyl-terminal polyhistidine tag was added to one Arabidopsis MTHFR, and used to purify the enzyme 640-fold to apparent homogeneity. Size exclusion chromatography and denaturing gel electrophoresis of the recombinant enzyme indicate that it exists as a dimer of approximately 66-kDa subunits. Unlike mammalian MTHFR, the plant enzymes strongly prefer NADH to NADPH, and are not inhibited by S-adenosylmethionine. An NADH-dependent MTHFR reaction could be reversible in plant cytosol, where the NADH/NAD ratio is 10(-3). Consistent with this, leaf tissues metabolized [methyl-(14)C]methyltetrahydrofolate to serine, sugars, and starch. A reversible MTHFR reaction would obviate the need for inhibition by S-adenosylmethionine to prevent excessive conversion of methylene- to methyltetrahydrofolate.
Assuntos
DNA Complementar/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , DNA Complementar/isolamento & purificação , Metilenotetra-Hidrofolato Redutase (NADPH2) , Dados de Sequência Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/isolamento & purificação , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Alinhamento de Sequência , Análise de SequênciaRESUMO
We have characterized three cDNAs from a gene family encoding lipid transfer proteins, LTP, from Arabidopsis thaliana (Wassilewskija). In addition to the already characterized Ltp1, our analysis includes Ltp2 and Ltp3, two sequences previously known as expressed sequence tags (EST) only. The deduced amino acid sequences of the three cDNAs share 56 to 57% identity and show unique tissue- and cell-specific expression. Genes Ltp1 and LTp2 are located within approximately 1.4 kb of each other in tandem orientation. RNA hydridizations showed that all three LTP are expressed in flowering meristems, flowers and developing seeds. Ltp1 is expressed in leaves in addition. Ltp3, though not Ltp2, is also expressed in a short segment of the stem close to the flowering meristem. In contrast to the epidermis-specific Ltp1, both Ltp2 and Ltp3 are not restricted to the epidermis, but are also expressed in sub-epidermal layers of the organs in which they are found. In the upper stem segment, Ltp3 is predominantly cortical. It appears that the expression of these three cDNAs is sufficient to account for the formation of LTP in all meristematic and expanding cells of the aboveground plant. Evolutionary analysis allows the conclusion that each Ltp belongs to a different sub-family of genes. Additionally, parsimony analysis provides evidence that several copies of Ltp genes already existed in ancestors of the Brassicaceae family.
Assuntos
Arabidopsis/genética , Proteínas de Transporte/genética , Genes de Plantas , Família Multigênica/genética , Sequência de Aminoácidos , Antígenos de Plantas , Arabidopsis/crescimento & desenvolvimento , Proteínas de Transporte/biossíntese , Proteínas de Transporte/classificação , Evolução Molecular , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Ten transcripts (Mpc1-10) homologous to protein phosphatases of the 2C family have been isolated from the halophyte Mesembryanthemum crystallinum (common ice plant). Transcripts range in size from 1.6 to 2.6 kb, and encode proteins whose catalytic domains are between 24% and 62% identical to that of the Arabidopsis PP2C, ABI1. Transcript expression is tissue specific. Two isoforms are present only in roots (Mpc1 and Mpc5), three in young leaves (Mpc6, 8 and 9), two in old leaves (Mpc6 and Mpc8), and two in post-flowering leaves (Mpc8 and Mpc9). Mpc2 is strongly expressed in roots and also in seeds, meristematic tissues and mature flowers. Mpc3 is specific for leaf meristems, and Mpc4 is found in root and leaf meristems. Mpc7 is restricted to meristematic tissues. Mpc10 is only present in mature flowers. Mpc2 (in roots and leaves), Mpc5 (in roots) and Mpc8 (weakly in leaves) are induced by salinity stress and drought conditions with different kinetics in different tissues, but other Mpcs are downregulated by stress. Cold stress (4 degrees C) leads to a decline in Mpc5 and Mp6, but low temperature provoked a long-term (days) increase in Mpc2 levels in leaves and a transient increase (less than 24 h) in roots. Four full-length transcripts have been obtained. In each case, after over-expression in E. coli, the isolated proteins exhibited (Mg2+-dependent, okadeic acid-insensitive) protein phosphatase activity, although activity against 32P-phosphocasein varied among different PP2Cs. Determination of tissue developmental and stress response specificity of PP2C will facilitate functional studies of signal-transducing enzymes in this halophytic organism.
Assuntos
Fosfoproteínas Fosfatases/genética , Plantas/enzimologia , Plantas/genética , Proteínas de Saccharomyces cerevisiae , Sequência de Bases , DNA de Plantas , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Fosfoproteínas Fosfatases/classificação , Fosfoproteínas Fosfatases/metabolismo , Proteína Fosfatase 2 , Proteína Fosfatase 2C , RNA de Plantas , Saccharomyces cerevisiaeRESUMO
A salt-tolerant stable cell-suspension culture from the halophyte Mesembryanthemum crystallinum L. has been established from calli generated from leaves of 6-week-old well-watered plants. Optimal cell growth was observed in the presence of 200 mM NaCl, and within 7 d cells were able to concentrate Na+ to levels exceeding those in the growth medium. Accumulation of Na+ was paralled by increases in the compatible solute pinitol and myo-inositol methyl transferase (IMT), a key enzyme in pinitol biosynthesis. Increasing concentrations of NaCl stimulated the activities of tonoplast and plasma-membrane H(+)-ATPases. Immunodetection of the ATPases showed that the increased activity was not due to changes in protein amount that could be attributed to treatment conditions. A specific role for these mechanisms in salt-adaptation is supported by the inability of mannitol-induced water stress to elicit the same responses, and the absence of enzyme activity and protein expression associated with Crassulacean acid metabolism in the cells. Results demonstrate that these M. crystallinum cell suspensions show a halophytic growth response, comparable to that of the whole plant, and thus provide a valuable tool for studying signaling and biochemical pathways involved in salt recognition and response.