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In plants with crassulacean acid metabolism (CAM), it has been proposed that the requirement for nocturnal provision of phosphoenolpyruvate as a substrate for CO2 uptake has resulted in a re-routing of chloroplastic starch degradation from the amylolytic route to the phosphorolytic route. To test this hypothesis, we generated and characterized four independent RNAi lines of the obligate CAM species Kalanchoë fedtschenkoi with >10-fold reduction in transcript abundance of plastidic α-glucan phosphorylase (PHS1). The rPHS1 lines showed diminished nocturnal starch degradation, reduced dark CO2 uptake, a reduction in diel water use efficiency (WUE) and an overall reduction in growth. A re-routing of starch degradation via the hydrolytic/amylolytic pathway was indicated by hyper-accumulation of maltose in all rPHS1 lines. Further examination indicated that whilst operation of the core circadian clock was not compromised, plasticity in modulating net dark CO2 uptake in response to changing photoperiods was curtailed. The data show that phosphorolytic starch degradation is critical for efficient operation of the CAM cycle and for optimizing water-use efficiency. This finding has clear relevance for on-going efforts to engineer CAM into non-CAM species as a means of boosting crop water use efficiency for a warmer, drier future.

Background and Aims Crassulacean acid metabolism (CAM) is a specialised type of photosynthesis characterised by a diel pattern of stomatal opening at night and closure during the day, which increases water-use efficiency. Starch degradation is a key regulator of CAM, providing phosphoenolpyruvate as substrate in the mesophyll for nocturnal assimilation of CO2. Growing recognition of a key role for starch degradation in C3 photosynthesis guard cells for mediating day-time stomatal opening presents the possibility that starch degradation might also impact CAM by regulating the provision of energy and osmolytes to increase guard cell turgor and drive stomatal opening at night. This study tested the hypothesis that the timing of diel starch turnover in CAM guard cells has been re-programmed during evolution to enable nocturnal stomatal opening and day-time closure. Methods Biochemical and genetic characterisation of wild type and starch-deficient RNAi lines of Kalanchoë fedtschenkoi with reduced activity of plastidic phosphoglucomutase (PGM) constituted a preliminary approach for the understanding of starch metabolism and its implications for stomatal regulation in CAM plants. Key results Starch deficiency reduced nocturnal net CO2 uptake, but had negligible impact on nocturnal stomatal opening. In contrast, day-time stomatal closure was reduced in magnitude and duration in the starch-deficient rPGM RNAi lines, and their stomata were unable to remain closed in response to elevated concentrations of atmospheric CO2 administered during the day. Curtailed day-time stomatal closure was linked to higher soluble sugar contents in the epidermis and mesophyll. Conclusions Nocturnal stomatal opening is not reliant upon starch degradation, but starch biosynthesis is an important sink for carbohydrates, ensuring day-time stomatal closure in this CAM species.