Dissociation, reassociation, and purification of plastid and cytosolic phosphoglucose isomerase isozymes

The plastid and cytosolic isozymes of the dimeric enzyme phosphoglucose isomerase (EC 5.3.1.9) from spinach (Spinacia oleracea) and cauliflower (Brassica oleracea) were purified to apparent homogeneity. The isozymes from sunflower (Helianthus annuus) and Clarkia xantiana were partially purified. When subunits from two electrophoretically distinguishable cytosolic isozymes, either from the same or from different species, were dissociated and allowed to reassociate in each other's presence, an active hybrid enzyme, consisting of one subunit of each type, was formed in addition to the two original homodimers. Active hybrid enzymes were also formed by dissociation and reassociation of plastid isozymes. Hybrid molecules were not produced between the plastid and cytosolic subunits, suggesting that they are not able to bind with each other. Additional differences between the plastid and cytosolic isozymes are described.     

Cytosolic phosphoglucose isomerase is essential for microsporogenesis and embryogenesis in Arabidopsis

Phosphoglucose isomerase (PGI) catalyzes the interconversion of fructose-6-phosphate and glucose-6-phosphate, which impacts cell carbon metabolic flow. Arabidopsis (Arabidopsis thaliana) contains two nuclear PGI genes respectively encoding plastidial PGI1 and cytosolic PGI (cPGI). The loss of PGI1 impairs the conversion of F6P of the Calvin–Benson cycle to G6P for the synthesis of transitory starch in leaf chloroplasts. Since cpgi knockout mutants have not yet been obtained, they are thought to be lethal. The cpgi lethality can be rescued by expressing CaMV 35S promoter (p35S)-driven cPGI; however, the complemented line is completely sterile due to pollen degeneration. Here, we generated a cpgi mutant expressing p35S::cPGI-YFP in which YFP fluorescence in developing anthers was undetectable specifically in the tapetum and in pollen, which could be associated with male sterility. We also generated RNAi-cPGI knockdown lines with strong cPGI repression in floral buds that exhibited reduced male fertility due to the degeneration of most pollen. Histological analyses indicated that the synthesis of intersporal callose walls was impaired, causing microsporocytes to fail to separate haploid daughter nuclei to form tetrads, which might be responsible for subsequent pollen degeneration. We successfully isolated cpgi knockout mutants in the progeny of a heterozygous cpgi mutant floral-dipped with sugar solutions. The rescued cpgi mutants exhibited diminished young vegetative growth, reduced female fertility, and impaired intersporal callose wall formation in a meiocyte, and, thus, male sterility. Collectively, our data suggest that cPGI plays a vital role in carbohydrate partitioning, which is indispensable for microsporogenesis and early embryogenesis.

The cPGI-mediated carbohydrate partition is essential for early pollen and embryo development in Arabidopsis.     

Oat leaf phosphoglucose isomerase: competitive inhibition by erythrose-4-phosphate

Phosphoglucose isomerase was partially purified from oat leaves and shown to be strongly inhibited by erythrose-4-P. Estimated Ki values were between 0.4 and 4.0 M. The inhibition was of the competitive type with respect to either of the substrates glucose-6-P and fructose-6-P. Several other plant phosphoglucose isomerases were found to be similarly sensitive to erythrose-4-P.     

Competitive inhibition of spinach leaf phosphoglucose isomerase isoenzymes by erythrose 4-phosphate

In plant cells, the reversible isomerization between fructose 6-phosphate (Fru6P) and glucose 6-phosphate (Glc6P) is catalyzed by a cytosolic and a chloroplastic isoenzyme of phosphoglucose isomerase (PGI, EC 5.3.1.9). The extractable activities of both PGI isoenzymes are in large excess compared with the flux required for product synthesis, but the measured Glu6P/Fru6P ratio in illuminated chloroplasts and in whole leaves is always displaced from equilibrium. Cytosolic (PGI 2) and stromal (PGI 1) isoenzymes were purified from spinach leaves and used to investigate the possibility of metabolic regulation at this step. Several metabolites were found to inhibit PGI, but within the physiological concentration range, only erythrose 4-phosphate (Ery4P) inhibited significantly. The inhibition was competitive, with K_i values below 10 μM for PGI 2 and 1. The physiological significance of the inhibition of PGI by Ery4P was assessed in isolated intact spinach chloroplasts. We conclude that, in vivo, this inhibition is probably responsible for the observed displacement from equilibrium in the chloroplasts, but limits the carbon flow towards starch synthesis only when Fru6P is low. In contrast, the inhibition by Ery4P is unlikely to play any role in the cytosolic carbon metabolism because both Fru6P concentration and PGI activity, are much higher than in the chloroplast stroma.     

Mutations affecting starch synthase III in Arabidopsis alter leaf starch structure and increase the rate of starch synthesis

The role of starch synthase (SS) III (SSIII) in the synthesis of transient starch in Arabidopsis (Arabidopsis thaliana) was investigated by characterizing the effects of two insertion mutations at the AtSS3 gene locus. Both mutations, termed Atss3-1 and Atss3-2, condition complete loss of SSIII activity and prevent normal gene expression at both the mRNA and protein levels. The mutations cause a starch excess phenotype in leaves during the light period of the growth cycle due to an apparent increase in the rate of starch synthesis. In addition, both mutations alter the physical structure of leaf starch. Significant increases were noted in the mutants in the frequency of linear chains in amylopectin with a degree of polymerization greater than approximately 60, and relatively small changes were observed in chains of degree of polymerization 4 to 50. Furthermore, starch in the Atss3-1 and Atss3-2 mutants has a higher phosphate content, approximately two times that of wild-type leaf starch. Total SS activity is increased in both Atss3 mutants and a specific SS activity appears to be up-regulated. The data indicate that, in addition to its expected direct role in starch assembly, SSIII also has a negative regulatory function in the biosynthesis of transient starch in Arabidopsis.     

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.     

ABI3 affects plastid differentiation in dark-grown Arabidopsis seedlings

The Arabidopsis ABSCISIC ACID-INSENSITIVE3 (ABI3) protein has been identified previously as a crucial regulator of late seed development. Here, we show that dark-grown abi3 plants, or abi3 plants returned to the dark after germination in the light, developed and maintained an etioplast with a prominent prolamellar body at developmental stages in which the wild type did not. Overexpression of ABI3 led to the preservation of the plastid ultrastructure that was present at the onset of darkness. These observations suggest that ABI3 plays a role in plastid differentiation pathways in vegetative tissues. Furthermore, the analysis of deetiolated (det1) abi3 double mutants revealed that DET1 and ABI3 impinge on a multitude of common processes. During seed maturation, ABI3 required DET1 to achieve its full expression. Mature det1 abi3 seeds were found to be in a highly germinative state, indicating that germination is controlled by both DET1 and ABI3. During plastid differentiation in leaves of dark-grown plants, DET1 is required for the action of ABI3 as it is during seed development. Together, the results suggest that ABI3 is at least partly regulated by light.     

The histochemical demonstration of phosphoglucose isomerase

Summary A technique for the histochemical demonstration of phosphoglucose isomerase, using an indirect tetrazolium method, is described. The enzyme is shown to be widely distributed, thus confirming biochemical findings. The wide distribution is of significance because phosphoglucose isomerase occupies a position of considerable importance in carbohydrate metabolism, particularly in the conversion of fructose to glucose by means of intermediate phosphates.     

A variant of rabbit phosphoglucose isomerase

An autosomally inherited variant of the rabbit enzyme phosphoglucose isomerase that differs both electrophoretically and kinetically from the usual (wild-type) rabbit enzyme has been investigated. Animals homozygous for this character have an isomerase with considerably decreased activity (in the erythrocytes), an increased K(m) for fructose 6-phosphate, an increased K(i) for 6-phosphogluconate and a decreased stability towards heat and urea. The variant enzyme has been demonstrated in erythrocytes, leucocytes and tissues from the affected rabbits. The kinetic evidence suggests that the mutation present in the variant enzyme affects a histidine residue.     

Day length affects the dynamics of leaf expansion and cellular development in Arabidopsis thaliana partially through floral transition timing

Background and Aims: Plant aerial development is well known to be affected by daylength in terms of the timing and developmental stage of floraltransition. Arabidopsis thaliana is a ‘long day’plant in which the time to flower is delayed by short days andleaf number is increased. The aim of the work presented herewas to determine the effects of different day lengths on individualleaf area expansion. The effect of flower emergence per se onthe regulation of leaf expansion was also tested in this study. Methods: Care was taken to ensure that day length was the only sourceof micro-meteorological variation. The dynamics of individualleaf expansion were analysed in Ler and Col-0 plants grown underfive day lengths in five independent experiments. Responsesat cellular level were analysed in Ler plants grown under variousday lengths and treatments to alter the onset of flowering. Key Results: When the same leaf position was compared, the final leaf areaand both the relative and absolute rates of leaf expansion weredecreased by short days, whereas the duration of leaf expansionwas increased. Epidermal cell number and cell area were alsoaltered by day-length treatments and some of these responsescould be mimicked by manipulating the date of flowering. Conclusions: Both the dynamics and cellular bases of leaf development arealtered by differences in day length even when visible phenotypesare absent. To some extent, cell area and its response to daylength are controlled by whole plant control mechanisms associatedwith the onset of flowering.     

The cdc30 mutation in Saccharomyces cerevisiae affects phosphoglucose isomerase, the cell cycle and sporulation

Spontaneous revertants of the cdc30 mutation in Saccharomyces cerevisiae simultaneously regained the ability to grow and divide at 36.5 degrees C on glucose-containing media along with a more thermostable phosphoglucose isomerase (PGI). An independently isolated allele of cdc30 gave a similar phenotype to that previously described including temperature-sensitivity of PGI. Isoelectric focussing allowed the separation of two isoenzymes of PGI. These results all support the idea that two genes--PGI1 and CDC30--are responsible for PGI activity in yeast. Diploid strains homozygous for the cdc30 mutation sporulated poorly in potassium acetate irrespective of whether the cells had previously been cultured at a temperature that was permissive or restrictive for cell cycle progression. This was not surprising because a strain defective in PGI would not be expected to be able to complete the gluconeogenic events of sporulation.     

Rice debranching enzyme isoamylase3 facilitates starch metabolism and affects plastid morphogenesis

Debranching enzymes, which hydrolyze α-1 and 6-glucosidic linkages in α-polyglucans, play a dual role in the synthesis and degradation of starch in plants. A transposon-inserted rice mutant of isoamylase3 (isa3) contained an increased amount of starch in the leaf blade at the end of the night, indicating that ISA3 plays a role in the degradation of transitory starch during the night. An epitope-tagged ISA3 expressed in Escherichia coli exhibited hydrolytic activity on β-limit dextrin and amylopectin. We investigated whether ISA3 plays a role in amyloplast development and starch metabolism in the developing endosperm. ISA3-green fluorescent protein (GFP) fusion protein expressed under the control of the rice ISA3 promoter was targeted to the amyloplast stroma in the endosperm. Overexpression of ISA3 in the sugary1 mutant, which is deficient in ISA1 activity, did not convert water-soluble phytoglycogen to starch granules, indicating that ISA1 and ISA3 are not functionally redundant. Both overexpression and loss of function of ISA3 in the endosperm generated pleomorphic amyloplasts and starch granules. Furthermore, chloroplasts in the leaf blade of isa3 seedlings were large and pleomorphic. These results suggest that ISA3 facilitates starch metabolism and affects morphological characteristics of plastids in rice.     

Mutation of a termination codon affects src initiation.

The four Rous sarcoma virus messages gag, gag-pol, env, and src all derive from a full-length RNA precursor. All four messages contain the same 5' leader segment. Three of the messages, gag, gag-pol, and env, use an AUG present in this leader to initiate translation. The src AUG initiation codon lies 3' of the leader segment, 90 bases downstream of the gag initiation codon in the spliced src message. However, in the spliced src message a UGA termination codon lies between the gag AUG and the src AUG. All three codons are in the same reading frame. By using oligonucleotide-directed mutagenesis, the UGA termination codon has been converted to CGA. Cells infected with the mutant (called 1057 CGA) were spindle shaped, distinct from the rounded shape of cells infected with the parental Rous sarcoma virus. The mutant virus initiates src translation at the gag AUG, producing a 63,000-dalton src protein. We suggest that the wild-type src message produces two polypeptides, a very small (nine-amino acid) peptide that is initiated at the gag AUG and the 60,000-dalton src protein that is initiated at the src AUG.