Molecular evolution of starch degradation in the guard cells of CAM plants

The water conserving properties of CAM photosynthesis have identified the pathway as a target for synthetic biology. CAM conserves water by shifting net CO2 uptake to the night when rates of leaf evapotranspiration are reduced compared to the day. CAM improves water-use efficiency (CO2 fixed per unit water lost) some 5 to 10 fold over that in plants with other types of photosynthesis. Thus, bioengineering CAM into non-CAM crops offers the potential to sustain plant productivity for food, feed, fibre, and biofuel production whilst curtailing water inputs (Borland et al, 2014). The rationale for CAM-bioengineering is rooted in the knowledge that since CAM emerged frequently from C3 photosynthesis throughout evolutionary history, all of the enzymes required for CAM are homologues of ancestral forms found in C3 species. Thus, understanding the molecular evolution of key genes required for CAM is a crucial component for informing CAM engineering.
Engineering CAM into C3 plants requires a day/night rescheduling of stomatal movement so that stomata (leaf pores) open at night and close during the day. Recent unpublished work in the Borland lab has suggested a fundamental difference in guard cell metabolism between CAM and C3 plants which merits further study. Specifically, starch breakdown in stomatal guard cells, which enhances guard cell turgor and accelerates day-time stomatal opening in C3 plants, is suppressed in CAM guard cells. Starch degradation in C3 guard cells requires a different enzymatic route to that in the leaf mesophyll and key enzymes required for C3 guard cell starch turnover have recently been identified in Arabidopsis. Our observations for CAM guard cells provide two hypotheses: 1) CAM guard cells lack key enzymes required for starch degradation; 2) Guard cell starch degradation is subject to contrasting modes of regulation in CAM and C3 plants. This project will test these hypotheses using a range of wet and dry lab approaches that include; a) analysis of the guard cell-enriched proteome of the model CAM plant Kalanchoë fedtschenkoi and how it compares to published guard cell proteomes of Arabidopsis, with particular focus on proteins implicated in starch and osmolyte turnover; b) genetic manipulation of selected candidate genes implicated in CAM guard cell starch metabolism and c) bioinformatics interrogation of published C3 and CAM genomes to establish molecular phylogenies of candidate genes implicated in guard cell starch degradation and to test if these genes have evolved differently in CAM and C3 plants.