Starch Turnover Research Articles

β-amylase 1 (BAM1) degrades transitory starch to sustain proline biosynthesis during drought stress.

During photosynthesis of higher plants, absorbed light energy is converted into chemical energy that, in part, is accumulated in the form of transitory starch within chloroplasts. In the following night, transitory starch is mobilized to sustain the heterotrophic metabolism of the plant. β-amylases are glucan hydrolases that cleave α-1,4-glycosidic bonds of starch and release maltose units from the non-reducing end of the polysaccharide chain. In Arabidopsis, nocturnal degradation of transitory starch involves mainly β-amylase-3 (BAM3). A second β-amylase isoform, β-amylase-1 (BAM1), is involved in diurnal starch degradation in guard cells, a process that sustains stomata opening. However, BAM1 also contributes to diurnal starch turnover in mesophyll cells under osmotic stress. With the aim of dissecting the role of β-amylases in osmotic stress responses in Arabidopsis, mutant plants lacking either BAM1 or BAM3 were subject to a mild (150mM mannitol) and prolonged (up to one week) osmotic stress. We show here that leaves of osmotically-stressed bam1 plants accumulated more starch and fewer soluble sugars than both wild-type and bam3 plants during the day. Moreover, bam1 mutants were impaired in proline accumulation and suffered from stronger lipid peroxidation, compared with both wild-type and bam3 plants. Taken together, these data strongly suggest that carbon skeletons deriving from BAM1 diurnal degradation of transitory starch support the biosynthesis of proline required to face the osmotic stress. We propose the transitory-starch/proline interplay as an interesting trait to be tackled by breeding technologies aimingto improve drought tolerance in relevant crops.

Blue Light Induces a Distinct Starch Degradation Pathway in Guard Cells for Stomatal Opening

Stomatal pores form a crucial interface between the leaf mesophyll and the atmosphere, controlling water and carbon balance in plants [1]. Major advances have been made in understanding the regulatory networks and ion fluxes in the guard cells surrounding the stomatal pore [2]. However, our knowledge on the role of carbon metabolism in these cells is still fragmentary [3-5]. In particular, the contribution of starch in stomatal opening remains elusive [6]. Here, we used Arabidopsis thaliana as a model plant to provide the first quantitative analysis of starch turnover in guard cells of intact leaves during the diurnal cycle. Starch is present in guard cells at the end of night, unlike in the rest of the leaf, but is rapidly degraded within 30 min of light. This process is critical for the rapidity of stomatal opening and biomass production. We exploited Arabidopsis molecular genetics to define the mechanism and regulation of guard cell starch metabolism, showing it to be mediated by a previously uncharacterized pathway. This involves the synergistic action of β-amylase 1 (BAM1) and α-amylase 3 (AMY3)-enzymes that are normally not required for nighttime starch degradation in other leaf tissues. This pathway is under the control of the phototropin-dependent blue-light signaling cascade and correlated with the activity of the plasma membrane H(+)-ATPase. Our results show that guard cell starch degradation has an important role in plant growth by driving stomatal responses to light.

ß-amylase1 mutant Arabidopsis plants show improved drought tolerance due to reduced starch breakdown in guard cells.

In plants, drought stress is a major growth limiting factor causing cell water loss through open stomata. In this study, guard cell-specific transcripts from drought-stressed Arabidopsis plants were analysed and a down-regulation of β-amylase 1 (BAM1) was found. In previous studies, BAM1 was shown to be involved in stomatal starch degradation under ambient conditions. Impaired starch breakdown of bam1 mutant plants was accompanied by decreased stomatal opening. Here, it is shown that drought tolerance of bam1 mutant plants is improved as compared with wild-type controls. Microarray analysis of stomata-specific transcripts from bam1 mutant plants revealed a significant down-regulation of genes encoding aquaporins, auxin- and ethylene-responsive factors, and cell-wall modifying enzymes. This expression pattern suggests that reduced water uptake and limited cell wall extension are associated with the closed state of stomata of bam1 mutant plants. Together these data suggest that regulation of stomata-specific starch turnover is important for adapting stomata opening to environmental needs and its breeding manipulation may result in drought tolerant crop plants.

Improvement of enzymatic saccharification yield in Arabidopsis thaliana by ectopic expression of the rice SUB1A-1 transcription factor.

Saccharification of polysaccharides releases monosaccharides that can be used by ethanol-producing microorganisms in biofuel production. To improve plant biomass as a raw material for saccharification, factors controlling the accumulation and structure of carbohydrates must be identified. Rice SUB1A-1 is a transcription factor that represses the turnover of starch and postpones energy-consuming growth processes under submergence stress. Arabidopsis was employed to test if heterologous expression of SUB1A-1 or SUB1C-1 (a related gene) can be used to improve saccharification. Cellulolytic and amylolytic enzymatic treatments confirmed that SUB1A-1 transgenics had better saccharification yield than wild-type (Col-0), mainly from accumulated starch. This improved saccharification yield was developmentally controlled; when compared to Col-0, young transgenic vegetative plants yielded 200–300% more glucose, adult vegetative plants yielded 40–90% more glucose and plants in reproductive stage had no difference in yield. We measured photosynthetic parameters, starch granule microstructure, and transcript abundance of genes involved in starch degradation (SEX4, GWD1), juvenile transition (SPL3-5) and meristematic identity (FUL, SOC1) but found no differences to Col-0, indicating that starch accumulation may be controlled by down-regulation of CONSTANS and FLOWERING LOCUS T by SUB1A-1 as previously reported. SUB1A-1 transgenics also offered less resistance to deformation than wild-type concomitant to up-regulation of AtEXP2 expansin and BGL2 glucan-1,3,-beta-glucosidase. We conclude that heterologous SUB1A-1 expression can improve saccharification yield and softness, two traits needed in bioethanol production.

Profiling of spatial metabolite distributions in wheat leaves under normal and nitrate limiting conditions

The control and interaction between nitrogen and carbon assimilatory pathways is essential in both photosynthetic and non-photosynthetic tissue in order to support metabolic processes without compromising growth. Physiological differences between the basal and mature region of wheat (Triticum aestivum) primary leaves confirmed that there was a change from heterotrophic to autotrophic metabolism. Fourier Transform Infrared (FT-IR) spectroscopy confirmed the suitability and phenotypic reproducibility of the leaf growth conditions. Principal Component–Discriminant Function Analysis (PC–DFA) revealed distinct clustering between base, and tip sections of the developing wheat leaf, and from plants grown in the presence or absence of nitrate. Gas Chromatography-Time of Flight/Mass Spectrometry (GC-TOF/MS) combined with multivariate and univariate analyses, and Bayesian network (BN) analysis, distinguished different tissues and confirmed the physiological switch from high rates of respiration to photosynthesis along the leaf. The operation of nitrogen metabolism impacted on the levels and distribution of amino acids, organic acids and carbohydrates within the wheat leaf. In plants grown in the presence of nitrate there was reduced levels of a number of sugar metabolites in the leaf base and an increase in maltose levels, possibly reflecting an increase in starch turnover. The value of using this combined metabolomics analysis for further functional investigations in the future are discussed.

Loss of cytosolic phosphoglucose isomerase affects carbohydrate metabolism in leaves and is essential for fertility of Arabidopsis.

Carbohydrate metabolism in plants is tightly linked to photosynthesis and is essential for energy and carbon skeleton supply of the entire organism. Thus, the hexose phosphate pools of the cytosol and the chloroplast represent important metabolic resources that are maintained through action of phosphoglucose isomerase (PGI) and phosphoglucose mutase interconverting glucose 6-phosphate, fructose 6-phosphate, and glucose 1-phosphate. Here, we investigated the impact of disrupted cytosolic PGI (cPGI) function on plant viability and metabolism. Overexpressing an artificial microRNA targeted against cPGI (amiR-cpgi) resulted in adult plants with vegetative tissue essentially free of cPGI activity. These plants displayed diminished growth compared with the wild type and accumulated excess starch in chloroplasts but maintained low sucrose content in leaves at the end of the night. Moreover, amiR-cpgi plants exhibited increased nonphotochemical chlorophyll a quenching during photosynthesis. In contrast to amiR-cpgi plants, viable transfer DNA insertion mutants disrupted in cPGI function could only be identified as heterozygous individuals. However, homozygous transfer DNA insertion mutants could be isolated among plants ectopically expressing cPGI. Intriguingly, these plants were only fertile when expression was driven by the ubiquitin10 promoter but sterile when the seed-specific unknown seed protein promoter or the Cauliflower mosaic virus 35S promoter were employed. These data show that metabolism is apparently able to compensate for missing cPGI activity in adult amiR-cpgi plants and indicate an essential function for cPGI in plant reproduction. Moreover, our data suggest a feedback regulation in amiR-cpgi plants that fine-tunes cytosolic sucrose metabolism with plastidic starch turnover.

Relationship between starch degradation and carbon demand for maintenance and growth in Arabidopsis thaliana in different irradiance and temperature regimes.

Experiments were designed to compare the relationship between starch degradation and the use of carbon for maintenance and growth in Arabidopsis in source-limited and sink-limited conditions. It is known that starch degradation is regulated by the clock in source-limited plants, which degrade their starch in a linear manner such that it is almost but not completely exhausted at dawn. We asked whether this response is maintained under an extreme carbon deficit. Arabidopsis was subjected to a sudden combination of a day of low irradiance, to decrease starch at dusk, and a warm night. Starch was degraded in a linear manner through the night, even though the plants became acutely carbon starved. We conclude that starch degradation is not increased to meet demand in carbon-limited plants. This network property will allow stringent control of starch turnover in a fluctuating environment. In contrast, in sink-limited plants, which do not completely mobilize their starch during the night, starch degradation was accelerated in warm nights to meet the increased demand for maintenance and growth. Across all conditions, the rate of growth at night depends on the rate of starch degradation, whereas the rate of maintenance respiration decreases only when starch degradation is very slow.

Functions of autophagy in plant carbon and nitrogen metabolism.

Carbon and nitrogen are essential components for plant growth. Although models of plant carbon and nitrogen metabolisms have long been established, certain gaps remain unfilled, such as how plants are able to maintain a flexible nocturnal starch turnover capacity over various light cycles, or how nitrogen remobilization is achieved during the reproductive growth stage. Recent advances in plant autophagy have shed light on such questions. Not only does autophagy contribute to starch degradation at night, but it participates in the degradation of chloroplast proteins and even chloroplasts after prolonged carbon starvation, thus help maintain the free amino acid pool and provide substrate for respiration. The induction of autophagy under these conditions may involve transcriptional regulation. Large-scale transcriptome analyses revealed that ATG8e belongs to a core carbon signaling response shared by Arabidopsis accessions, and the transcription of Arabidopsis ATG7 is tightly co-regulated with genes functioning in chlorophyll degradation and leaf senescence. In the reproductive phase, autophagy is essential for bulk degradation of leaf proteins, thus contributes to nitrogen use efficiency (NUE) both under normal and low-nitrogen conditions.

Phosphorylation of transitory starch by α‐glucan, water dikinase during starch turnover affects the surface properties and morphology of starch granules

Glucan, water dikinase (GWD) is a key enzyme of starch metabolism but the physico-chemical properties of starches isolated from GWD-deficient plants and their implications for starch metabolism have so far not been described. Transgenic Arabidopsis thaliana plants with reduced or no GWD activity were used to investigate the properties of starch granules. In addition, using various in vitro assays, the action of recombinant GWD, β-amylase, isoamylase and starch synthase 1 on the surface of native starch granules was analysed. The internal structure of granules isolated from GWD mutant plants is unaffected, as thermal stability, allomorph, chain length distribution and density of starch granules were similar to wild-type. However, short glucan chain residues located at the granule surface dominate in starches of transgenic plants and impede GWD activity. A similarly reduced rate of phosphorylation by GWD was also observed in potato tuber starch fractions that differ in the proportion of accessible glucan chain residues at the granule surface. A model is proposed to explain the characteristic morphology of starch granules observed in GWD transgenic plants. The model postulates that the occupancy rate of single glucan chains at the granule surface limits accessibility to starch-related enzymes.

Regulatory principles and experimental approaches to the circadian control of starch turnover

In many plants, starch is synthesized during the day and degraded during the night to avoid carbohydrate starvation in darkness. The circadian clock participates in a dynamic adjustment of starch turnover to changing environmental condition through unknown mechanisms. We used mathematical modelling to explore the possible scenarios for the control of starch turnover by the molecular components of the plant circadian clock. Several classes of plausible models were capable of describing the starch dynamics observed in a range of clock mutant plants and light conditions, including discriminating circadian protocols. Three example models of these classes are studied in detail, differing in several important ways. First, the clock components directly responsible for regulating starch degradation are different in each model. Second, the intermediate species in the pathway may play either an activating or inhibiting role on starch degradation. Third, the system may include a light-dependent interaction between the clock and downstream processes. Finally, the clock may be involved in the regulation of starch synthesis. We discuss the differences among the models’ predictions for diel starch profiles and the properties of the circadian regulators. These suggest additional experiments to elucidate the pathway structure, avoid confounding results and identify the molecular components involved.

Adjustment of carbon fluxes to light conditions regulates the daily turnover of starch in plants: a computational model

In the light, photosynthesis provides carbon for metabolism and growth. In the dark, plant growth depends on carbon reserves that were accumulated during previous light periods. Many plants accumulate part of their newly-fixed carbon as starch in their leaves in the day and remobilise it to support metabolism and growth at night. The daily rhythms of starch accumulation and degradation are dynamically adjusted to the changing light conditions such that starch is almost but not totally exhausted at dawn. This requires the allocation of a larger proportion of the newly fixed carbon to starch under low carbon conditions, and the use of information about the carbon status at the end of the light period and the length of the night to pace the rate of starch degradation. This regulation occurs in a circadian clock-dependent manner, through unknown mechanisms. We use mathematical modelling to explore possible diurnal mechanisms regulating the starch level. Our model combines the main reactions of carbon fixation, starch and sucrose synthesis, starch degradation and consumption of carbon by sink tissues. To describe the dynamic adjustment of starch to daily conditions, we introduce diurnal regulators of carbon fluxes, which modulate the activities of the key steps of starch metabolism. The sensing of the diurnal conditions is mediated in our model by the timer α and the "dark sensor"β, which integrate daily information about the light conditions and time of the day through the circadian clock. Our data identify the β subunit of SnRK1 kinase as a good candidate for the role of the dark-accumulated component β of our model. The developed novel approach for understanding starch kinetics through diurnal metabolic and circadian sensors allowed us to explain starch time-courses in plants and predict the kinetics of the proposed diurnal regulators under various genetic and environmental perturbations.

Metabolic analysis of kiwifruit (Actinidia deliciosa) berries from extreme genotypes reveals hallmarks for fruit starch metabolism.

Tomato, melon, grape, peach, and strawberry primarily accumulate soluble sugars during fruit development. In contrast, kiwifruit (Actinidia Lindl. spp.) and banana store a large amount of starch that is released as soluble sugars only after the fruit has reached maturity. By integrating metabolites measured by gas chromatography–mass spectrometry, enzyme activities measured by a robot-based platform, and transcript data sets during fruit development of Actinidia deliciosa genotypes contrasting in starch concentration and size, this study identified the metabolic changes occurring during kiwifruit development, including the metabolic hallmarks of starch accumulation and turnover. At cell division, a rise in glucose (Glc) concentration was associated with neutral invertase (NI) activity, and the decline of both Glc and NI activity defined the transition to the cell expansion and starch accumulation phase. The high transcript levels of β-amylase 9 (BAM9) during cell division, prior to net starch accumulation, and the correlation between sucrose phosphate synthase (SPS) activity and sucrose suggest the occurrence of sucrose cycling and starch turnover. ADP-Glc pyrophosphorylase (AGPase) is identified as a key enzyme for starch accumulation in kiwifruit berries, as high-starch genotypes had 2- to 5-fold higher AGPase activity, which was maintained over a longer period of time and was also associated with enhanced and extended transcription of the AGPase large subunit 4 (APL4). The data also revealed that SPS and galactinol might affect kiwifruit starch accumulation, and suggest that phloem unloading into kiwifruit is symplastic. These results are relevant to the genetic improvement of quality traits such as sweetness and sugar/acid balance in a range of fruit species.

Feedback inhibition of starch degradation in Arabidopsis leaves mediated by trehalose 6-phosphate.

Many plants accumulate substantial starch reserves in their leaves during the day and remobilize them at night to provide carbon and energy for maintenance and growth. In this paper, we explore the role of a sugar-signaling metabolite, trehalose-6-phosphate (Tre6P), in regulating the accumulation and turnover of transitory starch in Arabidopsis (Arabidopsis thaliana) leaves. Ethanol-induced overexpression of trehalose-phosphate synthase during the day increased Tre6P levels up to 11-fold. There was a transient increase in the rate of starch accumulation in the middle of the day, but this was not linked to reductive activation of ADP-glucose pyrophosphorylase. A 2- to 3-fold increase in Tre6P during the night led to significant inhibition of starch degradation. Maltose and maltotriose did not accumulate, suggesting that Tre6P affects an early step in the pathway of starch degradation in the chloroplasts. Starch granules isolated from induced plants had a higher orthophosphate content than granules from noninduced control plants, consistent either with disruption of the phosphorylation-dephosphorylation cycle that is essential for efficient starch breakdown or with inhibition of starch hydrolysis by β-amylase. Nonaqueous fractionation of leaves showed that Tre6P is predominantly located in the cytosol, with estimated in vivo Tre6P concentrations of 4 to 7 µm in the cytosol, 0.2 to 0.5 µm in the chloroplasts, and 0.05 µm in the vacuole. It is proposed that Tre6P is a component in a signaling pathway that mediates the feedback regulation of starch breakdown by sucrose, potentially linking starch turnover to demand for sucrose by growing sink organs at night.

Starch synthase 4 is essential for coordination of starch granule formation with chloroplast division during Arabidopsis leaf expansion

Arabidopsis thaliana mutants lacking the SS4 isoform of starch synthase have strongly reduced numbers of starch granules per chloroplast, suggesting that SS4 is necessary for the normal generation of starch granules. To establish whether it plays a direct role in this process, we investigated the circumstances in which granules are formed in ss4 mutants.Starch granule numbers and distribution and the accumulation of starch synthase substrates and products were investigated during ss4 leaf development, and in ss4 mutants carrying mutations or transgenes that affect starch turnover or chloroplast volume.We found that immature ss4 leaves have no starch granules, but accumulate high concentrations of the starch synthase substrate ADPglucose. Granule numbers are partially restored by elevating the capacity for glucan synthesis (via expression of bacterial glycogen synthase) or by increasing the volumes of individual chloroplasts (via introduction of arc mutations). However, these granules are abnormal in distribution, size and shape.SS4 is an essential component of a mechanism that coordinates granule formation with chloroplast division during leaf expansion and determines the abundance and the flattened, discoid shape of leaf starch granules.

Reduced Carbohydrate Availability Enhances the Susceptibility of Arabidopsis toward Colletotrichum higginsianum

Colletotrichum higginsianum is a hemibiotrophic ascomycete fungus that is adapted to Arabidopsis (Arabidopsis thaliana). After breaching the host surface, the fungus establishes an initial biotrophic phase in the penetrated epidermis cell, before necrotrophic growth is initiated upon further host colonization. We observed that partitioning of major leaf carbohydrates was shifted in favor of sucrose and at the expense of starch during necrotrophic fungal growth. Arabidopsis mutants with impaired starch turnover were more susceptible toward C. higginsianum infection, exhibiting a strong negative correlation between diurnal carbohydrate accumulation and fungal proliferation for the tested genotypes. By altering the length of the light phase and employing additional genotypes impaired in nocturnal carbon mobilization, we revealed that reduced availability of carbon enhances susceptibility in the investigated pathosystem. Systematic starvation experiments resulted in two important findings. First, we showed that carbohydrate supply by the host is dispensable during biotrophic growth of C. higginsianum, while carbon deficiency was most harmful to the host during the necrotrophic colonization phase. Compared with the wild type, the increases in the total salicylic acid pool and camalexin accumulation were reduced in starch-free mutants at late interaction stages, while an increased ratio of free to total salicylic acid did not convey elevated pathogenesis-related gene expression in starch-free mutants. These observations suggest that reduced carbon availability dampens induced defense responses. In contrast, starch-free mutants were more resistant toward the fungal biotroph Erysiphe cruciferarum, indicating that reduced carbohydrate availability influences susceptibility differently in the interaction with the investigated hemibiotrophic and biotrophic fungal pathogens.

A dual role of tobacco hexokinase 1 in primary metabolism and sugar sensing

Hexokinase (HXK) is present in all virtually living organisms and is central to carbohydrate metabolism catalysing the ATP-dependent phosphorylation of hexoses. In plants, HXKs are supposed to act as sugar sensors and/or to interact with other enzymes directly supplying metabolic pathways such as glycolysis, the nucleotide phosphate monosaccharide (NDP-glucose) pathway and the pentose phosphate pathway. We identified nine members of the tobacco HXK gene family and observed that among RNAi lines of these nine NtHXKs, only RNAi lines of NtHXK1 showed an altered phenotype, namely stunted growth and leaf chlorosis. NtHXK1 was also the isoform with highest relative expression levels among all NtHXKs. GFP-tagging and immunolocalization indicated that NtHXK1 is associated with mitochondrial membranes. Overexpression of NtHXK1 resulted in elevated glucose phosphorylation activity in leaf extracts or chloroplasts. Moreover, NtHXK1 was able to complement the glucose-insensitive Arabidopsis mutant gin2-1 suggesting that NtHXK1 can take over glucose sensing functions. RNAi lines of NtHXK1 showed severely damaged leaf and chloroplast structure, coinciding with an excess accumulation of starch. We conclude that NtHXK1 is not only essential for maintaining glycolytic activity during respiration but also for regulating starch turnover, especially during the night.

Fruit production in three masting tree species does not rely on stored carbon reserves

Fruiting is typically considered to massively burden the seasonal carbon budget of trees. The cost of reproduction has therefore been suggested as a proximate factor explaining observed mast-fruiting patterns. Here, we used a large-scale, continuous (13)C labeling of mature, deciduous trees in a temperate Swiss forest to investigate to what extent fruit formation in three species with masting reproduction behavior (Carpinus betulus, Fagus sylvatica, Quercus petraea) relies on the import of stored carbon reserves. Using a free-air CO2 enrichment system, we exposed trees to (13)C-depleted CO2 during 8 consecutive years. By the end of this experiment, carbon reserve pools had significantly lower δ(13)C values compared to control trees. δ(13)C analysis of new biomass during the first season after termination of the CO2 enrichment allowed us to distinguish the sources of built-in carbon (old carbon reserves vs. current assimilates). Flowers and expanding leaves carried a significant (13)C label from old carbon stores. In contrast, fruits and vegetative infructescence tissues were exclusively produced from current, unlabeled photoassimilates in all three species, including F. sylvatica, which had a strong masting season. Analyses of δ(13)C in purified starch from xylem of fruit-bearing shoots revealed a complete turn-over of starch during the season, likely due to its usage for bud break. This study is the first to directly demonstrate that fruiting is independent from old carbon reserves in masting trees, with significant implications for mechanistic models that explain mast seeding.

Altered Starch Turnover in the Maternal Plant Has Major Effects on Arabidopsis Fruit Growth and Seed Composition

Mature seeds of both the high-starch starch-excess1 (sex1) mutant and the almost starchless phosphoglucomutase1 mutant of Arabidopsis (Arabidopsis thaliana) have 30% to 40% less lipid than seeds of wild-type plants. We show that this is a maternal effect and is not attributable to the defects in starch metabolism in the embryo itself. Low lipid contents and consequent slow postgerminative growth are seen only in mutant embryos that develop on maternal plants with mutant phenotypes. Mutant embryos that develop on plants with wild-type starch metabolism have wild-type lipid contents and postgerminative growth. The maternal effect on seed lipid content is attributable to carbohydrate starvation in the mutant fruit at night. Fruits on sex1 plants grow more slowly than those on wild-type plants, particularly at night, and have low sugars and elevated expression of starvation genes at night. Transcript levels of the transcription factor WRINKLED1, implicated in lipid synthesis, are reduced at night in sex1 but not in wild-type seeds, and so are transcript levels of key enzymes of glycolysis and fatty acid synthesis. sex1 embryos develop more slowly than wild-type embryos. We conclude that the reduced capacity of mutant plants to convert starch to sugars in leaves at night results in low nighttime carbohydrate availability in the developing fruit. This in turn reduces the rate of development and expression of genes encoding enzymes of storage product accumulation in the embryo. Thus, the supply of carbohydrate from the maternal plant to the developing fruit at night can have an important influence on oilseed composition and on postgerminative growth.

Inactivation of thioredoxin f1 leads to decreased light activation of ADP‐glucose pyrophosphorylase and altered diurnal starch turnover in leaves of Arabidopsis plants

Chloroplast thioredoxin f (Trx f) is an important regulator of primary metabolic enzymes. However, genetic evidence for its physiological importance is largely lacking. To test the functional significance of Trx f in vivo, Arabidopsis mutants with insertions in the trx f1 gene were studied, showing a drastic decrease in Trx f leaf content. Knockout of Trx f1 led to strong attenuation in reductive light activation of ADP-glucose pyrophosphorylase (AGPase), the key enzyme of starch synthesis, in leaves during the day and in isolated chloroplasts, while sucrose-dependent redox activation of AGPase in darkened leaves was not affected. The decrease in light-activation of AGPase in leaves was accompanied by a decrease in starch accumulation, an increase in sucrose levels and a decrease in starch-to-sucrose ratio. Analysis of metabolite levels at the end of day shows that inhibition of starch synthesis was unlikely due to shortage of substrates or changes in allosteric effectors. Metabolite profiling by gas chromatography-mass spectrometry pinpoints only a small number of metabolites affected, including sugars, organic acids and ethanolamine. Interestingly, metabolite data indicate carbon shortage in trx f1 mutant leaves at the end of night. Overall, results provide in planta evidence for the role played by Trx f in the light activation of AGPase and photosynthetic carbon partitioning in plants.

Metabolism and growth in Arabidopsis depend on the daytime temperature but are temperature-compensated against cool nights.

Diurnal cycles provide a tractable system to study the response of metabolism and growth to fluctuating temperatures. We reasoned that the response to daytime and night temperature may vary; while daytime temperature affects photosynthesis, night temperature affects use of carbon that was accumulated in the light. Three Arabidopsis thaliana accessions were grown in thermocycles under carbon-limiting conditions with different daytime or night temperatures (12 to 24 °C) and analyzed for biomass, photosynthesis, respiration, enzyme activities, protein levels, and metabolite levels. The data were used to model carbon allocation and growth rates in the light and dark. Low daytime temperature led to an inhibition of photosynthesis and an even larger inhibition of growth. The inhibition of photosynthesis was partly ameliorated by a general increase in protein content. Low night temperature had no effect on protein content, starch turnover, or growth. In a warm night, there is excess capacity for carbon use. We propose that use of this capacity is restricted by feedback inhibition, which is relaxed at lower night temperature, thus buffering growth against fluctuations in night temperature. As examples, the rate of starch degradation is completely temperature compensated against even sudden changes in temperature, and polysome loading increases when the night temperature is decreased.