RESUMO
Glycine betaine (GB) is a compatible solute accumulated by many plants under various abiotic stresses. GB is synthesized in two steps, choline â betaine aldehyde â GB, where a functional choline-oxidizing enzyme has only been reported in Amaranthaceae (a chloroplastic ferredoxin-dependent choline monooxygenase) thus far. Here, we have cloned a cDNA encoding a choline monooxygenase (CMO) from barley (Hordeum vulgare) plants, HvCMO. In barley plants under non-stress condition, GB had accumulated in all the determined organs (leaves, internodes, awn and floret proper), mostly in the leaves. The expression of HvCMO protein was abundant in the leaves, whereas the expression of betaine aldehyde dehydrogenase (BADH) protein was abundant in the awn, floret proper and the youngest internode than in the leaves. The accumulation of HvCMO mRNA was increased by high osmotic and low-temperature environments. Also, the expression of HvCMO protein was increased by the presence of high NaCl. Immunofluorescent labeling of HvCMO protein and subcellular fractionation analysis showed that HvCMO protein was localized to peroxisomes. [(14)C]choline was oxidized to betaine aldehyde and GB in spinach (Spinacia oleracea) chloroplasts but not in barley, which indicates that the subcellular localization of choline-oxidizing enzyme is different between two plant species. We investigated the choline-oxidizing reaction using recombinant HvCMO protein expressed in yeast (Saccharomyces cerevisiae). The crude extract of HvCMO-expressing yeast coupled with recombinant BBD2 protein converted [(14)C]choline to GB when NADPH was added as a cofactor. These results suggest that choline oxidation in GB synthesis is mediated by a peroxisomal NADPH-dependent choline monooxygenase in barley plants.
Assuntos
Betaína/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Hordeum/enzimologia , Oxigenases/metabolismo , Peroxissomos/enzimologia , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Betaína-Aldeído Desidrogenase/genética , Betaína-Aldeído Desidrogenase/metabolismo , Colina/metabolismo , Temperatura Baixa , DNA Complementar/genética , Regulação da Expressão Gênica de Plantas/genética , Hordeum/genética , Dados de Sequência Molecular , Pressão Osmótica , Oxirredução , Oxigenases/genética , Folhas de Planta/enzimologia , Folhas de Planta/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , RNA Mensageiro/genética , RNA de Plantas/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Spinacia oleracea/genética , Spinacia oleracea/metabolismoRESUMO
The accumulation of glycinebetaine (GB) is one of the adaptive strategies to adverse salt stress conditions. Although it has been demonstrated that barley plants accumulate GB in response to salt stress and various studies focused on GB synthesis were performed, its transport mechanism is still unclear. In this study, we identified a novel gene, HvProT2, encoding Hordeum vulgare GB/proline transporter from barley plants. Heterologous expression in yeast (Saccharomyces cerevisiae) mutant demonstrated that the affinity of HvProT2 was highest for GB, intermediate for proline and lowest for gamma-aminobutyric acid. Transient expression of fusions of HvProT2 and green fluorescent protein in onion epidermal cells revealed that HvProT2 is localized at the plasma membrane. Relative quantification of mRNA level of HvProT2 using semi-quantitative reverse transcription-polymerase chain reaction analysis showed that HvProT2 is constitutively expressed in both leaves and roots, and the expression level was higher in old leaves than young leaves and roots. Moreover, we found that HvProT2 was expressed in the mestome sheath and lateral root cap cells. We discussed the possible involvement of HvProT2 for salt stress tolerance.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros/genética , Betaína/metabolismo , Glicina/metabolismo , Hordeum/genética , Raízes de Plantas/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Clonagem Molecular , DNA Complementar , Hibridização In Situ , Cinética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/genética , Frações Subcelulares/metabolismoRESUMO
Since sweetness is one of the most important qualities of many fruits, and since sugars are translocated from leaves to fruits, the present study investigates photosynthetic activity, activity of sugar metabolizing enzymes, sugar content in leaves and fruits and endogenous levels of hydrogen peroxide in leaves of melon plants treated with various dilutions of hydrogen peroxide, a nonspecific signaling molecule in abiotic stress. For this purpose, 4-month-old melon plants were treated with various concentrations (<50mM) of hydrogen peroxide by applying 300 mL per day to the soil of potted plants. The treatments resulted in increased fructose, glucose, sucrose and starch in the leaves and fruits. The most effective concentration of hydrogen peroxide was 20mM. During the day, soluble sugars in leaves were highest at 12:00 h and starch at 15:00 h. Furthermore, the peroxide treatment increased the photosynthetic activity and the activities of chloroplastic and cytosolic fructose-1,6-bisphosphatase, sucrose phosphate synthase and invertases. Thus, our data show that exogenous hydrogen peroxide, applied to the soil, can increase the soluble sugar content of melon fruits.