Rising rates of starch degradation during daytime and trehalose 6-phosphate optimize carbon availability.

Many plants, including Arabidopsis (Arabidopsis thaliana), accumulate starch in the light and remobilize it to support maintenance and growth at night. Starch synthesis and degradation are usually viewed as temporally separate processes. Recently, we reported that starch is also degraded in the light. Degradation rates are generally low early in the day but rise with time. Here, we show that the rate of degradation in the light depends on time relative to dawn rather than dusk. We also show that degradation in the light is inhibited by trehalose 6-phosphate, a signal for sucrose availability. The observed responses of degradation in the light can be simulated by a skeletal model in which the rate of degradation is a function of starch content divided by time remaining until dawn. The fit is improved by extension to include feedback inhibition of starch degradation by trehalose 6-phosphate. We also investigate possible functions of simultaneous starch synthesis and degradation in the light, using empirically parameterized models and experimental approaches. The idea that this cycle buffers growth against falling rates of photosynthesis at twilight is supported by data showing that rates of protein and cell wall synthesis remain high during a simulated dusk twilight. Degradation of starch in the light may also counter over-accumulation of starch in long photoperiods and stabilize signaling around dusk. We conclude that starch degradation in the light is regulated by mechanisms similar to those that operate at night and is important for stabilizing carbon availability and signaling, thus optimizing growth in natural light conditions.

Distinct plastid fructose bisphosphate aldolases function in photosynthetic and non-photosynthetic metabolism in Arabidopsis.

Plastid metabolism is critical in both photoautotrophic and heterotrophic plant cells. In chloroplasts, fructose-1,6-bisphosphate aldolase (FBA) catalyses the formation of both fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate within the Calvin-Benson cycle. Three Arabidopsis genes, AtFBA1-AtFBA3, encode plastidial isoforms of FBA, but the contribution of each isoform is unknown. Phylogenetic analysis indicates that FBA1 and FBA2 derive from a recently duplicated gene, while FBA3 is a more ancient paralog. fba1 mutants are phenotypically indistinguishable from the wild type, while both fba2 and fba3 have reduced growth. We show that FBA2 is the major isoform in leaves, contributing most of the measurable activity. Partial redundancy with FBA1 allows both single mutants to survive, but combining both mutations is lethal, indicating a block of photoautotrophy. In contrast, FBA3 is expressed predominantly in heterotrophic tissues, especially the leaf and root vasculature, but not in the leaf mesophyll. We show that the loss of FBA3 affects plastidial glycolytic metabolism of the root, potentially limiting the biosynthesis of essential compounds such as amino acids. However, grafting experiments suggest that fba3 is dysfunctional in leaf phloem transport, and we suggest that a block in photoassimilate export from leaves causes the buildup of high carbohydrate concentrations and retarded growth.

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.

Changes in resource partitioning between and within organs support growth adjustment to neighbor proximity in Brassicaceae seedlings.

In shade-intolerant plants, the perception of proximate neighbors rapidly induces architectural changes resulting in elongated stems and reduced leaf size. Sensing and signaling steps triggering this modified growth program have been identified. However, the underlying changes in resource allocation that fuel stem growth remain poorly understood. Through 14CO2 pulse labeling of Brassica rapa seedlings, we show that perception of the neighbor detection signal, low ratio of red to far-red light (R:FR), leads to increased carbon allocation from the major site of photosynthesis (cotyledons) to the elongating hypocotyl. While carbon fixation and metabolite levels remain similar in low R:FR, partitioning to all downstream carbon pools within the hypocotyl is increased. Genetic analyses using Arabidopsis thaliana mutants indicate that low-R:FR-induced hypocotyl elongation requires sucrose transport from the cotyledons and is regulated by a PIF7-dependent metabolic response. Moreover, our data suggest that starch metabolism in the hypocotyl has a growth-regulatory function. The results reveal a key mechanism by which metabolic adjustments can support rapid growth adaptation to a changing environment.

Repression of Sex4 and Like Sex Four2 Orthologs in Potato Increases Tuber Starch Bound Phosphate With Concomitant Alterations in Starch Physical Properties.

To examine the roles of starch phosphatases in potatoes, transgenic lines were produced where orthologs of SEX4 and LIKE SEX FOUR2 (LSF2) were repressed using RNAi constructs. Although repression of either SEX4 or LSF2 inhibited leaf starch degradation, it had no effect on cold-induced sweetening in tubers. Starch amounts were unchanged in the tubers, but the amount of phosphate bound to the starch was significantly increased in all the lines, with phosphate bound at the C6 position of the glucosyl units increased in lines repressed in StSEX4 and in the C3 position in lines repressed in StLSF2 expression. This was accompanied by a reduction in starch granule size and an alteration in the constituent glucan chain lengths within the starch molecule, although no obvious alteration in granule morphology was observed. Starch from the transgenic lines contained fewer chains with a degree of polymerization (DP) of less than 17 and more with a DP between 17 and 38. There were also changes in the physical properties of the starches. Rapid viscoanalysis demonstrated that both the holding strength and the final viscosity of the high phosphate starches were increased indicating that the starches have increased swelling power due to an enhanced capacity for hydration.

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.

Leaf Starch Turnover Occurs in Long Days and in Falling Light at the End of the Day.

We investigated whether starch degradation occurs at the same time as starch synthesis in Arabidopsis (Arabidopsis thaliana) leaves in the light. Starch accumulated in a linear fashion for about 12 h after dawn, then accumulation slowed and content plateaued. Following decreases in light intensity, the rate of accumulation of starch declined in proportion to the decline in photosynthesis if the decrease occurred <10 h after dawn, but accumulation ceased or loss of starch occurred if the same decrease in light intensity was imposed more than 10 h after dawn. These changes in starch accumulation patterns after prolonged periods in the light occurred at both high and low starch contents and were not related to time-dependent changes in either the rate of photosynthesis or the partitioning of assimilate between starch and Suc, as assessed from metabolite measurements and 14CO2 pulse experiments. Instead, measurements of incorporation of 13C from 13CO2 into starch and of levels of the starch degradation product maltose showed that substantial starch degradation occurred simultaneously with synthesis at time points >14 h after dawn and in response to decreases in light intensity that occurred >10 h after dawn. Starch measurements in circadian clock mutants suggested that the clock influences the timing of onset of degradation. We conclude that the propensity for leaf starch to be degraded increases with time after dawn. The importance of this phenomenon for efficient use of carbon for growth in long days and for prevention of starvation during twilight is discussed.

Analysis of genes involved in glycogen degradation in Escherichia coli.

Escherichia coli accumulate or degrade glycogen depending on environmental carbon supply. Glycogen phosphorylase (GlgP) and glycogen debranching enzyme (GlgX) are known to act on the glycogen polymer, while maltodextrin phosphorylase (MalP) is thought to remove maltodextrins released by GlgX. To examine the roles of these enzymes in more detail, single, double and triple mutants lacking all their activities were produced. GlgX and GlgP were shown to act directly on the glycogen polymer, while MalP most likely catabolised soluble malto-oligosaccharides. Interestingly, analysis of a triple mutant lacking all three enzymes indicates the presence of another enzyme that can release maltodextrins from glycogen.

A Pragmatic Approach to Getting Published: 35 Tips for Early Career Researchers.

It is trite to say "publish or perish," yet many early career researchers are often at a loss on how to best get their work published. With strong competition and many manuscripts submitted, it is difficult to convince editors and reviewers to opt for acceptance. A pragmatic approach to publishing may increase one's odds of success. Here, we - a group of postdocs in the field of plant science - present specific recommendations for early career scientists on advanced levels. We cannot provide a recipe-like set of instructions with success guaranteed, but we come from a broad background in plant science, with experience publishing in a number of journals of varying topics and impact factors. We provide tips, tricks, and tools for collaboration, journal selection, and achieving acceptance.

Diverse Exopolysaccharide Producing Bacteria Isolated from Milled Sugarcane: Implications for Cane Spoilage and Sucrose Yield.

Bacterial deterioration of sugarcane during harvesting and processing is correlated with significant loss of sucrose yield and the accumulation of bacterial polysaccharides. Dextran, a homoglucan produced by Leuconostoc mesenteroides, has been cited as the primary polysaccharide associated with sugarcane deterioration. A culture-based approach was used to isolate extracellular polysaccharide (EPS) producing bacterial strains from milled sugarcane stalks. Ribosomal RNA sequencing analysis grouped 25 isolates into 4 genera. This study identified 2 bacterial genera not previously associated with EPS production or sucrose degradation. All isolates produced polysaccharide when grown in the presence of sucrose. Monosaccharide analysis of purified polymers by Gas Chromatography revealed 17 EPSs consisting solely of glucose (homoglucans), while the remainder contained traces of mannose or fructose. Dextranase treatment of polysaccharides yielded full digestion profiles for only 11 extracts. Incomplete hydrolysis profiles of the remaining polysaccharides suggest the release of longer oligosaccharides which may interfere with sucrose crystal formation.

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.

Manipulation of L-ascorbic acid biosynthesis pathways in Solanum lycopersicum: elevated GDP-mannose pyrophosphorylase activity enhances L-ascorbate levels in red fruit.

Ascorbate (AsA) plays a fundamental role in redox homeostasis in plants and animals, primarily by scavenging reactive oxygen species. Three genes, representing diverse steps putatively involved in plant AsA biosynthesis pathways, were cloned and independently expressed in Solanum lycopersicum (tomato) under the control of the CaMV 35S promoter. Yeast-derived GDP-mannose pyrophosphorylase (GMPase) and arabinono-1,4-lactone oxidase (ALO), as well as myo-inositol oxygenase 2 (MIOX2) from Arabidopsis thaliana, were targeted. Increases in GMPase activity were concomitant with increased AsA levels of up to 70% in leaves, 50% in green fruit, and 35% in red fruit. Expression of ALO significantly pulled biosynthetic flux towards AsA in leaves and green fruit by up to 54 and 25%, respectively. Changes in AsA content in plants transcribing the MIOX2 gene were inconsistent in different tissue. On the other hand, MIOX activity was strongly correlated with cell wall uronic acid levels, suggesting that MIOX may be a useful tool for the manipulation of cell wall composition. In conclusion, the Smirnoff-Wheeler pathway showed great promise as a target for biotechnological manipulation of ascorbate levels in tomato.

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.