Mutations affecting starch synthase III in Arabidopsis alter leaf starch structure and increase the rate of starch synthesis

Abstract

The role of starch synthase (SS) III (SSIII) in the synthesis of transient starch in Arabidopsis (Arabidopsis thaliana) was investigated by characterizing the effects of two insertion mutations at the AtSS3 gene locus. Both mutations, termed Atss3-1 and Atss3-2, condition complete loss of SSIII activity and prevent normal gene expression at both the mRNA and protein levels. The mutations cause a starch excess phenotype in leaves during the light period of the growth cycle due to an apparent increase in the rate of starch synthesis. In addition, both mutations alter the physical structure of leaf starch. Significant increases were noted in the mutants in the frequency of linear chains in amylopectin with a degree of polymerization greater than approximately 60, and relatively small changes were observed in chains of degree of polymerization 4 to 50. Furthermore, starch in the Atss3-1 and Atss3-2 mutants has a higher phosphate content, approximately two times that of wild-type leaf starch. Total SS activity is increased in both Atss3 mutants and a specific SS activity appears to be up-regulated. The data indicate that, in addition to its expected direct role in starch assembly, SSIII also has a negative regulatory function in the biosynthesis of transient starch in Arabidopsis.

Figure 1.

Characterization of Atss3 mutations. A, Gene map. The scaled linear map depicts the 14 exons as boxes and the 13 introns as lines between the boxes. The positions of the translational start and stop codons in exons 1 and 14, respectively, are noted. The locations of specific PCR primer sequences are noted, as well as the locations of the two T-DNA insertions in the gene (insertions are not drawn to scale). B, Analysis of AtSS3 5′-end transcripts. Control genomic DNA from wild-type leaves (lane 1) was amplified by PCR and total leaf RNA from Atss3-1 and Atss3-2 mutant plants (lanes 2 and 3, respectively) was amplified by RT-PCR using the gene-specific primers SS3-U2 and SS3-L1. C, Analysis of transcripts from the Atss3-1 mutant. Total RNA from leaves of wild-type plants (lane 1) and Atss3-1 plants (lanes 2 and 3) was amplified by RT-PCR. Primer pairs are SS3-TU1 and SS3-TL1 (lanes 1 and 3). Arabidopsis SSII gene-specific primers SS2-RP1 and SS2-LP1 served as a positive control (lane 2). D, Analysis of transcripts from the Atss3-2 mutant. Total RNA from leaves of wild-type plants (lane 1) and Atss3-2 plants (lanes 2 and 3) was amplified by RT-PCR. Primer pairs were SS3-RP2 and SS3-LP2 (lanes 1 and 3) or Arabidopsis SSII gene-specific primers SS2-RP2 and SS2-LP2 as a positive control (lane 2). E, Analysis of SSIII protein accumulation. Total soluble leaf extracts from wild-type, Atss3-1, and Atss3-2 plants were separated by SDS-PAGE and probed in immunoblot analysis with α-AtSSIII monoclonal antibody.

Figure 2.

Analysis of SSIII activity. A, Two-dimensional zymogram analysis. Proteins from equivalent fresh weights of leaves of wild-type (top) and Atss3-1 (bottom) plants were separated by anion-exchange chromatography. Proteins in equivalent volumes of the indicated HiTrapQ column fractions were separated in native polyacrylamide gels containing 0.3% glycogen, then stained with iodine solution. Identifiable SS activity bands are indicated. The asterisk in the Atss3-1 analysis indicates the position that corresponds to band A in the wild-type analysis. B, Immunoblot analysis. Duplicate gels to those shown in (A) were probed for the presence of SSIII with α-AtSSIII monoclonal antibody. C, One-dimensional zymogram analysis. Total soluble protein extracts from leaves of wild-type, Atss3-1, and Atss3-2 plants were analyzed for SS activity as described for (A). In this instance, there was no prior anion-exchange fractionation step.

Figure 3.

Qualitative analysis of leaf starch content. Wild-type, Atss3-1, and Atss3-2 Arabidopsis plants grown under long-day (16-h light/8-h dark) conditions (A–C) or short-day (12-h light/12-h dark) conditions (D and E) were decolorized and stained with iodine solution, then washed with water and photographed. A, Atss3-1 and wild-type leaves harvested at the end of the light phase. B, Atss3-1 and wild-type leaves harvested at the end of the dark phase. C, Atss3-2 and wild-type leaves harvested at the end of the light phase. D, Atss3-1 and wild-type leaves harvested at the end of the light phase. E, Atss3-1 and wild-type leaves harvested at the end of the dark phase.

Figure 4.

Starch accumulation in Arabidopsis leaves over the course of a diurnal cycle. Starch was harvested at 4-h intervals from wild-type plants (solid line) and Atss3-1 plants (dashed line) grown under a long-day photoperiod of 16-h light (white bar)/8-h dark (black bar). Starch quantification is in units of mg/g fresh weight of tissue. Each data point is the average of six independent plants and the se is indicated.

Figure 5.

Size fractionation of glucan polymers from starch granules. Starch from wild-type and Atss3-1 plants was separated by GPC on a Sepharose CL-2B column, and the Glc equivalents in each column fraction were determined.

Figure 6.

FACE analysis of leaf starch chain length distributions. Starch from leaves of wild-type, Atss3-1, and Atss3-2 plants was debranched with commercial isoamylase. The reducing ends of the linear chains were fluorescently labeled and separated by FACE. The frequency of individual chain lengths in each starch sample was normalized to total peak area. In the two bottom images, the normalized value for each chain length from the wild-type starch was subtracted from that of either the Atss3-1 or the Atss3-2 mutant. Values are the average of three independent determinations and se is indicated.

Figure 7.

Size fractionation of linear glucan chains. Starch from leaves of wild-type (solid line), Atss3-1 (dotted line), and Atss3-2 (dashed line) plants was debranched to completion with commercial isoamylase and the resulting linear chains were separated by GPC on a Sepharose CL-2B column. The Glc equivalents in each column fraction were quantified and plotted as a percentage of total Glc equivalents.

Figure 8.

Starch granule morphology. Starch granules from leaves of wild-type and Atss3-1 plants were coated with gold particles, visualized by scanning electron microscopy, and photographed.