Sugar-responsive Genes Research Articles

OsbZIP38/87-mediated activation of OsHXK7 improves the viability of rice cells under hypoxic conditions

Maintenance of energy metabolism is critical for rice (Oryza sativa) tolerance under submerged cultivation. Here, OsHXK7 was the most actively induced hexokinase gene in the embryos of hypoxically germinating rice seeds. Suspension-cultured cells established from seeds of T-DNA null mutants for the OsHXK7 locus did not regrow after 3-d-hypoxic stress and showed increased susceptibility to low-oxygen stress—in terms of viability—and decreased alcoholic fermentation activities compared to those of the wild-type. The promoter element containing the TGACG-motif, a well-known target site for the basic leucine zipper (bZIP) transcription factors, was responsible for sugar regulation of the OsHXK7 promoter activity. Systematic screening of the OsbZIP genes showing the similar expression patterns to that of OsHXK7 in the transcriptomic datasets produced two bZIP genes, OsbZIP38 and 87, belonging to the S1 bZIP subfamily as the candidate for the activator for this gene expression. Gain- and loss-of-function experiments through transient expression assays have demonstrated that these two bZIP proteins are indeed involved in the induction of OsHXK7 expression under starvation or low-energy conditions. Our finding suggests that C/S1 bZIP network-mediated hypoxic deregulation of sugar-responsive genes may work in concert for the molecular adaptation of rice cells to submergence.

Arabidopsis ACT DOMAIN REPEAT9 represses glucose signaling pathways.

Nutrient sensing and signaling are critical for plants to coordinate growth and development in response to nutrient availability. Plant ACT DOMAIN REPEAT (ACR) proteins have been proposed to serve as nutrient sensors, but their functions remain largely unknown. Here, we showed that Arabidopsis (Arabidopsis thaliana) ACR9 might function as a repressor in glucose (Glc) signaling pathways. ACR9 was highly expressed in the leaves, and its expression was downregulated by sugars. Interestingly, the acr9-1 and acr9-2 T-DNA insertion mutants were hypersensitive to Glc during seedling growth, development, and anthocyanin accumulation. Nitrogen deficiency increased the mutants' sensitivity to Glc. The expression of sugar-responsive genes was also significantly enhanced in the acr9 mutants. By contrast, the 35S:ACR9 and 35S:ACR9-GFP overexpression (OE) lines were insensitive to Glc during early seedling development. The Glc signaling pathway is known to interact with the plant hormone abscisic acid (ABA). Notably, the acr9 mutants were also hypersensitive to ABA during early seedling development. The Glc sensor HEXOKINASE1 (HXK1) and the energy sensor SUCROSE NON-FERMENTING1 (SNF1)-RELATED PROTEIN KINASE1 (SnRK1) are key components of the Glc signaling pathways. The acr9-1/hxk1-3 and acr9-1/snrk1 double mutants were no longer hypersensitive to Glc, indicating that functional HXK1 and SnRK1 were required for the acr9-1 mutant to be hypersensitive to Glc. Together, these results suggest that ACR9 is a repressor of the Glc signaling pathway, which may act independently or upstream of the HXK1-SnRK1 signaling module.

Triose phosphate export from chloroplasts and cellular sugar content regulate anthocyanin biosynthesis during high light acclimation

Plants have evolved multiple strategies to cope with rapid changes in the environment. During high light (HL) acclimation, the biosynthesis of photoprotective flavonoids, such as anthocyanins, is induced. However, the exact nature of the signal and downstream factors for HL induction of flavonoid biosynthesis (FB) is still under debate. Here, we show that carbon fixation in chloroplasts, subsequent export of photosynthates by triose phosphate/phosphate translocator (TPT), and rapid increase in cellular sugar content permit the transcriptional and metabolic activation of anthocyanin biosynthesis during HL acclimation. In combination with genetic and physiological analysis, targeted and whole-transcriptome gene expression studies suggest that reactive oxygen species and phytohormones play only a minor role in rapid HL induction of the anthocyanin branch of FB. In addition to transcripts of FB, sugar-responsive genes showed delayed repression or induction in tpt-2 during HL treatment, and a significant overlap with transcripts regulated by SNF1-related protein kinase 1 (SnRK1) was observed, including a central transcription factor of FB. Analysis of mutants with increased and repressed SnRK1 activity suggests that sugar-induced inactivation of SnRK1 is required for HL-mediated activation of anthocyanin biosynthesis. Our study emphasizes the central role of chloroplasts as sensors for environmental changes as well as the vital function of sugar signaling in plant acclimation.

Metabolic gene regulation by Drosophila GATA transcription factor Grain.

Nutrient-dependent gene regulation critically contributes to homeostatic control of animal physiology in changing nutrient landscape. In Drosophila, dietary sugars activate transcription factors (TFs), such as Mondo-Mlx, Sugarbabe and Cabut, which control metabolic gene expression to mediate physiological adaptation to high sugar diet. TFs that correspondingly control sugar responsive metabolic genes under conditions of low dietary sugar remain, however, poorly understood. Here we identify a role for Drosophila GATA TF Grain in metabolic gene regulation under both low and high sugar conditions. De novo motif prediction uncovered a significant over-representation of GATA-like motifs on the promoters of sugar-activated genes in Drosophila larvae, which are regulated by Grain, the fly ortholog of GATA1/2/3 subfamily. grain expression is activated by sugar in Mondo-Mlx-dependent manner and it contributes to sugar-responsive gene expression in the fat body. On the other hand, grain displays strong constitutive expression in the anterior midgut, where it drives lipogenic gene expression also under low sugar conditions. Consistently with these differential tissue-specific roles, Grain deficient larvae display delayed development on high sugar diet, while showing deregulated central carbon and lipid metabolism primarily on low sugar diet. Collectively, our study provides evidence for the role of a metazoan GATA transcription factor in nutrient-responsive metabolic gene regulation in vivo.

WRKY18 and WRKY53 Coordinate with HISTONE ACETYLTRANSFERASE1 to Regulate Rapid Responses to Sugar.

Sugars provide a source of energy; they also function as signaling molecules that regulate gene expression, affect metabolism, and alter growth in plants. Rapid responses to sugar signaling and metabolism are essential for optimal growth and fitness, but the regulatory mechanisms underlying these are largely unknown. In this study, we found that the rapid induction of sugar responses in Arabidopsis (Arabidopsis thaliana) requires the W-box cis-elements in the promoter region of GLC 6-PHOSPHATE/PHOSPHATE TRANSLOCATOR2, a well-studied sugar response marker gene. The transcription factors WRKY18 and WRKY53 directly bind to the W-Box cis-elements in the promoter region of sugar response genes and activate their expression. In addition, HISTONE ACETYLTRANSFERASE 1 (HAC1) is recruited to the WRKY18 and WRKY53 complex that resides on the promoters. In this complex, HAC1 facilitates the acetylation of histone 3 Lys 27 (H3K27ac) on the sugar-responsive genes. Taken together, our findings demonstrate a mechanism by which sugar regulates chromatin modification and gene expression, thus helping plants to adjust their growth in response to environmental changes.

Transcriptome dynamics at Arabidopsis graft junctions reveal an intertissue recognition mechanism that activates vascular regeneration

The ability for cut tissues to join and form a chimeric organism is a remarkable property of many plants; however, grafting is poorly characterized at the molecular level. To better understand this process, we monitored genome-wide gene expression changes in grafted Arabidopsis thaliana hypocotyls. We observed a sequential activation of genes associated with cambium, phloem, and xylem formation. Tissues above and below the graft rapidly developed an asymmetry such that many genes were more highly expressed on one side than on the other. This asymmetry correlated with sugar-responsive genes, and we observed an accumulation of starch above the graft junction. This accumulation decreased along with asymmetry once the sugar-transporting vascular tissues reconnected. Despite the initial starvation response below the graft, many genes associated with vascular formation were rapidly activated in grafted tissues but not in cut and separated tissues, indicating that a recognition mechanism was activated independently of functional vascular connections. Auxin, which is transported cell to cell, had a rapidly elevated response that was symmetric, suggesting that auxin was perceived by the root within hours of tissue attachment to activate the vascular regeneration process. A subset of genes was expressed only in grafted tissues, indicating that wound healing proceeded via different mechanisms depending on the presence or absence of adjoining tissues. Such a recognition process could have broader relevance for tissue regeneration, intertissue communication, and tissue fusion events.

Chloroplasts Are Central Players in Sugar-Induced Leaf Growth.

Leaves are the plant's powerhouses, providing energy for all organs through sugar production during photosynthesis. However, sugars serve not only as a metabolic energy source for sink tissues but also as signaling molecules, affecting gene expression through conserved signaling pathways to regulate plant growth and development. Here, we describe an in vitro experimental assay, allowing one to alter the sucrose (Suc) availability during early Arabidopsis (Arabidopsis thaliana) leaf development, with the aim to identify the affected cellular and molecular processes. The transfer of seedlings to Suc-containing medium showed a profound effect on leaf growth by stimulating cell proliferation and postponing the transition to cell expansion. Furthermore, rapidly after transfer to Suc, mesophyll cells contained fewer and smaller plastids, which are irregular in shape and contain fewer starch granules compared with control mesophyll cells. Short-term transcriptional responses after transfer to Suc revealed the repression of well-known sugar-responsive genes and multiple genes encoded by the plastid, on the one hand, and up-regulation of a GLUCOSE-6-PHOSPHATE TRANSPORTER (GPT2), on the other hand. Mutant gpt2 seedlings showed no stimulation of cell proliferation and no repression of chloroplast-encoded transcripts when transferred to Suc, suggesting that GPT2 plays a critical role in the Suc-mediated effects on early leaf growth. Our findings, therefore, suggest that induction of GPT2 expression by Suc increases the import of glucose-6-phosphate into the plastids that would repress chloroplast-encoded transcripts, restricting chloroplast differentiation. Retrograde signaling from the plastids would then delay the transition to cell expansion and stimulate cell proliferation.

Hexokinase 1 is required for glucose-induced repression of bZIP63, At5g22920, and BT2 in Arabidopsis.

Simple sugars, like glucose (Glc) and sucrose (Suc), act as signals to modulate the expression of hundreds of genes in plants. Frequently, however, it remains unclear whether this regulation is induced by the sugars themselves or by their derivatives generated in the course of carbohydrate (CH) metabolism. In the present study, we tested the relevance of different CH metabolism and allocation pathways affecting expression patterns of five selected sugar-responsive genes (bZIP63, At5g22920, BT2, MGD2, and TPS9) in Arabidopsis thaliana. In general, the expression followed diurnal changes in the overall sugar availability. However, under steady growth conditions, this response was hardly impaired in the mutants for CH metabolizing/ transporting proteins (adg1, sex1, sus1-4, sus5/6, and tpt2), including also hexokinase1 (HXK1) loss- and gain-of-function plants—gin2.1 and oe3.2, respectively. In addition, transgenic plants carrying pbZIP63::GUS showed no changes in reporter-gene-expression when grown on sugar under steady-state conditions. In contrast, short-term treatments of agar-grown seedlings with 1% Glc or Suc induced pbZIP63::GUS repression, which became even more apparent in seedlings grown in liquid media. Subsequent analyses of liquid-grown gin2.1 and oe3.2 seedlings revealed that Glc -dependent regulation of the five selected genes was not affected in gin2.1, whereas it was enhanced in oe3.2 plants for bZIP63, At5g22920, and BT2. The sugar treatments had no effect on ATP/ADP ratio, suggesting that changes in gene expression were not linked to cellular energy status. Overall, the data suggest that HXK1 does not act as Glc sensor controlling bZIP63, At5g22920, and BT2 expression, but it is nevertheless required for the production of a downstream metabolic signal regulating their expression.

Functional dissection of sugar signals affecting gene expression in Arabidopsis thaliana.

BackgroundSugars modulate expression of hundreds of genes in plants. Previous studies on sugar signaling, using intact plants or plant tissues, were hampered by tissue heterogeneity, uneven sugar transport and/or inter-conversions of the applied sugars. This, in turn, could obscure the identity of a specific sugar that acts as a signal affecting expression of given gene in a given tissue or cell-type.Methodology/Principal FindingsTo bypass those biases, we have developed a novel biological system, based on stem-cell-like Arabidopsis suspension culture. The cells were grown in a hormone-free medium and were sustained on xylose as the only carbon source. Using functional genomics we have identified 290 sugar responsive genes, responding rapidly (within 1 h) and specifically to low concentration (1 mM) of glucose, fructose and/or sucrose. For selected genes, the true nature of the signaling sugar molecules and sites of sugar perception were further clarified using non-metabolizable sugar analogues. Using both transgenic and wild-type A. thaliana seedlings, it was shown that the expression of selected sugar-responsive genes was not restricted to a specific tissue or cell type and responded to photoperiod-related changes in sugar availability. This suggested that sugar-responsiveness of genes identified in the cell culture system was not biased toward heterotrophic background and resembled that in whole plants.ConclusionsAltogether, our research strategy, using a combination of cell culture and whole plants, has provided an unequivocal evidence for the identity of sugar-responsive genes and the identity of the sugar signaling molecules, independently from their inter-conversions or use for energy metabolism.

EXO modifies sucrose and trehalose responses and connects the extracellular carbon status to growth

Plants have the capacity to adapt growth to changing environmental conditions. This implies the modulation of metabolism according to the availability of carbon (C). Particular interest in the response to the C availability is based on the increasing atmospheric levels of CO2. Several regulatory pathways that link the C status to growth have emerged. The extracellular EXO protein is essential for cell expansion and promotes shoot and root growth. Homologous proteins were identified in evolutionarily distant green plants. We show here that the EXO protein connects growth with C responses. The exo mutant displayed altered responses to exogenous sucrose supplemented to the growth medium. Impaired growth of the mutant in synthetic medium was associated with the accumulation of starch and anthocyanins, altered expression of sugar-responsive genes, and increased abscisic acid levels. Thus, EXO modulates several responses related to the C availability. Growth retardation on medium supplemented with 2-deoxy-glucose, mannose, and palatinose was similar to the wild type. Trehalose feeding stimulated root growth and shoot biomass production of exo plants whereas it inhibited growth of the wild type. The phenotypic features of the exo mutant suggest that apoplastic processes coordinate growth and C responses.

The Mysterious Rescue of adg1-1/tpt-2 – an Arabidopsis thaliana Double Mutant Impaired in Acclimation to High Light – by Exogenously Supplied Sugars

An Arabidopsis thaliana double mutant (adg1-1/tpt-2) defective in the day- and night-path of photoassimilate export from the chloroplast due to a knockout in the triose phosphate/phosphate translocator (TPT; tpt-2) and a lack of starch [mutation in ADP glucose pyrophosphorylase (AGPase); adg1-1] exhibits severe growth retardation, a decrease in the photosynthetic capacity, and a high chlorophyll fluorescence (HCF) phenotype under high light conditions. These phenotypes could be rescued when the plants were grown on sucrose (Suc) or glucose (Glc). Here we address the question whether Glc-sensing hexokinase1 (HXK1) defective in the Glc insensitive 2 (gin2-1) mutant is involved in the sugar-dependent rescue of adg1-1/tpt-2. Triple mutants defective in the TPT, AGPase, and HXK1 (adg1-1/tpt-2/gin2-1) were established as homozygous lines and grown together with Col-0 and Landsberg erecta (Ler) wild-type plants, gin2-1, the adg1-1/tpt-2 double mutant, and the adg1-1/tpt-2/gpt2-1 triple mutant [additionally defective in the glucose 6-phosphate/phosphate translocator 2 (GPT2)] on agar in the presence or absence of 50 mM of each Glc, Suc, or fructose (Fru). The growth phenotype of the double mutant and both triple mutants could be rescued to a similar extent only by Glc and Suc, but not by Fru. All three sugars were capable of rescuing the HCF and photosynthesis phenotype, irrespectively of the presence or absence of HXK1. Quantitative RT-PCR analyses of sugar-responsive genes revealed that plastidial HXK (pHXK) was up-regulated in adg1-1/tpt-2 plants grown on sugars, but showed no response in adg1-1/tpt-2/gin2-1. It appears likely that soluble sugars are directly taken up by the chloroplasts and enter further metabolism, which consumes ATP and NADPH from the photosynthetic light reaction and thereby rescues the photosynthesis phenotype of the double mutant. The implication of sugar turnover and probably signaling inside the chloroplasts for the concept of retrograde signaling is discussed.

Abscisic Acid Plays an Important Role in the Regulation of Strawberry Fruit Ripening

The plant hormone abscisic acid (ABA) has been suggested to play a role in fruit development, but supporting genetic evidence has been lacking. Here, we report that ABA promotes strawberry (Fragaria ananassa) fruit ripening. Using a newly established Tobacco rattle virus-induced gene silencing technique in strawberry fruit, the expression of a 9-cis-epoxycarotenoid dioxygenase gene (FaNCED1), which is key to ABA biosynthesis, was down-regulated, resulting in a significant decrease in ABA levels and uncolored fruits. Interestingly, a similar uncolored phenotype was observed in the transgenic RNA interference (RNAi) fruits, in which the expression of a putative ABA receptor gene encoding the magnesium chelatase H subunit (FaCHLH/ABAR) was down-regulated by virus-induced gene silencing. More importantly, the uncolored phenotype of the FaNCED1-down-regulated RNAi fruits could be rescued by exogenous ABA, but the ABA treatment could not reverse the uncolored phenotype of the FaCHLH/ABAR-down-regulated RNAi fruits. We observed that down-regulation of the FaCHLH/ABAR gene in the RNAi fruit altered both ABA levels and sugar content as well as a set of ABA- and/or sugar-responsive genes. Additionally, we showed that exogenous sugars, particularly sucrose, can significantly promote ripening while stimulating ABA accumulation. These data provide evidence that ABA is a signal molecule that promotes strawberry ripening and that the putative ABA receptor, FaCHLH/ABAR, is a positive regulator of ripening in response to ABA.

Arabidopsis plants acclimate to water deficit at low cost through changes of carbon usage: an integrated perspective using growth, metabolite, enzyme, and gene expression analysis.

Growth and carbon (C) fluxes are severely altered in plants exposed to soil water deficit. Correspondingly, it has been suggested that plants under water deficit suffer from C shortage. In this study, we test this hypothesis in Arabidopsis (Arabidopsis thaliana) by providing an overview of the responses of growth, C balance, metabolites, enzymes of the central metabolism, and a set of sugar-responsive genes to a sustained soil water deficit. The results show that under drought, rosette relative expansion rate is decreased more than photosynthesis, leading to a more positive C balance, while root growth is promoted. Several soluble metabolites accumulate in response to soil water deficit, with K(+) and organic acids as the main contributors to osmotic adjustment. Osmotic adjustment costs only a small percentage of the daily photosynthetic C fixation. All C metabolites measured (not only starch and sugars but also organic acids and amino acids) show a diurnal turnover that often increased under water deficit, suggesting that these metabolites are readily available for being metabolized in situ or exported to roots. On the basis of 30 enzyme activities, no in-depth reprogramming of C metabolism was observed. Water deficit induces a shift of the expression level of a set of sugar-responsive genes that is indicative of increased, rather than decreased, C availability. These results converge to show that the differential impact of soil water deficit on photosynthesis and rosette expansion results in an increased availability of C for the roots, an increased turnover of C metabolites, and a low-cost C-based osmotic adjustment, and these responses are performed without major reformatting of the primary metabolism machinery.

The Arabidopsis bZIP1 Transcription Factor Is Involved in Sugar Signaling, Protein Networking, and DNA Binding

Sugar signaling is a mechanism that plants use to integrate various internal and external cues to achieve nutrient homeostasis, mediate developmental programs, and articulate stress responses. Many bZIP transcription factors are known to be involved in nutrient and/or stress signaling. An Arabidopsis S1-group bZIP gene, AtbZIP1, was identified as a sugar-sensitive gene in a previous gene expression profiling study (Plant Cell. 16, 2128-2150). In this report, we show that the expression of AtbZIP1 is repressed by sugars in a fast, sensitive, and reversible way. The sugar repression of AtbZIP1 is affected by a conserved sugar signaling component, hexokinase. Besides being a sugar-regulated gene, AtbZIP1 can mediate sugar signaling and affect gene expression, plant growth, and development. When carbon nutrients are limited, gain or loss of function of AtbZIP1 causes changes in the rates of early seedling establishment. Results of phenotypic analyses indicate that AtbZIP1 acts as a negative regulator of early seedling growth. Using gain- and loss-of-function plants in a microarray analysis, two sets of putative AtbZIP1-regulated genes have been identified. Among them, sugar-responsive genes are highly over-represented, implicating a role of AtbZIP1 in sugar-mediated gene expression. Using yeast two-hybrid (Y-2-H) screens and bimolecular fluorescence complementation (BiFC) analyses, we are able to recapitulate extensive C/S1 AtbZIP protein interacting network in living cells. Finally, we show that AtbZIP1 can bind ACGT-based motifs in vitro and that the binding characteristics appear to be affected by the heterodimerization between AtbZIP1 and the C-group AtbZIPs, including AtbZIP10 and AtbZIP63.

Luminal fructose inhibits rat intestinal sodium-phosphate cotransporter gene expression and phosphate uptake

BackgroundWhile searching by microarray for sugar-responsive genes, we inadvertently discovered that sodium-phosphate cotransporter 2B (NaPi-2b) mRNA concentrations were much lower in fructose-perfused than in glucose-perfused intestines of neonatal rats. Changes in NaPi-2b mRNA abundance by sugars were accompanied by similar changes in NaPi-2b protein abundance and in rates of inorganic phosphate (Pi) uptake. ObjectiveWe tested the hypothesis that luminal fructose regulates NaPi-2b. DesignWe perfused into the intestine fructose, glucose, and nonmetabolizable or poorly transported glucose analogs as well as phlorizin. ResultsNaPi-2b mRNA concentrations and Pi uptake rates in fructose-perfused intestines were ≈30% of those in glucose and its analogs. NaPi-2b inhibition by fructose is specific because the mRNA abundance and activity of the fructose transporter GLUT5 (glucose transporter 5) increased with fructose perfusion, whereas those of other transporters were independent of the perfusate. Plasma Pi after 4 h of perfusion was independent of the perfusate, probably because normal kidneys can maintain normophosphatemia. Inhibiting glucose-6-phosphatase, another fructose-responsive gene, with tungstate or vanadate nonspecifically inhibited NaPi-2b mRNA expression and Pi uptake in both glucose- or fructose-perfused intestines. The AMP kinase (AMPK)–activator AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) enhanced and the fatty acid synthase–AMPK inhibitor C75 (3-carboxy-4-octyl-2-methylenebutyrolactone trans-4-carboxy-5-octyl-3-methylenebutyrolactone) prevented fructose inhibition of NaPi-2b but had no effect on expression of other transporters. NaPi-2b expression decreased markedly with age and was inhibited by fructose in all age groups. ConclusionsEnergy levels in enterocytes may play a role in NaPi-2b inhibition by luminal fructose. Consumption of fructose that supplies ≈10% of caloric intake by Americans clearly affects absorption of Pi and may promote Pi homeostasis in patients with impaired renal function.

Signaling from an Altered Cell Wall to the Nucleus Mediates Sugar-Responsive Growth and Development inArabidopsis thaliana

Sugars such as glucose function as signal molecules that regulate gene expression, growth, and development in plants, animals, and yeast. To understand the molecular mechanisms of sugar responses, we isolated and characterized an Arabidopsis thaliana mutant, high sugar response8 (hsr8), which enhances sugar-responsive growth and gene expression. Light-grown hsr8 plants exhibited increased starch and anthocyanin and reduced chlorophyll content in response to glucose treatment. Dark-grown hsr8 seedlings showed glucose-hypersensitive hypocotyl elongation and development. The HSR8 gene, isolated using map-based cloning, was allelic to the MURUS4 (MUR4) gene involved in arabinose synthesis. Dark-grown mur1 and mur3 seedlings also exhibited similar sugar responses to hsr8/mur4. The sugar-hypersensitive phenotypes of hsr8/mur4, mur1, and mur3 were rescued by boric acid, suggesting that alterations in the cell wall cause hypersensitive sugar-responsive phenotypes. Genetic analysis showed that sugar-hypersensitive responses in hsr8 mutants were suppressed by pleiotropic regulatory locus1 (prl1), indicating that nucleus-localized PRL1 is required for enhanced sugar responses in hsr8 mutant plants. Microarray analysis revealed that the expression of many cell wall-related and sugar-responsive genes was altered in mur4-1, and the expression of a significant proportion of these genes was restored to wild-type levels in the mur4-1 prl1 double mutant. These findings reveal a pathway that signals changes in the cell wall through PRL1 to altered gene expression and sugar-responsive metabolic, growth, and developmental changes.

Multilevel genomics analysis of carbon signalling during low carbon availability: coordinating the supply and utilisation of carbon in a fluctuating environment.

Plants alternate between a net surplus of carbon in the light and a net deficit at night. This is buffered by accumulating starch in the light and degrading it at night. Enough starch is accumulated to support degradation throughout the night, with a small amount remaining at the end of the 24-h diurnal cycle. This review discusses how this balance between the supply and utilisation of carbon is achieved in Arabidopsis. It is important to regulate starch turnover to avoid an acute carbon deficiency. A 2-4 h extension of the night leads to exhaustion of starch, a collapse of sugars, a switch from biosynthesis to catabolism and an acute inhibition of growth by low carbon, which is not immediately reversed when carbon becomes available again. In starchless pgm mutants, where sugars are depleted each night, this leads to a recurring inhibition of growth that is not reversed until 5-6 h into the following light period. Several lines of evidence show that starch accumulation is regulated in response to events that are initiated during periods of low carbon. Starch accumulation is decreased when small amounts of sucrose are included in the growth medium. Sets of sugar-responsive genes were identified by supplying sugars to carbon-starved seedlings, or by illuminating 5-week-old plants in the presence of 350 or 50 ppm [CO2]. Almost all of these genes show large diurnal changes in starchless pgm mutants, which are driven by the depletion of carbon during the night. Many show significant diurnal changes in wild type plants, showing that 'anticipatory' changes in signalling pathways occur before acute carbon limitation develops. However, these diurnal changes of transcripts do not lead to immediate changes of enzyme activities. Whereas an extension of the night leads to major changes of transcripts within 4-6 h, changes in enzyme activities require several days. In pgm, enzyme activities and the levels of >150 metabolites resemble those found in wild type plants after several days in the dark. It is concluded that diurnal changes in transcript levels are integrated, over days, as changes in the levels of enzymes. We hypothesise that this facilitates an adjustment of metabolism to a mid-term shift in the conditions, while ignoring noise due to diurnal changes and day-to-day fluctuations. The rapid adjustment of starch synthesis after a period of acute carbon depletion is a consequence of the transient inhibition of growth. This leads to accumulation of sugars when carbon becomes available again, which triggers a large increase in trehalose-6-phosphate. This signal metabolite promotes thioredoxin-dependent post-translational activation of ADP glucose pyrophosphorylase. Mid-term acclimation to a decreased carbon supply may be mediated by a combination of post-translational regulation, longer-term changes in enzyme activities, and a decrease in the rate of growth.

A Microarray Analysis of Rice Near-Isogenic Lines that Cover QTLs Associated with Callus Proliferation

Near-isogenic lines (NILs) were applied for the first time to microarray analysis of 8,987 randomly selected expressed sequence tags (ESTs) aiming at screening rice genes associated with seed callus proliferation. Callus proliferation of the variety Koshihikari is poor, since its callus tends to become brown with time. We developed ten NILs that each had a chromosomal segment of the variety Kasalath on chromosome 1 in a Koshihikari background, to cover a region that contains two QTLs for subcultured callus color, qSc1-1 and qSc1-2. The existence of QTLs was verified through tissue culture of the seed calli of these NILs. mRNAs from the calli of three NILs that had the Kasalath allele in qSc1-1 and/or qSc1-2 were applied to microarray analysis. A comparison of expression profiles between Koshihikari and each NIL followed by Northern hybridization showed that 22 unique genes were induced and 15 unique genes were repressed in the calli of these Kasalath allele-containing NILs on chromosome 1. The results of a cis-element search using 500 bp of genomic sequence upstream of each gene suggested that the expression profiles of GA- and/or sugar-responsive genes were different between each NIL and Koshihikari, and that defense-related genes and genes that act negatively in metabolism were repressed in the NILs compared with Koshihikari.

Sugars and Circadian Regulation Make Major Contributions to the Global Regulation of Diurnal Gene Expression in Arabidopsis

The diurnal cycle strongly influences many plant metabolic and physiological processes. Arabidopsis thaliana rosettes were harvested six times during 12-h-light/12-h-dark treatments to investigate changes in gene expression using ATH1 arrays. Diagnostic gene sets were identified from published or in-house expression profiles of the response to light, sugar, nitrogen, and water deficit in seedlings and 4 h of darkness or illumination at ambient or compensation point [CO(2)]. Many sugar-responsive genes showed large diurnal expression changes, whose timing matched that of the diurnal changes of sugars. A set of circadian-regulated genes also showed large diurnal changes in expression. Comparison of published results from a free-running cycle with the diurnal changes in Columbia-0 (Col-0) and the starchless phosphoglucomutase (pgm) mutant indicated that sugars modify the expression of up to half of the clock-regulated genes. Principle component analysis identified genes that make large contributions to diurnal changes and confirmed that sugar and circadian regulation are the major inputs in Col-0 but that sugars dominate the response in pgm. Most of the changes in pgm are triggered by low sugar levels during the night rather than high levels in the light, highlighting the importance of responses to low sugar in diurnal gene regulation. We identified a set of candidate regulatory genes that show robust responses to alterations in sugar levels and change markedly during the diurnal cycle.

ACTIVATOR of Spomin::LUC1/WRINKLED1 of Arabidopsis thaliana Transactivates Sugar-inducible Promoters

We isolated an enhancer activation-tagged mutant of Arabidopsis thaliana line sGsL carrying the luciferase (LUC) gene under control of a short sugar-inducible promoter derived from a sweet potato sporamin gene (Spomin) that showed high level expression of LUC under non-inducing conditions. The activator of Spomin::LUC1 (ASML1) gene located downstream of the enhancer encoded an APETALA2 (AP2)-type AP2 domain protein, and this gene was shown recently to be responsible for the wrinkled1 mutation which causes defective accumulation of seed storage oil. Overexpression of ASML1 cDNA in sGsL plants resulted in enhanced expression of not only the LUC reporter but also endogenous sugar-inducible genes including Atbeta-Amy encoding beta-amylase. Transient co-expression of 35S::ASML1 with Spomin::LUC or Atbeta-Amy::LUC reporters in protoplasts resulted in an approximately 10-fold transactivation of LUC expression. This transactivation was lost when the C-terminal acidic region of ASML1 was deleted. Expression of ASML1 was high in reproductive organs, and ASML1 mRNA showed transient accumulation in leaves after treatment with 6% sucrose, whereas it did not respond to abscisic acid. These results suggest that ASML1/WRI1 is a transcriptional activator involved in the activation of a subset of sugar-responsive genes and the control of carbon flow from sucrose import to oil accumulation in developing seeds.