Fig 1.
Schematic representation of the chimeric RNAi hairpin construct.
Fig 2.
Relative expression of isoamylase isogenes in wild type (WT) and selected transgenic lines 7, 16 and 39 as determined by qRT-PCR.
Relative expression of ISA1, ISA2 and ISA3 were determined in (A) source leaves, (B) mature potato tubers, (C) in sprouting tubers. Values were normalized to Ubiquitin and displayed relative to the expression level of the wild type which was set to one. Values displayed are the mean +/- SD of three to four independent biological samples each with two technical replicates. Significant differences to wild type were calculated using a two-tailed t-test assuming equal variances and are indicated by asterisks (***p<0.001; ** p< 0.01, *p<0.05).
Fig 3.
Impact of reduced expression of ISA isogenes on soluble sugars and starch content in source leaves.
Metabolite contents in wild type and ISA- silenced plants were determined at the end of the light period (after 16h light) (light grey) and at the end of the dark period (after 8h darkness) (dark grey). A) starch B) sucrose, C) hexoses. Values represent the mean +/- SE of 5 biological and 2 technical replicates each.
Fig 4.
Effect of reduced ISA gene expression on tuber yield and content of sugars.
Parameters were determined from greenhouse-grown plants at harvest A) Tuber yield was determined from 5 plants each. B) starch content, C) sucrose content and D) hexose levels were determines from tuber parenchyma. Values represent the mean +/- SE of 5 biological and 2 technical replicates Significant differences to wild type were calculated using two-tailed t-test assuming equal variances and are indicated by asterisks (***p<0.001, *p<0.05).
Fig 5.
Determination of the starch granule size.
Starch granules from freshly harvested tubers were extracted, stained with iodine and inspected under the microscope. A) representative micrographs of iodine-stained starch granules derived from tubers of wild-type and ISA-silenced lines 7, 16 and 39. B) Representative image illustrating the segmentation of images by means of the ImageJ software tool to determine the relative size of each 2D starch granule. The software allows for the manual separation of granules connected to each other in the image.
Fig 6.
Starch granule size distribution.
Size distribution was determined as pixels’ size from ca. 150 starch granules of each replicate using ImageJ software as schematically shown in Fig 5. A) Granules were compiled into groups and the number of granules within a specific range (e.g. >25, >50, >75, etc.) were counted, calculated as relative percentage of total number and values cumulatively plotted. The inset shows the median granule size of each genotype. B) Bar charts of selected groups of relative starch granule sizes subjected to t-test test analysis. Values represent means +/- SE from 3 individual tubers (n = 3). Significant differences to wild type are indicated by asterisks (*p<0.05). Line 7 (light grey), line 16 (dark grey), line 39 (white) wild-type control (WT; black).
Fig 7.
Impact of ISA silencing on tuber sprouting.
Tubers of 5 plants each from wild type (WT) and transgenic lines 7, 16 and 39 were stored after harvest at room temperature in darkness. A) Sprouting kinetics. To monitor the impact on dormancy length, 2–5 similar sized tubers from each plant were picked (n = 13–20) and their sprouting behaviour was regularly scored over a 15-week period until 100% sprouting had been reached in wild-type tubers. A tuber was considered to sprout when sprouts of 2 mm length became visible. B) Photographs of transgenic (lines 7, 16, 39) and control tubers taken after 13 weeks of storage showing that the transgenic lines sprout earlier than the wild-type controls (WT). C) Number of sprouts per tuber. Number of sprouts formed per tuber were counted from 13–20 individual tubers. Values represent the mean +/- SE. Significant differences to wild type were calculated using two-tailed t-test assuming equal variances and are indicated by asterisks (**p<0.01, *p<0.05).
Fig 8.
Contents of sucrose and starch in tuber parenchyma tissue associated with the sprout.
Samples from wild-type and transgenic tubers were taken after 15 weeks of storage, when sprouts became visible in wild-type tubers. A) Schematic representation of a potato tuber to illustrate the sampling. A cork borer (# 2) was used to isolate the tissue approximately 5 mm below the bud indicated in red. It can be assumed that this region is enriched in vascular tissue. B) Starch content and C) sucrose content in the tissue below the growing sprout. Values represent the mean +/- SE of 5 independent tubers. Significant differences to wild type were calculated using two-tailed t-test assuming equal variances and are indicated by asterisks (**p<0.01, *p<0.05).
Fig 9.
Content of soluble glucans (phytoglycogen) in tubers.
The amount was determined at harvest (light grey) and after 4 months of storage in the parenchyma around growing sprout (dark grey). Error bars represent standard errors (n = 4–5). Asterisks indicate statistically significant differences (* p < 0.05 and ** p < 0.01).
Fig 10.
Structure of soluble glucan polymers determined as relative chain length distribution.
A) in sprout-associated tuber parenchyma and B) in tuber parenchyma not associated with a sprout. Line 7 (blue), line 16 (red), line 39 (grey) and the wild-type control (WT; black). Values represent mean +/- SE of 3–4 biological replicates (n = 3–4).
Fig 11.
Starch structure determined as relative chain length distribution.
Starch from sprouting tubers was either isolated from parenchyma associated with the sprout (A) or (B) from parenchyma not associated with the sprout. Line 7 (blue), line 16 (red), line 39 (grey) and the wild-type control (WT; black). Values represent mean +/- SE of 3–4 biological replicates.