The starch-deficient plastidic PHOSPHOGLUCOMUTASE mutant of the constitutive crassulacean acid metabolism (CAM) species Kalanchoë fedtschenkoi impacts diel regulation and timing of stomatal CO2 responsiveness.

Crassulacean acid metabolism (CAM) is a specialized type of photosynthesis characterized 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 a 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 daytime 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. In this study, we tested the hypothesis that the timing of diel starch turnover in CAM guard cells has been reprogrammed during evolution to enable nocturnal stomatal opening and daytime closure. Biochemical and genetic characterization 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. Starch deficiency reduced nocturnal net CO2 uptake but had negligible impact on nocturnal stomatal opening. In contrast, daytime 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 daytime stomatal closure was linked to higher soluble sugar contents in the epidermis and mesophyll. Nocturnal stomatal opening is not reliant upon starch degradation, but starch biosynthesis is an important sink for carbohydrates, ensuring daytime stomatal closure in this CAM species.

Hurtado-Castano N ，Atkins E ，Barnes J ，Boxall SF ，Dever LV ，Kneřová J ，Hartwell J ，Cushman JC ，Borland AM ... -  《-》

Peeling back the layers of crassulacean acid metabolism: functional differentiation between Kalanchoë fedtschenkoi epidermis and mesophyll proteomes.

Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that offers the potential to engineer improved water-use efficiency (WUE) and drought resilience in C3 plants while sustaining productivity in the hotter and drier climates that are predicted for much of the world. CAM species show an inverted pattern of stomatal opening and closing across the diel cycle, which conserves water and provides a means of maintaining growth in hot, water-limited environments. Recent genome sequencing of the constitutive model CAM species Kalanchoë fedtschenkoi provides a platform for elucidating the ensemble of proteins that link photosynthetic metabolism with stomatal movement, and that protect CAM plants from harsh environmental conditions. We describe a large-scale proteomics analysis to characterize and compare proteins, as well as diel changes in their abundance in guard cell-enriched epidermis and mesophyll cells from leaves of K. fedtschenkoi. Proteins implicated in processes that encompass respiration, the transport of water and CO2 , stomatal regulation, and CAM biochemistry are highlighted and discussed. Diel rescheduling of guard cell starch turnover in K. fedtschenkoi compared with that observed in Arabidopsis is reported and tissue-specific localization in the epidermis and mesophyll of isozymes implicated in starch and malate turnover are discussed in line with the contrasting roles for these metabolites within the CAM mesophyll and stomatal complex. These data reveal the proteins and the biological processes enriched in each layer and provide key information for studies aiming to adapt plants to hot and dry environments by modifying leaf physiology for improved plant sustainability.

Abraham PE ，Hurtado Castano N ，Cowan-Turner D ，Barnes J ，Poudel S ，Hettich R ，Flütsch S ，Santelia D ，Borland AM ... -  《-》

Kalanchoë PPC1 Is Essential for Crassulacean Acid Metabolism and the Regulation of Core Circadian Clock and Guard Cell Signaling Genes.

Unlike C3 plants, Crassulacean acid metabolism (CAM) plants fix CO2 in the dark using phosphoenolpyruvate carboxylase (PPC; EC 4.1.1.31). PPC combines phosphoenolpyruvate with CO2 (as HCO3 -), forming oxaloacetate. The oxaloacetate is converted to malate, leading to malic acid accumulation in the vacuole, which peaks at dawn. During the light period, malate decarboxylation concentrates CO2 around Rubisco for secondary fixation. CAM mutants lacking PPC have not been described. Here, we employed RNA interference to silence the CAM isogene PPC1 in Kalanchoë laxiflora Line rPPC1-B lacked PPC1 transcripts, PPC activity, dark period CO2 fixation, and nocturnal malate accumulation. Light period stomatal closure was also perturbed, and the plants displayed reduced but detectable dark period stomatal conductance and arrhythmia of the CAM CO2 fixation circadian rhythm under constant light and temperature free-running conditions. By contrast, the rhythm of delayed fluorescence was enhanced in plants lacking PPC1 Furthermore, a subset of gene transcripts within the central circadian oscillator was upregulated and oscillated robustly in this line. The regulation of guard cell genes involved in controlling stomatal movements was also perturbed in rPPC1-B These findings provide direct evidence that the regulatory patterns of key guard cell signaling genes are linked with the characteristic inverse pattern of stomatal opening and closing during CAM.

Boxall SF ，Kadu N ，Dever LV ，Kneřová J ，Waller JL ，Gould PJD ，Hartwell J ... -  《-》

Phosphorolytic degradation of leaf starch via plastidic α-glucan phosphorylase leads to optimized plant growth and water use efficiency over the diel phases of Crassulacean acid metabolism.

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 a >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 hyperaccumulation 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 WUE. This finding has clear relevance for ongoing efforts to engineer CAM into non-CAM species as a means of boosting crop WUE for a warmer, drier future.

Ceusters N ，Ceusters J ，Hurtado-Castano N ，Dever LV ，Boxall SF ，Kneřová J ，Waller JL ，Rodick R ，Van den Ende W ，Hartwell J ，Borland AM ... -  《-》

Crassulacean acid metabolism guard cell anion channel activity follows transcript abundance and is suppressed by apoplastic malate.

Plants utilising crassulacean acid metabolism (CAM) concentrate CO2 around RuBisCO while reducing transpirational water loss associated with photosynthesis. Unlike stomata of C3 and C4 species, CAM stomata open at night for the mesophyll to fix CO2 into malate (Mal) and store it in the vacuole. CAM plants decarboxylate Mal in the light, generating high CO2 concentrations within the leaf behind closed stomata for refixation by RuBisCO. CO2 may contribute to stomatal closure but additional mechanisms, plausibly including Mal activation of anion channels, ensure closure in the light. In the CAM species Kalanchoë fedtschenkoi, we found that guard cell anion channel activity, recorded under voltage clamp, follows KfSLAC1 and KfALMT12 transcript abundance, declining to near zero by the end of the light period. Unexpectedly, however, we found that extracellular Mal inhibited the anion current of Kalanchoë guard cells, both in wild-type and RNAi mutants with impaired Mal metabolism. We conclude that the diurnal cycle of anion channel gene transcription, rather than the physiological signal of Mal release, is a key factor in the inverted CAM stomatal cycle.

Lefoulon C ，Boxall SF ，Hartwell J ，Blatt MR ... -  《-》