Most of ADP x glucose linked to starch biosynthesis occurs outside the chloroplast in source leaves

Abstract

Sucrose and starch are end products of two segregated gluconeogenic pathways, and their production takes place in the cytosol and chloroplast of green leaves, respectively. According to this view, the plastidial ADP.glucose (ADPG) pyrophosphorylase (AGP) is the sole enzyme catalyzing the synthesis of the starch precursor molecule ADPG. However, a growing body of evidences indicates that starch formation involves the import of cytosolic ADPG to the chloroplast. This evidence is consistent with the idea that synthesis of the ADPG linked to starch biosynthesis takes place in the cytosol by means of sucrose synthase, whereas AGP channels the glucose units derived from the starch breakdown. To test this hypothesis, we first investigated the subcellular localization of ADPG. Toward this end, we constructed transgenic potato plants that expressed the ADPG-cleaving adenosine diphosphate sugar pyrophosphatase (ASPP) from Escherichia coli either in the chloroplast or in the cytosol. Source leaves from plants expressing ASPP in the chloroplast exhibited reduced starch and normal ADPG content as compared with control plants. Most importantly however, leaves from plants expressing ASPP in the cytosol showed a large reduction of the levels of both ADPG and starch, whereas hexose phosphates increased as compared with control plants. No pleiotropic changes in photosynthetic parameters and maximum catalytic activities of enzymes closely linked to starch and sucrose metabolism could be detected in the leaves expressing ASPP in the cytosol. The overall results show that, essentially similar to cereal endosperms, most of the ADPG linked to starch biosynthesis in source leaves occurs in the cytosol.

Fig. 1.

Suggested pathways of starch synthesis in source leaves. (A) “Classic model” according to which the starch biosynthetic process takes place exclusively in the chloroplast, segregated from the sucrose biosynthetic process taking place in the cytosol. (B) The alternative model in which both sucrose and starch biosynthetic pathways are interconnected by means of the ADPG-synthesizing activity catalyzed by SuSy. In A, starch is shown to be the end product of a unidirectional pathway, whereas B shows starch as an intermediate component of a cyclic gluconeogenic pathway. The enzyme activities that are involved are numbered as follows: 1 and 1′, fructose-1,6-bisphosphate aldolase; 2 and 2′, fructose 1,6-bisphosphatase; 3, PPi:fructose-6-phosphate phosphotransferase; 4 and 4′, phosphoglucoisomerase; 5 and 5′, phosphoglucomutase; 6, UGP; 7, SPS; 8, sucrose phosphate phosphatase; 9, AGP; 10, SS; 11, SP; 12, SuSy.

Fig. 2.

ADPG hydrolytic activity in source leaves of control (WT and 35S-GUS-int) and ASPP plants (35S-ASPP-NOS and 35S-ChlTPASPP-NOS). Results are given as mean ± SEM of 10 independent plants per line. FW, fresh weight.

Fig. 3.

ADPG and starch levels in fully expanded source (fifth) leaves from 6-week-old control and ASPP-expressing plants. Leaf samples were taken and quenched in liquid nitrogen at 7 h after the beginning of the light period. Results are given as mean ± SEM of 10 independent plants per line.