Doctorate thesis of National Institute of Further Education in Agricultural Science

Friday, December 16, 2016

Control of leaf transpiration in rice: molecular and functional analysis of K+ transport in stomata

Thanh Hao Nguyen
BPMP, équipes ELSA & TICER

Jury :
Hervé SENTENAC, Directeur de recherche, Biochimie & physiologie moléculaire des plantes, CoDirecteur de thèse
Anne-Aliénor VERY, Chargé de recherche, Biochimie & physiologie moléculaire des plantes, CoDirecteur de thèse
Nathalie LEONHARDT, Cadre scientifique des EPIC, CEA Cadarache, Rapporteur
Rob ROELFSEMA, Assistant de Recherche Senior, Université de Würzburg, Rapporteur
Pierre CHARNET, Directeur de recherche, CNRS, Montpellier, Examinateur
Thierry SIMONNEAU, Directeur de recherche, INRA, Montpellier, Examinateur

Abstract :
Stomata form pores on leaf surface which control the CO2 uptake and the water loss by transpiration. In Arabidopsis, Shaker channels have important roles in the stomatal movement by mediating influxes and effluxes of K+ across the guard cell plasma membrane. Rice has a different stomatal structure from that in Arabidopsis and the mechanism controlling the movement of this grass-type stoma is yet to be unraveled. In this study, the tissue specific expression analysis using GUS reporter gene under the control of candidate gene promoters identified the expression of three inward Shaker channels (OsK2.1, OsK2.2, and OsK2.3) and two outward Shaker channels (OsK5.1 and OsK5.2) in rice stomatal complexes. Among these genes, OsK2.2 and OsK5.2 showed the highest expression level in stomata. Apart from Shaker channels, two HKT transporter genes, OsHKT3 and OsHKT9, belonging to the subfamily of potassium-permeable members, were also identified in rice stomata. The electrophysiological characterization of OsK2.1, OsK2.2, and OsK2.3 in the Xenopus laevis oocyte system showed that they encoded inwardly-rectifying potassium channels with low affinities for potassium, which differed in voltage activation threshold, cation selectivity, and pH sensitivity. The electrophysiology experiments performed in intact plant guard cells of wild type and mutant genotypes evidenced the central role of OsK5.2 channel in the release of potassium from these cells. The analysis of plant transpiration suggested different roles for these channels and transporters in the regulation of stomatal movement. OsK5.2 was proved to participate in both stomatal opening and closure. osk5.2 knock-out mutant displayed higher levels of transpiration in darkness as in light conditions and was severely affected in the stomatal closure kinetics. OsK2.2 and OsK2.3 might redundantly function in rice stomata since individual knock-out mutants had weak transpiration phenotype. OsK5.1 could indirectly contribute to the control of stomatal movement by transporting the potassium from the roots to the leaves. OsHKT3 and OsHKT9 transporters could provide an additional pathway for potassium transport uptake during stomatal opening and closure.