Samuel C. Zeeman

Starch: its metabolism, evolution, and biotechnological modification in plants.

Starch is the most widespread and abundant storage carbohydrate in plants. We depend upon starch for our nutrition, exploit its unique properties in industry, and use it as a feedstock for bioethanol production. Here, we review recent advances in research in three key areas. First, we assess progress in identifying the enzymatic machinery required for the synthesis of amylopectin, the glucose polymer responsible for the insoluble nature of starch. Second, we discuss the pathways of starch degradation, focusing on the emerging role of transient glucan phosphorylation in plastids as a mechanism for solubilizing the surface of the starch granule. We contrast this pathway in leaves with the degradation of starch in the endosperm of germinated cereal seeds. Third, we consider the evolution of starch biosynthesis in plants from the ancestral ability to make glycogen. Finally, we discuss how this basic knowledge has been utilized to improve and diversify starch crops.

Diurnal Changes in the Transcriptome Encoding Enzymes of Starch Metabolism Provide Evidence for Both Transcriptional and Posttranscriptional Regulation of Starch Metabolism in Arabidopsis Leaves

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 turnover: pathways, regulation and role in growth

Many plants store part of their photosynthate as starch during the day and remobilise it to support metabolism and growth at night. Mutants unable to synthesize or degrade starch show strongly impaired growth except in long day conditions. In rapidly growing plants, starch turnover is regulated such that it is almost, but not completely, exhausted at dawn. There is increasing evidence that premature or incomplete exhaustion of starch turnover results in lower rates of plant growth. This review provides an update on the pathways for starch synthesis and degradation. We discuss recent advances in understanding how starch turnover and the use of carbon for growth is regulated during diurnal cycles, with special emphasis on the role of the biological clock. Much of the molecular and genetic research on starch turnover has been performed in the reference system Arabidopsis. This review considers to what extent information gained in this weed species maybe applicable to annual crops and perennial species.

The diurnal metabolism of leaf starch

Starch is a primary product of photosynthesis in leaves. In most plants, a large fraction of the carbon assimilated during the day is stored transiently in the chloroplast as starch for use during the subsequent night. Photosynthetic partitioning into starch is finely regulated, and the amount of carbohydrate stored is dependent on the environmental conditions, particularly day length. This regulation is applied at several levels to control the flux of carbon from the Calvin cycle into starch biosynthesis. Starch is composed primarily of branched glucans with an architecture that allows the formation of a semi-crystalline insoluble granule. Biosynthesis has been most intensively studied in non-photosynthetic starch-storing organs, such as developing seeds and tubers. Biosynthesis in leaves has received less attention, but recent reverse-genetic studies of Arabidopsis (thale cress) have produced data generally consistent with what is known for storage tissues. The pathway involves starch synthases, which elongate the glucan chains, and branching enzymes. Remarkably, enzymes that partially debranch glucans are also required for normal amylopectin synthesis. In the last decade, our understanding of starch breakdown in leaves has advanced considerably. Starch is hydrolysed to maltose and glucose at night via a pathway that requires recently discovered proteins in addition to well-known enzymes. These sugars are exported from the plastid to support sucrose synthesis, respiration and growth. In the present review we provide an overview of starch biosynthesis, starch structure and starch degradation in the leaves of plants. We focus on recent advances in each area and highlight outstanding questions.

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

Starch is the major storage carbohydrate in higher plants and of considerable importance for the human diet and for numerous technical applications. In addition, starch can be accumulated transiently in chloroplasts as a temporary deposit of carbohydrates during ongoing photosynthesis. This transitory starch has to be mobilized during the subsequent dark period. Mutants defective in starch mobilization are characterized by high starch contents in leaves after prolonged periods of darkness and therefore are termed starch excess (sex) mutants. Here we describe the molecular characterization of the Arabidopsis sex1 mutant that has been proposed to be defective in the export of glucose resulting from hydrolytic starch breakdown. The mutated gene in sex1 was cloned using a map-based cloning approach. By complementation of the mutant, immunological analysis, and analysis of starch phosphorylation, we show that sex1 is defective in the Arabidopsis homolog of the R1 protein and not in the hexose transporter. We propose that the SEX1 protein (R1) functions as an overall regulator of starch mobilization by controlling the phosphate content of starch.

A Mutant of Arabidopsis Lacking a Chloroplastic Isoamylase Accumulates Both Starch and Phytoglycogen

In this study, our goal was to evaluate the role of starch debranching enzymes in the determination of the structure of amylopectin. We screened mutant populations of Arabidopsis for plants with alterations in the structure of leaf starch by using iodine staining. The leaves of two mutant lines stained reddish brown, whereas wild-type leaves stained brownish black, indicating that a more highly branched polyglucan than amylopectin was present. The mutants were allelic, and the mutation mapped to position 18.8 on chromosome 1. One mutant line lacked the transcript for a gene with sequence similarity to higher plant debranching enzymes, and both mutants lacked a chloroplastic starch-hydrolyzing enzyme. This enzyme was identified as a debranching enzyme of the isoamylase type. The loss of this isoamylase resulted in a 90% reduction in the accumulation of starch in this mutant line when compared with the wild type and in the accumulation of the highly branched water-soluble polysaccharide phytoglycogen. Both normal starch and phytoglycogen accumulated simultaneously in the same chloroplasts in the mutant lines, suggesting that isoamylase has an indirect rather than a direct role in determining amylopectin structure.

Quantification of starch in plant tissues

This protocol describes a simple means of measuring the starch content of plant tissues by solubilizing the starch, converting it quantitatively to glucose and assaying the glucose. Plant tissue must initially be frozen rapidly to stop metabolism, then extracted to remove free glucose. Starch is solubilized by heating, then digested to glucose by adding glucan hydrolases. Glucose is assayed enzymatically. The method is more sensitive and accurate than iodine-based protocols, and is suitable for tissues that have a wide range of starch contents. Measurements on multiple samples can be completed within a day.

β-AMYLASE4, a Noncatalytic Protein Required for Starch Breakdown, Acts Upstream of Three Active β-Amylases in Arabidopsis Chloroplasts

This work investigated the roles of β-amylases in the breakdown of leaf starch. Of the nine β-amylase (BAM)–like proteins encoded in the Arabidopsis thaliana genome, at least four (BAM1, -2, -3, and -4) are chloroplastic. When expressed as recombinant proteins in Escherichia coli, BAM1, BAM2, and BAM3 had measurable β-amylase activity but BAM4 did not. BAM4 has multiple amino acid substitutions relative to characterized β-amylases, including one of the two catalytic residues. Modeling predicts major differences between the glucan binding site of BAM4 and those of active β-amylases. Thus, BAM4 probably lost its catalytic capacity during evolution. Total β-amylase activity was reduced in leaves of bam1 and bam3 mutants but not in bam2 and bam4 mutants. The bam3 mutant had elevated starch levels and lower nighttime maltose levels than the wild type, whereas bam1 did not. However, the bam1 bam3 double mutant had a more severe phenotype than bam3, suggesting functional overlap between the two proteins. Surprisingly, bam4 mutants had elevated starch levels. Introduction of the bam4 mutation into the bam3 and bam1 bam3 backgrounds further elevated the starch levels in both cases. These data suggest that BAM4 facilitates or regulates starch breakdown and operates independently of BAM1 and BAM3. Together, our findings are consistent with the proposal that β-amylase is a major enzyme of starch breakdown in leaves, but they reveal unexpected complexity in terms of the specialization of protein function.

Plastidial α-Glucan Phosphorylase Is Not Required for Starch Degradation in Arabidopsis Leaves But Has a Role in the Tolerance of Abiotic Stress

To study the role of the plastidial α-glucan phosphorylase in starch metabolism in the leaves of Arabidopsis, two independent mutant lines containing T-DNA insertions within the phosphorylase gene were identified. Both insertions eliminate the activity of the plastidial α-glucan phosphorylase. Measurement of other enzymes of starch metabolism reveals only minor changes compared with the wild type. The loss of plastidial α-glucan phosphorylase does not cause a significant change in the total accumulation of starch during the day or its remobilization at night. Starch structure and composition are unaltered. However, mutant plants display lesions on their leaves that are not seen on wild-type plants, and mesophyll cells bordering the lesions accumulate high levels of starch. Lesion formation is abolished by growing plants under 100% humidity in still air, but subsequent transfer to circulating air with lower humidity causes extensive wilting in the mutant leaves. Wilted sectors die, causing large lesions that are bordered by starch-accumulating cells. Similar lesions are caused by the application of acute salt stress to mature plants. We conclude that plastidial phosphorylase is not required for the degradation of starch, but that it plays a role in the capacity of the leaf lamina to endure a transient water deficit.

STARCH-EXCESS4 Is a Laforin-Like Phosphoglucan Phosphatase Required for Starch Degradation in Arabidopsis thaliana

Starch is the major storage carbohydrate in plants. It is comprised of glucans that form semicrystalline granules. Glucan phosphorylation is a prerequisite for normal starch breakdown, but phosphoglucan metabolism is not understood. A putative protein phosphatase encoded at the Starch Excess 4 (SEX4) locus of Arabidopsis thaliana was recently shown to be required for normal starch breakdown. Here, we show that SEX4 is a phosphoglucan phosphatase in vivo and define its role within the starch degradation pathway. SEX4 dephosphorylates both the starch granule surface and soluble phosphoglucans in vitro, and sex4 null mutants accumulate phosphorylated intermediates of starch breakdown. These compounds are linear α-1,4-glucans esterified with one or two phosphate groups. They are released from starch granules by the glucan hydrolases α-amylase and isoamylase. In vitro experiments show that the rate of starch granule degradation is increased upon simultaneous phosphorylation and dephosphorylation of starch. We propose that glucan phosphorylating enzymes and phosphoglucan phosphatases work in synergy with glucan hydrolases to mediate efficient starch catabolism.