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

Abstract

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.

Leaf vein density correlates with crassulacean acid metabolism, but not hydraulic capacitance, in the genus Clusia

“…One physiological adaptation that is thought to have a substantial effect on transpiration rates of plants is Crassulacean acid metabolism (CAM). CAM is a photosynthetic mode that minimizes water loss via reorganized stomatal dynamics, so that stomata open at night and close during the day (Borland et al ., 2014 , 2015 ; Yang et al , 2015 ; Hurtado-Castano et al , 2023 ). By keeping stomata closed during the middle of the day (when the air is hottest and driest), CAM plants can decrease transpiration and conserve water.…”

Prospects and perspectives: inferring physiological and regulatory targets for CAM from molecular and modelling approaches

“…This provided more evidence for the potential connection between carbohydrate supply and circadian clock expression ( Borland and Griffiths, 1997 ; Borland et al ., 1999 ; Boxall et al ., 2017 ; Gilman et al ., 2022 ). Subsequently more specific studies have developed RNAi lines, either for plastidic starch phosphorylase ( PHS1 : Ceusters et al ., 2021 ) or phosphoglucomutase ( PGM : Hurtado-Castano et al ., 2023 ). These have consolidated insights into the biochemical and molecular regulation of CAM evolution, with a reversal to those in controlling starch partitioning in the C 3 pathway ( Boxall et al ., 2020 ).…”

“…In transgenic CAM plants, transcripts for genes controlling the amylolytic pathway (C 3 ) were upregulated as compared to the phosphorolytic pathway normally associated with starch storage during CAM ( Borland et al ., 2009 , 2016 ; Ceusters et al ., 2021 ). Also, starch degradation was not required to drive stomatal opening in the dark in the PGM mutant ( Hurtado-Castano et al ., 2023 ).…”

“…The regulation of stomatal behaviour also brings an additional level of complexity – whether due to contrasting responses to light intensity/quality following CAM induction, or developmental stages ( Lee & Assmann, 1992 ; Tallman et al ., 1997 ; Gotoh et al ., 2019 ; Hurtado-Castano et al ., 2023 ). On the one hand, the stomata of facultative CAM species ( Mesembryanthemum crystallinum and Portulacaria afra ) were reported to not respond to light once induced into the CAM mode ( Lee and Assmann, 1992 ; Tallman et al ., 1997 ).…”

“…The extent that an inverse cycle of guard cell PEPC activity and malate metabolism could contribute to stomatal solute balance and stomatal opening at night has not been resolved ( Santelia and Lawson, 2016 ; Males and Griffiths, 2017 ). Recent studies suggest a role for diurnal anion channel transcription in regulating stomatal opening at night ( Lefoulon et al ., 2020 ), and the absence of any role for guard cell starch accumulation ( Hurtado-Castano et al ., 2023 ). Additionally, increased transcripts associated with abscisic acid (ABA) synthesis and signalling before dawn suggested a possible role in triggering daytime stomatal closure ( Abraham et al ., 2016 ), although the direct effect of ABA on the inverted timing of CAM stomatal behaviour remains elusive ( Holtum and Winter, 1982 ; Chu et al ., 1990 ; Bastide et al ., 1993 ; Dai et al ., 1994 ; Taybi et al ., 1995 ; Taybi and Cushman, 1999 ).…”