Publicação
Role of PIP5K in heat-stress signalling and tolerance in Arabidopsis thaliana
| Resumo: | Plants are exposed to a wide variety of biotic and abiotic stresses that disturb their development and productivity, such as extreme temperatures that can gravely affect the plant morphology, physiology and metabolism. On its turn, plants have developed responses to cope with heat stress, a process termed thermotolerance. Basal thermotolerance refers to the innate capacity of plants to survive when exposed to high temperatures while acquired thermotolerance involves a prior acclimation to a moderate level of heat. Plants’ perception of the heat stress involves multiple pathways and signalling molecules such as the phosphoinositides, which are derived from the structural membrane lipid phosphatidylinositol. Previous work on phospholipid signalling of heat stress have demonstrate a dramatic increase in PtdIns(4,5)P2, as a consequence of an increase in PIP5K activity, and by 32Pphospholipid labelling it was discovered to involve PIP5K7 and PIP5K8 (Mishkind et al., 2009; Munnik lab, unpublished). These kinases are associated to the PtdIns(4,5)P2 generation pathway, throughout the phosphorylation of the head group of another phosphoinositide, PtdInds4P. The principal aim of this work is understanding what are the role of these kinases in heat signalling. For that, T-DNA insertion knock-out mutants (single and double pip5k7 and pip5k8 mutants) have been generated to study the role of these genes in basal- and acquired-heat tolerance and evaluation of the gene expression analysis by Promoter-GUS assays. For evaluating seedlings survival to heat, 3 days-old seedlings were subjected to a heat shock of 44 °C for 36 min, for basal thermotolerance, or 45 °C for 2.5 h with a prior treatment of 37 °C for 1 h, for acquired thermotolerance. Root elongation and GUS-staining analysis were performed with 6 days-old seedlings subjected to 40 °C for 30 min. Seed germination was assessed after seeds being subjected to a heat shock of 50 °C for 2 hours. Phenotypic analysis has showed that pip5k8 loss-offunction seedlings exhibit a better recovery from heat shock, compared to wild-type ones, with greener leaves and a more developed root system. The better performance of the pip5k8 loss-of-function mutants in basal thermotolerance may indicate that it is not the PIP5K8 but the PIP5K7 that is involved in the tolerance to heat stress for basal thermotolerance. This was also corroborated by the histologic GUS-staining analysis of the pip5k7 promoter, being displayed an intensive blue staining in non-vascular cells of the cotyledons, which was not observed for untreated seedlings. For acquired thermotolerance, it was observable a phenotype for the pip5k7 loss-of-function seedlings, that showed higher survival from the heat shock than the wild-type. The higher viability of pip5k7-1 loss-of-function mutant suggest that the PIP5K8 is involved in acquired thermotolerance of young seedlings, promoting an improvement of seedlings survival. Looking at the germination patterns after subjecting the seeds to heat treatments (basal and acquired), the pip5k8 loss-of-function seeds showed a percentage of germination significantly inferior than the wild-type ones. This phenotype may indicate a role for PIP5K8 in heat tolerance during seed germination, either for basal and acquired thermotolerance. The approaches used in this work to investigate the PIP5K7 and PIP5K8 function have disclosed the role of these PIP5K isoforms in heat-stress responses (basal and acquired thermotolerance) during seed germination and early plant development. Further research on the expression of these kinases by Q-PCR will strengthen the current knowledge about their role in heat signalling. |
|---|---|
| Autores principais: | Alves, Rui Jorge Andrade |
| Assunto: | Ciências Naturais::Ciências Biológicas |
| Ano: | 2018 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Plants are exposed to a wide variety of biotic and abiotic stresses that disturb their development and productivity, such as extreme temperatures that can gravely affect the plant morphology, physiology and metabolism. On its turn, plants have developed responses to cope with heat stress, a process termed thermotolerance. Basal thermotolerance refers to the innate capacity of plants to survive when exposed to high temperatures while acquired thermotolerance involves a prior acclimation to a moderate level of heat. Plants’ perception of the heat stress involves multiple pathways and signalling molecules such as the phosphoinositides, which are derived from the structural membrane lipid phosphatidylinositol. Previous work on phospholipid signalling of heat stress have demonstrate a dramatic increase in PtdIns(4,5)P2, as a consequence of an increase in PIP5K activity, and by 32Pphospholipid labelling it was discovered to involve PIP5K7 and PIP5K8 (Mishkind et al., 2009; Munnik lab, unpublished). These kinases are associated to the PtdIns(4,5)P2 generation pathway, throughout the phosphorylation of the head group of another phosphoinositide, PtdInds4P. The principal aim of this work is understanding what are the role of these kinases in heat signalling. For that, T-DNA insertion knock-out mutants (single and double pip5k7 and pip5k8 mutants) have been generated to study the role of these genes in basal- and acquired-heat tolerance and evaluation of the gene expression analysis by Promoter-GUS assays. For evaluating seedlings survival to heat, 3 days-old seedlings were subjected to a heat shock of 44 °C for 36 min, for basal thermotolerance, or 45 °C for 2.5 h with a prior treatment of 37 °C for 1 h, for acquired thermotolerance. Root elongation and GUS-staining analysis were performed with 6 days-old seedlings subjected to 40 °C for 30 min. Seed germination was assessed after seeds being subjected to a heat shock of 50 °C for 2 hours. Phenotypic analysis has showed that pip5k8 loss-offunction seedlings exhibit a better recovery from heat shock, compared to wild-type ones, with greener leaves and a more developed root system. The better performance of the pip5k8 loss-of-function mutants in basal thermotolerance may indicate that it is not the PIP5K8 but the PIP5K7 that is involved in the tolerance to heat stress for basal thermotolerance. This was also corroborated by the histologic GUS-staining analysis of the pip5k7 promoter, being displayed an intensive blue staining in non-vascular cells of the cotyledons, which was not observed for untreated seedlings. For acquired thermotolerance, it was observable a phenotype for the pip5k7 loss-of-function seedlings, that showed higher survival from the heat shock than the wild-type. The higher viability of pip5k7-1 loss-of-function mutant suggest that the PIP5K8 is involved in acquired thermotolerance of young seedlings, promoting an improvement of seedlings survival. Looking at the germination patterns after subjecting the seeds to heat treatments (basal and acquired), the pip5k8 loss-of-function seeds showed a percentage of germination significantly inferior than the wild-type ones. This phenotype may indicate a role for PIP5K8 in heat tolerance during seed germination, either for basal and acquired thermotolerance. The approaches used in this work to investigate the PIP5K7 and PIP5K8 function have disclosed the role of these PIP5K isoforms in heat-stress responses (basal and acquired thermotolerance) during seed germination and early plant development. Further research on the expression of these kinases by Q-PCR will strengthen the current knowledge about their role in heat signalling. |
|---|