RESUMO
There are two major methodical approaches with which changes of status in stomatal pores are addressed: indirectly by measurement of leaf transpiration, and directly by measurement of stomatal apertures. Application of the former method requires special equipment, whereas microscopic images are utilized for the direct measurements. Due to obscure visualization of cell boundaries in intact leaves, a certain degree of invasive leaf manipulation is often required. Our aim was to develop a protocol based on the minimization of leaf manipulation and the reduction of analysis completion time, while still producing consistent results. We applied rhodamine 6G staining of Arabidopsis thaliana leaves for stomata visualization, which greatly simplifies the measurement of stomatal apertures. By using this staining protocol, we successfully conducted analyses of stomatal responses in Arabidopsis leaves to both closure and opening stimuli. We performed long-term monitoring of living stomata and were able to document the same leaf before and after treatment. Moreover, we developed a protocol for rapid-fixation of epidermal peels, which enables high throughput data analysis. The described method allows analysis of stomatal apertures with minimal leaf manipulation and usage of the same leaf for sequential measurements, and will facilitate the analysis of several lines in parallel.
Assuntos
Arabidopsis/ultraestrutura , Microscopia de Fluorescência/métodos , Folhas de Planta/ultraestrutura , Estômatos de Plantas/ultraestrutura , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Corantes Fluorescentes/análise , Histidina Quinase/genética , Microscopia de Fluorescência/economia , Folhas de Planta/genética , Folhas de Planta/fisiologia , Estômatos de Plantas/genética , Estômatos de Plantas/fisiologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/ultraestrutura , Rodaminas/análise , Coloração e Rotulagem/economia , Coloração e Rotulagem/métodos , Fatores de TempoRESUMO
Rice is one of the most important staple foods worldwide, but it often contains inorganic arsenic, which is toxic and gives rise to severe health problems. Rice plants take up arsenate As(V) via the phosphate transport pathways, though it is not known how As(V), as compared to phosphate, modifies the expression of phosphate transporters (PTs). Therefore, the impact of As(V) or phosphate (Pi) on the gene expression of PTs and several Pi signaling regulators was investigated. Rice plants were grown on medium containing different As(V) or Pi concentrations. Growth was evaluated and the expression of tested genes was quantified at different time points, using quantitative RT-PCR (qPCR). The As and P content in plants was determined using inductively coupled plasma mass spectrometry (ICP-MS). As(V) elicited diverse and opposite responses of different PTs in roots and shoots, while Pi triggered a more shallow and uniform transcriptional response in several tested genes. Only a restricted set of genes, including PT2, PT3, PT5 and PT13 and two SPX-MFS family members, was particularly responsive to As(V). Despite some common reactions, the responses of the analyzed genes were predominantly ion-specific. The possible reasons and consequences are discussed.
