Phosphoglucoisomerase Is an Important Regulatory Enzyme in Partitioning Carbon out of the Calvin-Benson Cycle

Abstract

Phosphoglucoisomerase (PGI) isomerizes fructose 6-phosphate (F6P) and glucose 6-phosphate (G6P) in starch and sucrose biosynthesis. Both plastidic and cytosolic isoforms are found in plant leaves. Using recombinant enzymes and isolated chloroplasts, we have characterized the plastidic and cytosolic isoforms of PGI. We have found that the Arabidopsis plastidic PGI K _m for G6P is three-fold greater compared to that for F6P and that erythrose 4-phosphate is a key regulator of PGI activity. Additionally, the K _m of spinach plastidic PGI can be dynamically regulated in the dark compared to the light and increases by 200% in the dark. We also found that targeting Arabidopsis cytosolic PGI into plastids of Nicotiana tabacum disrupts starch accumulation and degradation. Our results, in combination with the observation that plastidic PGI is not in equilibrium, indicates that PGI is an important regulatory enzyme that restricts flow and acts as a one-way valve preventing backflow of G6P into the Calvin-Benson cycle. We propose the PGI may be manipulated to improve flow of carbon to desired targets of biotechnology.

Keywords: Calvin-Benson cycle; carbon partitioning; erythrose 4-phosphate; phosphoglucoisomerase; starch.

FIGURE 1

Comparison of specific activity of plastidic and cytosolic AtPGI with various metabolites. Each bar represents mean and error bars represent SD, (n = 3). All metabolites were screened at 1:1 F6P substrate to metabolite. Data with an asterisk (*) are significantly different from the control as determined by Student’s t-test (P < 0.05) (two tailed and assuming unequal variance). 3PGA, 3-phosphoglyceric acid; E4P, erythrose 4-phosphate; DHAP, dihydroxyacetone phosphate; 6PG, 6 phosphogluconate.

FIGURE 2

Effect of erythrose 4-phosphate (E4P) and 6-phosphogluconate (6PG) on plastidic AtPGI. We measured the effect of E4P (A,B) and 6PG (C,D) on AtPGI. Different symbols represent different concentrations of inhibitor. PGI was more inhibited by E4P than by 6PG. Lines represent data fit to Eq. 2. F6P, fructose 6-phosphate; G6P, glucose 6-phosphate.

FIGURE 3

Comparison of fructose 6-phosphate (F6P) and glucose 6-phosphate (G6P) K_m in plastidic phosphoglucoisomerase in dark and light-treated isolated spinach chloroplasts. Each bar represents mean and error bars represent SE, (n = 3 independent isolations of chloroplasts). The K_m for G6P increased in dark treated compared to light treated isolated chloroplasts. Bars with an asterisk (*) are significantly different from corresponding light treated samples as determined by Student’s t-test (P < 0.05) (two tailed and assuming unequal variance).

FIGURE 4

Localization of modified plastid phosphoglucoisomerase (cPGI) to the chloroplast. Confocal images of Nicotiana tabacum epidermal cell transiently expressing N- or C-terminal tagged mislocalized PGI. Both fluorescent fusions localize at the chloroplasts (cyan) as the co-localization (merge channel) shows with the chloroplast autofluorescence (magenta). Scale bar 10 μm.

FIGURE 5

Starch amounts at end of day and end of night (A) and degradation of starch (B) in Nicotiana tabacum expressing mislocalized plastid phosphoglucoisomerase (pPGI). Starch in plants with mislocalized pPGI was significantly higher than empty vector control plants. Starch breakdown in empty vector plants was linear through the night, however, starch breakdown was non-linear in mislocalized pPGI plants. Each bar or data point represents mean and error bars represent SE, (n = 5 biological replicates). Bars or data points with an asterisk (*) are significantly different from corresponding empty vector samples as determined by Student’s t-test (P < 0.05) (two tailed and assuming unequal variance). Significance for linearity was determined by Prob(F) of the linear regressions.