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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

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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Arabidopsis Seedlings Deficient in a Plastidic Pyruvate Kinase Are Unable to Utilize Seed Storage Compounds for Germination and Establishment1,[OA]

Carl Andre and Christoph Benning*

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


   ABSTRACT
TOP
 ABSTRACT
RESULTS
 DISCUSSION
 CONCLUSION
 MATERIALS AND METHODS
 LITERATURE CITED
 
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, 1997Go). 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., 2005Go; Cernac et al., 2006Go).

Carbohydrates in the form of Suc stored in Arabidopsis embryos (Baud et al., 2002Go) are likely used to fuel germination. The breakdown of Suc proceeds through glycolysis, and the induction of this pathway during germination is necessary for and is reflected by rapid increases in the amounts of downstream biosynthetic products, e.g. organic acids and amino acids (Fait et al., 2006Go). Additionally, the ATP produced by glycolysis could be an important source of energy in the absence of photosynthesis. The breakdown of sugar within the embryo may also contribute to relief from dormancy as excess Glc is known to delay germination (Zhou et al., 1998Go; Dekkers et al., 2004Go). Seedling establishment and hypocotyl elongation in the dark are driven by the catabolism of seed storage oil (TAG). Fatty acids cleaved from the glycerol backbone of TAG enter the glyoxysome, where they are subjected to β-oxidation. Arabidopsis mutants defective in β-oxidation (Germain et al., 2001Go; Footitt et al., 2002Go; Lawand et al., 2002Go; Pinfield-Wells et al., 2005Go) typically fail to progress beyond germination and do not establish unless provided with an exogenous carbon source. Similar phenotypes are observed in glyoxylate cycle and gluconeogenesis mutants, which are unable to convert acetyl-CoA into TCA intermediates and ultimately carbohydrates (Eastmond et al., 2000Go; Rylott et al., 2003Go; Cornah et al., 2004Go; Penfield et al., 2004Go; Pracharoenwattana et al., 2005Go). Of course, mutants that do not accumulate seed oil to the same extent as the wild type, such as wrinkled1 and triacylglycerol1, are also impaired in their ability to establish in the absence of an exogenous sugar source (Lu and Hills, 2002Go; Cernac et al., 2006Go).

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, 2006Go). For example, Pyr transported into mitochondria for entry into respiration is most likely derived from PKc, especially when considering the lack of a reported plastidic Pyr transporter (Weber, 2004Go). Plastid-localized metabolic pathways that use PEP and Pyr are no doubt influenced by PKp activity. Two enzymes in the shikimate pathway, 3-deoxy-D-arabino-heptulosonate-7-P synthase and 5-enolpyruvylshikimate-3-P synthase, use PEP as a substrate (Herrmann and Weaver, 1999Go). The final product of this pathway, chorismate, is metabolized into aromatic amino acids that themselves are the starting points for the synthesis of a variety of secondary metabolites (e.g. anthocyanins). The plastidic Pyr dehydrogenase complex, which produces acetyl-CoA for fatty acid synthesis, acts on Pyr. The first enzyme of the methylerythritol-4-P (MEP) pathway, 1-deoxy-D-xylulose-5-P synthase, uses Pyr and glyceraldehyde-3-P to synthesize the first intermediate of plastidic isoprenoid synthesis (Lichtenthaler, 1999Go). Plastid-derived isoprenoids include the carotenoids and phytol used in the biosynthesis of chlorophyll and tocopherol. In addition, Pyr is a substrate in the biosynthetic pathways of Val, Lys, and Ile, and can be directly converted to Ala by a transaminase. Clearly, the ratio of PEP to Pyr must be balanced, in large part by PKp, such that these biochemical pathways function properly.

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., 2007Go). The pkp1 mutant did not synthesize fatty acids at the same rate as the wild type and this was reflected by a 60% reduction in the amount of seed oil. This lack of seed oil caused an establishment defect when sown on soil that could be rescued by growth on agar plates containing Suc. A more recent paper confirmed these seed phenotypes using several new PKp-deficient mutants and touched on the germination defect of the seedlings (Baud et al., 2007Go). Here, we extended previous analyses of the pkp1 mutant and discovered that delayed germination is accompanied by an apparent defect in storage compound and sugar catabolism. Additionally, we found changes in specific metabolite pools in pkp1 that highlight the importance of PKp in regulating the supply of precursors for plastid metabolism by participating in the breakdown in carbohydrates.


   RESULTS
TOP
 ABSTRACT
 RESULTS
DISCUSSION
 CONCLUSION
 MATERIALS AND METHODS
 LITERATURE CITED
 

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., 2007Go). During routine growth of these plants it was observed that pkp1 seedlings do not establish (defined as development of true leaves and root elongation) when sown directly on soil and will not unless provided with Suc on agar medium. Application of Ala, aromatic, or branched-chain amino acids in the presence or absence of Suc did not result in improved establishment. Growth of pkp1 was retarded even when provided with Suc. Root elongation assays were performed with 55 mM Suc in the medium to quantify this defect. As shown in Figure 1 , pkp1 roots did not grow at the same rate as those of the wild type or either of the rescued lines, suggesting an inability to fully utilize the available carbon source. The initiation of root growth itself may have been delayed in pkp1 as at 3 d after sowing (DAS) its roots had not elongated past 1 mm. One possible explanation for the delay is that germination itself was inhibited in these seedlings.


Figure 1
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  		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 PKp-β1 (Rβ1-23; black squares), and pkp1 rescued with cauliflower mosaic virus 35S-driven expression of a cDNA encoding PKp-β2 (Rβ2-3; black triangles) in the presence of 55 mM Suc. Values are the mean ± SD, n = 25.

 
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.


Figure 2
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  		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.

 
Developing seeds of pkp1 accumulate carbohydrates in the form of Suc and starch late during embryogenesis (Andre et al., 2007Go). 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.


Figure 3
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  		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.

 
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., 2006Go). 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.


Figure 4
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  		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.

 

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., 2006Go). However, even when provided with 55 mM Suc, pkp1 hypocotyls do not elongate, a phenotype that is rescued in the complemented lines (Fig. 5A). Dark-grown seedlings must generate ATP mainly by glycolysis and respiration, whether fueled by endogenous storage reserves or by uptake of an exogenous carbon source. In either case, PK is important for the substrate-level phosphorylative generation of ATP and for the production of respiratory precursors. Thus, a reduction in PK activity could help explain the hypocotyl elongation phenotype of pkp1 when grown with Suc. Indeed, PK specific activity is relatively low in pkp1 etiolated seedlings when grown with or without exogenous Suc (Fig. 5B). However, this effect is not extended to ATP content as the amount of ATP in dark-grown pkp1 seedlings (on 55 mM Suc medium) was higher than that of the wild type at both 1 and 5 DAS (Table I ). Thus, a defect in the conversion of sugars into biosynthetic precursors is a more likely explanation for the pkp1 seedling phenotype.


Figure 5
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  		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.

 

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  		Table I. ATP content (nmol/g FW) of seedlings grown on agar plates with 55 mM Suc

Whole seedlings were harvested at the DAS indicated. Values represent the mean ± SD, n = 6. WT, Wild type.

 
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., 1990Go), 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.


Figure 6
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  		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).

 
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., 2004Go). 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 {gamma}-tocopherol, whereas {alpha}- and {delta}-tocopherol are actually increased in pkp1.


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  		Table II. Wild-type and pkp1 seed tocopherol content (pg/seed)

Values are the mean ± SD, n = 3.

 

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.


Figure 7
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  		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).

 

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  		Table III. Leaf pigments in 25-d-old plants grown on soil or on agar plates

Values represent the mean ± SD of at least three repeats. FW, Fresh weight.

 
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.


Figure 8
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  		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.

 

   DISCUSSION
TOP
 ABSTRACT
 RESULTS
 DISCUSSION
CONCLUSION
 MATERIALS AND METHODS
 LITERATURE CITED
 

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., 2007Go). In germinating wild-type seeds, a rapid increase of PKp specific activity was observed coincident with radicle emergence (0–1 DAS; Fig. 2B). In pkp1 there was a delay in this induction, which could have resulted from or caused the impaired germination rate (Fig. 2B). The eventual increase in PKp activity in pkp1 can be explained by two possibilities: (1) enhanced expression of the PKp-β2-encoding gene or (2) induction of a cytosolic enzyme with higher than normal pH optimum. The first hypothesis is supported by elevated expression of the PKp-β2-encoding gene in dark-grown seedlings of a plastidic ATP/ADP transporter mutant, supposedly to compensate for reduced ATP import into plastids (Reiser et al., 2004Go). Induction of a PKc is also not unreasonable. In fact, Baud et al. (2007)Go showed an induction of PKc activity at 2 DAS in other PKp-deficient mutants.

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, 2002Go; Cernac et al., 2006Go). However, pkp1 differs from these mutants in that increasing the osmotic potential of the medium had no effect on germination (Fig. 3B). It is possible that in pkp1 an accumulation of carbohydrates in the seedling brought on by a reduction in glycolytic activity is responsible for delayed germination and increased sensitivity to sugar in the medium. Indeed, in pkp1 PK activity was only 50% that of the wild type at 1 DAS (Fig. 2B) and soluble sugars were in excess (Fig. 4). The sugar content determined for pkp1 seedlings agrees with the buildup of carbohydrates observed in developing seeds, and apparently the switch from a high hexose-to-Suc to a high Suc-to-hexose ratio is maintained through germination (Fig. 4; Andre et al., 2007Go). In both the wild-type and pkp1 seedlings, there was approximately 5-fold more Suc than hexose (on a molar basis; Fig. 4). There was a strong correlation between Suc content and the delayed germination of pkp1. There was twice as much Suc relative to the wild type at 0 DAS, and by 3 DAS, when all of the pkp1 seeds have germinated, the Suc content had dropped to wild-type levels while Glc was still in gross excess (Fig. 4). While it is possible that the excess sugar in pkp1 inhibits germination, it could also be that the loss of PKp activity results in an inability to catabolize endogenous sugars to fuel germination. The inability of pkp1 to utilize applied Suc for root (Fig. 1) or hypocotyl (Fig. 5A) elongation bolsters this idea. Impaired Suc metabolism could also be inferred from the fact that at 1 DAS pkp1 contains less ATP than the wild type (Table I).


Storage Compound Utilization Is Dependent on PKp

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., 2007Go). In contrast to β-oxidation, glyoxylate cycle, and gluconeogenesis mutants, we and Baud et al. (2007)Go observed that exogenous Suc does not rescue this phenotype. We investigated this phenotype further by measuring PKp activity in actively elongating hypocotyls. Without Suc pkp1 hypocotyl elongation is likely inhibited due to a lack of storage reserves. However, on 55 mM Suc the reduction in PKp activity (Fig. 5B) may contribute to the apparent inability to utilize the supplied sugar. The amount of ATP in pkp1 etiolated seedlings is actually increased in the presence of Suc (Table I), suggesting that unfavorable energy status is not the cause of the pkp1 hypocotyl phenotype. A similar increase in ATP content was observed in pkp1 developing seeds and as with that tissue, the phenotype could be explained by a reciprocal induction of PKc (Andre et al., 2007Go). It could also be the result of less demand for ATP due to substrate-limited flux through various biosynthetic pathways, which likewise provides an explanation for the reduced hypocotyl elongation. A major sink for ATP in seedlings is the synthesis of lipids (e.g. fatty acids) for expansive growth. Indeed, pkp1 seedlings do not synthesize linolenic acid (18:3) as fast as the wild type (Fig. 6B). On the other hand, amino acid biosynthesis is probably not affected to any great extent in pkp1 as exogenous amino acids did not rescue any of the seedling phenotypes.

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., 2002Go). Tocopherol (vitamin E) has also been shown to have a role in regulating storage lipid catabolism (Sattler et al., 2004Go). Tocopherol is synthesized in the plastid and uses phytyldiphosphate as a precursor (Collakova and DellaPenna, 2001Go). Phytyldiphosphate is synthesized exclusively in the plastid as a downstream product of the MEP pathway and as a salvage product released during chlorophyll degradation (Ischebeck et al., 2006Go). Developing seeds of pkp1 expectedly have lower flux through PKp and therefore less Pyr for entry into the MEP pathway. Additionally, seed chlorophyll content is drastically reduced in developing seeds possibly due to a lack of phytol (Andre et al., 2007Go). Moreover, the cloroplastos alterado1 (cla1) mutant of Arabidopsis, deficient in 1-deoxy-D-xylulose-5-P synthase that catalyzes the first step of the MEP pathway (using Pyr as one substrate), has retarded germination and reduced tocopherol content at least in leaves (Estevez et al., 2001Go). The combined data are in agreement with reduced flux through the MEP pathway leading to lower tocopherol content in pkp1 seeds. As expected pkp1 has reduced seed tocopherol content (Table II), which could explain some of the seedling phenotypes. The effect of the pkp1 mutation on tocopherol content is subtle probably because PKp activity peaks just prior to the maximum rate of oil biosynthesis and steadily declines (Andre et al., 2007Go), whereas tocopherol is synthesized later once all of the oil has been deposited and PKp activity is low (Valentin et al., 2006Go). Tocopherol composition is altered in pkp1 and, interestingly, also in the Rβ2-3 line despite it being rescued to nearly the wild-type total tocopherol level (Table II). The same phenomenon is seen in seed oil from these two lines, where Rβ2-3 has wild-type total fatty acid content but the same composition as pkp1 (Andre et al., 2007Go). A correlation between tocopherol composition and seed fatty acid profile has been observed in Zea mays (Goffman and Bohme, 2001Go). Our data suggest that a similar coordination could be happening in Arabidopsis.


Arabidopsis Depends on PKp to Coordinate Anabolic Metabolism in the Plastid

The effects of the pkp1 mutation are evident in mature plants. As noted by Baud and coworkers (Baud et al., 2007Go), growth and overall morphology are relatively unaffected in PKp-deficient mutants (Fig. 7), when taking into account the delayed germination. However, more thorough analysis revealed that leaves of pkp1 contain 30% to 60% less chlorophyll than the wild type, depending on if they were grown on soil or on agar plates (Table III). Reduced PKp activity (Fig. 7) in leaves and resultant substrate-limited impairment of isoprenoid biosynthesis could explain this. Such substrate-limitation could also be a reason for the low chlorophyll phenotype of the cue1 mutant of Arabidopsis deficient in a plastidic PEP transporter (Li et al., 1995Go; Voll et al., 2003Go). Also, the cla1 mutant supports this hypothesis in that it too has reduced chlorophyll content in leaves (Mandel et al., 1996Go). There are more anthocyanins in pkp1 leaves (Table III), and this may also be related to PKp activity as PEP is a substrate for the shikimic acid pathway, which gives rise to precursors for anthocyanin biosynthesis. Inversely, the cue1 mutant, which presumably has less plastidic PEP, has decreased anthocyanin content in leaves (Streatfield et al., 1999Go; Voll et al., 2003Go). Hexose content is reduced in pkp1 leaves (Fig. 8) and likely has no role in repression of chlorophyll biosynthesis. The cue1 mutant shares this carbohydrate phenotype as well (Streatfield et al., 1999Go). Reduced photosynthesis due to a lack of chlorophyll might be the reason for altered hexose content and reduced biomass in pkp1 (Fig. 7A). Suc and starch appear to be unaffected in pkp1, suggesting that any problems with carbohydrate metabolism are localized in or around glycolysis (Fig. 8). In any case, the phenotype of mature pkp1 plants is less severe than that of young (<10 DAS) tissue and this is probably a result of different metabolic demands. For instance, the supply of Pyr for the MEP pathway and for fatty acid synthesis is important for plastids in new and expanding leaves, whereas fully mature chloroplasts have fewer metabolic demands and, at least in the case of isoprenoids, are satisfied by import of intermediates generated in the cytosol (Heintze et al., 1990Go).


   CONCLUSION
TOP
 ABSTRACT
 RESULTS
 DISCUSSION
 CONCLUSION
MATERIALS AND METHODS
 LITERATURE CITED
 
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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 RESULTS
 DISCUSSION
 CONCLUSION
 MATERIALS AND METHODS
LITERATURE CITED
 

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.


Root and Hypocotyl Elongation Assays

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., 2004Go). Once exposed to the light, hypocotyls were immediately measured.


Seed Germination Assays

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.


PK Enzyme Activity Measurements

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).


Metabolite Analysis

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 {alpha}-naphthol. Lipid composition was determined by fatty acid methyl ester analysis as described previously (Focks and Benning, 1998Go). Ten seedlings or two whole leaves were used for each sample. Glc, Fru, and Suc were extracted and quantified as described previously (Focks and Benning, 1998Go). Five 25-d-old plants or bulked seeds or seedlings were homogenized and approximately 50 mg (fresh weight) of tissue was used for each extraction. Soluble sugars were resuspended in 200 µL of water and 15 µL were used for each measurement. Insoluble carbohydrate pellets were resuspended in 300 µL of 0.2 N KOH and the remaining volumes were adjusted proportionally. Starch assays were done with 15 µL of the final preparation. Seed tocopherols were extracted from dry seeds as described previously (Tian et al., 2003Go) and were quantified by HPLC using a published protocol (Gilliland et al., 2006Go). ATP was extracted with perchloric acid (Hausler et al., 2000Go) and was quantified with an ATP bioluminescence assay kit (Sigma).


Leaf Pigment Quantification

Chlorophyll was extracted from leaves and seedlings using 100 volumes (µL/mg) of 80% acetone and was quantified as described previously (Lichtenthaler, 1987Go). Anthocyanins were extracted and quantified using a published protocol (Martin et al., 2002Go).


   ACKNOWLEDGMENTS
 
We thank Maria Magallanes-Lundback for her help with determining tocopherol contents.

Received August 29, 2007; accepted October 24, 2007; published October 26, 2007.


   FOOTNOTES
 
1 This work was supported in part by grants from the Michigan Agriculture Experiment Station and by BASF Plant Sciences LLC. Back

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. Back

www.plantphysiol.org/cgi/doi/10.1104/pp.107.108340

* Corresponding author; e-mail benning{at}msu.edu.


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