Transcriptomic analysis of starch biosynthesis in the developing grain of hexaploid wheat

Abstract

The expression of genes involved in starch synthesis in wheat was analyzed together with the accumulation profiles of soluble sugars, starch, protein, and starch granule distribution in developing caryopses obtained from the same biological materials used for profiling of gene expression using DNA microarrays. Multiple expression patterns were detected for the different starch biosynthetic gene isoforms, suggesting their relative importance through caryopsis development. Members of the ADP-glucose pyrophosphorylase, starch synthase, starch branching enzyme, and sucrose synthase gene families showed different expression profiles; expression of some members of these gene families coincided with a period of high accumulation of starch while others did not. A biphasic pattern was observed in the rates of starch and protein accumulation which paralleled changes in global gene expression. Metabolic and regulatory genes that show a pattern of expression similar to starch accumulation and granule size distribution were identified, suggesting their coinvolvement in these biological processes.

Figure 1

Soluble sugar content during caryopsis development. Starch and sugar content of the developing wheat caryopsis. Starch and sugar were measured by HPLC in dried whole caryopses; the same batch of biomaterial used for the microarray analysis. Values represent the percentage of each sugar in caryopsis tissue expressed as a percent dry weight (% DWT) of caryopsis tissue. Values are the mean ± Standard Error of the Mean (SEM) of 3-4 independent measurements of 3 biological replicates per time-point. The starch data are the mean of 2–5 biological replicates per time-point.

Figure 2

Amount and rate of storage product accumulation in the developing wheat grain. For both starch and protein assays, three determinations were made per biological sample, and 2–5 samples were used per time-point. The amylose data is the mean of the measurements of 3-4 biological replicates per time-point. (a) Level of starch, protein, amylose, fresh weight (FW) and dry weight (DW) per caryopis (mg/C) where C denotes caryopsis. (b) Rate of starch, protein and amylose accumulation (mg/C/day). Rate of accumulation was determined for each time interval.

Figure 3

Starch granule size distribution in developing grain. (a) Column 1—granules as percent of total volume; column 2—as percent of total number. (b) Relative granule proportions in terms of their % number (num.) and % volume (vol.). Starch granules bigger than 10 μm in diameter were considered A-granules and granules smaller or equal to 10 μm in diameter were considered B-granules, except at 7 DPA when all granules were considered A-granules. For volume calculations all granules bigger than 5 μm in diameter were considered oblate spheroid with thickness of 5 μm and varying equatorial diameters.

Figure 3

Starch granule size distribution in developing grain. (a) Column 1—granules as percent of total volume; column 2—as percent of total number. (b) Relative granule proportions in terms of their % number (num.) and % volume (vol.). Starch granules bigger than 10 μm in diameter were considered A-granules and granules smaller or equal to 10 μm in diameter were considered B-granules, except at 7 DPA when all granules were considered A-granules. For volume calculations all granules bigger than 5 μm in diameter were considered oblate spheroid with thickness of 5 μm and varying equatorial diameters.

Figure 4

SEM of developing starch granules. Developing starch granules at different stages were viewed at the same magnification (4000X); the bar size represents 10 μm. Starch granules at 7 DPA were 10 μm or less in size. Arrow points to an oblate spheroid shaped A-granule at 14 DPA with less than 10 μm in size. Starch granules with 5 μm or less in size are present throughout the developing caryopsis.

Figure 5

Expression profile of genes involved in starch metabolism during caryopsis development organized by pattern of expression. RNA samples used for the hybridization of the cDNA arrays were extracted from T. aestivum cv. Bobwhite caryopses. The expression profiles of genes were organized by pattern of expression: Early, Middle, or Late expresser. The x-axis shows the developmental stage time-points, and the y-axis represents the relative gene expression. Expression values are given as log 2-transformed normalized relative signal intensities, so that one unit on the y-axis represents an expression ratio of a factor of 2. GenBank accession numbers of the clones surveyed on the array are displayed on each chart.

Figure 5

Expression profile of genes involved in starch metabolism during caryopsis development organized by pattern of expression. RNA samples used for the hybridization of the cDNA arrays were extracted from T. aestivum cv. Bobwhite caryopses. The expression profiles of genes were organized by pattern of expression: Early, Middle, or Late expresser. The x-axis shows the developmental stage time-points, and the y-axis represents the relative gene expression. Expression values are given as log 2-transformed normalized relative signal intensities, so that one unit on the y-axis represents an expression ratio of a factor of 2. GenBank accession numbers of the clones surveyed on the array are displayed on each chart.

Figure 6

Expression profiles of major starch biosynthetic genes using gene specific oligoprobes. RNA samples used for hybridization of the oligoarrays were extracted from T. aestivum cv. Hereward caryopses. The x-axis shows the developmental stage time-points, the y-axis represents the relative gene expression (log 2). All the probesets are gene-specific based on Affymetrix annotation except for enolase and SuSy1, which could potentially cross-hybridize with other similar genes.

Figure 6

Expression profiles of major starch biosynthetic genes using gene specific oligoprobes. RNA samples used for hybridization of the oligoarrays were extracted from T. aestivum cv. Hereward caryopses. The x-axis shows the developmental stage time-points, the y-axis represents the relative gene expression (log 2). All the probesets are gene-specific based on Affymetrix annotation except for enolase and SuSy1, which could potentially cross-hybridize with other similar genes.

Figure 7

Expression profile of other starch biosynthetic genes using the oligoarrays. Other key starch biosynthetic genes present on the oligoarray but not the cDNA array platform.

Figure 8

Transcript profiles of regulatory genes. The x-axis shows the developmental stage. The y-axis represents the relative gene expression. Expression values are given as log 2-transformed normalized relative signal intensities, so that one unit on the y-axis represent an expression ratio of a factor of 2. GenBank accession numbers of the clones surveyed on the array are displayed on the chart. Transcript expression profiles of (a) 14-3-3 proteins; (b) kinases; (c) pyruvate orthophosphate dikinase; (d) WRKY TFs.

Figure 9

Schematic of the metabolic flux and expression of genes involved in sucrose to starch pathway in developing wheat seed. Black arrows indicate enzymatic reactions. Blue broken arrows represent transport of metabolites across amyloplast membranes. Enzyme and transporter names are in green. Upper box legend indicates level of gene expression and lower box legend indicates the time or developmental stage as days postanthesis. Only the expression of genes represented on the cDNA array is shown.