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First published online October 26, 2007; 10.1104/pp.107.108340 Plant Physiology 145:1670-1680 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
Arabidopsis Seedlings Deficient in a Plastidic Pyruvate Kinase Are Unable to Utilize Seed Storage Compounds for Germination and Establishment1,[OA]Department of Energy Plant Research Laboratory (C.A.), Department of Plant Biology (C.A.), and Department of Biochemistry and Molecular Biology (C.B.), Michigan State University, East Lansing, Michigan 48824
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 (PKp) 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 PKp 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 PKp is required for the efficient conversion of sugar into precursors for different anabolic pathways.
For plants, the challenges of reproduction vary and are greatly influenced by the environment in which they take root. Regardless of such factors, the packaging of nutrient reserves in seeds has evolved as a major strategy to ensure the survival of the next generation. These reserves (oil in the form of triacylglycerol [TAG], proteins, and carbohydrates) are used to fuel the germination and establishment of a plant's offspring. Germination initiates with release from dormancy and seed imbibition, and is finished when the radicle emerges through the seed coat (Bewley, 1997  ). Establishment involves the transition from a heterotrophic to a photoautotrophic seedling. In wild-type Arabidopsis (Arabidopsis thaliana) germination is typically completed after 24 h and is largely driven by the metabolism of storage reserves other than lipids, while seed oil is used for subsequent seedling establishment (Penfield et al., 2005; Cernac et al., 2006).
Carbohydrates in the form of Suc stored in Arabidopsis embryos (Baud et al., 2002
Establishment and growth of a seedling not only require the breakdown of energy-rich compounds, but also the synthesis of an array of metabolites used for growth. Glycolysis is very important for this process as it produces precursors for a variety of biosynthetic pathways. The final enzyme of glycolysis, pyruvate kinase (PK; EC 2.7.1.40), which converts phosphoenolpyruvate (PEP) into pyruvate (Pyr) with the concomitant production of ATP, occupies one of the most connected nodes in the plant metabolic network (AraCyc metabolic map; www.arabidopsis.org/tools/aracyc/). The occurrence of plant PKs in the plastid (PKp) and cytosol (PKc) almost certainly reflects the unique roles for the respective enzymes (Plaxton and Podesta, 2006
We recently described the seed-specific phenotype of an Arabidopsis T-DNA insertion mutant (pkp1) disrupted in the gene (At5g52920) encoding the PKp-β1 subunit of a plastid-localized PK complex (Andre et al., 2007
Seed Germination and Seedling Establishment Are Aberrant in pkp1
The seed-specific phenotypes of the pkp1 mutant and of pkp1 rescued by ectopic overexpression of PKp-β1 (Rβ1-23) or by ectopic overexpression of PKp-β2 (Rβ2-3) have been reported (Andre et al., 2007
Germination assays were conducted to explore the possibility that pkp1 seeds do not germinate as fast as wild-type seeds. Figure 2A shows the germination rates of the wild type, pkp1, and the rescued lines determined from independent experiments performed in the presence of 55 mM Suc using seeds of the same age (see "Materials and Methods" for details). After 1 d almost 100% of wild-type and Rβ1-23 and about 75% of Rβ2-3 seeds had germinated, whereas in pkp1 less than 20% of the radicles had emerged. Not until 5 DAS did pkp1 reach its maximum germination percentage, which is about 90%. Again, exogenous amino acids did not rescue the pkp1 phenotype. PK activity was measured at pH 8.0 to ascertain if reductions in enzyme activity are correlated with the delayed germination in pkp1. Imbibed and stratified seeds served as the 0-DAS time point and at this time all of the lines had about the same PK specific activity (Fig. 2B). Twenty-four hours later, there was a doubling of PK specific activity in the wild type and a similar, although less intense, induction in the rescued lines. By 5 DAS, activity had returned to that at the starting point, where it remained for the rest of the time course. The pattern of PK induction was skewed in pkp1 and did not peak until 3 DAS, after which it steadily declined. There is indeed a correlation between delayed induction of PK specific activity and germination in pkp1.
Developing seeds of pkp1 accumulate carbohydrates in the form of Suc and starch late during embryogenesis (Andre et al., 2007 ). Therefore, it is reasonable to postulate that the mutant will be more sensitive to exogenous sugar in the medium. To test this hypothesis, germination assays were performed on agar plates containing 0, 55, or 110 mM Suc. A very subtle delay in germination was observed for the wild type with increasing sugar concentration (Fig. 3A
, top). In all treatments, though, germination of wild-type seeds reached a maximum by 2 DAS. As predicted, pkp1 is more sensitive to sugar in the medium. In the absence of Suc, the maximum number of pkp1 seeds had germinated by 3 DAS (Fig. 3A, bottom). With 55 mM, this time was extended to 5 DAS. On 110 mM Suc only 50% of the pkp1 seeds had germinated at 5 DAS and not until 10 DAS was the maximum achieved. To control for effects of osmoticum, pkp1 germination was measured in the presence of the nonmetabolizable sugar alcohol sorbitol. The germination rate of the wild type (not shown) and pkp1 (Fig. 3B) was unaffected by any of the tested concentrations of sorbitol, indicating that the inhibition caused by Suc is indeed a result of the sugar itself and not differences in osmotic potential.
Accumulation of soluble sugars in seed tissue is presumably a prerequisite for the sugar-induced inhibition of germination. The pkp1 germination rate is lower than the wild type in the absence of sugar and is more easily inhibited by the application of Suc. Based on this observation, we hypothesized that imbibed and germinating pkp1 seeds have increased sugar levels relative to the wild type. Figure 4 shows the results of sugar measurements performed on seeds germinated in the absence of exogenous Suc. In the wild type, hexose content slightly increased after 1 d and was lowest at 3 DAS, while Suc steadily declined over the time course. These results for the wild type follow the same trends that were observed in a previous study (Fait et al., 2006 ). The pkp1 mutant contained roughly 2.5 and 1.5 times as much hexose and Suc, respectively, at 0 DAS, both of which also decreased over time. By 3 DAS, Suc in pkp1 was at the same level as the wild type, while hexoses remained in about 2.5-fold excess.
Storage Lipid Metabolism Is Defective in pkp1
The accumulation of excess soluble sugar and the fact that root growth is not fully restored by the application of Suc suggests that pkp1 may be defective in either catabolizing its storage reserves or in synthesizing cellular components needed for growth. Hypocotyl elongation assays in the dark are a standard means of examining storage oil metabolism in germinated seeds. When grown in the dark in the presence or absence of Suc, wild-type hypocotyls elongate (Fig. 5A
). The hypocotyls of pkp1 do not elongate in medium without an exogenous carbon source, which is typical of oil-deficient mutants such as wrinkled1 (Cernac et al., 2006
To examine whether storage lipid metabolism was affected in pkp1, we measured the rate of oil degradation in seedlings grown on 55 mM Suc. Very-long-chain fatty acids, which in Arabidopsis are specific to seed TAG (Lemieux et al., 1990 ), were used as markers for storage oil content. In the wild type and the rescued lines, storage oil began to be metabolized at 2 DAS and was essentially depleted by 6 DAS (Fig. 6A
). Interestingly, storage lipids were not used by pkp1 seedlings (Fig. 6A). An increase in the proportion of linolenic acid (18:3) in membrane lipids, which is a major component of thylakoid membranes, was used as a marker for anabolic lipid metabolism in seedlings. By 5 DAS, the proportion of 18:3 to other fatty acids had increased by about 30% in the wild type and the rescued lines (Fig. 6B). In pkp1 the increase was limited to about 5%. From these data it is clear that lipid metabolism is abnormal in pkp1 seedlings.
The root growth and lipid phenotypes of pkp1 seedlings are reminiscent of Arabidopsis mutants that lack seed tocopherol, vitamin e1 (vte1) and vte2 (Sattler et al., 2004 ). Therefore, tocopherols were extracted from dry seeds and were quantified with HPLC. Table II
shows that pkp1 seeds contained 25% less total tocopherols than the wild type. All of this reduction can be accounted for by decreased  -tocopherol, whereas  - and  -tocopherol are actually increased in pkp1.
PKp Is Required for Normal Plant Growth A plant growth time course was recorded to determine if the pkp1 metabolic defect has an impact beyond seed germination and establishment. Seeds were germinated on 55 mM Suc and were transferred to soil at 10 DAS. The pkp1 seedlings that successfully established continued to experience difficulties in growth after being transferred to soil. Biomass production was limited and around the time of flowering initiation (25 DAS) pkp1 aerial parts weighed approximately half compared to the wild type (Fig. 7A ). When taking into account the delay in germination, the difference in growth was much less severe and pkp1 followed the same developmental time course as the wild type. By 35 DAS both genotypes were well into flowering and pkp1 was nearly equal to the wild type in size (Fig. 7B). Another noticeable phenotype of pkp1 was slight chlorosis. When grown on soil, total chlorophyll content was reduced by 30%, accompanied by a moderate increase in anthocyanins (Table III ). This phenotype was exacerbated in plants propagated on agar plates in lower light conditions (photon flux densities of 100–120 and 60–80 µmol m–2 s–1 for soil and agar growth, respectively). PK enzyme assays were performed to see if the morphological differences of pkp1 are correlated with a reduction in activity. The data in Figure 7C establish that PKp activity in pkp1 leaves only reached about 60% of wild-type levels.
Lipid metabolism is aberrant in pkp1 seeds and seedlings and so it seemed reasonable to presume that there also is an effect in leaves of mature plants. However, examination of leaf lipids revealed very little qualitative, if any, difference between the wild type and pkp1. Total leaf lipids were extracted from 25-DAS plants and analyzed by TLC and there were no differences between the membrane lipid profiles of the wild type and pkp1. Analysis of the fatty acid composition of pkp1 revealed a very subtle 5% decrease in 18:3 content, which was accompanied by 3% increases in each of oleic acid (18:1) and linoleic acid (18:2). This result is not totally unexpected as the same trend was seen in seedlings, although to a much greater extent (Fig. 6). Reduced biomass production and chlorophyll content of pkp1 could reflect altered carbohydrate metabolism. Therefore, soluble sugars and starch were extracted from leaves during the day and night and were quantified. Hexose content (Glc and Fru only) was reduced by more than half in pkp1 during both the day and the night (Fig. 8 ). On the other hand, Suc and starch accumulation were unaffected. Collectively, the effects of loss of PKp activity in pkp1 were more pronounced in germinating seeds and seedlings than in mature plant tissue.
PKp Activity Is Required for Germination
We observed delayed germination of pkp1 seeds that was intermediate to what was observed for several other PKp-deficient lines (Fig. 2A; Baud et al., 2007
Addition of an exogenous carbon source does not rescue the germination defect of pkp1. In fact, increasing the amount of Suc actually inhibits germination (Fig. 3A). The oil-deficient wrinkled1 and triacylglycerol1 mutants have a similar phenotype and in those cases it was attributed to heightened sensitivity to osmolarity (Lu and Hills, 2002
The pkp1 mutant had 60% less seed oil than the wild type and did not elongate its hypocotyls in the absence of Suc (Fig. 5A; Baud et al., 2007
The pkp1 mutant does not efficiently metabolize its seed oil reserves (Fig. 6A) and this could also contribute to faulty seedling establishment and hypocotyl elongation. Lipid breakdown may be inhibited in pkp1 by the presence of excess sugar, which has been shown to inhibit oil catabolism in Arabidopsis (Martin et al., 2002
The effects of the pkp1 mutation are evident in mature plants. As noted by Baud and coworkers (Baud et al., 2007
Here, we have provided evidence that PKp has an important role in catabolizing storage compounds in germinating seeds, apparently for the purpose of feeding various plastid-localized biosynthetic pathways. Similar to what was observed in developing seeds, cytosolic glycolysis was unable to compensate for the loss of PKp activity. Specific and regulated metabolite exchange between cellular compartments is a function of membrane-localized transporters enabling cells to maintain distinct subcellular metabolite pools. At the same time, compartment-specific isoforms of enzymes contribute to the maintenance of subcellular metabolite pools. As such, PKp described here provides an elegant example of the exquisite partitioning of metabolism in plant cells.
Plant Growth Conditions Wild-type plants were of the Columbia-2 ecotype, while pkp1 and the respective rescued lines were in the Columbia-0 background. All seeds were sterilized with 20% bleach, 0.05% Triton X-100 for 15 min and were rinsed five times in sterile water. Medium used for germination and growth on agar plates was full-strength Linsmaier and Skoog (Caisson Laboratories), pH 5.7, 0.9% agar and included 0, 55, or 110 mM Suc when appropriate. Seeds were stratified at 4°C for 3 d prior to being put into an incubator with a photon flux density of 60 to 80 µmol m–2 s–1 and a light period of 16 h (22°C) and a dark period of 8 h (18°C). After 10 d, seedlings were either transferred to soil or to fresh agar plates. Soil-grown plants were put into a 16-h photoperiod with a day temperature of 22°C and a night temperature of 20°C at a photon flux density of 100 to 120 µmol m–2 s–1. Plant growth measurements were done on plants transferred to soil at 10 DAS. The aerial portions of six individuals were used for each time point.
For these assays, seeds were sown in a straight line and the agar plates were arranged vertically. Root lengths were measured to the nearest millimeter every 24 h. The same set of agar plates was used throughout the experiment. Hypocotyl elongation assays were performed using 7-DAS seedlings as described previously (Penfield et al., 2004
Seeds from various genetic backgrounds were produced from mother plants grown in identical conditions, were harvested at the same time, and were after-ripened for 3 months. Germination assays were routinely performed using the plant growth procedures mentioned above. When appropriate, 0, 55, or 110 mM Suc (or sorbitol) was added to the medium prior to being autoclaved. Germination was scored as radicle emergence from the seed coat and was determined every 24 h using the same set of horizontally grown agar plates. Amino acids were suspended in water and were filter sterilized and added to the medium at a final concentration of 0.3 mM.
Crude protein extracts were prepared by grinding tissue in approximately 10 volumes (µL/mg) of buffer containing 50 mM Tris-Cl, pH 7.5, 5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1% Triton X-100, 10% glycerol, and a protease inhibitor mix (Complete mini; Roche). PK activity was coupled to the conversion of NADH to NAD+ by lactate dehydrogenase. Reactions were kept at 25°C, were started by the addition of enzyme mix, and were linear for at least 5 min. Absorbance at 340 nm was measured using a FLUOstar Optima 96-well plate reader (BMG Labtech). The PK reaction mixtures contained 50 mM HEPES-KOH, pH 8.0, 5% PEG-8000, 50 mM KCl, 15 mM MgCl2, 1 mM dithiothreitol, 2 mM PEP, 1 mM ADP, 0.2 mM NADH, and 2 units mL–1 desalted rabbit muscle lactate dehydrogenase. PEP phosphatase activity was corrected for by omitting ADP from the reaction. Reactions were conducted at pH 8.0 to preferentially detect PKp activity. Protein was quantified using Bradford reagent (Sigma).
Total leaf lipids were extracted from preweighed tissue by vigorously shaking for 5 min in 500 µL of methanol:chloroform:formate (2:1:0.1, v/v). Then 250 µL of 1 M KCl, 0.2 M H3PO4 was added and the tubes were vortexed. The phases were separated by centrifugation at 16,000g for 5 min. The organic phase was loaded quantitatively onto a treated [soaked in 0.15 M (NH4)2SO4 and dried, then heated to 120°C for 2.5 h] silica-250 TLC plate (Baker). The solvent system used was acetone:toluene:water (91:30:7, v/v), and staining was done with iodine and
Chlorophyll was extracted from leaves and seedlings using 100 volumes (µL/mg) of 80% acetone and was quantified as described previously (Lichtenthaler, 1987
We thank Maria Magallanes-Lundback for her help with determining tocopherol contents. Received August 29, 2007; accepted October 24, 2007; published October 26, 2007.
1 This work was supported in part by grants from the Michigan Agriculture Experiment Station and by BASF Plant Sciences LLC.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Christoph Benning (benning{at}msu.edu).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.108340 * Corresponding author; e-mail benning{at}msu.edu.
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