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First published online September 3, 2004; 10.1104/pp.104.044347 Plant Physiology 136:2687-2699 (2004) © 2004 American Society of Plant Biologists Diurnal Changes in the Transcriptome Encoding Enzymes of Starch Metabolism Provide Evidence for Both Transcriptional and Posttranscriptional Regulation of Starch Metabolism in Arabidopsis Leaves1Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh EH9 3JH, United Kingdom (S.M.S., D.C.F., D.T., H.D.); Department of Metabolic Biology, John Innes Centre, Colney, Norwich NR4 7UH, United Kingdom (T.C., A.C., C.H., A.M.S.); and Institute of Plant Sciences, University of Bern, CH–3013 Bern, Switzerland (S.C.Z.)
To gain insight into the synthesis and functions of enzymes of starch metabolism in leaves of Arabidopsis L. Heynth, Affymetrix microarrays were used to analyze the transcriptome throughout the diurnal cycle. Under the conditions employed, transitory leaf starch is degraded progressively during a 12-h dark period, and then accumulates during the following 12-h light period. Transcripts encoding enzymes of starch synthesis changed relatively little in amount over 24 h except for two starch synthases, granule bound starch synthase and starch synthase II, which increased appreciably during the transition from dark to light. The increase in RNA encoding granule-bound starch synthase may reflect the extensive destruction of starch granules in the dark. Transcripts encoding several enzymes putatively involved in starch breakdown showed a coordinated decline in the dark followed by rapid accumulation in the light. Despite marked changes in their transcript levels, the amounts of some enzymes of starch metabolism do not change appreciably through the diurnal cycle. Posttranscriptional regulation is essential in the maintenance of amounts of enzymes and the control of their activities in vivo. Even though the relationships between transcript levels, enzyme activity, and diurnal metabolism of starch metabolism are complex, the presence of some distinctive diurnal patterns of transcripts for enzymes known to be involved in starch metabolism facilitates the identification of other proteins that may participate in this process.
Starch is the major form in which carbon is stored in plants, the major source of calories in the human diet, and an important industrial commodity (Jobling, 2004  ). However, our understanding of the nature and regulation of the pathways of starch synthesis and degradation is incomplete. The Arabidopsis genome sequence, together with extensive functional genomics resources, is facilitating investigations to better understand starch metabolism in plants (Zeeman et al., 2002; Smith et al., 2003). Starch is synthesized in many organs of Arabidopsis, including leaves, flowers, developing seeds, and root caps, and the structure and composition of starch isolated from leaves are similar to that from crop plants (Zeeman et al., 2002). Use of leaves enables both synthesis and degradation to be studied within a 24-h period independently of changes in plant development. The two processes are integrated with each other, and their rates are related to the duration of day and night. Characteristic changes in the content of sugars and maltooligosaccharides are also observed throughout the diurnal cycle, showing a complex integration of starch and sugar metabolism. The Arabidopsis leaf thus provides an excellent model system with which to elucidate the pathways and regulatory mechanisms of starch synthesis and breakdown in the plastids of living cells (Zeeman et al., 2002; Smith et al., 2003). The Arabidopsis genome sequence reveals many genes encoding enzymes that may be involved in starch synthesis and degradation, and in the synthesis of Suc from starch. Some of these enzymes have been extensively studied in other species, and their functions are well established (such as starch synthases and branching enzymes), while the functions of others are uncertain. In many cases, several genes in Arabidopsis encode different putative isoforms of the same enzyme, in some instances with different subcellular targeting information. With the increasing availability of functional genomic resources in Arabidopsis, the comprehensive analysis of the function of all putative enzymes of starch metabolism is now possible.
Isolation of mutants using both forward and reverse genetics approaches has provided important information about some of these enzymes. The results have led to some surprising discoveries: enzymes previously assumed to be important have no obvious role and proteins of previously unknown function have been shown to be important. A mutant (dbe1) identified as a result of unusual iodine staining of leaf starch was found to contain both starch and phytoglycogen (Zeeman et al., 1998a
Microarray analyses have revealed that transcripts for at least some of these enzymes show strong diurnal changes (Harmer et al., 2000
Physiological Context of Transcriptome Analysis
Conditions chosen for investigation of the starch transcriptome throughout the diurnal cycle were those used routinely in studies of starch metabolism in our laboratories (e.g. Zeeman et al., 1998a
Experimental Design of Transcriptome Analysis We harvested leaves at the 11 time points we routinely use for metabolite analyses (Fig. 1). The harvesting is particularly focused on the periods immediately after the transitions from dark to light and from light to dark, since changes in carbohydrate metabolism are most pronounced at these times. The first sample was taken at the end of the light period, and subsequent samples were taken after 1, 2, 4, 8, and 12 h of darkness, then after 1, 2, 4, 8, and 12 h of light. Thus, the last sample is a replicate of the first. For each sample, we harvested three leaves from each of eight plants to reduce biological variability as far as realistically possible. We used single microarrays at each time point because the Affymetrix genome arrays are highly reproducible (less than 0.6% of genes show a 2-fold difference in expression due to technical variability; http://nasc.nott.ac.uk/). Evidence for the reliability of the data was obtained by observing progressive changes in the levels of individual transcripts at successive time points in the diurnal cycle and by observing very similar expression patterns for coordinately regulated genes (see below). Finally, by repeating the whole experiment on plants grown in the same growth chamber, we were able to demonstrate the reproducibility of the results (see below).
We focused our analysis on genes encoding enzymes involved in starch synthesis from the Calvin cycle intermediate Fru-6-P, and in the conversion of starch to maltose and Glc in the dark (Table I). For starch synthesis, we included genes encoding chloroplastic phosphoglucoisomerase (PGI) and phosphoglucomutase (PGM), subunits of ADP-Glc pyrophosphorylase (AGPase), starch synthase (STS) and starch branching enzyme (SBE) isoforms, and the two isoamylases (ISA1 and ISA2) homologous to the isoamylase proteins that constitute the major isoamylase activity of the potato tuber (Hussain et al., 2003
The pathway by which starch is degraded in leaves at night is far from understood (Smith et al., 2003 ; Zeeman et al., 2004b), so we have examined transcript levels of all of the enzymes either shown to be involved in this pathway, or predicted from their sequences or known activities to be capable of catalyzing relevant reactions. Thus, we include genes encoding GWD and GWD-like enzymes, AMYs, BAMs, disproportionating enzyme (DPE1) and the related DPE2 protein, a third isoamylase (ISA3), the pullulanase or limit dextrinase (LDA), glucan (starch) phosphorylases (PHS), and putative  -glucosidases (AGL). For some of these enzymes there is strong evidence that they occur in the plastid, and some are encoded by genes including predicted transit peptides for plastid targeting (Table I). AMY2 and some putative AGLs are apparently synthesized with putative signal sequences for endomembrane targeting. Other proteins contain no apparent targeting information and are therefore assumed to be cytosolic. The functions of these nonplastidial enzymes is unknown, but they could potentially be involved in metabolism of products of starch breakdown exported from the plastid—or in the degradation of starch in lysosome-like vacuoles, or in the metabolism of extracellular glucans.
We also included genes encoding transporters on the chloroplast envelope that are capable of transferring metabolites related to starch metabolism between the chloroplast and the cytosol, including a recently discovered maltose transporter (Niittylä et al., 2004
Microarray analysis using the Affymetrix ATH1 genome array was carried out by the Nottingham Arabidopsis Stock Centre (NASC) using RNA delivered to them. The microarray data are available for public access on the NASC Web site (http://nasc.nott.ac.uk/). To obtain an overview of the trends in expression of the transcriptome in the first experiment, genes with a low expression signal (arbitrarily chosen as 6 or more time points with values below 50) were excluded from the total set of 22,810 genes, leaving a subset of 9,437 genes. The expression values for each gene in the subset were normalized to the value at time zero (0 h), and expressed as fold change in signal at each subsequent time point. The results (Fig. 2A) reveal several striking features. First, changes in gene expression are most pronounced immediately after the transitions from light to dark and from dark to light. Some genes in this set show changes in expression of up to 50-fold. Others not included in this set show even greater changes in gene expression throughout the diurnal cycle (data not shown). Second, some transcripts increase in amount prior to the light period and thereafter continue to increase, suggesting that they could be under circadian control (Harmer et al., 2000
For a very small number of genes, the signal at 24 h is severalfold greater than that at 0 h, in both experiments. Some of these genes, such as those encoding VSP1 (Guerineau et al., 2003
Next, we sought some indicators of the reliability of the signals produced by each genome array and of the comparability of the two experiments. First, we examined expression patterns of genes that have previously been extensively studied in the context of diurnal or circadian regulation. For these purposes and for all subsequent evaluation of expression profiles, we normalized the data for each gene as a percentage relative to the mean of its expression level throughout the diurnal cycle (Harmer et al., 2000
To test the relevance of relatively small changes in apparent levels of gene expression between time points, we focused initially on the gene encoding the triose phosphate transporter (TPT1). The expression level of this gene changes little in magnitude but exhibits a complex pattern of change. GeneSpring software (http://www.silicongenetics.com) was used to search for genes with similar expression profiles in the first experiment. The seven genes with profiles most similar to TPT1 (Fig. 2D) all encode chloroplast proteins, all with functions in photosynthesis. One of these genes encodes glyceraldehyde 3-phosphate dehydrogenase, which produces the substrate for TPT1. The same seven genes show coexpression in the second experiment (Fig. 2E), although the pattern differed in detail from that of the first. The small differences between experiments indicate that an aspect of the physiology of the leaves may have been different in each experiment. However, the expression patterns of genes not directly related to photosynthesis was remarkably consistent between experiments (Fig. 2, B and C; see below). The coincidence of successive values for these related genes throughout the diurnal cycle provides confidence that individual values for each gene are reliable and that even relatively small changes in transcript level at successive time points can be meaningful. A similar conclusion was reached by Menges et al. (2003)
Transcript signal strengths for the 48 genes under investigation varied widely (Table I). Individual values for some genes were appreciably below the commonly accepted cutoff value of 100. Caution should be exercised when interpreting results with low signal strengths. The relationship between signal strength and mRNA levels was investigated by comparing Affymetrix signal strengths to mRNA abundance estimated by Massively Parallel Sequence Signatures (MPSS; Brenner et al., 2000
Results for experiments 1 and 2 were very similar for all genes of starch metabolism, so only those of experiment 1 are shown in subsequent sections, except where indicated. There are single genes encoding chloroplastic PGI and PGM, the enzymes responsible for conversion of Fru-6-P to Glc-6-P and Glc-1-P for starch synthesis. The transcripts for these two enzymes show different diurnal patterns: PGI1 transcript changes relatively little, while PGM1 transcript decreases during the dark and increases during the light (Fig. 3A). A total of six genes encode the large and small subunits of AGPase: four large subunit (APL) genes and two small subunit (APS) genes. Mutational analysis shows that most of the activity in the leaf comes from the APS1 and APL1 genes. Mutations in APS1 result in plants (adg1 mutants) with no measurable AGPase activity in the leaves and essentially no leaf starch (Lin et al., 1988a
There are five genes for STS and three for SBE, representing classes that are conserved across higher plants (Ball and Morell, 2003 ). Mutational analysis in species other than Arabidopsis suggests that each isoform plays a specific role in determining the structure and composition of the starch granule during its synthesis (e.g. Edwards et al., 1999; Schwall et al., 2000; Fulton et al., 2002). Transcript levels for these eight genes do not change in a coordinated way over 24 h (Fig. 3, C and D). In fact, no two genes show exactly the same pattern. Transcripts for granule-bound starch synthase (GBS) show a dramatic diurnal change, far greater than for the other STS and SBE enzymes. This change has been observed in other species and has been shown to be under circadian control (Merida et al., 1999; Wang et al., 1999). GBS differs from the other starch synthases in being located solely within the matrix of the starch granule and active in the synthesis of the amylose component of starch in that location. We speculate that the profound diurnal change in transcript levels might reflect a need for resynthesis of the GBS protein following granule degradation at night. Immunoblot analysis of GBS levels in the insoluble (starch-containing) and soluble fractions of the leaf during a 12-h light period support this idea. GBS protein is not detectable at any point in the soluble fraction of the leaf (data not shown). On a fresh-weight basis, the amount in the insoluble fraction of the leaf is very low at the end of the night. It increases dramatically during the first 4 h of the day (Fig. 4A, compare time 0 and 4 h). On a starch-weight basis, the protein is most abundant at the end of the night and falls in amount throughout the day (Fig. 4). Taken together with the diurnal pattern of synthesis and degradation of starch (Fig. 1), these data suggest that GBS is destroyed as it is released from granules during degradation at night. At the end of the night, the amount in the leaf is very low. GBS synthesis occurs during the first part of the light period, then slows or stops while starch synthesis continues. Thus, GBS protein is apparently more abundant toward the center of the granule than at the periphery, and, hence, the residual starch at the end of the night has a very high GBS content. Therefore, the rise in GBS transcript level in the latter part of the night and the first 2 h of the day would be associated with the daily period of synthesis of this protein.
A second starch synthase gene STS2, representing the SSII class, also shows a marked diurnal change in transcript level. Although this isoform is probably active in amylopectin synthesis at the outer edge of the granule, in several plant organs it is known to become buried within starch granules as starch synthesis proceeds (e.g. Denyer et al., 1993 ). It is possible that the diurnal behavior of transcript and protein of this isoform is the same as that proposed for GBS above. Levels of transcripts for SBE1 and SBE2 show small changes at the start and end of the night, but SBE3 shows a distinctive pattern of decline through the night and a rapid increase after 4 h in the light period (Fig. 3D). We observed a pattern very similar to that of SBE3 for several enzymes with putative roles in starch degradation (see below). Levels of transcript for both of the isoamylases involved in starch synthesis (ISA1 and ISA2) are generally higher during the day than at night, and their patterns of diurnal change have some similar features (Fig. 5A). The pattern for the third isoamylase gene, ISA3, matches closely the pattern shown by several genes encoding enzymes putatively involved in starch degradation (see below).
A Coexpressed Gene Set Encoding Putative Enzymes of Starch Degradation
Transcripts encoding enzymes potentially involved in starch breakdown exhibit a wide range of expression profiles (Fig. 5). Emerging from this range is a distinctive pattern shown by nine genes, of slow decline during the night to a low level during the first few hours of the light, followed by a rapid increase between 4 and 8 h of the light (Fig. 6). This distinctive expression pattern is highly consistent between the two experiments (Fig. 6, A and B) and is suggestive of coordinate regulation. This set includes genes encoding three enzymes shown to be necessary for normal rates of starch degradation, namely GWD1 (Yu et al., 2001
Genes Encoding Other Enzymes Putatively Involved in Starch Degradation
Transcript levels of AMY1 and 2, GWD3, the debranching enzyme LDA1, and the AGLs do not conform to the coordinated pattern described above (Figs. 5 and 6). Each shows a distinctive pattern of change over 24 h. Transcript levels of AMY2 show a particularly strong diurnal change. There is a marked increase at the end of the night and a fall in the first few hours of the day. The functions of these enzymes are not yet known. LDA1 and GWD3 are predicted to be plastidial, but the others have no obvious plastid transit peptides. Knockout mutants of AMY2 and 3 and LDA1 are not impaired in leaf starch breakdown when grown under the conditions used here (H. Dunstan, D. Fulton, S. Zeeman, and S. Smith, unpublished data). There is no obvious requirement for
The transcript levels for the TPT1, GLT1, and MEX1 transporters show intricate and different patterns of diurnal change, of relatively low amplitude (Fig. 5H). Our recent work shows that most of the carbon derived from starch is exported by the maltose transporter MEX1, with a smaller contribution from GLT1 (Niittylä et al., 2004
The pathway by which maltose exported from the chloroplast is converted to Suc in the cytosol is not fully understood (Chia et al., 2004
Analysis of diurnal changes in the starch transcriptome reveals that it is very complex. For the most part, transcripts encoding enzymes thought to function in the same pathway do not show similar patterns of change over 24 h. For example, mutational and transgenic analysis in several species reveals that starch structure is determined by the coordinated actions of the multiple isoforms of starch synthase and starch branching enzyme (Schwall et al., 2000 ; Fulton et al., 2002; Jobling et al., 2002). In spite of this, no two isoforms of these enzymes show the same diurnal pattern of transcript change. Changes in transcript abundance are also not coordinated for the major two subunits of AGPase expressed in the leaf, or for the chloroplastic BAMs, disproportionating enzyme (DPE1), and the maltose and Glc transporters (MEX1 and GLT1, respectively), which together constitute a pathway of glucan degradation and export to the cytosol at night (Chia et al., 2004; Niittylä et al., 2004).
We identified a set of nine genes that appear to be coordinately expressed. Transcripts decline during the dark and characteristically increase rapidly between 4 and 8 h in the light. Transcripts for eight of these genes encode enzymes that are either known to be required for starch breakdown or could reasonably be expected to be. However, they do not constitute a complete pathway (for example, no BAMs are included), and they include proteins apparently not necessary for starch degradation (PHS1 and AMY3; Zeeman et al., 2004a
This lack of correspondence between RNA and protein for three members of the coordinately expressed group (AMY3, GWD1, and DPE2) indicates the importance of posttranscriptional control in regulating amounts of enzymes of starch metabolism and the fluxes through the key pathways. Control over amounts of protein may be exerted at the levels of both translation and protein stability. It is possible, for example, that the changes in transcript level for the coordinated set of enzymes reflect a substantial turnover of these proteins on a diurnal basis. The simultaneous occurrence of translation and protein degradation during a specific, limited period of the day would allow protein levels to be maintained at constant levels throughout the day. Control of flux through the pathways of starch synthesis and degradation is likely to be exerted primarily through modulation of enzyme activity. Little is known about control of starch degradation, but synthesis is thought to be controlled primarily via modulation of AGPase activity via metabolites and via redox-mediated changes in enzyme structure (Neuhaus and Stitt, 1990
The exception to this picture is GBS1 (and possibly STS2). Because of the location of this protein inside the starch granule and the requirement for the granule matrix for its activity and stability (Denyer et al., 1999 Although there is no obvious correspondence between transcript levels, levels of the encoded proteins, and fluxes in starch metabolism, the information obtained about individual transcripts may nonetheless help to direct future experiments to reveal the functions of the encoded proteins. For example, the isoamylase ISA3 is expressed coordinately with genes encoding degradative enzymes, while ISA1 and ISA2, shown to be required for starch synthesis, show a different pattern. Thus, ISA3 merits investigation as a debranching enzyme involved in starch degradation in the chloroplast. Changes in levels of transcript of BAM9 are strikingly similar to those of AMY2. Both enzymes are extraplastidial; perhaps both are involved in the degradation of same, as yet unknown glucan. A potential use of the coordinately expressed gene set is to search for other genes showing a similar pattern of expression. This may help to identify new proteins involved in starch metabolism. We searched the entire transcriptome for transcripts showing this same pattern of expression, expressed to an appreciable level, and encoding proteins with putative plastid transit peptides. This search identified a small number of genes encoding proteins of unknown function. The expression patterns for four of these genes are shown for experiments 1 and 2 (Fig. 6, C and D). Transcripts for these genes show a small increase in level between 1 and 2 h in experiment 2 but not in experiment 1 (Fig. 6, C and D). Transcripts for AMY 3 and PHS1 show a similar increase in level in experiment 2 but not in experiment 1 (Fig. 6, A and B). Such subtle differences in expression level observed between different experiments can be particularly helpful in identifying coexpressed genes. The possibility that these proteins of unknown function are involved in starch degradation is under investigation.
The coexpression of nine genes of starch metabolism suggests that they could be coregulated. The promoter sequences of these genes range from 550 to 4,680 bp (defined as the intergenic regions upstream of the ATG translation start codon). Searches for previously identified putative cis-acting regulatory elements, using programs such as PLACE (Higo et al., 1999 In summary, it is clear that the relationships between transcript levels, protein levels, enzyme activities, and fluxes of carbon into and out of starch are extremely complex. Nevertheless, analysis of the starch transcriptome can provide clues about the possible roles of individual proteins and can help to identify new proteins involved in starch synthesis and breakdown. Analyses of changes in the transcriptome in other situations, such as in response to sugar feeding of heterotrophic tissues or during the development of seeds, may provide yet further insight into the starch transcriptome and the regulation of starch synthesis and breakdown. Our results have already identified new targets for study and new hypotheses to test.
Plant Material
Arabidopsis Col-0 was obtained from the NASC (accession no. N1093). It was sown in a peat-based compost and incubated at 4°C for 3 d, then grown at 20°C, 75% relative humidity, in a 12-h-light/12-h-dark photoperiod with an irradiance of 180 µmol m–2 s–1. Plants were grown to growth stage 3.90 (Boyes et al., 2001
RNA was freeze-dried and sent to NASC, where quality control analysis was carried out before labeling and hybridization to 22,800 Affymetrix ATH1 genome arrays (Santa Clara, CA). Procedures for hybridization and data handling are given on the NASC Web site (http://nasc.nott.ac.uk/). Clustering of transcript patterns was carried out using Silicon Genetics GeneSpring 6 (http://www.silicongenetics.com; Redwood City, CA).
Immunoblotting for DPE2 was according to Chia et al. (2004)
We thank GARNet (http://www.york.ac.uk/res/garnet/garnet.htm) for supporting this use of Affymetrix microarrays and the NASC for carrying out microarray analysis. We thank Ryan Lister (University of Western Australia, Perth) for valuable help with the use of GeneSpring software. Received April 8, 2004; returned for revision June 9, 2004; accepted June 23, 2004.
1 This work was supported by the Biotechnology and Biological Science Research Council (BBSRC) of the United Kingdom (research grant nos. D11089 and D11090 to A.M.S. and S.M.S.), and by the National Centre of Competence in Research (Plant Survival), National Science Foundation, Switzerland (S.C.Z.). The John Innes Centre is supported by a core strategic grant from the BBSRC.  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.044347. * Corresponding author; e-mail s.smith{at}ed.ac.uk; fax 44 131 650 5392.
Ball SG, Morell MK (2003) From bacterial glycogen to starch: understanding the biogenesis of the plant starch granule. Annu Rev Plant Biol 54: 207–233[CrossRef][Medline]
Boyes DC, Zayed AM, Ascenzi R, McCaskill AJ, Hoffman NE, Davis KR, Gorlach J (2001) Growth stage-based phenotypic analysis of Arabidopsis: a model for high throughput functional genomics in plants. Plant Cell 13: 1499–1510 Brenner S, Johnson M, Bridgham J, Golda G, Lloyd DH, Johnson D, Luo S, McCurdy S, Foy M, Ewan M, et al (2000) Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays. Nat Biotechnol 18: 630–634[CrossRef][ISI][Medline]
Caspar T, Lin T-P, Monroe J, Bernhard W, Spilatro S, Preiss J, Somerville C (1989) Altered regulation of
Chandler JW, Apel K, Melzer S (2001) A novel putative Chia T, Thorneycroft D, Chapple A, Messerli G, Chen J, Zeeman SC, Smith SM, Smith AM (2004) A cytosolic glucosyltransferase is required for conversion of starch to sucrose in Arabidopsis leaves at night. Plant J 37: 853–863[CrossRef][ISI][Medline]
Crevillén P, Ballicora MA, Mérida A, Preiss J, Romero JM (2003) The different large subunit isoforms of Arabidopsis thaliana ADP-glucose pyrophosphorylase confer distinct kinetic and regulatory properties to the heterotetrameric enzyme. J Biol Chem 278: 28508–28515 Critchley JH, Zeeman SC, Takaha T, Smith AM, Smith SM (2001) A critical role for disproportionating enzyme in starch breakdown is revealed by a knock-out mutation in Arabidopsis. Plant J 26: 89–100[CrossRef][ISI][Medline] Denyer K, Sidebottom C, Hylton CM, Smith AM (1993) Soluble isoforms of starch synthase and starch-branching enzyme also occur within starch granules in developing pea embryos. Plant J 4: 191–198[CrossRef][Medline] Denyer K, Waite D, Motawia S, Lindberg Møller B, Smith AM (1999) Granule-bound starch synthase I in isolated starch granules elongates malto-oligosaccharides processively. Biochem J 340: 183–191 Edwards A, Borthakur A, Bornemann S, Venail J, Denyer K, Waite D, Fulton D, Smith AM, Martin C (1999) Specificity of starch synthase isoforms from potato. Eur J Biochem 266: 724–726[ISI][Medline]
Fulton DC, Edwards A, Pilling E, Robinson HL, Fahy B, Seale R, Kato L, Donald AM, Geigenberger P, Martin C, et al (2002) Role of granule-bound starch synthase in determination of amylopectin structure and starch granule morphology in potato. J Biol Chem 277: 10834–10841
Guerineau F, Benjdia M, Zhou DX (2003) A jasmonate-responsive element within the A. thaliana vsp1 promoter. J Exp Bot 54: 1153–1162
Harmer SL, Hogenesch JB, Straume M, Chang H-S, Han B, Zhu T, Wang X, Kreps JA, Kay SA (2000) Orchestrated transcription of key pathways in Arabidopsis by the circadian clock. Science 290: 2110–2113 Hayama R, Coupland G (2003) Shedding light on the circadian clock and the photoperiodic control of flowering. Curr Opin Plant Biol 6: 13–19[CrossRef][ISI][Medline]
Hendriks JH, Kolbe A, Gibon Y, Stitt M, Geigenberger P (2003) ADP-glucose pyrophosphorylase is activated by posttranslational redox-modification in response to light and to sugars in leaves of Arabidopsis and other plant species. Plant Physiol 133: 838–849
Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant cis-acting regulatory DNA elements (PLACE) database. Nucleic Acids Res 27: 297–300
Hussain H, Mant A, Seale R, Zeeman S, Hinchliffe E, Edwards A, Hylton C, Bornemann S, Smith AM, Martin C, et al (2003) Three isoforms of isoamylase contribute different catalytic properties for the debranching of potato glucans. Plant Cell 15: 133–149 Jobling S (2004) Improving starch for food and industrial applications. Curr Opin Plant Biol 7: 210–218[CrossRef][ISI][Medline] Jobling SA, Westcott RJ, Tayal A, Jeffcoat R, Schwall GP (2002) Production of a freeze-thaw-stable potato starch by antisense inhibition of three starch synthase genes. Nat Biotechnol 20: 295–299[Medline]
Laby RJ, Kim D, Gibson SI (2001) The ram1 mutant of Arabidopsis exhibits severely decreased
Lin T-P, Caspar T, Somerville C, Preiss J (1988a) Isolation and characterisation of a starchless mutant of Arabidopsis thaliana (L.) Heynth lacking ADPglucose pyrophosphorylase activity. Plant Physiol 86: 1131–1135
Lin T-P, Caspar T, Somerville C, Preiss J (1988b) A starch deficient mutant of Arabidopsis thaliana with low ADPglucose pyrophosphorylase activity lacks one of the two subunits of the enzyme. Plant Physiol 88: 1175–1181
Liu XL, Covington MF, Fankhauser C, Chory J, Wagner DR (2001) ELF3 encodes a circadian clock-regulated nuclear protein that functions in an Arabidopsis PHYB signal transduction pathway. Plant Cell 13: 1293–1304 Lorberth R, Ritte G, Willmitzer L, Kossmann J (1998) Inhibition of a starch-granule-bound protein leads to modified starch and repression of cold sweetening. Nat Biotechnol 16: 473–477[CrossRef][ISI][Medline] Lu Y, Sharkey TD (2004) The role of amylomaltase in maltose metabolism in the cytosol of photosynthetic cells. Planta 218: 466–473[CrossRef][ISI][Medline] Menges M, Hennig L, Gruissem W, Murray JA (2003) Genome-wide gene expression in an Arabidopsis cell suspension. Plant Mol Biol 53: 423–442[CrossRef][ISI][Medline]
Merida A, Rodriguez-Galan JM, Vincent C, Romero JM (1999) Expression of the granule-bound starch synthase I (Waxy) gene from snapdragon is developmentally and circadian clock regulated. Plant Physiol 120: 401–410
Millar AJ, Kay SA (1991) Circadian control of cab gene transcription and mRNA accumulation in Arabidopsis. Plant Cell 3: 541–550
Myers AM, Morell MK, James MG, Ball SG (2000) Recent progress toward understanding biosynthesis of the amylopectin crystal. Plant Physiol 122: 989–997 Neuhaus HE, Stitt M (1990) Control analysis of photosynthate partitioning—impact of reduced activity of ADP-glucose pyrophosphorylase or plastid phosphoglucomutase on the fluxes to starch and sucrose in Arabidopsis thaliana (L.) Heynh. Planta 182: 445–454[CrossRef]
Niittylä T, Messerli G, Trevisan M, Chen J, Smith AM, Zeeman SC (2004) A previously unknown maltose transporter essential for starch degradation in leaves. Science 303: 87–89
Ritte G, Lloyd JR, Eckermann N, Rottmann A, Kossmann J, Steup M (2002) The starch-related R1 protein is an
Rombauts S, Florquin K, Lescot M, Marchal K, Rouze P, van de Peer Y (2003) Computational approaches to identify promoters and cis-regulatory elements in plant genomes. Plant Physiol 132: 1162–1176
Schaffer R, Landgraf J, Accerbi M, Simon V, Larson M, Wisman E (2001) Microarray analysis of diurnal and circadian-regulated genes in Arabidopsis. Plant Cell 13: 113–123 Schwall GP, Safford R, Westcott RJ, Jeffcoat R, Tayal A, Shi YC, Gidley MJ, Jobling SA (2000) Production of very-high-amylose potato starch by inhibition of SBE A and B. Nat Biotechnol 18: 551–554[CrossRef][ISI][Medline]
Sehnke PC, Chung HJ, Wu K, Ferl RJ (2001) Regulation of starch accumulation by granule-associated plant 14-3-3 proteins. Proc Natl Acad Sci USA 98: 765–770
Smith AM, Zeeman SC, Thorneycroft D, Smith SM (2003) Starch mobilization in leaves. J Exp Bot 54: 577–583
Strayer C, Oyama T, Schultz TF, Raman R, Somers DE, Mas P, Panda S, Kreps JA, Kay SA (2000) Cloning of the Arabidopsis clock gene TOC1, an autoregulatory response regulator homolog. Science 289: 768–771 Tatge H, Marshall J, Martin C, Edwards EA, Smith AM (1999) Evidence that amylose synthesis occurs within the matrix of starch granules. Plant Cell Environ 22: 543–550[CrossRef] Terzaghi WB, Cashmore AR (1995) Light-regulated transcription. Annu Rev Plant Physiol Plant Mol Biol 46: 445–474[CrossRef][ISI]
Tetlow IJ, Wait R, Lu Z, Akkasaeng R, Bowsher CG, Esposito S, Kosar-Hashemi B, Morell MK, Emes MJ (2004) Protein phosphorylation in amyloplasts regulates starch branching enzyme activity and protein-protein interactions. Plant Cell 16: 694–708
Wang Q, Monroe J, Sjolund RD (1995) Identification and characterisation of a phloem-specific Wang SJ, Yeh KW, Tsai CY (1999) Molecular characterisation and expression of starch granule-bound starch synthase in the sink and source tissues of sweet potato. Physiol Plant 106: 253–261[CrossRef] Wang SM, Chu B, Lue WL, Yu TS, Eimert K, Chen J (1997) adg2-1 represents a missense mutation in the ADPG pyrophosphorylase large subunit gene of Arabidopsis thaliana. Plant J 11: 1121–1126[Medline] Wang SM, Lue WL, Yu TS, Long JH, Wang CN, Eimert K, Chen J (1998) Characterization of ADG1, an Arabidopsis locus encoding for ADPG pyrophosphorylase small subunit, demonstrates that the presence of the small subunit is required for large subunit stability. Plant J 13: 63–70[CrossRef][Medline] Wang ZY, Tobin EM (1998) Constitutive expression of the CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) gene disrupts circadian rhythms and suppresses its own expression. Cell 93: 1207–1217[CrossRef][ISI][Medline] Weise SE, Weber AP, Sharkey TD (2004) Maltose is the major form of carbon exported from the chloroplast at night. Planta 218: 474–482[CrossRef][ISI][Medline]
Yu TS, Kofler H, Hausler RE, Hille D, Flugge UI, Zeeman SC, Smith AM, Kossmann J, Lloyd J, Ritte G, et al (2001) The Arabidopsis sex1 mutant is defective in the R1 protein, a general regulator of starch degradation in plants, and not in the chloroplast hexose transporter. Plant Cell 13: 1907–1918
Zeeman SC, Umemoto T, Lue WL, Au-Yeung P, Martin C, Smith AM, Chen J (1998) A mutant of Arabidopsis lacking a chloroplastic isoamylase accumulates both starch and phytoglycogen. Plant Cell 10: 1699–1712
Zeeman SC, Tiessen A, Pilling E, Kato KL, Donald AM, Smith AM (2002) Starch synthesis in Arabidopsis. Granule synthesis, composition, and structure. Plant Physiol 129: 516–529
Zeeman SC, Thorneycroft D, Schupp N, Chapple A, Weck M, Dunstan H, Haldimann P, Bechtold N, Smith AM, Smith SM (2004a) Chloroplastic starch phosphorylase is not required for transitory starch degradation in Arabidopsis leaves but has a role in the tolerance of abiotic stress. Plant Physiol 135: 849–858 Zeeman SC, Smith SM, Smith AM (2004b) The breakdown of starch in leaves. New Phytol 163: 247–261[CrossRef] This article has been cited by other articles:
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ABSTRACT
RESULTS
-amylase (BAM) does appear to be required (D. Fulton, H. Dunstan, S. Zeeman, and S. Smith, unpublished data). However, significant progress has been made in establishing the pathway of metabolism of starch breakdown products. Disproportionating enzyme (DPE1) has been shown to be required for conversion of maltotriose into larger maltooligosaccharides, which can be further attacked by amylolytic enzymes, and Glc, which is exported from the plastid (Critchley et al., 2001
