RESUMO
Eugenia uniflora is an Atlantic Forest native species, occurring in contrasting edaphoclimatic environments. The identification of genes involved in response to abiotic factors is very relevant to help in understanding the processes of local adaptation. 1-Pyrroline-5-carboxylate synthetase (P5CS) is one interesting gene to study in this species since it encodes a key enzyme of proline biosynthesis, which is an osmoprotectant during abiotic stress. Applying in silico analysis, we identified one P5CS gene sequence of E. uniflora (EuniP5CS). Phylogenetic analysis, as well as, gene and protein structure investigation, revealed that EuniP5CS is a member of P5CS gene family. Plants of E. uniflora from two distinct environments (restinga and riparian forest) presented differences in the proline accumulation and P5CS expression levels under growth-controlled conditions. Both proline accumulation and gene expression level of EuniP5CS were higher in the genotypes from riparian forest than those from restinga. When these plants were submitted to drought stress, EuniP5CS gene was up-regulated in the plants from restinga, but not in those from riparian forest. These results demonstrated that EuniP5CS is involved in proline biosynthesis in this species and suggest that P5CS gene may be an interesting candidate gene in future studies to understand the processes of local adaptation in E. uniflora.
Assuntos
Eugenia/genética , Glutamato-5-Semialdeído Desidrogenase/genética , Complexos Multienzimáticos/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Aldeído Desidrogenase/genética , Aldeído Desidrogenase/metabolismo , Secas , Eugenia/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Glutamato-5-Semialdeído Desidrogenase/metabolismo , Ligases/metabolismo , Complexos Multienzimáticos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Filogenia , Plantas/metabolismo , Prolina/biossíntese , Pirróis/metabolismo , Estresse Fisiológico/genéticaRESUMO
Reed canary grass (RCG) is a perennial grass traditionally cultivated for forage. It is also used as fuel to produce energy in Finland and Sweden, and other countries have expressed interest in the cultivation of RCG. In China, arable land is limited. Salinity is considered to be a major factor limiting plant crop development and productivity. To boost biofuel production of RCG and extend its range in saline soil, we seek to improve its salt tolerance. Proline acts as an osmolyte that accumulates when plants are subjected to abiotic stress. P5CS plays a crucial role in proline biosynthesis. We isolated a P5CS gene from RCG, designated B231P5CS (GenBank accession No. JQ622685). B231P5CS is a fragment (971 bp) that encodes a 323-amino acid polypeptide. We also cloned an actin gene fragment from RCG as a reference gene in expression analysis of B231P5CS gene. Expression analysis revealed that B231P5CS transcripts were upregulated in leaves after treatment with salt (200 mM NaCl) and that transcript levels of B231P5CS reached a maximum 12 h after exposure, which was 14.69 times the level in control plants. The trends of expression were exactly opposite in roots; transcripts were downregulated after salt treatment. Proline concentration increased in leaves after stress. In contrast, proline content of roots decreased up to 3.6-fold relative to controls. Changes in proline concentration after stress were correlated with B231P5CS expression. Our results suggest that B231P5CS is a stress-inducible gene and plays a non-redundant role in plant development. This gene may be used to improve stress tolerance of RGC and other bioenergy feedstock.
Assuntos
Glutamato-5-Semialdeído Desidrogenase/genética , Complexos Multienzimáticos/genética , Phalaris/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Glutamato-5-Semialdeído Desidrogenase/classificação , Glutamato-5-Semialdeído Desidrogenase/metabolismo , Dados de Sequência Molecular , Complexos Multienzimáticos/classificação , Complexos Multienzimáticos/metabolismo , Phalaris/enzimologia , Phalaris/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/classificação , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Filogenia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/classificação , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Prolina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tolerância ao Sal/genética , Análise de Sequência de DNA , Cloreto de Sódio/farmacologia , Estresse Fisiológico/genética , Fatores de TempoRESUMO
Proline (Pro) is one of the most accumulated osmolytes in salinity and water deficit conditions in plants. In the present study, we measured the Pro content, the activity and the expression level of delta 1-pyrroline-5-carboxylate synthetase (P5CS: gamma-glutamyl kinase, EC 2.7.2.11 and glutamate-5-semialdehyde dehydrogenase, EC 1.2.1.41), a key regulatory enzyme involved in the biosynthesis of Pro, in cactus pear (Opuntia streptacantha) subjected to 6, 9 and 11 days of salt stress. Treatment with NaCl of O. streptacantha young plants resulted in a decrease in the cladode thickness and root length, and in a significant and gradual accumulation of Pro in young cladodes, in a time- and concentration-dependent manner. P5CS activity, studied as gamma-glutamyl kinase, was reduced at all times as a consequence of salt treatment, except at the sixth day at 75 and 150mM of NaCl, where a slight increase was observed. We isolated an open reading frame (ORF) fragment of p5cs gene. The deduced amino acid sequence of the P5CS protein exhibited 90.4% of identity with the P5CS protein from Mesembryanthemum crystallinum. RT-PCR analysis revealed that the Osp5cs gene of O. streptacantha was induced by salt stress at 9 and 11 days of treatment. Furthermore, ABA-induced Osp5cs gene expression was observed in cladodes of cactus pear young plants. We observed an evident correlation between the transcript up-regulation and the Pro accumulation under salt stress; however, these results do not parallel with the changes in P5CS enzymatic activity. This Pro accumulation might function as an osmolyte for the intracellular osmotic adjustment and might be playing a critical role in protecting photosynthetic activity in O. streptacantha plants under salt stress.