Genetic and isotope ratio mass spectrometric evidence for the occurrence of starch degradation and cycling in illuminated Arabidopsis leaves

<div><p>Although there is a great wealth of data supporting the occurrence of simultaneous synthesis and breakdown of storage carbohydrate in many organisms, previous ¹³CO₂ pulse-chase based studies indicated that starch degradation does not operate in illuminated Arabidopsis leaves. Here we show that leaves of <i>gwd</i>, <i>sex4</i>, <i>bam4</i>, <i>bam1/bam3</i> and <i>amy3/isa3/lda</i> starch breakdown mutants accumulate higher levels of starch than wild type (WT) leaves when cultured under continuous light (CL) conditions. We also show that leaves of CL grown <i>dpe1</i> plants impaired in the plastidic disproportionating enzyme accumulate higher levels of maltotriose than WT leaves, the overall data providing evidence for the occurrence of extensive starch degradation in illuminated leaves. Moreover, we show that leaves of CL grown <i>mex1/pglct</i> plants impaired in the chloroplastic maltose and glucose transporters display a severe dwarf phenotype and accumulate high levels of maltose, strongly indicating that the MEX1 and pGlcT transporters are involved in the export of starch breakdown products to the cytosol to support growth during illumination. To investigate whether starch breakdown products can be recycled back to starch during illumination through a mechanism involving ADP-glucose pyrophosphorylase (AGP) we conducted kinetic analyses of the stable isotope carbon composition (δ¹³C) in starch of leaves of ¹³CO₂ pulsed-chased WT and AGP lacking <i>aps1</i> plants. Notably, the rate of increase of δ¹³C in starch of <i>aps1</i> leaves during the pulse was exceedingly higher than that of WT leaves. Furthermore, δ¹³C decline in starch of <i>aps1</i> leaves during the chase was much faster than that of WT leaves, which provides strong evidence for the occurrence of AGP-mediated cycling of starch breakdown products in illuminated Arabidopsis leaves.</p></div

Amyloglucosidase releases carbon compounds other than starch glucose molecules from Arabidopsis leaf ethanol precipitates.

<p>The graphic shows the total carbon (TOC) and the starch carbon content in amyloglucosidase digests of WT (Col-<i>0</i>) and <i>aps1</i> leaves. Values represent the means ± SE determined from three independent experiments using 6 plants in each experiment.</p

Leaves of different starch breakdown mutants display a high starch content phenotype when cultured under continuous light conditions.

<p>(A) Iodine staining and (B) starch content in leaves of WT and the indicated starch breakdown mutants cultured under CL conditions. Leaves were harvested at the 18 days after sowing (DAS) growth stage. In “B” values represent the means ± SE determined from three independent experiments using 6 plants in each experiment. Asterisks indicate significant differences with respect to WT plants according to Student´s t-tests (<i>p</i><0.05).</p

Isotope ratio mass spectrometric evidence for the occurrence of starch cycling in illuminated leaves through a mechanism involving AGP.

<p>The graphics represent the values of δ¹³C in starch of leaves of (A) 26 DAS WT (Col-<i>0</i>) and <i>aps1</i> plants, and (B) 22 DAS WT (Ws-2) and <i>pgi1-2</i> plants exposed to ¹³C enriched CO₂ for 5 hours and then chased for 15 additional hours. Plants were cultured in growth cabinets under long day (LD) conditions. The grey area indicates the ¹³CO₂ pulse period. Starch content in leaves is shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171245#pone.0171245.s004" target="_blank">S4 Fig</a></b>. Values represent the means ± SE determined from three independent experiments using 6 plants in each experiment.</p

Suggested mechanism of starch metabolism in illuminated leaves of Arabidopsis involving simultaneous synthesis and breakdown of starch.

<p>During the day, photosynthetically fixed carbon is either exported to the cytosol as triose phosphates by means of TPT to be subsequently converted into sucrose, and/or retained within the chloroplast to fuel starch biosynthesis. Starch is then degraded to maltose and glucose molecules that are either exported to the cytosol via MEX1 and pGlcT, respectively, or recycled back to starch. This interpretation of leaf starch metabolism previews that (i) hexose-phosphates and/or ADPG occurring in the cytosol enter the chloroplast for subsequent conversion into starch, (ii) CBC and the pPGM-AGP-SS starch biosynthetic pathway are not connected by pPGI, and (iii) pPGM and AGP play important roles not only in the <i>de novo</i> synthesis of starch from the CBC, but also in the scavenging of starch breakdown products. The enzyme activities involved are numbered as follows: 1, pPGI; 2, pPGM; 3, AGP; 4, SS; 5, β-amylase; 6, AMY; 7, debranching enzymes; 8, DPE1; 9, SP; 10, hexokinase. Enzymatic reactions involved in starch cycling are indicated with red arrows.</p

Maltose content in leaves of WT (Col-<i>0</i>), <i>aps1</i>, <i>pgm</i>, <i>aps1/pgm</i>, <i>mex1/aps1</i> and <i>mex1/pgm</i> plants.

<p>Leaves of the indicated plants were harvested at the 18 DAS growth stage. Values obtained using HPAEC-PAD and GC-MS are represented with white and grey columns, respectively. Values represent the means ± SE determined from three independent experiments using 6 plants in each experiment. Asterisks indicate significant differences according to Student´s t-tests (*<i>P</i><0.05, <i>aps1</i>, <i>pgm</i> and <i>aps1/pgm</i> vs. Col-<i>0</i>; **<i>P</i><0.05, <i>mex1/aps1</i> vs. <i>aps1;</i> ***<i>P</i><0.05, <i>mex1/pgm</i> vs. <i>pgm</i>). Values correspond to plants cultured under continuous light (CL) conditions. Essentially the same results were obtained using plants cultured under long day (LD) conditions (not shown).</p

δ¹³C kinetics in starch is slower than that of sucrose in WT plants cultured in ¹³CO₂-enriched environment.

<p>The graphic represents the values of δ¹³C in starch and the indicated soluble sugars of leaves of 26 DAS WT (Col-<i>0</i>) plants exposed to ¹³C enriched CO₂ for 5 hours and then chased for 15 additional hours. Plants were cultured in growth cabinets under long day (LD) conditions. The grey area indicates the ¹³CO₂ pulse period. Values represent the means ± SE determined from three independent experiments using 6 plants in each experiment.</p

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> 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

<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