Scientific Correspondence The Debate on the Pathway of Starch Synthesis: A Closer Look at Low-Starch Mutants Lacking Plastidial Phosphoglucomutase Supports the Chloroplast-Localized Pathway

ADPglucose (ADPGlc) is the substrate for starch synthesis in the plastids of higher plants. The glucosyl moiety is used by starch synthases to elongate the glucans that comprise starch. Recently, there has been renewed debate about the ADPGlc synthesis, with the widely accepted or classical pathway questioned and a controversial new pathway proposed. Published experimental results are not entirely consistent with either pathway. Here we focus on starch synthesis in the Arabidopsis (Arabidopsis thaliana) leaf and present our opinion in the debate. The classical pathway of starch synthesis in Arabidopsis leaves involves the chloroplastic enzymes phosphoglucomutase (PGM), which generates Glc-1-P from Glc-6-P, and ADPGlc pyrophosphorylase (AGPase), which uses Glc-1-P and ATP to generate ADPGlc and inorganic pyrophosphate (Fig. 1A). Mutations affecting either enzyme cause low-starch phenotypes, providing support for their role in starch synthesis. Low-starch mutants lacking plastidial PGM have also been reported for tobacco (Nicotiana tabacum) and pea (Pisum sativum; Hanson and McHale, 1988; Harrison et al., 1998, 2000). However, results at odds with the classical pathway include the report that ADPGlc levels are unaffected in Arabidopsis mutants lacking chloroplastic PGM and AGPase (Muñoz et al., 2005). If the classical pathway of starch synthesis were the only source of ADPGlc, a substantial reduction or an absence of ADPGlc would be expected in both mutants. Furthermore, while frequently described as starchless, several publications report that starch is measurable in pgm (ranging from 1%–3% of the corresponding wild type; Caspar et al., 1985; Kofler et al., 2000; Gibon et al., 2004; Niittylä et al., 2004). Muñoz et al. (2006) reported levels as high as 15% of the corresponding wild-type levels upon the provision of exogenous sugars (although in this experiment the absolute levels in the wild type—7.2 mmol g fresh weight—were only 10%–20% of that typically seen in soil-grown plants). These data have been highlighted as support for a new pathway of starch synthesis whereby cytosolic Suc synthase (SuSy), using ADP and Suc, produces Fru and ADPGlc (Fig. 1B; see also Baroja-Fernández et al., 2004, 2005; Muñoz et al., 2005). This model proposes that ADPGlc is imported into the chloroplast for starch synthesis using a similar transport mechanism to that in the developing cereal endosperm (though in that case ADPGlc is synthesized by a cytosolic form of AGPase; see Tetlow et al., 2004, and refs. therein). If correct, this new model would force a major reconsideration of the pathways and regulation of photosynthetic carbon metabolism. An obvious inconsistency in this newly proposed pathway is the low-starch or starchless phenotypes of mutants lacking chloroplastic PGM and AGPase (pgm and adg1). These enzymes would not be required for starch synthesis as carbon exported from the chloroplast as triose-Ps would be converted to Suc (and thence to ADPGlc) in the cytosol. Reassessing the Arabidopsis pgm phenotype using electron microscopy reveals that most leaf mesophyll chloroplast sections did not contain starch granules, but in a few, tiny granules could be observed (Fig. 2, A and B). Tiny granules can also be purified and visualized by scanning electron microscopy (Fig. 2, C and D). Because of the size of the granules, most would be missed when making ultrathin sections. Thus, many or all pgm chloroplasts could contain such granules. The tiny granules are consistent with the very low starch contents measured in this material (0.3% of the wild type; the limit of detection in our analyses was approximately 0.03%, equivalent to 2 mg starch g fresh weight; Fig. 3). The mutation in pgm is a null mutation (Caspar et al., 1985; Periappuram et al., 2000). Thus, there must be another source of either Glc-1-P or ADPGlc, either produced inside the chloroplast, or imported from the cytosol. A simple explanation for the low levels of starch in pgm is that the alternative source of substrate limits the rate of starch synthesis. For example, chloroplast envelope transporters for triose-Ps, phosphoenolpyr1 This work was supported by ETH Zurich and the Swiss National Science Foundation (grant no. 3100AO–116434/1), and through the National Centre for Competence in Research—Plant Survival. * Corresponding author; e-mail [email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Samuel C. Zeeman ([email protected]). www.plantphysiol.org/cgi/doi/10.1104/pp.109.144931