Abstract
Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that improves water use efficiency by shifting part or all of net atmospheric CO2 uptake to the night. Genetic dissection of regulatory and metabolic attributes of CAM has been limited by the difficulty of identifying a reliable phenotype for mutant screening. We developed a novel and simple colorimetric assay to measure leaf pH to screen fast neutron-mutagenized populations of common ice plant (Mesembryanthemum crystallinum), a facultative CAM species, to detect CAM-deficient mutants with limited nocturnal acidification. The isolated CAM-deficient mutants showed negligible net dark CO2 uptake compared with wild-type plants following the imposition of salinity stress. The mutants and wild-type plants accumulated nearly comparable levels of sodium in leaves, but the mutants grew more slowly than the wild-type plants. The mutants also had substantially reduced seed set and seed weight relative to wild type under salinity stress. Carbon-isotope ratios of seed collected from 4-month-old plants indicated that C3 photosynthesis made a greater contribution to seed production in mutants compared to wild type. The CAM-deficient mutants were deficient in leaf starch and lacked plastidic phosphoglucomutase, an enzyme critical for gluconeogenesis and starch formation, resulting in substrate limitation of nocturnal C4 acid formation. The restoration of nocturnal acidification by feeding detached leaves of salt-stressed mutants with glucose or sucrose supported this defect and served to illustrate the flexibility of CAM. The CAM-deficient mutants described here constitute important models for exploring regulatory features and metabolic consequences of CAM.
Highlights
Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that improves water use efficiency by shifting part or all of net atmospheric CO2 uptake to the night
We investigated whether a simple visual screen using a colorimetric pH indicator dye could be used for high-throughput screening of ice plant mutants with a reduced ability to conduct nocturnal acidification, a key feature of CAM
We have shown that a novel, yet simple, visual screen using a colorimetric pH indicator dye can be used for high-throughput screening and isolation of ice plant mutants with a reduced ability to conduct nocturnal acidification, a key feature of CAM (Fig. 5A)
Summary
Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that improves water use efficiency by shifting part or all of net atmospheric CO2 uptake to the night. Genetic dissection of the regulatory and metabolic attributes of CAM in common ice plant has been limited by the difficulty of identifying a reliable phenotype for mutant screening. The CAM-deficient mutants isolated here failed to express plastidic phosphoglucomutase (PGM), the enzyme that catalyzes the interconversion of Glc-6-P4Glc-1-P and is critical to subsequent gluconeogenesis and starch formation. Validation of this genetic lesion was demonstrated by the restoration of the CAM phenotype by feeding detached leaves with Glc or Suc. We discuss how the availability of such CAM-deficient mutants could improve our understanding of the complex day/night metabolic and circadian regulatory processes that govern CAM
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