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First published online November 9, 2007; 10.1104/pp.107.108423 Plant Physiology 146:277-288 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
Functional Analyses of Cytosolic Glucose-6-Phosphate Dehydrogenases and Their Contribution to Seed Oil Accumulation in Arabidopsis1,[OA]Department of Biochemistry and Molecular Biology (S.W., C.B.), Department of Plant Biology (C.A.), and United States Department of Energy-Plant Research Laboratory (C.A.), Michigan State University, East Lansing, Michigan 48824
Glucose-6-phosphate dehydrogenase (G6PDH) has been implicated in the supply of reduced nicotine amide cofactors for biochemical reactions and in modulating the redox state of cells. In plants, identification of its role is complicated due to the presence of several isoforms in the cytosol and plastids. Here we focus on G6PDHs in the cytosol of Arabidopsis (Arabidopsis thaliana) using single and double mutants disrupted in the two cytosolic G6PDHs. Only a single G6PDH isoform remained in the double mutant and was present in chloroplasts, consistent with a loss of cytosolic G6PDH activity. The activities of the cytosolic isoforms G6PD5 and G6PD6 were reciprocally increased in single mutants with no increase of their respective transcript levels. We hypothesized that G6PDH plays a role in supplying NADPH for oil accumulation in developing seeds in which photosynthesis may be light limited. G6PDH activity in seeds derived from G6PD6 and a plastid G6PDH isoform and showed a similar temporal activity pattern as oil accumulation. Seeds of the double mutant but not of the single mutants had higher oil content and increased weight compared to those of the wild type, with no alteration in the carbon to nitrogen ratio or fatty acid composition. A decrease in total G6PDH activity was observed only in the double mutant. These results suggest that loss of cytosolic G6PDH activity affects the metabolism of developing seeds by increasing carbon substrates for synthesis of storage compounds rather than by decreasing the NADPH supply specifically for fatty acid synthesis.
Glc-6-P dehydrogenase (G6PDH) is the first of the two NADPH-generating enzymes of the oxidative pentose phosphate pathway (OPPP), G6PDH and 6-phosphogluconate dehydrogenase. All eukaryotic G6PDHs studied are feedback inhibited by NADPH, hence they are presumed to act as cellular redox sensors. This together with the fact that this enzyme catalyzes the committing step makes G6PDH the regulatory enzyme of the OPPP. Its role in oxidative stress responses has been suggested for various organisms. In humans, certain alleles of the G6PDH gene are associated with hypersensitivity of erythrocytes to oxidative stress such as exposure to drugs, infection, and ingestion of fava beans (Vicia faba; for review, see Vulliamy et al., 1992  ; Martini and Ursini, 1996). In yeast (Saccharomyces cerevisiae) and Escherichia coli, genes coding for G6PDH are up-regulated by oxidative stress (Kletzien et al., 1994). In mammalian cells, cytosolic G6PDH is also regulated through alternative splicing (Tao et al., 2002) in response to hormonal and nutritional cues such as sugar and lipids (Salati et al., 2004). Increase in G6PDH gene expression has been associated with increasing glutathione levels and resistance to oxidative stress (Salvemini et al., 1999) or the protein modulating the activity of a DNA repair protein (Ayene et al., 2002).
Plants are unique in that the OPPP resides in the cytosol and the plastid (Schnarrenberger et al., 1973
Aside from nitrogen assimilation, G6PDH has been speculated to be an important source of NADPH in nonphotosynthetic tissues (Emes and Neuhaus, 1997
The unique attributes of the plant OPPP, such as enzyme isoforms in different compartments, complicate the interpretation of its role in whole cell physiology. Few studies address such questions. Maize mutants with the cytosolic 6-phosphogluconate dehydrogenases disrupted were affected in the capacity of nitrogen assimilation (Averill et al., 1998 Previously we have conducted a genome-wide characterization of Arabidopsis G6PDH isoforms. In this study, by taking advantage of T-DNA insertion lines, we attempted to determine the unique and redundant roles of the cytosolic G6PDHs, particularly in seed metabolism.
Cytosolic Localization of G6PD5 and G6PD6 To test that G6PD5 and G6PD6 indeed encode cytosolic isoforms the subcellular localization of the proteins was examined by transient expression of the respective cDNAs fused to a GFP gene. For both constructs containing G6PD5 and G6PD6, green fluorescence was observed dispersed in the cytosol and surrounding what is presumably the nucleus (Fig. 1, A and B ). The same patterns were observed in multiple experiments. A similar pattern was observed in cells expressing GFP alone, which localizes to the cytosol and to the nucleus (Fig. 1C). This result together with the lack of a potential targeting sequence in the proteins suggests that G6PD5 and G6PD6 are both likely to be cytosolic proteins.
There are three major active isoforms in vivo, G6PD5, G6PD6, and an unidentified isoform that is ubiquitous (Wakao and Benning, 2005 ). To rule out the possibility that the third isoform is localized in the cytosol, we examined G6PDH activity in isolated chloroplasts from Arabidopsis leaves. As shown in Figure 1D, isolated chloroplasts contain a single band on a zymogram with similar mobility as the unidentified band detected in protein extract from buds. This result strongly suggests that the unidentified ubiquitous isoform is localized in the plastid and that G6PD5 and G6PD6 are the only cytosolic isoforms with major activity in Arabidopsis.
To specifically examine the in vivo roles of the cytosolic G6PDHs, T-DNA insertion lines (SALK Institute) for the two genes were obtained. Their insertion sites were identified as previously described (Wakao and Benning, 2005
G6PDH activity was examined in plants of different genotypes using zymograms from bud tissue in which all three isoforms were easily detected (Fig. 2C). We chose zymograms to detect the activity of G6PDH rather than immunoblot analysis because the cytosolic isoforms have similar amino acid sequence (92%; data not shown) and have different sensitivity to redox effectors in vitro (Wakao and Benning, 2005 ). Moreover, we have been successful in reproducibly separating and detecting relative activity levels of G6PDH isoforms from Arabidopsis tissues using this method (Wakao and Benning, 2005). The zymogram pattern of the plants homozygous for one T-DNA insertion and heterozygous for the other looked similar to the respective single mutants, which were distinguishable by the loss of one band and the increase of another (Fig. 2C, compare lanes 2 and 5, 3 and 6). This result suggests there is no gene dosage effect on cytosolic G6PDH activity. In the double mutant, the increase of both bands was lost, indicating that the intensified bands observed in g6pd5 and g6pd6 were indeed G6PD6 and G6PD5 and not an alternative G6PDH. Residual G6PD5 activity was detected in bud tissue of the double mutant and not the single mutant (Fig. 2C, lanes 3 and 4), similarly to that in wild-type leaf tissues in which G6PD5 activity is the predominant isoform (Wakao and Benning, 2005). The T-DNA insertion site in g6pd5 is 3 bp downstream of the stop codon (Wakao and Benning, 2005) and thus a transcript containing the entire coding sequence and protein could be made. The activity of G6PD5 in the g6pd5 and double mutant may become more necessary in g6pd5 leaves where G6PD5 is presumed to be the major cytosolic activity, and when both cytosolic isoforms are lost in the double mutant. Surprisingly the double mutant was also indistinguishable in growth from the wild-type plant (Fig. 2A), despite the loss of most of the cytosolic G6PDH activity as observed in zymograms. Therefore we conclude that a nearly complete loss of cytosolic G6PDH activity does not result in severe morphological phenotypes of the plant under laboratory growth conditions. Attempts to complement the single and double mutants with a cDNA or G6PDH fused to GFP at the N terminus were unsuccessful (data not shown). Only when genomic fragments containing G6PD5 or G6PD6 were introduced did we observe the recovery of the lost bands on zymograms (Fig. 2D). This result together with the zymogram pattern of the double mutant proves the previously defined zymogram bands (Wakao and Benning, 2005) were indeed due to G6PD5 and G6PD6.
Unexpectedly, an apparent decrease in the activity of the plastidic isoform was observed in the single and double mutants (Fig. 2C; also in Wakao and Benning, 2005
To address whether the reciprocal increase in activity of the cytosolic G6PDH occurs in other tissues and at the mRNA level, activity and gene expression were examined in seedlings by zymogram and quantitative reverse transcription (RT)-PCR, respectively. G6PD5 activity increased in the g6pd6 mutant as was observed in bud tissues (compare Figs. 3A and 2C). In contrast, the increase in G6PD6 activity in the g6pd5 mutant was not as pronounced as that in bud tissues (Figs. 3A and 2C). This may be explained by the low activity of G6PD5 in wild-type seedlings compared to buds (wild type in Figs. 2C and 3A), hence no compensation for its loss by G6PD6 is necessary in the g6pd5 mutant. When primers were designed against regions upstream of the T-DNA insertion, no large increase of transcript levels of G6PD5 and G6PD6, in g6pd6 and g6pd5 mutants was observed that accompanied any changes in their activity (Fig. 3B, top section). In fact, the transcripts of both cytosolic G6PDH genes were wild-type level in g6pd5 and the double mutant, only G6PD6 was slightly reduced in the g6pd6 and double mutant (Fig. 3B, top section). This indicates that the T-DNA insertions did not lead to degradation of the entire mRNA, while transcript regions downstream of the insertion appeared reduced (data not shown). The reduced activity of the isoform in which the T-DNA insertion is in the 3' end of the gene may be attributed to decreased protein levels from lower translational efficiency. Most importantly, this result showed that the increase in activity of the remaining cytosolic G6PDH in the single mutants did not occur at the level of mRNA.
Because cytosolic G6PDH transcript was found to increase in response to sugars in potato (Solanum tuberosum; Hauschild and von Schaewen, 2003 ), the sugar inducibility of the cytosolic G6PDH genes was tested in seedlings. In wild-type seedlings G6PD5 and G6PD6 showed small (1.7- and 2.0-fold, respectively) but significant induction by Suc, though not to the extent of a known sugar-inducible gene flavonoid 3'-hydroxylase (F3H; Solfanelli et al., 2006), which in this experiment increased 18-fold (Fig. 3B, bottom section). In g6pd5 and double mutant, the sugar induction of the genes was attenuated and altered, respectively, while the induction was retained in g6pd6 (Fig. 3B, bottom section). Although a relatively small Suc induction was detected for the G6PD5 and G6PD6, this did not translate into increased activity in wild type or mutants as seen in Figure 3A.
In light of recent findings that photosynthesis in seeds contributes to oil accumulation, the role of G6PDH was directly addressed using the single and double mutants. It was shown previously that G6PD6 and a ubiquitous plastidic isoform are active in Arabidopsis seeds 5 d after flowering (DAF; Wakao and Benning, 2005
It has been a long debate what the major source of reducing equivalents for biosynthesis of storage lipid is in green developing seeds. While several studies suggested G6PDH as a potential source of NADPH in light-limited embryos (Eastmond et al., 1996 ; Kang and Rawsthorne, 1996), recent reports have demonstrated the importance of photosynthesis in green seeds (Ruuska et al., 2004; Schwender et al., 2004; Goffman et al., 2005). G6PDH has a seemingly inverse correlation with photosynthesis due to its feedback inhibition by NADPH and the plastidic isoforms' sensitivity to reductive inactivation through the thioredoxin/ferredoxin system, and thus we expected that a loss of photosynthesis would lead to activation of G6PDH. To gain insight into the relative contributions of photosynthesis and G6PDH in supplying NADPH for seed oil accumulation in Arabidopsis, we used the pds1 mutant. This mutant is unable to desaturate phytoene and photobleaches due to the lack of tocopherol when homozygous for the mutation (Norris et al., 1995). In the siliques of heterozygous plants, a 1-to-3 segregation of white and green seeds was observed (Fig. 5A
). A time course of FA accumulation in developing seeds showed that white pds1 seeds contain 80% as much FA as wild-type green seeds at 15 DAF (Fig. 5B). No large change in the pattern of G6PDH activity was observed for the white seeds on zymograms (Fig. 5C). When total G6PDH activity was measured by liquid assay in these samples the white seeds were reduced by approximately 25% to 30% and approximately 50% compared to the green seeds in 10 to 13, and 14 to 15 DAF, respectively (data not shown). Apparently no compensation for the lack of photosynthesis occurred through G6PDH activity.
The pds1 seeds cannot be distinguished when they are mature due to the brown seed coat. The population of mature seeds from a PDS1/pds1 heterozygous plant shows a bimodal distribution when plotted for oil content (Fig. 6A ). The group with less oil consists of approximately 25% of the total number of seeds and its distribution is maximal at 5 to 5.5 µg/seed, in contrast to the larger group with a maximum in the distribution at 8.5 to 9 µg/seed. Extrapolating from this distribution the white seeds are likely to have accumulated approximately 60% wild-type level oil in the absence of photosynthesis by the time of maturation. Interestingly the seeds with less oil contain a higher ratio of very long-chain FAs (Fig. 6B), possibly due to increased FA elongation to compensate for reduced de novo FA synthesis. These results indicate that 60% seed oil of wild type can be produced in the absence of photosynthesis with little change in G6PDH activity.
Seed Oil Content per Seed Is Increased Only in the Double Mutant
Oil content in seeds of wild type, g6pd5, g6pd6, and double mutants was measured to examine the contribution of the cytosolic G6PDHs. The data consists of values obtained from seeds from five or six individual plants of each genotype that were grown at two separate times but under the exact same conditions in the same growth chamber. In a single experiment, approximately 50 seeds were analyzed per plant. The mean of the values for seed oil content from each plant and the SD are shown in Table I
. The single mutants and the double mutant were compared to wild type by Student's t test. For g6pd5, g6pd6, and the double mutant the probability of assuming the null hypothesis (that they are not different from wild type) were 0.52, 0.35, and 0.056, respectively. This result suggests a difference between wild type and the double mutant with a 94% confidence, close to the typically accepted 95% (Kimble, 1978
To address whether the possible increase in oil in the double mutant occurred at the expense of storage protein accumulation, the content of total carbon and nitrogen in the seeds was analyzed for wild type and double mutant. No significant difference was observed between the carbon to nitrogen ratios of the two genotypes (Table I). The seeds of wild type and the double mutant were counted to 500 each, dried, and weighed. For the double mutant, seeds were on average heavier than those of wild type (Table I). A plot for the two variables, seed weight and oil content, gives a r2 value of 0.54, suggesting some correlation. This may imply that the seeds of the double mutant contain more oil due to larger seeds rather than the effect being specific to oil accumulation. The number of seeds in siliques was counted and compared between the mutants and wild type to address whether a decrease in seed number accompanied the increase in seed mass and no difference between genotypes was observed (data not shown). Because FA elongation beyond C16 and C18 requires NADPH in the cytosol, it was possible that the accumulation of long-chain FAs would be affected in the mutant lines that lack cytosolic G6PDHs. The FA compositions of seeds of the four genotypes closely resembled each other (Table II ). The loss of either of the cytosolic G6PDH isoform did not affect FA content or FA composition, however, the loss of both isoforms increased FA content without altering the composition.
Total G6PDH Activity Is Reduced Only in the Double Mutant
Only seeds of the double mutant accumulated more oil per seed than wild type. We hypothesized that this was due to increased substrate availability for glycolysis and subsequent FA synthesis as a result of the block in the cytosolic OPPP and that the redundancy of G6PD5 and G6PD6 prevented such a phenotype in the single mutants. We analyzed G6PDH activity in whole siliques to explore this hypothesis. The typical reciprocal increase in activity of the cytosolic isoforms was observed in the single mutants by zymogram (Fig. 7A
). When G6PDH activity was measured in these extracts by liquid assay, a decrease in total activity was observed only for the double mutant (Fig. 7B). The large decrease in total activity could be due to the loss of both cytosolic or the plastidic isoform or all three (because the plastidic activity decreases pleiotropically in the mutants; Fig. 2D). Nonetheless, these results indicate that total G6PDH activity is reduced only in the double mutant and not in the single mutants, coincident with the increased seed oil content and mass. In this experiment whole siliques were used instead of isolated seeds to simplify the harvesting procedure. Although the isoforms present are the same, the temporal pattern differs in whole siliques from that observed in isolated seeds (compare Figs. 4B and 7B), which is likely to be due to the contribution of G6PDH activity in the silique walls. We have observed that the zymogram patterns comparing whole siliques and seeds of the same stage are similar (Fig. 4A; Wakao and Benning, 2005
Role of Cytosolic G6PDHs
The activity of cytosolic G6PDH shows tissue specificity in wild-type plants; G6PD6 activity is ubiquitous in all tissues except leaves, while that of G6PD5 is the dominant cytosolic activity in leaves and is also present in roots (Wakao and Benning, 2005
The two cytosolic isoforms possibly have little or no overlapping roles in wild-type plants reflected in their tissue-specific distribution (Wakao and Benning, 2005
A compensatory increase in activity for the two cytosolic G6PDHs has been observed in different tissues, though in some tissues of g6pd5 (seedlings, Fig. 3 and whole siliques, Fig. 7) the increase was less pronounced than others (buds, Fig. 2C). Because G6PD6 is the predominant isoform in all tissues examined except leaves and roots (Wakao and Benning, 2005
In wild-type seeds, the activities of two isoforms were detected: G6PD6 and the unidentified plastidic isoform (Fig. 4A). The activity in seeds between 5 to 16 DAF was lowest at 7 to 8 DAF and increased thereafter (Fig. 4B). Before 5 DAF, seeds are nonphotosynthetic and go through rapid cell division, which is likely to require NADPH for FA synthesis to support membrane biosynthesis. A plastidic G6PDH was speculated to be important during these stages, but could not be examined due to difficulty in isolating such young seeds in a sufficient amount to perform enzyme assays. Seeds of 5 to 6 DAF are still transparent to the eye and contained relatively high G6PDH activity (Fig. 4), which may indicate the importance of G6PDH in supplying NADPH during the earlier nonphotosynthetic stages of seed development.
The seeds of the double mutant but not the single mutants accumulated more oil per seed than those of wild type, which was associated with the increase in seed mass and not specifically oil content. This was unexpected since the wild-type appearance of whole plants of the single and double mutants indicated the presence of a compensatory mechanism to supply cytosolic NADPH possibly through other enzymes that utilize NADP+. In agreement, the composition of FAs was similar among the different genotypes (Table II), indicating that the loss of cytosolic G6PDH as a NADPH source did not specifically affect FA elongation. Moreover, it was surprising that the loss of cytosolic G6PDHs resulted in an increase of seed oil content and mass rather than a decrease. Total G6PDH activity was reduced only in the double mutant and this evidence supports our hypothesis that an impairment in the OPPP leads to increased substrate availability for glycolysis. Glycolysis has been shown to be essential for seed oil biosynthesis and increasing glycolytic flux could provide more precursors for FA synthesis (Focks and Benning, 1998
Despite the lack of photosynthesis, total G6PDH activity or the isoforms present during seed development did not change in the pds1 mutant. The white seeds are presumed to have accumulated approximately 60% oil compared to wild type by maturation (Figs. 5B and 6A). The significance of photosynthesis in oil accumulation in green seeds has been an emerging focus in recent years (Schwender et al., 2003 Intriguingly, the loss of photosynthesis is not accompanied by increase of G6PDH activity, suggesting compensation, if any, occurs through other mechanisms. To test whether loss of the plastidic G6PDH would affect seed oil accumulation similarly as loss of photosynthesis we have attempted to generate RNA interference lines specific to certain plastidic G6PDH isoforms, but with no success. Because of the high sequence similarity of the isoforms, RNA interference was targeted to a short stretch of sequence in the 3' untranslated region and this may have resulted in inefficient silencing (data not shown). Because of the pleiotropic decrease in the plastidic G6PDH activity in the single and double mutants, we have indirectly observed that decrease in plastidic G6PDH activity does not lead to reduced seed oil. It remains unknown whether the plastidic G6PDH activity in wild-type seeds, without the metabolic alteration due to the loss of cytosolic G6PDH, is critical in supplying NADPH in the plastid for FA synthesis. Our results indicated the presence of a highly dynamic metabolic network that compensates for the loss of one or both of the cytosolic G6PDHs. Overall, cytosolic G6PDHs are largely dispensable for plant growth under laboratory growth conditions. However, the increased seed oil content and mass in the double mutant suggests there is indeed a metabolic change, possibly an increase in glycolytic flux, caused by the disruption in cytosolic G6PDHs.
Plant Growth Conditions and Transformation
All seeds were surface sterilized by incubation in 20% bleach and 0.05% Triton X. The tubes containing the seeds were inverted for 15 min and washed three times with water. The seeds were suspended in 0.1% agar and plated onto 1x Murashige and Skoog medium (pH 5.8; Murashige and Skoog, 1962
The coding regions of G6PD5 and G6PD6 were amplified with the following primers: for G6PD5, (+) 5'-GGACTAGTATGGGTTCTGGTCAATGGCA, (–) 5'-GGACTAGTCAATGTAGGAGGGATCCAAA, and for G6PD6, (+) 5'-GGACTAGTATGGGATCTGGTCAATGGCA, (–) 5'-GGACTAGTTAGTGTAGGAGGGATCCAG. The cDNAs were cloned into the SpeI site of pCAMBIA1302 (GenBank accession no. AF234298). The orientation was confirmed by restriction analysis and sequencing. Onion (Allium cepa) epidermal peels were bombarded with the above constructs following the methods previously described (Varagona et al., 1992
For sugar response experiment seedlings were germinated and grown for 2 weeks without Suc on Murashige and Skoog 1.5% agar plate vertically then transferred to liquid media with 90 or 0 mM Suc for 24 h. RNA was extracted and cDNA synthesized as described previously (Wakao and Benning, 2005
The T-DNA insertion lines were transformed with cosmid clones containing the regions of G6PD5 and G6PD6 that were isolated from a genomic cosmid (pBIC20) library (Meyer et al., 1994
For tissues other than seeds, protein extraction and zymogram detection were carried out as previously described (Wakao and Benning, 2005
Plants of different genotypes were grown in the same growth chamber and the seeds were harvested after having dried in the siliques attached to the plant. Single mature seeds from a single plant of which the genotype was confirmed by PCR were ground using a 6 mm glass bead (Fischer Scientific) in a glass tube using a paint shaker for 3 min. Lipid extraction and sample preparation for quantification by gas chromatography was carried out as previously described (Focks and Benning, 1998
Seeds were sent to the Duke Environmental Stable Isotope Laboratory for elemental analysis. Between 2 to 5 mg of seeds were sent in three replicates for each plant, and for all the plants that seed oil content was analyzed by gas chromatography.
We thank Dean DellaPenna for suggesting the use of the pds1 mutant and John Ohlrogge for helpful discussions. Received August 29, 2007; accepted November 5, 2007; published November 9, 2007.
1 This work was supported in part by the U.S. Department of Agriculture (grant no. MICL08253) and by the Michigan State Agricultural Experiment Station. 
2 Present address: Department of Plant and Microbial Biology, 441 Koshland Hall, University of California, Berkeley, CA 94720. 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.107.108423 * Corresponding author; e-mail benning{at}msu.edu.
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