Table 1.
Carbohydrate content.
Figure 1.
Phenotype of Col-0 and pgm2/3 plants in 12 h light/12 h dark regime.
A, Growth phenotypes. Photographs were taken of six-week-old plants. Bar = 1 cm. B, Fresh weight of plant rosettes. Values are means ± SD (n = 29−30). Plants were harvested after five weeks. Asterisks indicate significant difference from Col-0 (Student Test, P≤0.01). C, Leaf form from Col-0 and transgenic plants. Leaves were harvested from the middle of rosettes from six-week-old plants. Bar = 1 cm. D, Phosphoglucomutase activity in Col-0 and pgm2/3 plants. Crude extracts were subjected to native PAGE and subsequent PGM activity staining. Separation gel was 7.5% [T] and 25 µg protein was loaded per lane.
Figure 2.
Carbohydrate analysis of Col-0 and pgm2/3 plants.
A–E, Plants were grown under 12 h light/12 h dark conditions and after five weeks 7–8 plants were collected and homogenized per line. Values are means of four technical replicates (A–C), and three technical parallels (D–E) ± SD, respectively. A, Starch content. B–C, Soluble sugar content. D–E, Sugar phosphate content. Asterisks denote the significance levels comparing pgm2/3 mutants to Co1-0: * p≤0.01;** p≤0.05.
Figure 3.
Overlay heat map of the metabolite changes in pgm2/3 mutants in comparison with control (Co1-0) using false-color scale.
Red or blue indicate that the metabolite content is increased or decreased, respectively. Five-week-old plants were grown under 12 h light/12 h dark conditions and harvested at the end of light phase (EL) and dark phase (ED), and three replicates represented 3–4 plants were analyzed (two technical replicates each); asterisks denote the significance levels as comparing pgm2/3 mutants to Co1-0 : * p≤0.01;** p≤0.05.
Figure 4.
Roots and siliques of Col-0 and pgm2/3 plants.
A, Root length and morphology of Col-0 and pgm2/3 lines. Plants were grown on vertical MS plates without any external sugar and antibiotics under long day conditions (16 h light/8 h dark). Plants were two-week-old. Length of central roots was measured. Values are means ± SD (n = 26−35). Asterisks indicate significant difference from Col-0 (Student Test, P≤0.01) B, Mature Col-0 and pgm2/3d plants. Col-0 and pgm2/3 plants were six and 11- week-old, respectively. C, Morphology of siliques of Col-0 and pgm2/3 lines. D, pgm2/3d silique. Siliques were destained in chloral hydrate solution (2.5 g in 1 mL distilled water). Black arrows indicate absence of seeds. C–D, Plants were grown under 14 h light/10 h dark regime.
Table 2.
Amount of crystalline cellulose and of cell wall matrix in Col-0 and pgm2/3.
Figure 5.
Characterization of knock-out mutants lacking one cytosolic and the plastidial PGM.
A, Analysis of PGM activity in the Col-0 and pgm3 pgm1 and pgm2 pgm1 mutants using native PAGE and PGM activity staining. Separation gel 7.5% [T]. 35 µg proteins were loaded per lane. 1– Col-0, 2– pgm3, 3– pgm2, 4– pgm1, 5– pgm3 pgm1, 6– pgm2 pgm1. B, Analysis of floral stems development in Col-0 and different PGM knock-out plants. Plants were grown under long day conditions (14 h light/10 h dark). Days after germination were registered, when plants developed floral stems 1 cm long. Values are means ± SD (n = 24). a - significant difference from Col-0 (Student Test, p≤0.01), b - significant difference from pgm1 (Student Test, p≤0.01).
Table 3.
Starch and soluble sugar content in Col-0 and PGM knock-out mutants.
Figure 6.
Growth phenotype of cp-pgm plants.
A, Seeds were sowed on MS medium containing sucrose and antibiotics (kanamycin [50 µg/mL], hygromycin [50 µg/mL]). Plants were grown under long day conditions (16 h light/8 h dark) and were two-week-old. Bar = 1 cm. B, cp-pgm plant before trypan blue staining. C, Col-0 and cp-pgm plants after trypan blue staining. The cp-pgm plant was five- week-old, germinated on MS plate (as above) and the two last weeks grown under continuous illumination. Leave of Col-0 from three-week-old plant grown under 12 h light/12 h dark conditions. Bars = 1 cm. D–F, Phenotype of cp-pgm plants under continuous illumination. Seeds were germinated on MS medium containing sucrose with antibiotics (kanamycin [50 µg/mL], hygromycin [50 µg/mL]). After four weeks plants were transferred to soil and grown further under continuous illumination. D, Plant was six-week-old. Bar = 1 cm. E–F, Flower buds of cp-pgm transgenic plants. Plant was six-week-old (E) and seven-week-old (F). Bars = 1 mm.