Spatiotemporal profiling of starch biosynthesis and degradation in the developing barley grain

Abstract

Barley (Hordeum vulgare) grains synthesize starch as the main storage compound. However, some starch is degraded already during caryopsis development. We studied temporal and spatial expression patterns of genes coding for enzymes of starch synthesis and degradation. These profiles coupled with measurements of selected enzyme activities and metabolites have allowed us to propose a role for starch degradation in maternal and filial tissues of developing grains. Early maternal pericarp functions as a major short-term starch storage tissue, possibly ensuring sink strength of the young caryopsis. Gene expression patterns and enzyme activities suggest two different pathways for starch degradation in maternal tissues. One pathway possibly occurs via alpha-amylases 1 and 4 and beta-amylase 1 in pericarp, nucellus, and nucellar projection, tissues that undergo programmed cell death. Another pathway is deducted for living pericarp and chlorenchyma cells, where transient starch breakdown correlates with expression of chloroplast-localized beta-amylases 5, 6, and 7, glucan, water dikinase 1, phosphoglucan, water dikinase, isoamylase 3, and disproportionating enzyme. The suite of genes involved in starch synthesis in filial starchy endosperm is much more complex than in pericarp and involves several endosperm-specific genes. Transient starch turnover occurs in transfer cells, ensuring the maintenance of sink strength in filial tissues and the reallocation of sugars into more proximal regions of the starchy endosperm. Starch is temporally accumulated also in aleurone cells, where it is degraded during the seed filling period, to be replaced by storage proteins and lipids.

Figure 1.

Starch accumulation in the developing barley grains. A and B, Starch, ADP-Glc, and amylose contents in pericarp (A) and endosperm (B). FW, Fresh weight. C to H, Spatial distribution of starch granules (visualized in black) in the developing grains in a longitudinal section (C) and in median transverse sections of developing grains at anthesis (C), 2 DAF (D), 4 DAF (E), 5 DAF (F), 8 DAF (G), and 14 DAF (H). al, Aleurone; em, embryo sac; es, endosperm; mvt, main vascular tissue; np, nucellar projection; nu, nucellus; p, pericarp; tc, transfer cells. Bars = 500 μm in C to E and 1 mm in F to H.

Figure 2.

Transcript profiles of genes involved in starch synthesis in pericarp (left) and endosperm (right) fractions of developing barley grains. Transcript levels were determined by cDNA macroarray or northern-blot analyses for AGPase (A), starch synthases (B), starch-branching enzymes (C), and starch-debranching enzymes (D). The relative values were calculated as means of at least two independent experiments and are shown in normalized signal intensities.

Figure 3.

In situ localization of transcripts of small subunits of AGPase in developing barley caryopses. The top left panel shows a median transverse section of a developing caryopsis with the regions shown in A to E. Hybridization sites in A to E are visualized as white signals (indicated by red arrows). A, AGP-S1a transcripts were not detected in the developing caryopsis at 4 DAF. B, AGP-S1a mRNA is localized exclusively in endosperm at 14 DAF. C and D, Expression patterns of the AGP-S1b gene at 4 DAF (C) and 6 DAF (D). E, Endosperm-specific localization of AGP-S2 mRNA at 14 DAF. es, Endosperm; np, nucellar projection; pe, pericarp. Bars = 500 μm.

Figure 4.

Transcript profiles of genes involved in starch degradation in pericarp (left) and endosperm (right) fractions of developing barley grains. Transcript levels were determined by macroarray or northern-blot analyses for α-amylases (A), β-amylases (B), α-glucan phosphorylases (C), glucan, water dikinases (D), and disproportionating enzyme (E). The bars represent relative values calculated from normalized signal intensities derived from at least two independent experiments.

Figure 5.

In situ localization of α-amylase AMY1 transcripts (visualized in black and indicated by red arrows) in the pericarp of developing barley grains at anthesis (A), 4 DAF (B), and 8 DAF (C). es, Endosperm; np, nucellar projection; pe, pericarp. Bars = 250 μm.

Figure 6.

Distribution of starch granules and in situ localization of α-amylase AMY4 transcripts in developing barley grains. A and D, Transverse sections of barley grains at 2 DAF (A) and 14 DAF (D), toluidine blue staining. B and E, Distribution of starch granules in nucellus (B) and nucellar projection (E; visualized in black and shown by red arrows). C, Localization of AMY4 transcripts in outer cell rows of the nucellus and developing vascular tissue at 2 DAF (indicated by red arrows), bright-field visualization. F, Localization of AMY4 transcripts in the nucellar projection at 14 DAF, dark field visualization. es, Endosperm; np, nucellar projection; nu, nucellus; pe, pericarp; tc, transfer cells. Bars = 120 μm in A, C, D, and F and 250 μm in B and E.

Figure 7.

Expression patterns of AMY1 and AMY4 α-amylase genes in different barley tissues as determined by northern-blot hybridization. Total RNA (10 μg per line) isolated from different barley tissues was subsequently hybridized with 3′ UTRs of AMY1 (A) and AMY4 (B) cDNA. Hybridization with a 25S rDNA probe (C) was performed as a loading control. HAI, Hours after imbibition.

Figure 8.

In situ localization of β-amylase BAM1 transcripts (A and B) and distribution of starch granules (C and D) in developing caryopses at 12 DAF (A and C) and 16 DAF (B and D). The BAM1 mRNA was detected (indicated by red arrows) in transfer cell layer (A) and in aleurone/subaleurone (A and B). These tissues contain only small or no starch granules (C and D). al, Aleurone; es, endosperm; np, nucellar projection; pe, pericarp; tc, transfer cells. Bars = 250 μm.

Figure 9.

Activities (in units per gram fresh weight [FW]) of α-amylase (AMY) and β-amylase (BAM) enzymes in pericarp (left) and endosperm (right) fractions of developing barley grains.

Figure 10.

Schemes illustrating the involvement of differing suites of isoforms of starch biosynthetic enzymes in pericarp and endosperm (A), and proposed starch degradation pathways in the pericarp of developing barley grains (B). A, Distinct genes encoding enzymes of starch synthesis are expressed in maternal and filial tissues of developing barley grains. Genes in red are those that are exclusively expressed in the respective tissue. AGP-L, Large subunit of AGPase; AGP-S, small subunit of AGPase; GBSS, granule-bound starch synthase; ISA, isoamylase; PHO, glucan phosphorylase; PUL, pullanase (limit dextrinase); SBE, starch-branching enzyme; SS, starch synthase. B, The proposed pathways of starch breakdown in different cells of the pericarp. In the cells undergoing programmed cell death, starch degradation possibly occurs via AMY1 and AMY4 α-amylases. Linear maltooligosaccharides released by the action of α-amylases and an unidentified DBE may provide substrates for the BAM2 β-amylase. The question mark indicates an unidentified enzyme converting maltose in Glc. In living cells of the pericarp, GWD1 and PWD may phosphorylate the surface of the starch granule, making it accessible for β-amylase action. BAM5, BAM6, and BAM7 β-amylases may be involved in maltose production, acting either at the granule surface or on linear maltooligosaccharides. The action of ISA3 on the granule may release soluble maltooligosaccharides. Short oligosaccharides can be metabolized by DPE1, liberating Glc and larger maltooligosaccharides for continued degradation. After transport in cytosol, maltose may be further converted to Glc by DPE2.