Fig 1.
Leaves of different starch breakdown mutants display a high starch content phenotype when cultured under continuous light conditions.
(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 (p<0.05).
Fig 2.
mex1 and mex1/pglct leaves accumulate higher levels of maltose than WT leaves when plants are cultured under continuous light conditions.
(A) Maltose content in leaves, (B) total chlorophyll content in leaves, (C) rosette FW and (D) external phenotype of 20 DAS WT (Col-0), mex1 and mex1/pglct plants cultured on soil under CL conditions. (E) External phenotype and (F) leaf maltose content in 20 DAS WT and mex1/pglct plants cultured on solid MS medium with or without 90 mM sucrose supplementation. In “A”, “B”, “C” and “F” 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 (p<0.05).
Fig 3.
Amyloglucosidase releases carbon compounds other than starch glucose molecules from Arabidopsis leaf ethanol precipitates.
The graphic shows the total carbon (TOC) and the starch carbon content in amyloglucosidase digests of WT (Col-0) and aps1 leaves. Values represent the means ± SE determined from three independent experiments using 6 plants in each experiment.
Fig 4.
Isotope ratio mass spectrometric evidence for the occurrence of starch cycling in illuminated leaves through a mechanism involving AGP.
The graphics represent the values of δ13C in starch of leaves of (A) 26 DAS WT (Col-0) and aps1 plants, and (B) 22 DAS WT (Ws-2) and pgi1-2 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 S4 Fig. Values represent the means ± SE determined from three independent experiments using 6 plants in each experiment.
Fig 5.
δ13C kinetics in starch is slower than that of sucrose in WT plants cultured in 13CO2-enriched environment.
The graphic represents the values of δ13C in starch and the indicated soluble sugars of leaves of 26 DAS WT (Col-0) 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.
Fig 6.
Maltose content in leaves of WT (Col-0), aps1, pgm, aps1/pgm, mex1/aps1 and mex1/pgm plants.
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 (*P<0.05, aps1, pgm and aps1/pgm vs. Col-0; **P<0.05, mex1/aps1 vs. aps1; ***P<0.05, mex1/pgm vs. pgm). 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).
Fig 7.
Incorporation of the gwd mutation enhances starch content in leaves of continuous light grown pgm and aps1 plants.
The graphic represents the starch content in 20 DAS aps1, aps1/gwd, pgm and pgm/gwd leaves. Values represent the means ± SE determined from three independent experiments using 6 plants in each experiment. Asterisks in aps1/gwd and pgm/gwd indicate significant differences with respect to aps1 and pgm plants according to Student´s t-tests (p<0.05).
Fig 8.
Suggested mechanism of starch metabolism in illuminated leaves of Arabidopsis involving simultaneous synthesis and breakdown of starch.
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 de novo 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.