Salt-dependent regulation of chloride channel transcripts in rice

Abstract

Rice OsCLC1 homologous to voltage-dependent Cl⁻ channels of the CLC-family was characterized to study the regulation of Cl⁻ homeostasis under salt stress conditions. By transcript analyses, expression of OsCLC1 was found in leaves and roots. Transcriptional regulation during salt stress was compared in the salt-sensitive Cl⁻-accumulating rice line IR29 and the salt-tolerant Cl⁻-excluding rice line Pokkali. In response to salt stress OsCLC1 transcript levels were repressed in leaves and roots of IR29 whereas in Pokkali expression was transiently induced. Under same conditions, in IR29 mRNA levels of the Na⁺/H⁺ antiporter OsNHX1 and of the vacuolar H⁺-ATPase subunit OsVHA-B decreased upon salt stress whereas Pokkali showed transient stimulation of OsVHA-B transcripts. Cell-specificity of OsCLC1 transcription was analyzed by in situ PCR. In leaves, signals were detected in mesophyll cells and stomata. In addition, expression occurred in xylem parenchyma cells and in the phloem whereas in salt-treated plants transcript amounts were reduced in mesophyll cells. Our results indicate coordinated regulation of anion and cation homeostasis in salt-treated rice and suggest function of OsCLC1 in osmotic adjustment at high salinity.

Introduction

Salinity is an environmental factor that greatly affects plant growth and development and is a major constraint for crop production. Recently, molecular key mechanisms involved in regulation of cellular cation homeostasis and osmotic balancing in salt-treated plants have been identified. Salt-induced transcriptional regulations of HKT1-type alkali cation transporters and of AKT1-type K⁺-channels have been shown that are likely to control influx of Na⁺ [1], [2], [3], [4], [5]. Expression of members of HAK-type K⁺-transporters that efficiently exclude Na⁺ when heteologously expressed in yeast is stimulated in the halotolerant Mesembryanthemum crystallinum in response to salt stress and these transporters become the major K⁺ uptake system under conditions of high salinity in this species [6]. In Arabidopsis thaliana, the plasma membrane Na⁺/H⁺ antiporter AtSOS1 has been identified that controls cellular Na⁺ efflux [7], [8]. Vacuolar sequestration of Na⁺ is mediated by secondary active NHX1-type Na⁺/H⁺ antiporters [9], [10], [11] that are energized by the vacuolar H⁺-ATPase [12], [13], [14]. In M. crystallinum, expression and enzyme activity of NADP-specific isocitrate dehydrogenase that links C and N metabolism and is involved in biosynthesis of the osmoprotective amino acid proline are salt-induced [15]. Increased expression of the plasma membrane localized transporter McNRT1 was shown in root epidermal cells of salt-treated M. crystallinum indicating enhanced nitrate uptake whereas the proline transporter McAAT2 showed up-regulation in the root vasculature and is supposed to be involved in proline translocation in response to salinity [16].

In contrast to the detailed knowledge on molecular regulation of K⁺/Na⁺ homeostasis and metabolism of osmoprotective compounds, regulation of anion transporters and particularly of Cl⁻ transport systems in salt-treated plants is not well understood yet. Voltage-gated chloride channels of the CLC-type family are found in prokaryotic and eukaryotic organisms ranging from bacteria to animals and plants and mediate passive Cl⁻ transport that is driven by the electrochemical gradient. In animals, CLC-type channels function in regulation of membrane potential and cellular pH homeostasis, besides, mutation of CLC-channels causes diseases as nephropathies [17]. Homologous plant CLC-channels have been identified in tobacco and Arabidopsis, and expression of the Arabidopsis channels AtCLC-c and -d could functionally complement the CLC-type yeast mutant gef1 [18], [19]. Heterologous expression of tobacco CLC-NT1 in Xenopus oocytes induced hyper-polarization activated chloride channels whereas no chloride currents were elicited by AtCLC-a, -b, -c, and -d [18], [20]. Function of plant CLC-type chloride channels in regulation of stomatal movement has been suggested [21].

In the present study, we compared expression of the rice CLC-type chloride channel homologue OsCLC1 in two rice lines with differences in salt tolerance to determine the role of Cl⁻ homeostasis in plant salt adaptation. Both rice lines showed significant differences in chloride accumulation and transcriptional regulation of OsCLC1 that were correlated with expressional changes of a V-ATPase subunit and OsNHX1. Analysis of cell-specificity was performed by in situ PCR focusing on salt-dependent differences of transcript abundance. The results presented here indicate correlation of gene expression controlling cation and anion homeostasis in salt-stressed plants and indicate a role of OsCLC1 in adaptation to salinity.