A Plant Ca2+ Pump, ACA2, Relieves Salt Hypersensitivity in Yeast: MODULATION OF CYTOSOLIC CALCIUM SIGNATURE AND ACTIVATION OF ADAPTIVE Na+HOMEOSTASIS

Veena S Anil,Premraj Rajkumar,Pavan Kumar,M.K Mathew

doi:10.1074/jbc.m700766200

Veena S Anil, Premraj Rajkumar + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m700766200

Copy DOI

Abstract

Stress responses in both plants and yeast utilize calcium-mediated signaling. A yeast strain, K616, which lacks Ca(2+) pumps, requires micromolar Ca(2+) for growth. In medium containing 100 microM Ca(2+), K616 can withstand osmotic stress (750 mM sorbitol) and ionic stress (300 mM KCl) but not hypersodic stress (300 mM NaCl). Heterologous expression of the endoplasmic reticulum-located Arabidopsis thaliana Ca(2+)-ATPase, ACA2, permits K616 to grow under NaCl stress even in Ca(2+)-depleted medium. All stresses tested generated transient elevation of cytosolic Ca(2+) in wild type yeast, K601, whereas NaCl alone induced prolonged elevation of cytosolic Ca(2+) in K616. Both the Ca(2+) transient and survival of cultures subjected to NaCl stress was similar for the ACA2 transformant and K601. However, whereas K601 maintained low cytosolic Na(+) predominantly by pumping it out across the plasma membrane, the transformant sequestered Na(+) in internal organelles. This sequestration requires the presence of an endomembrane Na(+)/H(+)-antiporter, NHX1, which does not play a significant role in salt tolerance of wild type yeast except at acidic pH. Transcript levels of the plasma membrane Na(+)-ATPase, ENA1, were strongly induced only in K601, whereas NHX1 was strongly induced in both K601 and the ACA2 transformant. The calmodulin kinase inhibitor KN62 significantly reduced the salt tolerance of the ACA2 transformant and the transcriptional induction of NHX1. Thus, the heterologous expression of a plant endomembrane Ca(2+) pump results in the rapid depletion of cytosolic Ca(2+) and the activation of an alternate mechanism for surviving saline stress.

Highlights

Eukaryotic cells regulate a variety of cellular processes, including responses to abiotic stresses using calcium-mediated processes
Ura-selected colonies expressing ACA2 exhibited growth on complementation plates comprising SC medium depleted of calcium with 10 mM EGTA, unlike K616 and K616 transformed with the vector alone (Fig. 1A)
The ACA2 transformant strain will be referred to as K616ACA2 for simplicity, whereas the strain transformed with vector alone will be referred to as K616-V

Summary

The abbreviations used are

CaM, calmodulin; SOS, salt overly sensitive; ER, endoplasmic reticulum; RT, reverse transcription; ACA, N-(p-amylcinnamoyl) anthranilic acid; MES, 4-morpholineethanesulfonic acid. We demonstrate that the ER-located ACA2 relieves the hypersensitivity of the salt-sensitive triple mutant yeast strain K616, in which genes encoding the Ca2ϩ pumps (PMC1, PMR1, and the regulatory subunit of calcineurin, CNB1) have been deleted [39]. This strain requires 10Ϫ4 M Ca2ϩ to grow and millimolar Ca2ϩ to survive hypertonic stress. Using the aequorin reporter system, we demonstrate that ACA2 alters the [Ca2ϩ]cyt transient induced by NaCl stress in K616 We propose that this action of the pump contributes to the triggering of an alternative pathway, which activates the endomembrane Naϩ/Hϩ-antiporter, NHX1, resulting in enhanced salt tolerance

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION