Functional proteomics of Arabidopsis thaliana guard cells uncovers new stomatal signaling pathways

The guard cell is a specialized cell type, located in the epidermes of higher plants. The guard cell has been used as a model system in plant cell biology for decades. Here, I isolated a total of 3x108 guard cell protoplasts from 22,000 Arabidopsis plants and identified 1,764 unique proteins using three complementary proteomic methods: protein spot identification from broad and narrow pH range 2D gels, and 2D LC-MALDI MudPIT. Proteomic study suggested that myrosinase 1 (TGG1) is the most abundant protein in guard cells. TGG1 catalyzes the production of toxic isothiocyanates from glucosinolates, and the glucosinolate-myrosinase system is well known as a defense system against biotic invaders. Phenotypic analysis showed that tgg1 mutants were hyposensitive to abscisic acid (ABA)-inhibition of guard cell inward K+ channels and stomatal opening, revealing that the glucosinolate-myrosinase system is also central to abiotic stress responses. TopGO analysis of the identified guard cell proteome revealed that proteins involved in energy production were enriched in the GC proteome. I further characterized mutants lacking the glycolytic enzyme iPGM (2,3-biphosphoglycerate-independent phosphoglycerate mutase). ipgm mutants showed defects in stomatal movements, growth, and pollen production in our study. Our study demonstrates that proteomic studies can make powerful contributions to the identification of novel signaling pathways.;In addition to the guard cell proteomic study, I also compared the proteomic patterns of Col and gpa1-4 guard cells with and without ABA treatment using iTARQ technology. The gpa1-4 mutant, lacking the heterotrimeric G protein alpha subunit, shows hyposensitivity to ABA inhibition of inward K+ channels and ABA inhibition of stomatal opening. This iTRAQ study showed that two and six proteins were significantly regulated by ABA in protein abundance in Col and gpa1-4 guard cells respectively, while the abundance of 18 proteins in guard cells was affected by mutation of GPA1. Novel signaling models were proposed on the basis of the iTRAQ results.;To study the correlation of the transcriptome and proteome in guard cells, we also pursued microarray experiments. Comparison of transcriptome to proteome revealed that the correlation between mRNA and protein levels is poor in Arabidopsis guard cells, suggesting that the protein abundance in guard cells is not primarily regulated at the transcriptional level.