Investigation Of Mechanism Of Arabidopsis AtNRGA1and AtGPK1in Drought Stress Response

Drought causes severe irreversible damage to the plants, and eventually affects plant growth and crop yield. With climate change, drought is becoming a global crisis today, and threatens the agricultural production and food security. Most of the water absorbed by the plant roots, is lost through stomatal as transpiration. Thus, to search the functional genes involved in plant drought response, and to reveal their molecular mechanism in regulation of stomatal movements, will provide theoretical guidance and functional genes for breeding of crops with high water-use efficiency.In this study, two key functional genes of Arabidopsis thaliana, were found to be involved in plant drought response, and their loss-of-function mutants are with enhanced drought tolerance than their wild type background. These genes are named as AtNRGA1(Arabidopsis Negative Regulator of Guard cell ABA signaling) and AtGPKl (Arabidopsis Guard cell Protein Kinase1), respectively. By using genetics, molecular biology, physiology, electrophysiology and yeast two-hybrid techniques, we did a thorough investigation of their molecular mechanism, and the results and the conclusion drawn are indicated below:1, Functional study of AtNRGAl in stomatal movements and plant drought responseThe high expression of NRGA1in guard cells has been proved by the data collected from proteomics, GUS staining and Real-time PCR. The nrgal, the loss-of-function T-DNA insertion mutant of AtNRGAl, showed higher survival rate than Col-0under drought, and which is consistent with the observation from further water-loss experiment of detached leaves, the water loss is slower in mutant leaves.ABA is the best known phytohormone in plant drought response, as well as in guard cell signaling and stomatal movements. Under water deficient, ABA inhibits stomatal opening and promotes stomatal closure, and thus reducing water loss. Peel bioassays proved that nrgal is hypersensitive to ABA induced stomatal closure and ABA inhibited stomatal opening than wild-type. The guard cell patch clamp data revealed that, the inward potassium currents (Kin+) and slow anion currents of nrgal are more sensitive to ABA than those of the wild type. The hypersensitivity to ABA of the nrgalmutnat could be restored in the nrgal/NRGAl functional complementation lines (NRGA1-C-1, NRGA1-C-7). Furthermore, the NRGA1overexpressing lines (NRGA1-OE-5, NRGA1-OE-7) showed less sensitivity in ABA regulation of guard cell K+and anion currents, and stomatal movements. These findings suggest that NRGA1is a negative regulator in ABA regulation of guard cell ion channels and consequent stomatal movements, and thus to participate in plant drought response.Bioinformatics analysis showed that NRGA1has an Uncharacterised Protein Family (UPF0041) domain, and also has higher amino acid identity with a predicted mitochondrial pyruvate carrier2(MPC2)-like protein. Complementation analysis in yeast showed that NRGA1was able to restore the growth of yeast cells lacking MPC activity, suggest that NRGA1may be a functional MPC in plant.In conclusion, our data showed that NRGA1may function as a MPC to negatively participate in ABA regulation of guard cell ion transport, stomatal movement, and plant drought response.2, Functional study of AtGPKl in stomatal movement and plant drought resistanceGPK1is also highly expressed in guard cells and is evoked in plant drought response. The two T-DNA insertion mutants of GPK1, gpk1-1and gpk1-2, showed higher drought tolerance than wild type, and the water loss is also slower in mutant leaves.The experimental results show that the GPK1is a negative element in ABA regulation of guard cells ion channels, stomatal movements, and thus to contribute in plant drought response, gpk1-1and gpk1-2mutants are hypersensitive to ABA regulation of guard cell K+and anion currents, and stomatal movements. The functional complementation lines gpkl-1/GPKl (GPK1-C-1, GPK1-C-4) restored the phenotypes of these mutants. Furthermore, GPK1overexpression lines (GPK1-OE-1, GPK1-OE-2) showed opposite stomatal responses, with reduced drought tolerance and ABA sensitivity in guard cell K+and anion channel regulation. The subcellular localization indicated that GPK1is enriched in the plasma membrane, suggesting the potential membrane-delimited pathway of GPK1regulation of ion channels.Sequence analysis showed that GPK1belongs to the CDPK (Ca2+Dependent Protein Kinase) family. The in vitro GPK1kinase activity was analyzed with32P showed that, GPK1exhibits auto-phosphorylation activity with a Ca2+-dependent manner. To further explore the molecular mechanism of GPK1, yeast two-hybrid system was used for searching its functional partners, and the GIP1(GPK1Interacting Protein1) was identified, and further confirmed with the co-localization, In addition, the kinase activity of GPK1was also inhibited by GIP1in a concentration dependent in vitro. The GIP1overexpressing lines (GIP1-OE-8, GIP1-OE-14) were hypersensitive to ABA regulation of stomata1movements, consistent with the phenotypes of GPKl mutants, suggesting the GIP1could bind and inhibit the GPKl activity in ABA signaling.To summary, GPK1, a functional Ca2+dependent plant protein kinase, which kinase activity could be activated by Ca2+while inhibited by GIP1,and serves a negative element in ABA regulation of K+, anion currents and stomata1movements, and consequent to be involved in plant drought response.