Phosphorylation of Phosphoenolpyruvate Carboxylase Is Essential for Maximal and Sustained Dark CO2 Fixation and Core Circadian Clock Operation in the Obligate Crassulacean Acid Metabolism Species Kalanchoe fedtschenkoi

Phosphoenolpyruvate carboxylase (PPC; EC 4.1.1.31) catalyzes primary nocturnal CO2 fixation in Crassulacean acid metabolism (CAM) species. CAM PPC is regulated posttranslationally by a circadian clock-controlled protein kinase called phosphoenolpyruvate carboxylase kinase (PPCK). PPCK phosphorylates PPC during the dark period, reducing its sensitivity to feedback inhibition by malate and thus enhancing nocturnal CO2 fixation to stored malate. Here, we report the generation and characterization of transgenic RNAi lines of the obligate CAM species Kalanchoë fedtschenkoi with reduced levels of KfPPCK1 transcripts. Plants with reduced or no detectable dark phosphorylation of PPC displayed up to a 66% reduction in total dark period CO2 fixation. These perturbations paralleled reduced malate accumulation at dawn and decreased nocturnal starch turnover. Loss of oscillations in the transcript abundance of KfPPCK1 was accompanied by a loss of oscillations in the transcript abundance of many core circadian clock genes, suggesting that perturbing the only known link between CAM and the circadian clock feeds back to perturb the central circadian clock itself. This work shows that clock control of KfPPCK1 prolongs the activity of PPC throughout the dark period in K. fedtschenkoi, optimizing CAM-associated dark CO2 fixation, malate accumulation, CAM productivity, and core circadian clock robustness

Kalanchoe PPC1 Is Essential for Crassulacean Acid Metabolism and the Regulation of Core Circadian Clock and Guard Cell Signaling Genes([CC-BY])

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

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

ABSTRACT Unlike C 3 plants, Crassulacean acid metabolism (CAM) plants fix CO 2 in the dark using phosphoenolpyruvate carboxylase (PPC; EC 4.1.1.31). PPC combines PEP with CO 2 (as HCO 3 − ), 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 CO 2 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 CO 2 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 CO 2 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

Oxygen requirement and inhibition of C4 photosynthesis

The basis for O2 sensitivity of C4 photosynthesis was evaluated using a C4-cycle-limited mutant of Amaranthus edulis (a phosphoenolpyruvate carboxylase-deficient mutant), and a C3-cyclelimited transformant of Flaveria bidentis (an antisense ribulose-1,5- bisphosphate carboxylase/oxygenase [Rubisco] small subunit transformant). Data obtained with the C4-cycle-limited mutant showed that atmospheric levels of O2 (20 kPa) caused increased inhibition of photosynthesis as a result of higher levels of photorespiration. The optimal O2 partial pressure for photosynthesis was reduced from approximately 5 kPa O2 to 1 to 2 kPa O2, becoming similar to that of C3 plants. Therefore, the higher O2 requirement for optimal C4 photosynthesis is specifically associated with the C4 function. With the Rubisco-limited F. bidentis, there was less inhibition of photosynthesis by supraoptimal levels of O2 than in the wild type. When CO2 fixation by Rubisco is limited, an increase in the CO2 concentration in bundle-sheath cells via the C4 cycle may further reduce the oxygenase activity of Rubisco and decrease the inhibition of photosynthesis by high partial pressures of O2 while increasing CO2 leakage and overcycling of the C4 pathway. These results indicate that in C4 plants the investment in the C3 and C4 cycles must be balanced for maximum efficiency

Insights into the phytochemical composition of selected genotypes of organic kale (Brassica oleracea L. var. acephala)

Seven genotypes of kale (B. oleracea L. var. acephala), selected from a collection set up in the framework of the BRESOV H2020 Project, aimed at breeding under organic conditions, were analysed for the content in the characteristic phytochemicals of Brassica spp. The presence of prebiotic oligosaccharides was an important characteristic of this crop, with values ranging from 4.2 to 10.8 g/100 g d.w. The other detected soluble sugars were sucrose, glucose, and fructose, with glucose predominating (3.9–9.0 g/100 g d.w.). Sulphur compounds, such as sulphoxides, were detected in the form of pyruvic acid, their catabolic product (36–154 mg/100 g d.w.). In addition, the levels of main breakdown products of glucosinolates, such as sulforaphane and indole-3-carbinol, were found to be in average contents of 6.7 and 3.9 μmol/g d.w., respectively. Finally, the presence of major phytochemicals in kale, such as polyphenols and carotenoids, reported considerable concentrations (around 1400 mg/100 g d.w. and 19 mg/100 g d.w, respectively), typical for this Brassica crop

The Kalanchoe genome provides insights into convergent evolution and building blocks of crassulacean acid metabolism

Crassulacean acid metabolism (CAM) is a water-use efficient adaptation of photosynthesis that has evolved independently many times in diverse lineages of flowering plants. We hypothesize that convergent evolution of protein sequence and temporal gene expression underpins the independent emergences of CAM from C3 photosynthesis. To test this hypothesis, we generate a de novo genome assembly and genome-wide transcript expression data for Kalanchoë fedtschenkoi, an obligate CAM species within the core eudicots with a relatively small genome (~260 Mb). Our comparative analyses identify signatures of convergence in protein sequence and re-scheduling of diel transcript expression of genes involved in nocturnal CO2 fixation, stomatal movement, heat tolerance, circadian clock, and carbohydrate metabolism in K. fedtschenkoi and other CAM species in comparison with non-CAM species. These findings provide new insights into molecular convergence and building blocks of CAM and will facilitate CAM-into-C3 photosynthesis engineering to enhance water-use efficiency in crops

Control of C4 photosynthesis: effects of reduced activities of phosphoenolpyruvate carboxylase on CO2 assimilation in Amaranthus edulis L.

Heterozygous mutants of Amaranthus edulis deficient in PEP carboxylase (PEPC) have been used to study the control of photosynthetic carbon assimilation. A reduction in PEPC activity led to a decrease in the initial slope of the relationship between the CO2 assimilation rate and the intercellular CO2 concentration and to a decrease in photosynthesis at high light intensities, consistent with a decrease in the capacity of the C4 cycle in high light. PEPC exerted appreciable control on photosynthetic flux in the wild-type, with a relatively high flux control coefficient of 0.35 in saturating light and ambient CO2. The flux control coefficient was decreased in low light or increased in low CO2 or in plants containing lower PEPC activity. However, the rate of CO2 assimilation decreased down to about 55% PEPC, followed by an up-turn in the light-saturated photosynthetic rate as PEPC was further reduced, suggesting the existence of a mechanism that compensates for the loss of PEPC activity. The amounts of photosynthetic metabolites, including glycine and serine, also showed a biphasic response to decreasing PEPC. There was a linear relationship between the activity of PEPC and the activation state of the enzyme. A possible mechanism of compensation involving photorespiratory intermediates is discussed

White Paper describing the route to improved crop yields in Europe

The realisation of the full objectives of international policies targeting global food security and climate change mitigation, including the European Green Deal, United Nation’s Sustainable Development Goals (SDGs), the Paris Climate Agreement COP21 and transition away from a fossil-carbon industrial base to one that is more bio-based, requires that we(i) sustainably increase the yield, nutritional quality, and biodiversity of major crop species,(ii) select climate-ready crops that are adapted to future weather dynamic and(iii) increase the resource use efficiency of crops to preserve natural resources, such as fresh water and phosphate and reducing the environmental burden arising from the application of nitrogenous fertiliser.Advanced scientific knowledge and tools for research and crop breeding already provide an excellent platform to build on. A long-term Strategic Research Agenda now needs to be agreed and priorities set to deliver blueprints for climate resilient future-proofed crops. This strategy should then be implemented through an innovative collaborative approach that combines the joint knowledge base of the research and industrial communities, with multi-stakeholder involvement and strong support from policy makers.This white paper has been compiled from contributions from several experts across the field of plant sciences. The CropBooster-P project has developed this strategic research agenda for a crop improvement programme that will provide the genetic innovation to improve and future proof our crop plants. It builds a strong collaboration between plant scientists and modellers, physicists, soil scientists, engineers and coders, biomathematicians, agronomists, plant breeders and farmers. The goal is to exploit the largely untapped genetic diversity that exists within the wild relatives and ancient and heirloom varieties of our crop plants to improve our crops so they will be more resilient, high yielding, resource efficient, nutritious, and ready for the future climate of Europe. The knowledge and technology to reach this goal springs from the transformative developments that have occurred in the last 20 years in genomics, phenomics, crop sciences, molecular plant sciences, agronomy, and plant breeding