Enhanced Expression of Vacuolar H+-ATPase Subunit E in the Roots Is Associated with the Adaptation of Broussonetia papyrifera to Salt Stress

University College Dublin, Ireland
PLoS ONE (Impact Factor: 3.23). 10/2012; 7(10):e48183. DOI: 10.1371/journal.pone.0048183
Source: PubMed


Vacuolar H(+)-ATPase (V-H(+)-ATPase) may play a pivotal role in maintenance of ion homeostasis inside plant cells. In the present study, the expression of V-H(+)-ATPase genes was analyzed in the roots and leaves of a woody plant, Broussonetia papyrifera, which was stressed with 50, 100 and 150 mM NaCl. Moreover, the expression and distribution of the subunit E protein were investigated by Western blot and immunocytochemistry. These showed that treatment of B. papyrifera with NaCl distinctly changed the hydrolytic activity of V-H(+)-ATPase in the roots and leaves. Salinity induced a dramatic increase in V-H(+)-ATPase hydrolytic activity in the roots. However, only slight changes in V-H(+)-ATPase hydrolytic activity were observed in the leaves. In contrast, increased H(+) pumping activity of V-H(+)-ATPase was observed in both the roots and leaves. In addition, NaCl treatment led to an increase in H(+)-pyrophosphatase (V-H(+)-PPase) activity in the roots. Moreover, NaCl treatment triggered the enhancement of mRNA levels for subunits A, E and c of V-H(+)-ATPase in the roots, whereas only subunit c mRNA was observed to increase in the leaves. By Western blot and immunocytological analysis, subunit E was shown to be augmented in response to salinity stress in the roots. These findings provide evidence that under salt stress, increased V-H(+)-ATPase activity in the roots was positively correlated with higher transcript and protein levels of V-H(+)-ATPase subunit E. Altogether, our results suggest an essential role for V-H(+)-ATPase subunit E in the response of plants to salinity stress.

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    • "Furthermore, the regulations of V-H + - ATPase activity are proposed to be operated in parallel with the alterations in transcript levels and/or the amounts of different protein subunits of V-H + -ATPase under salt stress (Silva and Geros 2009). Enhanced expression of V-H + -ATPase subunit E is paralleled by the increase of V-H + -ATPase hydrolytic activity which improves the adaptation of Broussonetia papyrifera to salt stress (Zhang et al. 2012), while the transcript level of subunit A of V-H + -ATPase has also been shown to be induced by salt and osmotic stresses in Arabidopsis and barley (Magnotta and Gogarten 2002; Fukuda et al. 2004). Moreover, overexpression of Spartina alterniflora V-H + - ATPase subunit c in rice brings an enhanced tolerance to salt stress (Baisakh et al. 2012), and transgenic Arabidopsis with wheat V-H + -ATPase subunit B exhibited higher salt tolerance than the wild type (Wang et al. 2011). "
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    ABSTRACT: Vacuolar H(+)-ATPase (V-H(+)-ATPase) has been proved to be of importance in maintenance of ion homeostasis inside plant cells under NaCl stress. In this study, the expression levels and salt-tolerate function of V-H(+)-ATPase genes were investigated in the roots and leaves of a halotolerate peppermint (Mentha × piperita L.) Keyuan-1 treated with different concentrations of NaCl. Results showed that the expressions of V-H(+)-ATPase in the transcriptional, protein and activity levels were significantly enhanced in the halotolerate peppermint Keyuan-1 compared to the wild-type (WT) peppermint under 50, 100, and 150 mM NaCl treatment. Moreover, inhibition experiments exhibited that V-H(+)-ATPase activity played vital roles in the salt tolerance of peppermint Keyuan-1 to 150 mM NaCl stress through increasing the vacuolar H(+) pumping activity and Na(+) compartmentalization capacity. Furthermore, results of Western blots showed that the activity of a mitogen-activated protein kinase (MAPK) was significantly increased under different concentrations of NaCl in the halotolerate peppermint Keyuan-1, which was much higher than that of WT peppermint. Further experiments with inhibitors suggested that this MAPK protein was responsible for the enhanced expression of V-H(+)-ATPase in the halotolerate peppermint Keyuan-1. In response to NaCl stress, increase of cytoplasmic calcium concentration ([Ca(2+)]cyt) occurred upstream of MAPK activation in the halotolerate peppermint Keyuan-1. In all, these findings demonstrated that increased V-H(+)-ATPase activity was positively correlated with the enhanced salt tolerance in the halotolerate peppermint Keyuan-1, providing the theoretic basis for the further development and utilization of peppermint in saline areas.
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    • "The resulting pellets were plasma membrane protein and then resuspended in the above-described resuspended buffer involving 1 mM EDTA. Moreover, the crude microsomal membrane protein was also used to the extraction of tonoplast vesicles [44]. The microsomal membrane pellet was resuspended in a buffer containing 2 mM BTP/Mes, pH 7.0, 250 mM sucrose, 0.2% BSA, 10% glycerol and 1 mM DTT, covered with a 25/38% (w/w) discontinuous sucrose density gradient, and then centrifuged with 100,000 g for 2 h. "
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    ABSTRACT: Stomatal movement plays a key role in plant development and response to drought and salt stress by regulating gas exchange and water loss. A number of genes have been demonstrated to be involved in the regulation of this process. Using inverse genetics approach, we characterized the function of a rice (Oryza sativa L.) vacuolar H(+)-ATPase subunit A (OsVHA-A) gene in stomatal conductance regulation and physiological response to salt and osmotic stress. OsVHA-A was constitutively expressed in different rice tissues, and the fusion protein of GFP-OsVHA-A was exclusively targeted to tonoplast when transiently expressed in the onion epidermal cells. Heterologous expression of OsVHA-A was able to rescue the yeast mutant vma1Δ (lacking subunit A activity) phenotype, suggesting that it partially restores the activity of V-ATPase. Meanwhile, RNAi-directed knockdown of OsVHA-A led to a reduction of vacuolar H(+)-ATPase activity and an enhancement of plasma membrane H(+)-ATPase activity, thereby increasing the concentrations of extracellular H(+) and intracellular K(+) and Na(+) under stress conditions. Knockdown of OsVHA-A also resulted in the upregulation of PAM3 (plasma membrane H(+)-ATPase 3) and downregulation of CAM1 (calmodulin 1), CAM3 (calmodulin 3) and YDA1 (YODA, a MAPKK gene). Altered level of the ion concentration and the gene expression by knockdown of OsVHA-A probably resulted in expanded aperture of stomatal pores and increased stomatal density. In addition, OsVHA-A RNAi plants displayed significant growth inhibition under salt and osmotic stress conditions. Taken together, our results suggest that OsVHA-A takes part in regulating stomatal density and opening via interfering with pH value and ionic equilibrium in guard cells and thereby affects the growth of rice plants.
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