Kcl Treatment Suaeda Salsa Vacuolar Membrane H +-atpase And Of H ~~ +-ppase Activity

Potassium is an essential macroelement that plays vital roles in various aspects ofplant growth and metabolism and is needed in large quantities. K~+ has been consideredto play an important role in salt tolerance. In non-halophytes, Na~+ salinity induces adecrease in K~+ content of the tissue, which has been proposed to contribute to salttoxicity. However, high concentrations of external K~+ above certain threshold levelinhibit plant growth. It is accepted that plants can tolerate NaCl stress, but can nottolerat isotonic KCl. Plants tolerate high concentration of external Na~+ through thecapacity of sequestrating Na~+ into the vacuole. The transportation of Na~+ against theconcentration gradient from the cytoplasm into the vacuole via the tonoplast Na~+/H~+antiporter is dependent on the activities of vacuolar H~+-ATPase (V-ATPase) andH~+-pyrophosphatase (V-PPase), which establish an electrochemical H~+-gradient acrossthe tonoplast that energizes the transporters. Our lab had proved that NaCl treatmentleaded to an increase in tonoplast Na~+/H~+ antiport activity as well as V-ATPaseactivity. In this study, salt-dilution halophyte S. salsa plants exposed to 100 mmol/LKCl and NaCl were used to investigate the activities and protein and mRNAexpression of V-ATPase and V-PPase of S. salsa by Western and Northern blotanalysis in addition to the determination of some physiological parameters (growth,photosynthesis, stomata and so on). The aim was to determine whether there wassome relationship between KCl toxicity to S. salsa and V-ATPase and V-PPase. Themain results were shown as follows.1. Effects of NaCl and KCl treatments on the growth of S. salsa.Plants exposed to 100 mmol/L KCl did not grow well compared with control plantsand had effects on plant morphology. Stressed young leaves were chlorotic and lateralbranches developed slowly. Mature leaves became narrow and necrotic at the leaf tip.While plants exposed to 100 mmol/L NaCl growed better than control plants.Compared with control plants, the fresh weight and water content significantlyincreased with NaCl treatment, while the dry weight and relative water content had anobvious change. This indicates that the increase of fresh weight was mainly caused bythe increase of water content. KCl Treatment had no distinct effects on fresh weightand dry weight and led to a significant decrease in water content and relative watercontent. The results show that KCl treatment induced water deficit. Cell sap osmoticpotential decreased markedly with NaCl and KCl treatment. This indicates that plantstreated with NaCl was likely to maintain water uptake via the decrease of osmoticpotential, while plants treated with KCl had not a greater ability for osmoticadjustment though the osmotic potential also decreased.2. Effects of NaCl and KCl treatments on the relative photosynthetic parameters of S.salsa. In mature leaves, the total chlorophyll content and chlorophyll a content decreasedmarkedly with NaCl and KCl treatments. This effect was more evident with NaCltreatment. The high water content and fresh weight possibly led to the decrease ofrelative chlorophyll content (mg g-1FW) in plants exposed to NaCl. The decrease ofchlorophyll content was likely to be the result of chlorophyll degradation and/orsynthesis deficiency in plants exposed to KCl. In young leaves, the total chlorophyllcontent, chlorophyll a content and chlorophyll b content all significantly reduced withNaCl and KCl treatments. The decreae was more evident in plants exposed to KCl.This can be explained by the fact that succulence is more evident in mature leaves ofS. salsa plants exposed to NaCl. The result that KCl treatment led to a drasticalreduction of chlorophyll content probably shows that young leaves were moresensitive to KCl treatment than mature leaves. In mature leaves of plants exposed toNaCl and KCl, Fv/Fm, ФPSⅡand net photosynthetic rate were not affected. Thedecrease of chlorophyll content probably led to a decrease in leaf-absorbed lightenergy, but leaf-absorbed light still met the maximum utility ratio. Therefore, netphotosynthetic rate was not significantly affected by the decrease of chlorophyllcontent. On the other hand, it also shows that NaCl and KCl treatments in this studydid not affect the Photosystem Ⅱ(PSⅡ).3. Effects of NaCl and KCl treatments on the leaf stomata of S. salsa. The stomatal aperture in plants exposed to NaCl was bigger than that in controlplants. S. salsa plants were likely to promote uptake of water via strongertranspiration. KCl treatment caused a decrease in the stomatal aperture, even theclosure of stomata. This probably shows that KCl treatment induced water stress andplants reduced water loss by closing stomata. In addition, epicuticular waxmorphology had changed with KCl treatment. The plants presumedly saved water bydecreasing cuticular transpiration as a response to KCl treatment.4. Effects of KCl treatment on the microstructure of leaf tip of S. salsaFrom the vertical cut pictures of paraffin-embedded tissue sections of leaf tip of S.salsa plants exposed to KCl, the gradual wilting from leaf tip was observed. Thepictures also show that the central cells died earlier and peripheral cells died later.This probably indicates that the cells around the vascular bundle died earlier so thatperipheral cells could not gain the necessary water and nutrient and died eventually.5. Effects of NaCl and KCl treatments on the hydrolysis activities and proteinexpression of V-ATPase and V-PPase of S. salsa. In mature leaves, both NaCl and KCl treatments led to an increase in V-ATPasehydrolysis activity. This effect was more evident with KCl treatment. NaCl and KCltreatments also led to an increase in V-PPase hydrolysis activity. However, theincrease was more evident in plants exposed to NaCl. In young leaves, the V-ATPasehydrolysis activity also increased in plants exposed to NaCl and KCl, but to a lesserextent than mature leaves. The V-PPase hydrolysis activity increased in plantsexposed to NaCl, while decreased in plants exposed to KCl. In addition, the V-ATPasehydrolysis activity in mature leaves was higher than in young leaves and the V-PPasehydrolysis activity in young leaves was higher than in mature leaves. The results areconsistent with the view that V-PPase is the main proton pump of vacuolarmembranes in most young tissues, while V-ATPase is a major proton pump ofvacuolar membranes in mature tissues. Western blot analysis of proteins present in tonoplast-enriched membrane vesiclewith the antisera ATP95 and ATP88 directed against the V-ATPase holoenzymereached the similar results. On the whole, the V-ATPase protein amount increased inboth mature and young leaves, more evident in mature leaves. The V-ATPase proteinamount increase was more significant in plants exposed to KCl than in plants exposedto NaCl. Western blot analysis with the antiserum directed against the subunit A ofV-ATPase confirmed the above results. Western blot analysis of proteins present in tonoplast-enriched membrane vesiclewith the antiserum directed against the V-PPase revealed the protein amount in youngleaves was more than in mature leaves. NaCl and KCl treatments did not changeprotein expression in young leaves. In mature leaves, NaCl and KCl treatmentsenhanced protein expression, and the enhancement was more evident in plantsexposed to NaCl. There was a basically consistent relationship between the westernblot analysis and the hydrolysis activity. Northern blot analysis indicated that the transcription of V-ATPase subunit c was...