Arabidopsis seedlings deficient in a plastidic pyruvate kinase are unable to utilize seed storage compounds for germination and establishment

Abstract

Catabolism of storage reserves and biosynthesis of metabolites necessary for growth are essential for seed germination and establishment. An Arabidopsis (Arabidopsis thaliana) mutant (pkp1) deficient in plastidic pyruvate kinase (PK(p)) and unable to accumulate storage oil to the same extent as the wild type shows delayed germination and seedling establishment dependent on an exogenous sugar supply. It appears, however, as though these phenotypes are not entirely caused specifically by lack of seed oil and may be related to reduced PK(p) activity in germinating seeds. Increasing the sucrose concentration in the medium further inhibits germination of pkp1, possibly due to the accumulation of soluble sugars in seeds. Germinating seeds of pkp1 are unable to metabolize storage oil and cannot utilize applied sucrose for hypocotyl elongation in the dark. Moreover, pkp1 contains less tocopherol and chlorophyll than the wild type. Taken together, the results are consistent with a model in which PK(p) is required for the efficient conversion of sugar into precursors for different anabolic pathways.

Figure 1.

Root elongation of the wild type (WT; black diamonds), pkp1 (white squares), pkp1 rescued with cauliflower mosaic virus 35S-driven expression of a cDNA encoding PK_p-β₁ (Rβ₁-23; black squares), and pkp1 rescued with cauliflower mosaic virus 35S-driven expression of a cDNA encoding PK_p-β₂ (Rβ₂-3; black triangles) in the presence of 55 mm Suc. Values are the mean ± sd, n = 25.

Figure 2.

Delayed germination and induction of PK activity in pkp1. A, Seed germination in the presence of 55 mm Suc. Plant lines and symbols are as described in Figure 1. Values are the mean ± sd, n = 4. B, PK activity measured at pH 8.0 in germinating seeds and seedlings grown on 55 mm Suc. Plant lines and symbols are as described in Figure 1. One milliunit is 1 nmol Pyr formed per minute. Values are the mean ± sd, n = 4.

Figure 3.

Germination response of pkp1 to sugar and osmoticum. A, Germination rate of the wild type (WT; top) and pkp1 (bottom) in the presence of 0, 55, or 110 mm Suc. Values are the mean ± sd, n = 3. B, Effect of osmolarity on the germination rate of pkp1. Sorbitol (Srb) was given at 0, 55, or 110 mm. Values are the mean ± sd, n = 3.

Figure 4.

Soluble sugar amounts in the wild type (WT) and pkp1 imbibed (0 DAS) and germinating seeds (1 and 3 DAS). Hexose (Glc and Fru) content is shown in the top panel and Suc content in the bottom panel.

Figure 5.

Hypocotyl elongation and PK activity in dark-grown seedlings. A, Length of hypocotyls 7 DAS of plants grown in the dark in the presence (+) or absence (−) of 55 mm Suc. Plant lines are as described in Figure 1. Values are the mean ± sd, n = 25. B, PK activity measured at pH 8.0 of etiolated seedlings grown with 0 mm (top) or 55 mm (bottom) Suc. Plant lines and symbols are as described in Figure 1. One milliunit is 1 nmol Pyr formed per minute. Values are the mean ± sd, n = 4.

Figure 6.

Fatty acid composition of germinating seeds and seedlings. A, Seed oil-specific very-long-chain fatty acid (20:0, 20:1, 21:0, 21:1) content in seedlings grown on 55 mm Suc. Plant lines and symbols are as described in Figure 1. Values are the mean ± sd (n = 4). B, Amount (mol%) of linolenic acid (18:3) in seedlings grown on 55 mm Suc. Plant lines and symbols are as described in Figure 1. Values are the mean ± sd (n = 4).

Figure 7.

Tissue mass and PK activity in mature pkp1 plants. A, Plant growth time course of plants germinated on Murashige and Skoog with 55 mm Suc and transferred to soil at 10 DAS. At each time point, aerial portions of six plants were excised and weighed. Values are the mean ± sd. B, Wild-type (WT) and pkp1 plants 35 DAS. C, PK activity measured at pH 8.0. For each time point, five whole plants were homogenized and used to prepare protein extracts. Day = 8 h after lights on. Night = 8 h after lights off. One milliunit is 1 nmol Pyr formed per minute. Values are the mean ± sd (n = 4).

Figure 8.

Carbohydrate content of 25-d-old leaves. Hexose (Glc and Fru, top), Suc (middle), and starch (bottom) content of rosette leaves is shown. Five entire plants were homogenized and used for extraction. Day/night cycle used was 16 h light and 8 h dark. Day = 8 h after lights on. Night = 8 h after lights off. Values are the mean ± sd (n = 4). FW, Fresh weight.