A comparison of high-throughput techniques for assaying circadian rhythms in plants

Over the last two decades, the development of high-throughput techniques has enabled us to probe the plant circadian clock, a key coordinator of vital biological processes, in ways previously impossible. With the circadian clock increasingly implicated in key fitness and signalling pathways, this has opened up new avenues for understanding plant development and signalling. Our tool-kit has been constantly improving through continual development and novel techniques that increase throughput, reduce costs and allow higher resolution on the cellular and subcellular levels. With circadian assays becoming more accessible and relevant than ever to researchers, in this paper we offer a review of the techniques currently available before considering the horizons in circadian investigation at ever higher throughputs and resolutions

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

A roadmap for research on crassulacean acid metabolism (CAM) to enhance sustainable food and bioenergy production in a hotter, drier world.

Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that features nocturnal CO2 uptake, facilitates increased water-use efficiency (WUE), and enables CAM plants to inhabit water-limited environments such as semi-arid deserts or seasonally dry forests. Human population growth and global climate change now present challenges for agricultural production systems to increase food, feed, forage, fiber, and fuel production. One approach to meet these challenges is to increase reliance on CAM crops, such as Agave and Opuntia, for biomass production on semi-arid, abandoned, marginal, or degraded agricultural lands. Major research efforts are now underway to assess the productivity of CAM crop species and to harness the WUE of CAM by engineering this pathway into existing food, feed, and bioenergy crops. An improved understanding of CAM has potential for high returns on research investment. To exploit the potential of CAM crops and CAM bioengineering, it will be necessary to elucidate the evolution, genomic features, and regulatory mechanisms of CAM. Field trials and predictive models will be required to assess the productivity of CAM crops, while new synthetic biology approaches need to be developed for CAM engineering. Infrastructure will be needed for CAM model systems, field trials, mutant collections, and data management

A roadmap for research on crassulacean acid metabolism (CAM) to enhance sustainable food and bioenergy production in a hotter, drier world

Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that features nocturnal CO(2) uptake, facilitates increased water-use efficiency (WUE), and enables CAM plants to inhabit water-limited environments such as semi-arid deserts or seasonally dry forests. Human population growth and global climate change now present challenges for agricultural production systems to increase food, feed, forage, fiber, and fuel production. One approach to meet these challenges is to increase reliance on CAM crops, such as Agave and Opuntia, for biomass production on semi-arid, abandoned, marginal, or degraded agricultural lands. Major research efforts are now underway to assess the productivity of CAM crop species and to harness the WUE of CAM by engineering this pathway into existing food, feed, and bioenergy crops. An improved understanding of CAM has potential for high returns on research investment. To exploit the potential of CAM crops and CAM bioengineering, it will be necessary to elucidate the evolution, genomic features, and regulatory mechanisms of CAM. Field trials and predictive models will be required to assess the productivity of CAM crops, while new synthetic biology approaches need to be developed for CAM engineering. Infrastructure will be needed for CAM model systems, field trials, mutant collections, and data management

QCD analyses and determinations of alpha(s) in e+ e- annihilation at energies between 35-GeV and 189-GeV

We employ data taken by the JADE and OPAL experiments for an integrated QCD study in hadronic e+e- annihilations at c.m.s. energies ranging from 35 GeV through 189 GeV. The study is based on jet-multiplicity related observables. The observables are obtained to high jet resolution scales with the JADE, Durham, Cambridge and cone jet finders, and compared with the predictions of various QCD and Monte Carlo models. The strong coupling strength, alpha_s, is determined at each energy by fits of O(alpha_s^2) calculations, as well as matched O(alpha_s^2) and NLLA predictions, to the data. Matching schemes are compared, and the dependence of the results on the choice of the renormalization scale is investigated. The combination of the results using matched predictions gives alpha_s(MZ)=0.1187+{0.0034}-{0.0019}. The strong coupling is also obtained, at lower precision, from O(alpha_s^2) fits of the c.m.s. energy evolution of some of the observables. A qualitative comparison is made between the data and a recent MLLA prediction for mean jet multiplicities.Comment: 49 pages, 17 figures, submitted to Eur.Phys.J.