Identificação e caracterização de genes expressos em resposta ao estresse por baixa temperatura em cana-de-açucar

AUTOR(ES)
DATA DE PUBLICAÇÃO

2004

RESUMO

Environmental stresses represent one of the main factors limiting crop productivity, being very important studies that aim to analyze, at a molecular level, the effects caused by these stresses. In the present project, we analyzed the sugarcane gene expression profiles in response to low temperature. Initially, high-density filter arrays containing 1536 ESTs (Expressed Sequence Tags) from SUCEST (Sugarcane EST Project) were hybridized with P33-labeled cDNA probes obtained from total RNA of sugarcane plantlets growing at 4 oC during 3, 6, 12, 24, and 48 h. As a control, plantlets growing at 26 oC were utilized. The experiment was repeated twice, being total RNA extracted from novel cold-treated and untreated plantlet sets. Only those ESTs, which presented similar expression profiles in both experiments, were taken into consideration for a further analysis. The results allowed identifying 59 ESTs that were induced or repressed by chilling stress. Selected genes had their expression profiles confirmed by RNA-blot analysis. Using SUCEST database and protein sequences from NCBI, a database containing 33 sugarcane proteins related to cold temperatures was generated. Among those proteins, we identified a PUMP (Plant Uncoupling Mitochondrial Protein) gene family member. This protein was reported to be involved in oxidative stress responses. Further, in SUCEST database, four additional proteins belonging to PUMP and four proteins belonging to AOx family (alternative oxidase) were identified. Phylogenetic analyses confirmed the existence of novel members of both protein families in sugarcane (Saccharum sp. PUMPs and AOxs: SsPUMPs and SsAOxs) and Arabidopsis (AtPUMPs and AtAOxs). Gene expression profile analyses suggested that PUMPs and AOxs are expressed in different plant organs. RNAblot results suggested that two members of sugarcane PUMP gene family (SsPUMP4 and SsPUMP5) and one member of AOx family (SsAOx1c) were induced by low temperature (4 oC). Arabidopsis PUMP4 and PUMP5 orthologs and one member of AOx family (AtAOx1a) were also induced by cold stress. Interestingly, AtAOx1d was down-regulated after plantlets were exposed In silico analyses demonstrated the existence of 26 members of sugarcane NAC domain protein family, including the cold-inducible SsNAC23 (Saccharum sp. NAC23). This gene was completely sequenced and analyzed. RNA-blot results suggested that SsNAC23 is modulated by extreme low temperatures (4 oC), but not at moderate ones (12 oC). SsNAC23 transcripts also accumulated in response to herbivory (Diatraea saccharalis) and PEG-induced water stress. Nuclear localization essays demonstrated that SsNAC23 was targeted to nucleus of onion cells (Allium cepa). A molecular model of the NAC domain of SsNAC23 allowed analyzing the structural determinants putatively involved in DNA-binding, evidencing the SsNAC23 transcriptional activator nature. Alignment of other NAC domain proteins revealed high structural determinants conservation in different plant species. In summary, our results indicated that the SsNAC23 is a transcription factor involved in biotic and abiotic stress response.

ASSUNTO(S)

cana-de-açucar frio genetica

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