Dominant Negative Guard Cell K+ Channel Mutants Reduce Inward-Rectifying K+ Currents and Light-Induced Stomatal Opening in Arabidopsis

Abstract

Inward-rectifying potassium (K+ in) channels in guard cells have been suggested to provide a pathway for K+ uptake into guard cells during stomatal opening. To test the proposed role of guard cell K+ in channels in light-induced stomatal opening, transgenic Arabidopsis plants were generated that expressed dominant negative point mutations in the K+ in channel subunit KAT1. Patch-clamp analyses with transgenic guard cells from independent lines showed that K+ in current magnitudes were reduced by approximately 75% compared with vector-transformed controls at −180 mV, which resulted in reduction in light-induced stomatal opening by 38% to 45% compared with vector-transformed controls. Analyses of intracellular K+ content using both sodium hexanitrocobaltate (III) and elemental x-ray microanalyses showed that light-induced K+ uptake was also significantly reduced in guard cells of K+ in channel depressor lines. These findings support the model that K+ inchannels contribute to K+ uptake during light-induced stomatal opening. Furthermore, transpirational water loss from leaves was reduced in the K+ in channel depressor lines. Comparisons of guard cell K+ in current magnitudes among four different transgenic lines with different K+ in current magnitudes show the range of activities of K+ in channels required for guard cell K+ uptake during light-induced stomatal opening.

A Dominant Negative OsKAT2 Mutant Delays Light-Induced Stomatal Opening and Improves Drought Tolerance without Yield Penalty in Rice

“…Among these, some inward rectifier K + channels function in stomatal opening (Dietrich et al, 2001; Very and Sentenac, 2003). Although AtKAT1 was shown to play a role in light-dependent stomatal opening in Arabidopsis, the K + channel that functions in light-dependent stomatal opening in rice was previously unknown (Kwak et al, 2001). OsKAT2 is specifically expressed in rice guard cells and shows typical inward-rectifying K + channel activity, like that of Arabidopsis AtKAT1 (Hwang et al, 2013).…”

“…However, there is the quite significant difference of stomata conductance under the condition which transits from dark to light. In Arabidopsis, the expression of a dominant negative AtKAT1 mutant protein with T256R amino acid substitutions reduced light-induced stomatal opening in guard cells and resulted in a decline in water loss from the leaves (Baizabal-Aguirre et al, 1999; Kwak et al, 2001). Thus, plants overexpressing OsKTA2(T235R) mutant protein might be delayed in stomatal opening both in light induced condition and in water deficient condition.…”

“…The overexpression of AtKAT1 with two single point mutations (T256R) and G262K) in the GYGD motif and surrounding amino acids of this motif region suppressed time-dependent inward currents in Xenopus oocytes (Baizabal-Aguirre et al, 1999). Overexpressing these AtKAT1 mutant proteins in Arabidopsis depressed light-induced stomatal opening and reduced K + uptake, and therefore, these dominant negative mutant lines exhibited less water loss than the control (Kwak et al, 2001). In addition, a single point mutation of the threonine at position 306 in AtKAT1, which is a target site for ABA-activated SnRK2.6 protein kinase, reduced the K + transport uptake activity of AtKAT1 in Xenopus oocyte and yeast systems (Sato et al, 2009, 2010).…”

Knockout of the guard cell K + out channel and stomatal movements

“…It is further likely that the gork-1 mutant shows residual K (10,11). For example, sodium hexanitrocobaltate (III) K ϩ staining has been used to measure changes in Arabidopsis guard cell K ϩ content during light-induced stomatal opening in these inward-rectifying K ϩ uptake channel mutant lines (11) and could be used to analyze gork mutants.…”

“…For example, sodium hexanitrocobaltate (III) K ϩ staining has been used to measure changes in Arabidopsis guard cell K ϩ content during light-induced stomatal opening in these inward-rectifying K ϩ uptake channel mutant lines (11) and could be used to analyze gork mutants. Elemental x-ray microanalysis can also be used to compare K ϩ content in Vicia faba and WT and mutant Arabidopsis guard cells (2,11). The characterization of the gork-1 mutant will allow future analysis of the relative contributions of additional K ϩ , sucrose, and other possible solute transport and metabolic mechanisms to stimulus-induced stomatal closing.…”