The responses of stomatal aperture to light intensity and CO 2 concentration were studied in both... more The responses of stomatal aperture to light intensity and CO 2 concentration were studied in both Vicia faba (C 3) and Kalanchoë fedtschenkoi (Crassulacean acid metabolism; CAM), in material sampled from both light and dark periods. Direct comparison was made between intact leaf segments, epidermises grafted onto exposed mesophyll, and isolated epidermal peels, including transplantations between species and between diel periods. We reported the stomatal opening in response to darkness in isolated CAM peels from the light period, but not from the dark. Furthermore, we showed that C 3 mesophyll has stimulated CAM stomata in transplanted peels to behave as C 3 in response to light and CO 2. By using peels and mesophyll from plants sampled in the dark and the light period, we provided clear evidence that CAM stomata behaved differently from C 3. This might be linked to stored metabolites/ions and signalling pathway components within the guard cells, and/or a mesophyll-derived signal. Overall, our results provided evidence for both the involvement of guard cell metabolism and mesophyll signals in stomatal responses in both C 3 and CAM species.
Background and AimsCrassulacean acid metabolism (CAM) is a specialized type of photosynthesis cha... more Background and AimsCrassulacean 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.MethodsBiochemical and genetic characterization of wild-type and starch-deficient RNAi lines of Kalanchoë fedtschenkoi ...
Previously regarded as an intriguing photosynthetic curiosity, the occurrence of C4 and Crassulac... more Previously regarded as an intriguing photosynthetic curiosity, the occurrence of C4 and Crassulacean acid metabolism (CAM) photosynthesis within a single organism has recently emerged as a source of information for future biotechnological use. Among C4/CAM facultative species, Portulaca oleracea L. has been used as a model for biochemical and gene expression analysis of C4/CAM under field and laboratory conditions. In the present work, we focussed on developing molecular tools to facilitate functional genomics studies in this species, from the optimisation of RNA isolation protocols to a method for stable genetic transformation. Eleven variations of RNA extraction procedures were tested and compared for RNA quantity and quality. Also, 7 sample sets comprising total RNA from hormonal and abiotic stress treatments, distinct plant organs, leaf developmental stages, and subspecies were used to select, among 12 reference genes, the most stable reference genes for RT-qPCR analysis of each...
Portulaca oleracea is a C4 herb capable of performing CAM under drought stress. It is distributed... more Portulaca oleracea is a C4 herb capable of performing CAM under drought stress. It is distributed worldwide and is either considered a polymorphic species or a complex of subspecies, due to its numerous morphological variations. We evaluated CAM plasticity within P. oleracea genotypes since the complexity surrounding this species may be reflected in intraspecific variations in photosynthetic behavior. Eleven subspecies of P. oleracea from distant geographical locations and one cultivar were morphologically and physiologically characterized. C4 and CAM photosynthesis were monitored in plants exposed to well-watered, droughted and rewatered treatments, and data obtained were compared among individual genotypes. All subspecies expressed CAM in a fully-reversible manner. Transcript abundance of C4–CAM signature genes was shown to be a useful indicator of the C4–CAM–C4 switches in all genotypes. C4-related genes were down-regulated and subsequently fully expressed upon drought and rewate...
One of the best-characterized physiological rhythms in plants is the circadian rhythm of CO2 meta... more One of the best-characterized physiological rhythms in plants is the circadian rhythm of CO2 metabolism in Crassulacean acid metabolism (CAM) plants, which is the focus here. The central components of the plant circadian clock have been studied in detail only in Arabidopsis (Arabidopsis thaliana). Full-length cDNAs have been obtained encoding orthologs of CIRCADIAN CLOCK-ASSOCIATED1 (CCA1)/LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION1 (TOC1), EARLY FLOWERING4 (ELF4), ZEITLUPE (ZTL), FLAVIN-BINDING KELCH REPEAT F-BOX1 (FKF1), EARLY FLOWERING3 (ELF3), and a partial cDNA encoding GIGANTEA in the model stress-inducible CAM plant, Mesembryanthemum crystallinum (Common Ice Plant). TOC1 and LHY/CCA1 are under reciprocal circadian control in a manner similar to their regulation in Arabidopsis. ELF4, FKF1, ZTL, GIGANTEA, and ELF3 are under circadian control in C3 and CAM leaves. ELF4 transcripts peak in the evening and are unaffected by CAM induction. FKF1 shows an abrupt transcr...
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) performs most of the carbon fixation on... more Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) performs most of the carbon fixation on Earth. However, plant Rubisco is an intrinsically inefficient enzyme given its low carboxylation rate, representing a major limitation to photosynthesis. Replacing endogenous plant Rubisco with a faster Rubisco is anticipated to enhance crop photosynthesis and productivity. However, the requirement of chaperones for Rubisco expression and assembly has obstructed the efficient production of functional foreign Rubisco in chloroplasts. Here, we report the engineering of a Form 1A Rubisco from the proteobacterium Halothiobacillus neapolitanus in Escherichia coli and tobacco (Nicotiana tabacum) chloroplasts without any cognate chaperones. The native tobacco gene encoding Rubisco large subunit was genetically replaced with H. neapolitanus Rubisco (HnRubisco) large and small subunit genes. We show that HnRubisco subunits can form functional L8S8 hexadecamers in tobacco chloroplasts at high eff...
All in-text references underlined in blue are linked to publications on ResearchGate, letting you... more All in-text references underlined in blue are linked to publications on ResearchGate, letting you access and read them immediately.
Characterization of RNAi lines of Kalanchoë fedtschenkoi deficient in chloroplastic α-glucan phos... more Characterization of RNAi lines of Kalanchoë fedtschenkoi deficient in chloroplastic α-glucan phosphorylase demonstrates that the phosphorolytic route of starch degradation is of central importance for CAM in this model species.
For over 40 years, a circadian clock has been known to control the nocturnal fixation of CO2 in C... more For over 40 years, a circadian clock has been known to control the nocturnal fixation of CO2 in Crassulacean acid metabolism (CAM) plants. We now know that the observed rhythms of CO2-fixation are mainly due to circadian control of flux through phosphoenolpyruvate carboxylase (PEPc). Flux through PEPc is regulated by reversible protein phosphorylation, which is largely controlled by circadian fluctuations in the expression and activity of PEPc kinase. In order to obtain a more global picture of how the transcripts of CAM-associated genes vary over the diurnal and circadian cycle, we examined the transcript levels of a range of CAM-associated genes under diurnal and constant conditions in both young C3 and older CAM-induced Mesembryanthemum crystallinum plants. We designed sequence specific primers to a wide variety of CAM-associated genes. These primers were used to perform semi-quantitative RT-PCR analysis on each gene. Samples were collected every 6 h for 54 h from both C3 and CAM...
Light-induced phosphorylation of phosphoenolpyruvate carboxylase plays an important role in C4 ph... more Light-induced phosphorylation of phosphoenolpyruvate carboxylase plays an important role in C4 photosynthesis. It reduces the sensitivity of phosphoenolpyruvate carboxylase to malate and thereby allows this enzyme to fix bicarbonate even in the presence of a high concentration of malate in the mesophyll cell cytosol. Previous work has shown that light causes an increase in the activity of phosphoenolpyruvate carboxylase kinase in a process involving protein synthesis. Phosphoenolpyruvate carboxylase kinase genes have been cloned from a range of C3 and CAM species, but not so far from C4 species. We have identified a phosphoenolpyruvate carboxylase cDNA from the C4 plant Sorghum bicolor. It encodes a 307 residue protein of predicted Mr 32524, which, like other phosphoenolpyruvate carboxylase kinases, comprises a protein kinase catalytic domain with minimal extensions at the N- and C-terminal ends. However, relative to other phosphoenolpyruvate carboxylase kinases, the Sorghum enzyme ...
In grapevine, trans-Resveratrol (tR) is produced as a defence mechanism against stress or infecti... more In grapevine, trans-Resveratrol (tR) is produced as a defence mechanism against stress or infection. tR is also considered to be important for human health, which increases its interest to the scientific community. Transcriptomic analysis in grapevine cell cultures treated with the defence response elicitor methyl-β-cyclodextrin (CD) revealed that both copies of PHOSPHOENOLPYRUVATE CARBOXYLASE KINASE (PPCK) were down-regulated significantly. A role for PPCK in the defence response pathway has not been proposed previously. We therefore analysed the control of PPCK transcript levels in grapevine cell cultures and leaves elicited with CD. Moreover, phosphoenolpyruvate carboxylase (PPC), stilbene synthase (STS), and the transcription factors MYB14 and WRKY24, which are involved in the activation of STS transcription, were also analysed by RT-qPCR. The results revealed that under CD elicitation conditions PPCK down-regulation, increased stilbene production and loss of PPC activity occurs...
* Plants utilizing crassulacean acid metabolism (CAM) concentrate CO2 around RuBisCO while reduci... more * Plants utilizing 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.
Silencing phosphoenolpyruvate carboxylase in a Crassulacean acid metabolism species prevented noc... more Silencing phosphoenolpyruvate carboxylase in a Crassulacean acid metabolism species prevented nocturnal CO 2 fixation and malate accumulation and perturbed the circadian clock and guard cell signalling.
Although biochemically related, C 4 and crassulacean acid metabolism (CAM) systems are expected t... more Although biochemically related, C 4 and crassulacean acid metabolism (CAM) systems are expected to be incompatible. However, Portulaca species, including P. oleracea, operate C 4 and CAM within a single leaf, and the mechanisms behind this unique photosynthetic arrangement remain largely unknown. Here, we employed RNA-seq to identify candidate genes involved exclusively or shared by C 4 or CAM, and provided an in-depth characterization of their transcript abundance patterns during the drought-induced photosynthetic transitions in P. oleracea. Data revealed fewer candidate CAM-specific genes than those recruited to function in C 4. The putative CAMspecific genes were predominantly involved in night-time primary carboxylation reactions and malate movement across the tonoplast. Analysis of gene transcript-abundance regulation and photosynthetic physiology indicated that C 4 and CAM coexist within a single P. oleracea leaf under mild drought conditions. Developmental and environmental cues were shown to regulate CAM expression in stems, whereas the shift from C 4 to C 4-CAM hybrid photosynthesis in leaves was strictly under environmental control. Moreover, efficient starch turnover was identified as part of the metabolic adjustments required for CAM operation in both organs. These findings provide insights into C 4 /CAM connectivity and compatibility, contributing to a deeper understanding of alternative ways to engineer CAM into C 4 crop species.
The responses of stomatal aperture to light intensity and CO 2 concentration were studied in both... more The responses of stomatal aperture to light intensity and CO 2 concentration were studied in both Vicia faba (C 3) and Kalanchoë fedtschenkoi (Crassulacean acid metabolism; CAM), in material sampled from both light and dark periods. Direct comparison was made between intact leaf segments, epidermises grafted onto exposed mesophyll, and isolated epidermal peels, including transplantations between species and between diel periods. We reported the stomatal opening in response to darkness in isolated CAM peels from the light period, but not from the dark. Furthermore, we showed that C 3 mesophyll has stimulated CAM stomata in transplanted peels to behave as C 3 in response to light and CO 2. By using peels and mesophyll from plants sampled in the dark and the light period, we provided clear evidence that CAM stomata behaved differently from C 3. This might be linked to stored metabolites/ions and signalling pathway components within the guard cells, and/or a mesophyll-derived signal. Overall, our results provided evidence for both the involvement of guard cell metabolism and mesophyll signals in stomatal responses in both C 3 and CAM species.
Background and AimsCrassulacean acid metabolism (CAM) is a specialized type of photosynthesis cha... more Background and AimsCrassulacean 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.MethodsBiochemical and genetic characterization of wild-type and starch-deficient RNAi lines of Kalanchoë fedtschenkoi ...
Previously regarded as an intriguing photosynthetic curiosity, the occurrence of C4 and Crassulac... more Previously regarded as an intriguing photosynthetic curiosity, the occurrence of C4 and Crassulacean acid metabolism (CAM) photosynthesis within a single organism has recently emerged as a source of information for future biotechnological use. Among C4/CAM facultative species, Portulaca oleracea L. has been used as a model for biochemical and gene expression analysis of C4/CAM under field and laboratory conditions. In the present work, we focussed on developing molecular tools to facilitate functional genomics studies in this species, from the optimisation of RNA isolation protocols to a method for stable genetic transformation. Eleven variations of RNA extraction procedures were tested and compared for RNA quantity and quality. Also, 7 sample sets comprising total RNA from hormonal and abiotic stress treatments, distinct plant organs, leaf developmental stages, and subspecies were used to select, among 12 reference genes, the most stable reference genes for RT-qPCR analysis of each...
Portulaca oleracea is a C4 herb capable of performing CAM under drought stress. It is distributed... more Portulaca oleracea is a C4 herb capable of performing CAM under drought stress. It is distributed worldwide and is either considered a polymorphic species or a complex of subspecies, due to its numerous morphological variations. We evaluated CAM plasticity within P. oleracea genotypes since the complexity surrounding this species may be reflected in intraspecific variations in photosynthetic behavior. Eleven subspecies of P. oleracea from distant geographical locations and one cultivar were morphologically and physiologically characterized. C4 and CAM photosynthesis were monitored in plants exposed to well-watered, droughted and rewatered treatments, and data obtained were compared among individual genotypes. All subspecies expressed CAM in a fully-reversible manner. Transcript abundance of C4–CAM signature genes was shown to be a useful indicator of the C4–CAM–C4 switches in all genotypes. C4-related genes were down-regulated and subsequently fully expressed upon drought and rewate...
One of the best-characterized physiological rhythms in plants is the circadian rhythm of CO2 meta... more One of the best-characterized physiological rhythms in plants is the circadian rhythm of CO2 metabolism in Crassulacean acid metabolism (CAM) plants, which is the focus here. The central components of the plant circadian clock have been studied in detail only in Arabidopsis (Arabidopsis thaliana). Full-length cDNAs have been obtained encoding orthologs of CIRCADIAN CLOCK-ASSOCIATED1 (CCA1)/LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION1 (TOC1), EARLY FLOWERING4 (ELF4), ZEITLUPE (ZTL), FLAVIN-BINDING KELCH REPEAT F-BOX1 (FKF1), EARLY FLOWERING3 (ELF3), and a partial cDNA encoding GIGANTEA in the model stress-inducible CAM plant, Mesembryanthemum crystallinum (Common Ice Plant). TOC1 and LHY/CCA1 are under reciprocal circadian control in a manner similar to their regulation in Arabidopsis. ELF4, FKF1, ZTL, GIGANTEA, and ELF3 are under circadian control in C3 and CAM leaves. ELF4 transcripts peak in the evening and are unaffected by CAM induction. FKF1 shows an abrupt transcr...
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) performs most of the carbon fixation on... more Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) performs most of the carbon fixation on Earth. However, plant Rubisco is an intrinsically inefficient enzyme given its low carboxylation rate, representing a major limitation to photosynthesis. Replacing endogenous plant Rubisco with a faster Rubisco is anticipated to enhance crop photosynthesis and productivity. However, the requirement of chaperones for Rubisco expression and assembly has obstructed the efficient production of functional foreign Rubisco in chloroplasts. Here, we report the engineering of a Form 1A Rubisco from the proteobacterium Halothiobacillus neapolitanus in Escherichia coli and tobacco (Nicotiana tabacum) chloroplasts without any cognate chaperones. The native tobacco gene encoding Rubisco large subunit was genetically replaced with H. neapolitanus Rubisco (HnRubisco) large and small subunit genes. We show that HnRubisco subunits can form functional L8S8 hexadecamers in tobacco chloroplasts at high eff...
All in-text references underlined in blue are linked to publications on ResearchGate, letting you... more All in-text references underlined in blue are linked to publications on ResearchGate, letting you access and read them immediately.
Characterization of RNAi lines of Kalanchoë fedtschenkoi deficient in chloroplastic α-glucan phos... more Characterization of RNAi lines of Kalanchoë fedtschenkoi deficient in chloroplastic α-glucan phosphorylase demonstrates that the phosphorolytic route of starch degradation is of central importance for CAM in this model species.
For over 40 years, a circadian clock has been known to control the nocturnal fixation of CO2 in C... more For over 40 years, a circadian clock has been known to control the nocturnal fixation of CO2 in Crassulacean acid metabolism (CAM) plants. We now know that the observed rhythms of CO2-fixation are mainly due to circadian control of flux through phosphoenolpyruvate carboxylase (PEPc). Flux through PEPc is regulated by reversible protein phosphorylation, which is largely controlled by circadian fluctuations in the expression and activity of PEPc kinase. In order to obtain a more global picture of how the transcripts of CAM-associated genes vary over the diurnal and circadian cycle, we examined the transcript levels of a range of CAM-associated genes under diurnal and constant conditions in both young C3 and older CAM-induced Mesembryanthemum crystallinum plants. We designed sequence specific primers to a wide variety of CAM-associated genes. These primers were used to perform semi-quantitative RT-PCR analysis on each gene. Samples were collected every 6 h for 54 h from both C3 and CAM...
Light-induced phosphorylation of phosphoenolpyruvate carboxylase plays an important role in C4 ph... more Light-induced phosphorylation of phosphoenolpyruvate carboxylase plays an important role in C4 photosynthesis. It reduces the sensitivity of phosphoenolpyruvate carboxylase to malate and thereby allows this enzyme to fix bicarbonate even in the presence of a high concentration of malate in the mesophyll cell cytosol. Previous work has shown that light causes an increase in the activity of phosphoenolpyruvate carboxylase kinase in a process involving protein synthesis. Phosphoenolpyruvate carboxylase kinase genes have been cloned from a range of C3 and CAM species, but not so far from C4 species. We have identified a phosphoenolpyruvate carboxylase cDNA from the C4 plant Sorghum bicolor. It encodes a 307 residue protein of predicted Mr 32524, which, like other phosphoenolpyruvate carboxylase kinases, comprises a protein kinase catalytic domain with minimal extensions at the N- and C-terminal ends. However, relative to other phosphoenolpyruvate carboxylase kinases, the Sorghum enzyme ...
In grapevine, trans-Resveratrol (tR) is produced as a defence mechanism against stress or infecti... more In grapevine, trans-Resveratrol (tR) is produced as a defence mechanism against stress or infection. tR is also considered to be important for human health, which increases its interest to the scientific community. Transcriptomic analysis in grapevine cell cultures treated with the defence response elicitor methyl-β-cyclodextrin (CD) revealed that both copies of PHOSPHOENOLPYRUVATE CARBOXYLASE KINASE (PPCK) were down-regulated significantly. A role for PPCK in the defence response pathway has not been proposed previously. We therefore analysed the control of PPCK transcript levels in grapevine cell cultures and leaves elicited with CD. Moreover, phosphoenolpyruvate carboxylase (PPC), stilbene synthase (STS), and the transcription factors MYB14 and WRKY24, which are involved in the activation of STS transcription, were also analysed by RT-qPCR. The results revealed that under CD elicitation conditions PPCK down-regulation, increased stilbene production and loss of PPC activity occurs...
* Plants utilizing crassulacean acid metabolism (CAM) concentrate CO2 around RuBisCO while reduci... more * Plants utilizing 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.
Silencing phosphoenolpyruvate carboxylase in a Crassulacean acid metabolism species prevented noc... more Silencing phosphoenolpyruvate carboxylase in a Crassulacean acid metabolism species prevented nocturnal CO 2 fixation and malate accumulation and perturbed the circadian clock and guard cell signalling.
Although biochemically related, C 4 and crassulacean acid metabolism (CAM) systems are expected t... more Although biochemically related, C 4 and crassulacean acid metabolism (CAM) systems are expected to be incompatible. However, Portulaca species, including P. oleracea, operate C 4 and CAM within a single leaf, and the mechanisms behind this unique photosynthetic arrangement remain largely unknown. Here, we employed RNA-seq to identify candidate genes involved exclusively or shared by C 4 or CAM, and provided an in-depth characterization of their transcript abundance patterns during the drought-induced photosynthetic transitions in P. oleracea. Data revealed fewer candidate CAM-specific genes than those recruited to function in C 4. The putative CAMspecific genes were predominantly involved in night-time primary carboxylation reactions and malate movement across the tonoplast. Analysis of gene transcript-abundance regulation and photosynthetic physiology indicated that C 4 and CAM coexist within a single P. oleracea leaf under mild drought conditions. Developmental and environmental cues were shown to regulate CAM expression in stems, whereas the shift from C 4 to C 4-CAM hybrid photosynthesis in leaves was strictly under environmental control. Moreover, efficient starch turnover was identified as part of the metabolic adjustments required for CAM operation in both organs. These findings provide insights into C 4 /CAM connectivity and compatibility, contributing to a deeper understanding of alternative ways to engineer CAM into C 4 crop species.
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