Fig 1.
Growth phenotype and carbohydrate analyses of mutants and parental lines.
(A) Screening of generated double and triple mutants. One leaf (comparable sizes) from each plant was harvested and proteins were isolated in 120 μl of extraction buffer. A total of 25 μl of each was loaded per lane. DPE2 and PHS proteins were separated by discontinuous native PAGE, and their activities were detection. For GWD detection, crude extracts were separated by SDS-PAGE followed by western blot and immunodetection against GWD. (B) Growth phenotype of the generated mutants and wild type. Plants were six -weeks-old. (C) Starch content. All values are mean ± SD (n = 3–4 replicates from a mixture 4–10 plants each). Letters indicate samples that were not significantly different (P<0.05) according to one-way ANOVA with Holm-Sidak post hoc testing. The third bar indicates the level of starch degraded by the end of the night phase (starch content at the end of the day minus starch content at the end of the night). Triangles represent the amount of starch degraded at the end of the night [%]. (D) Semi quantitative starch determination. Plants were harvested at the end of the light phase (11 h in the light), decolorized in hot 80% (v/v) ethanol, and stained with iodine solution.
Fig 2.
Transmission electron microscopy analysis of generated lines.
Six-week-old plants were harvested in the middle of the light phase. Bars = 2 μm.
Fig 3.
Scanning electron microscopy analysis of native starch granules isolated from leaves of the various mutants and wild type.
Starch granules were isolated from the leaves of six-week-old plants harvested at the end of the light phase. Bars = 2 μm.
Fig 4.
Chain length distribution (CLD) pattern of starch granules isolated from dpe2-5/phs1b, dpe2-5/phs1b/sex1-8, and Col-0.
CLD profiles of isolated starch granules after heat solubilization and subsequent isoamylase treatment. All values are mean ± SD (n = 3). DP- degree of polymerization.
Fig 5.
Semi-quantitative starch determination in plants under prolonged dark conditions.
Plants were grown under 12 h light/12 h dark conditions for 5 weeks.
Table 1.
Estimated sizes of native starch granules.
Values represent average ± SD (n = 27–35). * Granules are deformed; the largest part of the granule was measured. Letters indicate samples that were not significantly different (p<0.05) according to one-way ANOVA with Holm-Sidak posthoc testing.