Plastidic Phosphoglucose Isomerase Is an Important Determinant of Starch Accumulation in Mesophyll Cells, Growth, Photosynthetic Capacity, and Biosynthesis of Plastidic Cytokinins in Arabidopsis

<div><p>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 <i>pgi1-3</i>, a mutant totally lacking pPGI activity as a consequence of aberrant intron splicing of the pPGI encoding gene, <i>PGI1</i>. Starch content in <i>pgi1-3</i> source leaves was ca. 10-15% of that of wild type (WT) leaves, which was similar to that of leaves of <i>pgi1-2</i>, a T-DNA insertion pPGI null mutant. Starch deficiency of <i>pgi1</i> leaves could be reverted by the introduction of a <i>sex1</i> null mutation impeding β-amylolytic starch breakdown. Although previous studies showed that starch granules of <i>pgi1-2</i> 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 <i>pgi1-2</i> and <i>pgi1-3</i> mesophyll cells. RT-PCR analyses showed high expression levels of plastidic and extra-plastidic β-amylase encoding genes in <i>pgi1</i> leaves, which was accompanied by increased β-amylase activity. Both <i>pgi1-2</i> and <i>pgi1-3</i> 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 <i>pgi1</i> 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 <i>pgi1</i> leaves were exceedingly lower than in WT leaves. Noteworthy, exogenous application of CKs largely reverted the low starch content phenotype of <i>pgi1</i> 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.</p></div

<i>pgi1–3</i> is a pPGI null allele.

<p>(A) RT-PCR of <i>PGI1</i> and (B) starch content in source leaves of WT (L<i>er</i>), <i>pgi1–3</i> and two independent lines each of <i>pgi1–3</i>::<i>PGI1</i> and <i>pgi1–3</i>::<i>PGI1*</i>. Plants were cultured on soil under LD conditions and leaves harvested from 30 DAG plants after 12 h of illumination. In “B” values represent the mean ± SE of determinations on five independent samples.</p

<i>pgi1–3</i> leaves lack pPGI activity.

<p>(A) PGI zymogram of proteins extracted from WT (L<i>er</i>) and <i>pgi1–3</i> leaves. (B) Q-sepharose chromatography profile of PGI activity in WT and <i>pgi1–3</i> leaves. In “B”, loaded WT extract contained 850 mU of total PGI activity, whereas <i>pgi1–3</i> extract loaded on the column contained 650 mU of PGI activity.</p

Introduction of <i>sex1</i> mutation into <i>pgi1</i> plants reverts the low starch content phenotype.

<p>(A) Iodine staining and (B) starch content of L<i>er</i>, <i>pgi1–3/sex1</i>, Ws-2 and <i>pgi1–2/sex1</i> leaves. Plants were cultured on soil under LD conditions and leaves harvested from 30 DAG plants after 12 h of illumination. In “B” values represent the mean ± SE of determinations on five independent samples.</p

<i>pgi1–2</i> leaves have reduced photosynthetic capacity and ETR.

<p>(A) CO₂ assimilation rates, and (B) photosynthetic electron transport at different intercellular CO₂ concentrations in WT and <i>pgi1–2</i> source leaves. Plants were cultured on soil under LD conditions. Values represent the mean ± SE (n = 5).</p

Metabolites content in mature leaves of WT (Ws-2 and L<i>er</i>), <i>pgi1–2</i> and <i>pgi1–3</i> plants cultured on soil under LD conditions.

<p>Fully developed, source leaves were harvested from 30 DAG plants after 12 h of illumination. Values represent the mean ± SE of determinations on five independent samples. Each sample included leaves from 3 different rosettes. Asterisks indicate significant differences based on Student’s t-tests. (*P<0.05, <i>pgi1–2</i> vs. Ws-2; **P<0.05, <i>pgi1–3</i> vs. L<i>er</i>).</p

<i>pgi1–2</i> and <i>pgi1–3</i> leaves display a slow growth phenotype under SD and CL photoperiod conditions.

<p>(A) Photographs of 25 DAG WT, <i>aps1</i>, <i>pgm</i>, <i>pgi1–2</i> and <i>pgi1–3</i> plants cultivated in growth cabinets under CL conditions. (B) Time-course of FW of rosettes of WT, <i>aps1</i>, <i>pgm</i>, <i>pgi1–2</i> and <i>pgi1–3</i> plants cultured on soil under SD and CL conditions. In “B”, values represent the mean ± SE of determinations on four independent samples.</p

Starch content (as percentage of starch occurring in WT leaves) in leaves of <i>pgi1–2</i> cultured in MS medium including the indicated concentrations of tZ.

<p>Values represent the mean ± SE of determinations on four independent samples.</p

<i>pgi1</i> leaves have reduced photosynthetic capacity.

<p>The graphics represent net CO₂ uptake (A) and stomatal conductance (<i>g</i>_<i>s</i>) in source leaves of WT (Ws-2 and L<i>er</i>), <i>pgi1–2</i> and <i>pgi1–3</i> plants cultured on soil under SD and CL conditions. Values represent the mean ± SE of determinations on four independent samples. Asterisks indicate significant differences based on Student’s t-tests. (*P<0.05, <i>pgi1–2</i> vs. Ws-2; **P<0.05, <i>pgi1–3</i> vs. L<i>er</i>).</p

Scheme of CK biosynthesis through plastidic MEP- and cytosolic MVA-pathway.

<p>Black arrows show the biosynthesis, interconversions and metabolism flow of CKs in <i>Arabidopsis</i> cell (adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119641#pone.0119641.ref121" target="_blank">121</a>]). The dashed arrow indicates the iPRMP-independent pathway of tZ biosynthesis [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119641#pone.0119641.ref104" target="_blank">104</a>]. The blue and the red arrows indicate a hypothetical exchange of common precursor(s) between the MEP and MVA pathways (adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119641#pone.0119641.ref101" target="_blank">101</a>]). GAP, glyceraldehyde 3-phosphate; PYR, pyruvate; DXP, 1-deoxy-D-xylulose 5-phosphate.</p