Plastidic Phosphoglucose Isomerase Research Articles

A structural basis for the functional differences between the cytosolic and plastid phosphoglucose isomerase isozymes.

Phosphoglucose isomerase (PGI) catalyzes the interconversion between glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P), thereby regulating sucrose synthesis in plant cells. In general, plants contain a pair of PGI isozymes located in two distinct compartments of the cell (cytosol and plastid) with differences in both the primary structure and the higher-order structure. Previously, we showed that the activity of cytosolic PGI (PGIc) is more robust (activity, thermal stability, substrate turnover rate, etc.) than that of the plastid counterpart (PGIp) in multiple organisms, including wheat, rice, and Arabidopsis. The crystal structures of apoTaPGIc (an isotype cytosol PGIc in Triticum aestivum), TaPGIc-G6P complex, and apoTaPGIp (an isotype plastid PGIp in Triticum aestivum) were first solved in higher plants, especially in crops. In this study, we detailed the structural characteristics related to the biochemical properties and functions of TaPGIs in different plant organelles. We found that the C-terminal domains (CTDs) of TaPGIc and TaPGIp are very different, which affects the stability of the dimerized enzyme, and that Lys213TaPGIc/Lys193TaPGIp and its surrounding residues at the binding pocket gateway may participate in the entrance and exit of substrates. Our findings provide a good example illuminating the evolution of proteins from primary to higher structures as a result of physical barriers and adaptation to the biochemical environment.

Arabidopsis Responds to Alternaria alternata Volatiles by Triggering Plastid Phosphoglucose Isomerase-Independent Mechanisms.

Volatile compounds (VCs) emitted by phylogenetically diverse microorganisms (including plant pathogens and microbes that do not normally interact mutualistically with plants) promote photosynthesis, growth, and the accumulation of high levels of starch in leaves through cytokinin (CK)-regulated processes. In Arabidopsis (Arabidopsis thaliana) plants not exposed to VCs, plastidic phosphoglucose isomerase (pPGI) acts as an important determinant of photosynthesis and growth, likely as a consequence of its involvement in the synthesis of plastidic CKs in roots. Moreover, this enzyme plays an important role in connecting the Calvin-Benson cycle with the starch biosynthetic pathway in leaves. To elucidate the mechanisms involved in the responses of plants to microbial VCs and to investigate the extent of pPGI involvement, we characterized pPGI-null pgi1-2 Arabidopsis plants cultured in the presence or absence of VCs emitted by Alternaria alternata We found that volatile emissions from this fungal phytopathogen promote growth, photosynthesis, and the accumulation of plastidic CKs in pgi1-2 leaves. Notably, the mesophyll cells of pgi1-2 leaves accumulated exceptionally high levels of starch following VC exposure. Proteomic analyses revealed that VCs promote global changes in the expression of proteins involved in photosynthesis, starch metabolism, and growth that can account for the observed responses in pgi1-2 plants. The overall data show that Arabidopsis plants can respond to VCs emitted by phytopathogenic microorganisms by triggering pPGI-independent mechanisms.

Plastidic phosphoglucose isomerase is an important determinant of starch accumulation in mesophyll cells, growth, photosynthetic capacity, and biosynthesis of plastidic cytokinins in Arabidopsis.

Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. It is involved in glycolysis and in the regeneration of glucose-6-P molecules in the oxidative pentose phosphate pathway (OPPP). In chloroplasts of illuminated mesophyll cells PGI also connects the Calvin-Benson cycle with the starch biosynthetic pathway. In this work we isolated pgi1-3, a mutant totally lacking pPGI activity as a consequence of aberrant intron splicing of the pPGI encoding gene, PGI1. Starch content in pgi1-3 source leaves was ca. 10-15% of that of wild type (WT) leaves, which was similar to that of leaves of pgi1-2, a T-DNA insertion pPGI null mutant. Starch deficiency of pgi1 leaves could be reverted by the introduction of a sex1 null mutation impeding β-amylolytic starch breakdown. Although previous studies showed that starch granules of pgi1-2 leaves are restricted to both bundle sheath cells adjacent to the mesophyll and stomata guard cells, microscopy analyses carried out in this work revealed the presence of starch granules in the chloroplasts of pgi1-2 and pgi1-3 mesophyll cells. RT-PCR analyses showed high expression levels of plastidic and extra-plastidic β-amylase encoding genes in pgi1 leaves, which was accompanied by increased β-amylase activity. Both pgi1-2 and pgi1-3 mutants displayed slow growth and reduced photosynthetic capacity phenotypes even under continuous light conditions. Metabolic analyses revealed that the adenylate energy charge and the NAD(P)H/NAD(P) ratios in pgi1 leaves were lower than those of WT leaves. These analyses also revealed that the content of plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP)-pathway derived cytokinins (CKs) in pgi1 leaves were exceedingly lower than in WT leaves. Noteworthy, exogenous application of CKs largely reverted the low starch content phenotype of pgi1 leaves. The overall data show that pPGI is an important determinant of photosynthesis, energy status, growth and starch accumulation in mesophyll cells likely as a consequence of its involvement in the production of OPPP/glycolysis intermediates necessary for the synthesis of plastidic MEP-pathway derived hormones such as CKs.

HPLC-MS/MS analyses show that the near-Starchless aps1 and pgm leaves accumulate wild type levels of ADPglucose: further evidence for the occurrence of important ADPglucose biosynthetic pathway(s) alternative to the pPGI-pPGM-AGP pathway.

In leaves, it is widely assumed that starch is the end-product of a metabolic pathway exclusively taking place in the chloroplast that (a) involves plastidic phosphoglucomutase (pPGM), ADPglucose (ADPG) pyrophosphorylase (AGP) and starch synthase (SS), and (b) is linked to the Calvin-Benson cycle by means of the plastidic phosphoglucose isomerase (pPGI). This view also implies that AGP is the sole enzyme producing the starch precursor molecule, ADPG. However, mounting evidence has been compiled pointing to the occurrence of important sources, other than the pPGI-pPGM-AGP pathway, of ADPG. To further explore this possibility, in this work two independent laboratories have carried out HPLC-MS/MS analyses of ADPG content in leaves of the near-starchless pgm and aps1 mutants impaired in pPGM and AGP, respectively, and in leaves of double aps1/pgm mutants grown under two different culture conditions. We also measured the ADPG content in wild type (WT) and aps1 leaves expressing in the plastid two different ADPG cleaving enzymes, and in aps1 leaves expressing in the plastid GlgC, a bacterial AGP. Furthermore, we measured the ADPG content in ss3/ss4/aps1 mutants impaired in starch granule initiation and chloroplastic ADPG synthesis. We found that, irrespective of their starch contents, pgm and aps1 leaves, WT and aps1 leaves expressing in the plastid ADPG cleaving enzymes, and aps1 leaves expressing in the plastid GlgC accumulate WT ADPG content. In clear contrast, ss3/ss4/aps1 leaves accumulated ca. 300 fold-more ADPG than WT leaves. The overall data showed that, in Arabidopsis leaves, (a) there are important ADPG biosynthetic pathways, other than the pPGI-pPGM-AGP pathway, (b) pPGM and AGP are not major determinants of intracellular ADPG content, and (c) the contribution of the chloroplastic ADPG pool to the total ADPG pool is low.

The role of plastidial glucose‐6‐phosphate/phosphate translocators in vegetative tissues of Arabidopsis thaliana mutants impaired in starch biosynthesis

Arabidopsis thaliana mutants impaired in starch biosynthesis due to defects in either ADP glucose pyrophosphorylase (adg1-1), plastidic phosphoglucose mutase (pgm) or a new allele of plastidic phosphoglucose isomerase (pgi1-2) exhibit substantial activity of glucose-6-phosphate (Glc6P) transport in leaves that is mediated by a Glc6P/phosphate translocator (GPT) of the inner plastid envelope membrane. In contrast to the wild type, GPT2, one of two functional GPT genes of A. thaliana, is strongly induced in these mutants during the light period. The proposed function of the GPT in plastids of non-green tissues is the provision of Glc6P for starch biosynthesis and/or the oxidative pentose phosphate pathway. The function of GPT in photosynthetic tissues, however, remains obscure. The adg1-1 and pgi1-2 mutants were crossed with the gpt2-1 mutant defective in GPT2. Whereas adg1-1/gpt2-1 was starch-free, residual starch could be detected in pgi1-2/gpt2-1 and was confined to stomatal guard cells, bundle sheath cells and root tips, which parallels the reported spatial expression profile of AtGPT1. Glucose content in the cytosolic heteroglycan increased substantially in adg1-1 but decreased in pgi1-2, suggesting that the plastidic Glc6P pool contributes to its biosynthesis. The abundance of GPT2 mRNA correlates with increased levels of soluble sugars, in particular of glucose in leaves, suggesting induction by the sugar-sensing pathway. The possible function of GPT2 in starch-free mutants is discussed in the background of carbon requirement in leaves during the light-dark cycle.

Mutation of Arabidopsis plastid phosphoglucose isomerase affects leaf starch synthesis and floral initiation.

We isolated pgi1-1, an Arabidopsis mutant with a decreased plastid phospho-glucose (Glc) isomerase activity. While pgi1-1 mutant has a deficiency in leaf starch synthesis, it accumulates starch in root cap cells. It has been shown that a plastid transporter for hexose phosphate transports cytosolic Glc-6-P into plastids and expresses restricted mainly to the heterotrophic tissues. The decreased starch content in leaves of the pgi1-1 mutant indicates that cytosolic Glc-6-P cannot be efficiently transported into chloroplasts to complement the mutant's deficiency in chloroplastic phospho-Glc isomerase activity for starch synthesis. We cloned the Arabidopsis PGI1 gene and showed that it encodes the plastid phospho-Glc isomerase. The pgi1-1 allele was found to have a single nucleotide substitution, causing a Ser to Phe transition. While the flowering times of the Arabidopsis starch-deficient mutants pgi1, pgm1, and adg1 were similar to that of the wild type under long-day conditions, it was significantly delayed under short-day conditions. The pleiotropic phenotype of late flowering conferred by these starch metabolic mutations suggests that carbohydrate metabolism plays an important role in floral initiation.

PHYLOGENETIC IMPLICATIONS OF DIFFERENCES IN NUMBER OF PLASTID PHOSPHOGLUCOSE ISOMERASE ISOZYMES IN NORTH AMERICAN COREOPSIS (ASTERACEAE: HELIANTHEAE: COREOPSIDINAE)

Enzyme electrophoresis was employed to ascertain the number of loci encoding plastid phosphoglucose isomerase (PGI) in species representing all sections of North American Coreopsis. Several species from each of the closely related genera Bidens, Coreocarpus, Cosmos, and Thelesperma were also examined. Species in nine of the 11 sections of North American Coreopsis have two isozymes for plastid PGI, and nearly all species examined in the four other genera also have two (one species has three) isozymes. Since most diploid vascular plants have one plastid PGI isozyme, a gene duplication probably occurred in an ancestor that is common to Coreopsis and the other four genera. That is, two isozymes represent the ancestral number for Coreopsis. The two sections (Electra and Anathysana) apparently lacking the duplication are closely related woody plants restricted largely to Mexico. One gene encoding plastid PGI ostensibly was silenced in a common ancestor of these two sections. This is concordant with other data suggesting a close relationship between the two sections, i.e., they appear to represent a monophyletic group. The electrophoretic data also indicate that 1) the enigmatic monotypic section Silphidium is more closely related to eastern North American sections and not derived from section Electra; and 2) section Anathysana is not ancestral to the three California sections Leptosyne, Pugiopappus, and Tuckermannia; rather, it represents a terminal element closely related to and possibly derived from section Electra.

Decreased-activity mutants of phosphoglucose isomerase in the cytosol and chloroplast of Clarkia xantiana. Impact on mass-action ratios and fluxes to sucrose and starch, and estimation of Flux Control Coefficients and Elasticity Coefficients.

1. Subcellular-compartment-specific decreased-activity mutants of phosphoglucose isomerase in Clarkia xantiana were used to analyse the control of sucrose and starch synthesis during photosynthesis. Mutants were available in which the plastid phosphoglucose isomerase complement is decreased to 75% or 50% of the wild-type level, and the cytosol complement to 64%, 36% or 18% of the wild-type level. 2. The effects on the [product]/[substrate] ratio and on fluxes to sucrose or starch and the rate of photosynthesis were studied with the use of saturating or limiting light intensity to impose a high or low flux through these pathways. 3. Removal of a small fraction of either phosphoglucose isomerase leads to a significant shift of the [product]/[substrate] ratio away, from equilibrium. We conclude that there is no 'excess' of enzyme over that needed to maintain its reactants reasonably close to equilibrium. 4. Decreased phosphoglucose isomerase activity can also alter the fluxes to starch or sucrose. However, the effect on flux does not correlate with the extent of disequilibrium, and also varies depending on the subcellular compartment and on the conditions. 5. The results were used to estimate Flux Control Coefficients for the chloroplast and cytosolic phosphoglucose isomerases. The chloroplast isoenzyme exerts control on the rate of starch synthesis and on photosynthesis in saturating light intensity and CO2, but not at low light intensity. The cytosolic enzyme only exerts significant control when its complement is decreased 3-5-fold, and differs from the plastid isoenzyme in exerting more control in low light intensity. It has a positive Control Coefficient for sucrose synthesis, and a negative Control Coefficient for starch synthesis. 6. The Elasticity Coefficients in vivo of the cytosolic phosphoglucose isomerase were estimated to lie between 5 and 8 in the wild-type. They decrease in mutants with a lowered complement of cytosolic phosphoglucose isomerase. 7. The implications of these results for regulation and for evolution are discussed.

Reduced-activity mutants of phosphoglucose isomerase in the cytosol and chloroplast of Clarkia xantiana

(i) We have studied the influence of reduced phosphoglucose-isomerase (PGI) activity on photosynthetic carbon metabolism in mutants of Clarkia xantiana Gray (Onagraceae). The mutants had reduced plastid (75% or 50% of wildtype) or reduced cytosolic (64%, 36% or 18% of wildtype) PGI activity. (ii) Reduced plastid PGI had no significant effect on metabolism in low light. In high light, starch synthesis decreased by 50%. There was no corresponding increase of sucrose synthesis. Instead glycerate-3-phosphate, ribulose-1,5-bisphosphate, reduction of QA (the acceptor for photosystem II) and energy-dependent chlorophyll-fluorescence quenching increased, and O2 evolution was inhibited by 25%. (iii) Decreased cytosolic PGI led to lower rates of sucrose synthesis, increased fructose-2,6-bisphosphate, glycerate-3-phosphate and ribulose-1,5-bisphosphate, and a stimulation of starch synthesis, but without a significant inhibition of O2 evolution. Partitioning was most affected in low light, while the metabolite levels changed more at saturating irradiances. (iv) These results provide decisive evidence that fructose-2,6-bisphosphate can mediate a feedback inhibition of sucrose synthesis in response to accumulating hexose phosphates. They also provide evidence that the ensuing stimulation of starch synthesis is due to activation of ADP-glucose pyrophosphorylase by a rising glycerate-3-phosphate: inorganic phosphate ratio, and that this can occur without any loss of photosynthetic rate. However the effectiveness of these mechanisms varies, depending on the conditions. (v) These results are analysed using the approach of Kacser and Burns (1973, Trends Biochem. Sci. 7, 1149-1161) to provide estimates for the elasticities and flux-control coefficient of the cytosolic fructose-1,6-bisphosphatase, and to estimate the gain in the fructose-2,6-bisphosphate regulator cycle during feedback inhibition of sucrose synthesis.

Reduced Enzyme Activity and Starch Level in an Induced Mutant of Chloroplast Phosphoglucose Isomerase

Ethyl methane sulfonate treatment was used to induce a mutation in the nuclear gene encoding the chloroplast isozyme of phosphoglucose isomerase in Clarkia xantiana. The mutation, which proved allelic to wild type activity, was backcrossed to wild type for five generations so that the two could be compared in a near isogenic background. An immunological analysis showed that the mutant, when homozygous, reduced the activity of the isozyme by about 50%. In contrast to wild type, the mutant showed little change in leaf starch level over a diurnal period or following a 72-hour continuous light treatment. By the end of the diurnal light period, the mutant accumulated only about 60% as much starch as wild type. However, mutant leaves had an increased sucrose level presumably because photosynthate was directly exported from the chloroplasts. The mutant also exhibited reduced leaf weight. These changes in metabolism and growth suggest that the wild type level of plastid phosphoglucose isomerase activity is necessary to achieve wild type carbohydrate status.

Immunological similarity between a cyanobacterial enzyme and a nuclear DNA-encoded plastid-specific isozyme from spinach

The immunochemical properties of the plastid and cytosolic isozymes of phosphoglucose isomerase (glucosephosphate isomerase; D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9) in spinach (Spinacia oleracea) and the single phosphoglucose isomerase enzyme from the cyanobacterium Synechococcus sp. were compared by an application of the enzyme-linked immunosorbent assay. Utilizing antibodies made in rabbits against subunits of purified plastid and cytosolic phosphoglucose isomerase isozymes from spinach, we demonstrate that the plastid isozyme is immunochemically more similar to the cyanobacterial enzyme than to the spinach cytosolic counterpart. The antiserum to plastid phosphoglucose isomerase crossreacted strongly with plastid phosphoglucose isomerases from other flowering plants. The antiserum to cytosolic phosphoglucose isomerase crossreacted with other plant cytosolic phosphoglucose isomerase isozymes. The results are consistent with the hypothesis [Weeden, N. F. (1981) J. Mol. Evol. 17, 133-139] that the nuclear gene specifying plastid phosphoglucose isomerase was derived from a prokaryote after the incorporation of a plastid-like symbiont into the ancestral plant cell.