Molecular characterisation of a new mutant allele of the plastid phosphoglucomutase in Arabidopsis, and complementation of the mutant with the wild-type cDNA

Abstract

Screening of transposon-associated mutants of Arabidopsis thaliana for altered starch metabolism resulted in the isolation of a mutant that did not accumulate starch in any tissue or at any developmental stage (starch-free mutant, stf1). Allelism tests with known mutants showed that stf1 represents a new mutant allele of the plastid isoform of the enzyme phosphoglucomutase (PGMp). The mutation was mapped to chromosome 5. An Arabidopsis EST that showed significant homology to the cytosolic isoform of phosphoglucomutase (PGM) from maize was able to complement the mutant phenotype. The Arabidopsis EST was transcribed and translated in vitro and the protein product was efficiently imported into isolated chloroplasts and processed to its mature form. The lack of starch biosynthesis in stf1 is accompanied by the accumulation of soluble sugars. The rate of CO2 assimilation measured in individual leaves was substantially diminished only under conditions of high CO2 and low O2. Remarkably, stf1 exhibits an increase rather than a decrease in total leaf PGM activity, suggesting an induction of the cytosolic isoform(s) in the mutant. The substrate for PGM, glucose 6-phosphate, accumulated in stf1 during the day, resulting in 10-fold higher content than in the wild type at the end of the photoperiod.

Differential metabolic networks unravel the effects of silent plant phenotypes

“…This phenomenon is often observed in plant molecular biology. In some cases, the total activity may even be found to have increased after suppression or knockout of a specific gene (25). In maize mutants, a loss of the sucrose synthase isoform sus1 was reported to have no phenotypic effect but was associated with ectopic expression of the other gene isoform (sh1 gene) complementing sus1 (26).…”

Diel Growth Cycle of Isolated Leaf Discs Analyzed with a Novel, High-Throughput Three-Dimensional Imaging Method Is Identical to That of Intact Leaves

“…On the other hand, only small replicate numbers are possible with this technique, as one camera can only visualize the growth of one leaf. The different growth kinematics of Ler and stf1 can be explained by the way that the stf1 mutation affects the diel carbohydrate availability (Kofler et al, 2000). In the daytime, the inability to synthesize starch causes an excess of hexoses to be available; thus, higher RGR in comparison with Ler can be maintained by stf1 mutants.…”

Analysis of the Plastidic phosphate translocator Gene Family in Arabidopsis and Identification of New phosphate translocator-Homologous Transporters, Classified by Their Putative Substrate-Binding Site

“…These include two dicarboxylate translocators that are involved in ammonia assimilation (Weber and Flü gge, 2002); a putative hexose transporter that exports hexoses, the product of hydrolytic starch degradation (Weber et al, 2000); an ADP/ATP translocator that supplies plastids with energy for biosynthesis of starch, fatty acids, and other compounds (Neuhaus et al, 1997); and a H ϩ /P i symporter (Pht2;1) that affects P i allocation within the plant (Daram et al, 1999;Versaw and Harrison, 2002).…”

“…In its functional form, pPT proteins are dimers composed of two identical subunits (Wagner et al, 1989). In this respect, the pPTs differ from other transporters of the plastid envelope membrane, which function as monomers that contain 12 transmembrane helices (Neuhaus et al, 1997;Weber et al, 2000;Weber and Flü gge, 2002). However, the pPT structures resemble the mitochondrial transporter superfamily without any significant similarity on the DNA or protein level (Walker and Runswick, 1993).…”

“…In heterotrophic tissues, the GPT mediates the uptake of carbon in the form of Glc-6-P into plastids, where it serves as substrate for starch synthesis, fatty acid synthesis, or the oxidative pentose phosphate pathway (Borchert et al, 1989;Bowsher et al, 1992;Flü gge, 1999). Analysis of starchless mutant lines that are deficient in the plastidic phosphoglucomutase (catalyzing the interconversion of Glc-6-P and Glc-1-P), led to the conclusion that in most plants, Glc-6-P is the sole precursor for starch synthesis (Harrison et al, 2000;Kofler et al, 2000).…”