Mutation of Arabidopsis plastid phosphoglucose isomerase affects leaf starch synthesis and floral initiation

Abstract

We isolated pgi1-1, an Arabidopsis mutant with a decreased plastid phospho-glucose (Glc) isomerase activity. While pgi1-1 mutant has a deficiency in leaf starch synthesis, it accumulates starch in root cap cells. It has been shown that a plastid transporter for hexose phosphate transports cytosolic Glc-6-P into plastids and expresses restricted mainly to the heterotrophic tissues. The decreased starch content in leaves of the pgi1-1 mutant indicates that cytosolic Glc-6-P cannot be efficiently transported into chloroplasts to complement the mutant's deficiency in chloroplastic phospho-Glc isomerase activity for starch synthesis. We cloned the Arabidopsis PGI1 gene and showed that it encodes the plastid phospho-Glc isomerase. The pgi1-1 allele was found to have a single nucleotide substitution, causing a Ser to Phe transition. While the flowering times of the Arabidopsis starch-deficient mutants pgi1, pgm1, and adg1 were similar to that of the wild type under long-day conditions, it was significantly delayed under short-day conditions. The pleiotropic phenotype of late flowering conferred by these starch metabolic mutations suggests that carbohydrate metabolism plays an important role in floral initiation.

Figure 1

Leaves and roots of the wild type, pgi1-1, and pgm1-1 mutants stained for starch with iodine. Plants of the wild type (A), pgi1-1 (B), and pgm1-1 (C) were de-pigmented and stained with an iodine solution. Starch can be seen to be present in the wild-type leaves and in root cap cells (arrowheads) of the wild type and the pgi1-1 mutant.

Figure 2

Activity gel assay for ADGase, PGM, and PGI in wild-type and mutant leaf extracts. Leaf extracts from the wild type (20 μL in lanes 1, 3, and 10; 10 μL in lane 7; and 0.4 μL in lane 5), TSY254 (20 μL in lanes 2, 4, and 8; 10 μL in lane 6), and TSY254/+ (20 μL in lane 9) were separated in a 7% (w/v) native PAGE and assayed for the ADGase, PGM, and PGI activity. ADGase activity was detected by calcium pyrophosphate precipitation; PGM and PGI activity were detected as the colored formazan formation in enzymatic coupling reactions. Arrowheads indicate the positions of the plastidial forms of the enzymes (Caspar et al., 1985).

Figure 3

Protein sequences of Arabidopsis PGIs. The top sequence is the plastid form and the bottom sequence is the cytosolic form. Identical amino acids are boxed, and the missense mutation (S166F) of pgi1-1 allele is labeled by an asterisk (*). Two conserved domains are underlined. The triangles indicate the positions of introns.

Figure 4

Northern-blot analysis of transcripts of plastidial PGI and tubulin in the wild type and pgi mutant. Northern blot of total leaf RNA (20 μg) isolated from the wild-type (WT) and pgi1-1 plants was probed with radioactive labeled Arabidopsis PGI1 and tubulin cDNA probes.

Figure 5

Immunoblot analysis of the plastidial PGI protein in wild-type (WT) and mutant leaf extracts. Leaf extracts from the wild type (containing 24, 18, and 12 μg of proteins in lanes 1–3, respectively), pgi1/+ (24 μg of proteins in lane 4), pgi1, pgm1, adg1 (24 μg of proteins in lanes 5–7), and prestained molecular mass markers (lane 8) were separated in a 10% (w/v) SDS PAGE, electroblotted onto a nylon membrane, and probed with the antiserum against Arabidopsis plastidial PGI protein. Bands of the expected sizes were detected by the antiserum, as indicated. There were cross-reactive bands detected by the antibodies. These may be related proteins sharing antigenic sites with the Arabidopsis plastidial PGI protein or E. coli-derived proteins.

Figure 6

Flowering time of the wild type and mutants under different growth conditions. Ten plants each of the wild type (WT; white columns), pgi1 (gray columns), pgm1 (stippled columns), and adg1 (black columns) mutants (all in Columbia ecotype) were grown under continuous light, 16-h-light, or 12-h-light conditions. Flowering time was recorded as the time the first floral stem appeared.