Influence of starch deficiency on photosynthetic and post-photosynthetic carbon isotope fractionations.

Carbon isotope (13C) fractionations occurring during and after photosynthetic CO2 fixation shape the carbon isotope composition (δ13C) of plant material and respired CO2. However, responses of 13C fractionations to diel variation in starch metabolism in the leaf are not fully understood. Here we measured δ13C of organic matter (δ13COM), concentrations and δ13C of potential respiratory substrates, δ13C of dark-respired CO2 (δ13CR), and gas exchange in leaves of starch-deficient plastidial phosphoglucomutase (pgm) mutants and wild-type plants of four species (Arabidopsis thaliana, Mesembryanthemum crystallinum, Nicotiana sylvestris, and Pisum sativum). The strongest δ13C response to the pgm-induced starch deficiency was observed in N. sylvestris, with more negative δ13COM, δ13CR, and δ13C values for assimilates (i.e. sugars and starch) and organic acids (i.e. malate and citrate) in pgm mutants than in wild-type plants during a diel cycle. The genotype differences in δ13C values could be largely explained by differences in leaf gas exchange. In contrast, the PGM-knockout effect on post-photosynthetic 13C fractionations via the plastidic fructose-1,6-bisphosphate aldolase reaction or during respiration was small. Taken together, our results show that the δ13C variations in starch-deficient mutants are primarily explained by photosynthetic 13C fractionations and that the combination of knockout mutants and isotope analyses allows additional insights into plant metabolism.

Design and Use of a Digitally Controlled Device for Accurate, Multiplexed Gas Exchange Measurements of the Complete Foliar Parts of Plants.

Performing accurate measurements of photosynthetic and respiration rates is vital to a large proportion of plant-based studies. While several commercial systems exist to perform such measurements, few are ideal for whole-plant measurements of small herbaceous plants such as Arabidopsis and none offer the capacity for simultaneous analysis of multiple plants. We, therefore, designed a multi-chamber, computer-controlled, infrared gas analyzer-coupled system for the continuous measurement of gas exchange in whole-plant shoots or rosettes. This system was called ETH Gas Exchange System-1 (EGES-1). We have subsequently expanded the device to accommodate a wider variety of species while providing precise control over environmental parameters. Critically, we have (1) increased the flow rates through each of the eight chambers, (2) introduced a computer-controlled feedback loop for the precise introduction of CO2, and (3) added an additional feedback loop for the introduction and control of humidity. The advantages of this new system (EGES-2) are illustrated here in the context of a variety of physiological experiments.

A whole-plant chamber system for parallel gas exchange measurements of Arabidopsis and other herbaceous species.

BACKGROUND: Photosynthetic assimilation of carbon is a defining feature of the plant kingdom. The fixation of large amounts of carbon dioxide supports the synthesis of carbohydrates, which make up the bulk of plant biomass. Exact measurements of carbon assimilation rates are therefore crucial due to their impact on the plants metabolism, growth and reproductive success. Commercially available single-leaf cuvettes allow the detailed analysis of many photosynthetic parameters, including gas exchange, of a selected leaf area. However, these cuvettes can be difficult to use with small herbaceous plants such as Arabidopsis thaliana or plants having delicate or textured leaves. Furthermore, data from single leaves can be difficult to scale-up for a plant shoot with a complex architecture and tissues in different physiological states. Therefore, we constructed a versatile system-EGES-1-to simultaneously measure gas exchange in the whole shoots of multiple individual plants. Our system was designed to be able record data continuously over several days. RESULTS: The EGES-1 system yielded comparable measurements for eight plants for up to 6 days in stable, physiologically realistic conditions. The chambers seals have negligible permeability to carbon dioxide and the system is designed so as to detect any bulk-flow air leaks. We show that the system can be used to monitor plant responses to changing environmental conditions, such as changes in illumination or stress treatments, and to compare plants with phenotypically severe mutations. By incorporating interchangeable lids, the system could be used to measure photosynthetic gas exchange in several genera such as Arabidopsis, Nicotiana, Pisum, Lotus and Mesembryanthemum. CONCLUSION: EGES-1 can be introduced into a variety of growth facilities and measure gas exchange in the shoots diverse plant species grown in different growth media. It is ideal for comparing photosynthetic carbon assimilation of wild-type and mutant plants and/or plants undergoing selected experimental treatments. The system can deliver valuable data for whole-plant growth studies and help understanding mutant phenotypes. Overall, the EGES-1 is complementary to the readily-available single leaf systems that focus more on the photosynthetic process in within the leaf lamina.

Virus-induced gene silencing as a scalable tool to study drought tolerance in plants.

Here we describe the methodology of using virus-induced gene silencing (VIGS) as a powerful and scalable tool to screen the function of genes that participate in adaptation to drought. Silencing of endogenous gene expression in Nicotiana benthamiana is achieved by systemic infection of the aerial parts of the plant with a virus engineered to contain homologous fragments of the target gene(s) of interest. Silenced plant material can be consistently produced with little optimization in less than 1 month without specialized equipment, using only simple cloning and transformation techniques. Although maximal silencing is localized to only a few leaves, when whole plants are subjected to water stress, the tissue from these silenced leaves can be characterized for physiological, biochemical, and transcriptional responses to determine the role of the candidate genes in drought tolerance.

Virus-induced multiple gene silencing to study redundant metabolic pathways in plants: silencing the starch degradation pathway in Nicotiana benthamiana.

Virus-induced gene silencing (VIGS) is a rapid technique that allows for specific and reproducible post-transcriptional degradation of targeted mRNA. The method has been proven efficient for suppression of expression of many single enzymes. The metabolic networks of plants, however, often contain isoenzymes and gene families that are able to compensate for a mutation and mask the development of a silencing phenotype. Here, we show the application of multiple gene VIGS repression for the study of these redundant biological pathways. Several genes in the starch degradation pathway [disproportionating enzyme 1; (DPE1), disproportionating enzyme 2 (DPE2), and GWD] were silenced. The functionally distinct DPE enzymes are present in alternate routes for sugar export to the cytoplasm and result in an increase in starch production when silenced individually. Simultaneous silencing of DPE1 and DPE2 in Nicotiana benthamiana resulted in a near complete suppression in starch and accumulation of malto-oligosaccharides.

Virus-induced gene silencing of plastidial soluble inorganic pyrophosphatase impairs essential leaf anabolic pathways and reduces drought stress tolerance in Nicotiana benthamiana.

The role of pyrophosphate in primary metabolism is poorly understood. Here, we report on the transient down-regulation of plastid-targeted soluble inorganic pyrophosphatase in Nicotiana benthamiana source leaves. Physiological and metabolic perturbations were particularly evident in chloroplastic central metabolism, which is reliant on fast and efficient pyrophosphate dissipation. Plants lacking plastidial soluble inorganic pyrophosphatase (psPPase) were characterized by increased pyrophosphate levels, decreased starch content, and alterations in chlorophyll and carotenoid biosynthesis, while constituents like amino acids (except for histidine, serine, and tryptophan) and soluble sugars and organic acids (except for malate and citrate) remained invariable from the control. Furthermore, translation of Rubisco was significantly affected, as observed for the amounts of the respective subunits as well as total soluble protein content. These changes were concurrent with the fact that plants with reduced psPPase were unable to assimilate carbon to the same extent as the controls. Furthermore, plants with lowered psPPase exposed to mild drought stress showed a moderate wilting phenotype and reduced vitality, which could be correlated to reduced abscisic acid levels limiting stomatal closure. Taken together, the results suggest that plastidial pyrophosphate dissipation through psPPase is indispensable for vital plant processes.