Silencing PHOSPHOENOLPYRUVATE CARBOXYLASE1 in the Obligate Crassulacean Acid Metabolism Species Kalanchoë laxiflora causes Reversion to C3-like Metabolism and Amplifies Rhythmicity in a Subset of Core Circadian Clock Genes

ABSTRACTUnlike C3 plants, Crassulacean acid metabolism (CAM) plants fix CO2 in the dark using phosphoenolpyruvate carboxylase (PPC; EC 4.1.1.31). PPC combines PEP with CO2 (as HCO3−), forming oxaloacetate that is rapidly converted to malate, leading to vacuolar malic acid accumulation that peaks phased to dawn. In the light period, malate decarboxylation concentrates CO2 around RuBisCO for secondary fixation. CAM mutants lacking PPC have not been described. Here, RNAi was employed to silence 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 (LL) 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 were up-regulated and oscillated robustly. The regulation guard cell genes involved controlling stomatal movements was also altered in rPPC1-B. This provided direct evidence that altered regulatory patterns of key guard cell signaling genes are linked with the characteristic inverse pattern of stomatal opening and closing during CAM.

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Crassulacean Acid Metabolism and Diurnal Variations of Internal CO2 and O2 Concentrations in Sedum praealtum DC

Functional Plant Biology ◽

10.1071/pp9790557 ◽

1979 ◽

Vol 6 (4) ◽

pp. 557 ◽

Cited By ~ 43

Author(s):

MH Spalding ◽

DK Stumpf ◽

MSB Ku ◽

RH Burris ◽

GE Edwards

Keyword(s):

Malic Acid ◽

Crassulacean Acid Metabolism ◽

Acid Metabolism ◽

Dark Period ◽

Stomatal Closure ◽

Co2 Concentration ◽

Co2 Exchange ◽

C3 Plants ◽

Cam Plants ◽

Internal Co2

Internal CO2 and O2 concentrations in Sedum praealtum DC. were determined by gas chromatography of 200-�l gas samples. Day-night monitoring showed that internal CO2 varied from a high of approximately 4000 �l/l during periods of daytime stomatal closure to a low of 270-280 �l/l during the dark period (stomata open). Internal O2 concentrations varied from a high of approximately 26 % at midday to a low of 20.8 % during the dark period. The calculated internal O2/CO2 ratio varied about 12-15-fold from 50-60 near midday to approximately 750 during the dark period (ratio in normal air is roughly 600). Day-night patterns of CO2 exchange and malic acid concentration were typical for a plant with crassulacean acid metabolism (CAM). Influx of CO2 during the late light period was inhibited by O2, but dark CO2 influx was O2-insensitive. Gas samples taken near midday from several CAM plants all showed elevated internal CO2 and O2 concentrations. Ratios of O2/CO2 in these plants ranged from 81 in Sedum praealtum to 285 in Hoya carnosa. The highest internal O2 concentration observed was 41.5% in Kalanchoe gastonis-bonnieri. The high CO2 concentration in leaves of CAM plants during daytime stomatal closure should provide a near- saturating level of this substrate for photosynthesis. In comparison to C3 plants, the relatively low O2/CO2 ratio in the CAM leaf during malic acid decarboxylation should be favourable for photosynthesis and unfavourable for O2 inhibition of photosynthesis.

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Degrees of crassulacean acid metabolism in tropical epiphytic and lithophytic ferns

Functional Plant Biology ◽

10.1071/pp99001 ◽

1999 ◽

Vol 26 (8) ◽

pp. 749 ◽

Cited By ~ 30

Author(s):

Joseph A.M. Holtum ◽

Klaus Winter

Keyword(s):

Carbon Isotope ◽

Crassulacean Acid Metabolism ◽

Acid Metabolism ◽

Dark Period ◽

Titratable Acidity ◽

Isotope Ratios ◽

Light Period ◽

Co2 Uptake ◽

Carbon Isotope Ratios ◽

Net Co2 Uptake

Crassulacean acid metabolism (CAM) was observed in three species of tropical ferns, the epiphytes Microsorium punctatum and Polypodium crassifolium and the lithophyte Platycerium veitchii. Polypodium crassifolium and P. veitchii exhibited characteristics of weak CAM. Although no net nocturnal CO2 uptake was observed, the presence of CAM was inferred from nocturnal increases in titratable acidity of 4.7 and 4.1 µequiv (g fr wt)–1 respectively, a reduction in the rates of net CO2 evolution during the first half of the dark period, and the presence of a CAM-like decrease in net CO2 uptake during the early light period. In M. punctatum net CO2 uptake during the first half of the dark period was accompanied by an increase in titratable acidity of 39.2 µequiv (g fr wt)–1 and a pronounced reduction in net CO2 uptake during the early light period. When water was withheld from P. crassifolium and M. punctatum, net CO2 uptake during the light was reduced markedly but there was no change in the extent or patterns of CO2 exhange in the dark. As a consequence, the proportion of carbon gained due to CO2 fixation in the dark increased from 2.8 and 10% to 63.5 and 49.3%, respectively (100% being net CO2 uptake during the light plus the estimated CO2 uptake during the dark). After 9 days without added water, dark CO2 uptake was responsible for the maintenance of a net 24 h carbon gain in P. crassifolium. Platycerium veitchii, P. crassifolium and M. punctatum exhibited carbon isotope ratios of between –25.9 and –22.6‰ indicating that carbon isotope ratios may not, by themselves, be sufficient for the identification of weak CAM. We suggest that CAM may be more prevalent in tropical epiphytic and lithophytic ferns than currently envisaged.

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Inference of Gene Regulatory Network Uncovers the Linkage Between Circadian Clock and Crassulacean Acid Metabolism in Kalanchoë fedtschenkoi

10.1101/745893 ◽

2019 ◽

Author(s):

Robert C. Moseley ◽

Francis Motta ◽

Gerald A. Tuskan ◽

Steve Haase ◽

Xiaohan Yang

Keyword(s):

Gene Expression ◽

Circadian Clock ◽

Gene Regulatory Network ◽

Regulatory Network ◽

Crassulacean Acid Metabolism ◽

Acid Metabolism ◽

Clock Genes ◽

Specific Gene ◽

Cam Plants ◽

Gene Regulatory

AbstractThe circadian clock drives time-specific gene expression, allowing for associated biological processes to be active during certain times of the 24 h day. Crassulacean acid metabolism (CAM) photosynthetic plants represent an interesting case of circadian regulation of gene expression as CO2 fixation and stomatal movement in CAM plants display strong circadian dynamics. The molecular mechanisms behind how the circadian clock enabled these physiological differences is not well understood. Therefore, we set out to investigate whether core circadian elements in CAM plants were re-phased during evolution, or whether networks of phase-specific genes were simply connected to different core elements. We utilized a new metric for identifying candidate core genes of a periodic gene network and then applied the Local Edge Machine (LEM) algorithm to infer regulatory relationships between the candidate core clock genes and orthologs of known core clock genes in K. fedtschenkoi. We also used LEM to identify stomata-related gene targets for K. fedtschenkoi core clock genes and constructed a subsequent gene regulatory network. Our results provide new insights into the mechanism of circadian control of CAM-related genes in K. fedtschenkoi, facilitating the engineering of CAM machinery into non-CAM plants for sustainable crop production in water-limited environments.

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Inference of Gene Regulatory Network Uncovers the Linkage between Circadian Clock and Crassulacean Acid Metabolism in Kalanchoë fedtschenkoi

Cells ◽

10.3390/cells10092217 ◽

2021 ◽

Vol 10 (9) ◽

pp. 2217

Author(s):

Robert C. Moseley ◽

Francis Motta ◽

Gerald A. Tuskan ◽

Steven B. Haase ◽

Xiaohan Yang

Keyword(s):

Gene Expression ◽

Circadian Clock ◽

Gene Regulatory Network ◽

Regulatory Network ◽

Crassulacean Acid Metabolism ◽

Acid Metabolism ◽

Clock Genes ◽

Stomatal Movement ◽

Cam Plants ◽

Gene Regulatory

The circadian clock drives time-specific gene expression, enabling biological processes to be temporally controlled. Plants that conduct crassulacean acid metabolism (CAM) photosynthesis represent an interesting case of circadian regulation of gene expression as stomatal movement is temporally inverted relative to stomatal movement in C3 plants. The mechanisms behind how the circadian clock enabled physiological differences at the molecular level is not well understood. Recently, the rescheduling of gene expression was reported as a mechanism to explain how CAM evolved from C3. Therefore, we investigated whether core circadian clock genes in CAM plants were re-phased during evolution, or whether networks of phase-specific genes were simply re-wired to different core clock genes. We identified candidate core clock genes based on gene expression features and then applied the Local Edge Machine (LEM) algorithm to infer regulatory relationships between this new set of core candidates and known core clock genes in Kalanchoë fedtschenkoi. We further inferred stomata-related gene targets for known and candidate core clock genes and constructed a gene regulatory network for core clock and stomata-related genes. Our results provide new insight into the mechanism of circadian control of CAM-related genes in K. fedtschenkoi, facilitating the engineering of CAM machinery into non-CAM plants for sustainable crop production in water-limited environments.

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Faculty Opinions recommendation of Kalanchoë PPC1 is essential for crassulacean acid metabolism and the regulation of core circadian clock and guard cell signaling genes.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.737370476.793573674 ◽

2020 ◽

Author(s):

Richard Leegood

Keyword(s):

Cell Signaling ◽

Circadian Clock ◽

Guard Cell ◽

Crassulacean Acid Metabolism ◽

Acid Metabolism

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How to tell the time: the regulation of phosphoenolpyruvate carboxylase in Crassulacean acid metabolism (CAM) plants

Biochemical Society Transactions ◽

10.1042/bst0310728 ◽

2003 ◽

Vol 31 (3) ◽

pp. 728-730 ◽

Cited By ~ 7

Author(s):

H.G. Nimmo

Keyword(s):

Circadian Rhythms ◽

Phosphoenolpyruvate Carboxylase ◽

Crassulacean Acid Metabolism ◽

Acid Metabolism ◽

Circadian Oscillator ◽

Cam Plants ◽

Reversible Phosphorylation ◽

Circadian Expression ◽

Phosphoenolpyruvate Carboxylase Kinase ◽

Co2 Metabolism

Crassulacean acid metabolism (CAM) plants exhibit persistent circadian rhythms of CO2 metabolism. These rhythms are driven by changes in the flux through phosphoenolpyruvate carboxylase, which is regulated by reversible phosphorylation in response to a circadian oscillator. This article reviews progress in our understanding of the circadian expression of phosphoenolpyruvate carboxylase kinase.

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

New Phytologist ◽

10.1111/nph.16640 ◽

2020 ◽

Vol 227 (6) ◽

pp. 1847-1857 ◽

Cited By ~ 1

Author(s):

Cécile Lefoulon ◽

Susanna F. Boxall ◽

James Hartwell ◽

Michael R. Blatt

Keyword(s):

Guard Cell ◽

Crassulacean Acid Metabolism ◽

Acid Metabolism ◽

Anion Channel ◽

Transcript Abundance ◽

Channel Activity

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Light-responsive expression atlas reveals the effects of light quality and intensity in Kalanchoë fedtschenkoi, a plant with crassulacean acid metabolism

GigaScience ◽

10.1093/gigascience/giaa018 ◽

2020 ◽

Vol 9 (3) ◽

Cited By ~ 1

Author(s):

Jin Zhang ◽

Rongbin Hu ◽

Avinash Sreedasyam ◽

Travis M Garcia ◽

Anna Lipzen ◽

...

Keyword(s):

Light Intensity ◽

White Light ◽

Crassulacean Acid Metabolism ◽

Light Quality ◽

Acid Metabolism ◽

Easy Access ◽

Cam Plants ◽

A Genome ◽

Acid Accumulation ◽

Expression Atlas

Abstract Background Crassulacean acid metabolism (CAM), a specialized mode of photosynthesis, enables plant adaptation to water-limited environments and improves photosynthetic efficiency via an inorganic carbon-concentrating mechanism. Kalanchoë fedtschenkoi is an obligate CAM model featuring a relatively small genome and easy stable transformation. However, the molecular responses to light quality and intensity in CAM plants remain understudied. Results Here we present a genome-wide expression atlas of K. fedtschenkoi plants grown under 12 h/12 h photoperiod with different light quality (blue, red, far-red, white light) and intensity (0, 150, 440, and 1,000 μmol m–2 s–1) based on RNA sequencing performed for mature leaf samples collected at dawn (2 h before the light period) and dusk (2 h before the dark period). An eFP web browser was created for easy access of the gene expression data. Based on the expression atlas, we constructed a light-responsive co-expression network to reveal the potential regulatory relationships in K. fedtschenkoi. Measurements of leaf titratable acidity, soluble sugar, and starch turnover provided metabolic indicators of the magnitude of CAM under the different light treatments and were used to provide biological context for the expression dataset. Furthermore, CAM-related subnetworks were highlighted to showcase genes relevant to CAM pathway, circadian clock, and stomatal movement. In comparison with white light, monochrome blue/red/far-red light treatments repressed the expression of several CAM-related genes at dusk, along with a major reduction in acid accumulation. Increasing light intensity from an intermediate level (440 μmol m−2 s−1) of white light to a high light treatment (1,000 μmol m–2 s–1) increased expression of several genes involved in dark CO2 fixation and malate transport at dawn, along with an increase in organic acid accumulation. Conclusions This study provides a useful genomics resource for investigating the molecular mechanism underlying the light regulation of physiology and metabolism in CAM plants. Our results support the hypothesis that both light intensity and light quality can modulate the CAM pathway through regulation of CAM-related genes in K. fedtschenkoi.

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CO2 starvation experiments provide support for the carbon-limited hypothesis on the evolution of CAM-like behaviour in Isoëtes

Annals of Botany ◽

10.1093/aob/mcaa153 ◽

2020 ◽

Vol 127 (1) ◽

pp. 135-141

Author(s):

Jacob S Suissa ◽

Walton A Green

Keyword(s):

Aquatic Ecosystems ◽

Crassulacean Acid Metabolism ◽

Acid Metabolism ◽

Selective Pressure ◽

Atmospheric Co2 ◽

Terrestrial Plants ◽

Cam Plants ◽

Acid Accumulation ◽

Use Efficiency ◽

Nocturnal Acid Accumulation

Abstract Background and Aims Crassulacean acid metabolism (CAM) is an adaptation to increase water use efficiency in dry environments. Similar biochemical patterns occur in the aquatic lycophyte genus Isoëtes. It has long been assumed and accepted that CAM-like behaviour in these aquatic plants is an adaptation to low daytime carbon levels in aquatic ecosystems, but this has never been directly tested. Methods To test this hypothesis, populations of Isoëtes engelmannii and I. tuckermanii were grown in climate-controlled chambers and starved of atmospheric CO2 during the day while pH was measured for 24 h. Key Results We demonstrate that terrestrial plants exposed to low atmospheric CO2 display diel acidity cycles similar to those in both xerophytic CAM plants and submerged Isoëtes. Conclusions Daytime CO2 starvation induces CAM-like nocturnal acid accumulation in terrestrial Isoëtes, substantiating the hypothesis that carbon starvation is a selective pressure for this physiological behaviour.

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