C. L. Hedley

The wrinkled-seed character of pea described by Mendel is caused by a transposon-like insertion in a gene encoding starch-branching enzyme

We describe the cloning of the r (rugosus) locus of pea (Pisum sativum L.), which determines whether the seed is round or wrinkled. Wrinkled (rr) seeds lack one isoform of starch-branching enzyme (SBEI), present in round (RR or Rr) seeds. A major polymorphism in the SBEI gene between near-isogenic RR and rr lines shows 100% cosegregation with the r locus, establishing that the SBEI gene is at the r locus. An aberrant transcript for SBEI is produced in rr embryos. In rr lines the SBEI gene is interrupted by a 0.8 kb insertion that is very similar to the Ac/Ds family of transposable elements from maize. Failure to produce SBEI has complex metabolic consequences on starch, lipid, and protein biosynthesis in the seed.

Mutations in the Gene Encoding Starch Synthase II Profoundly Alter Amylopectin Structure in Pea Embryos

Mutations at the rug5 (rugosus5) locus have been used to elucidate the role of the major soluble isoform of starch synthase II (SSII) in amylopectin synthesis in the developing pea embryo. The SSII gene maps to the rug5 locus, and the gene in one of three rug5 mutant lines has been shown to carry a base pair substitution that introduces a stop codon into the open reading frame. All three mutant alleles cause a dramatic reduction or loss of the SSII protein. The mutations have pleiotropic effects on the activities of other isoforms of starch synthase but apparently not on those of other enzymes of starch synthesis. These mutations result in abnormal starch granule morphology and amylopectin structure. Amylopectin contains fewer chains of intermediate length (B2 and B3 chains) and more very short and very long chains than does amylopectin from wild-type embryos. The results suggest that SSII may play a specific role in the synthesis of B2 and B3 chains of amylopectin. The extent to which these findings can be extrapolated to other species is discussed.

The granular structure of C-type pea starch and its role in gelatinization

We have used a combination of techniques to study the structure and properties of C-type starch from pea seeds. It was found that all C-type starch granules contain both types of polymorph; the B polymorphs are in the center of the granule and are surrounded by the A polymorphs. During heating in excess salt solution the A and B polymorphs within C-type granules melt independently, giving a double transition in heat capacity and a two-step swelling, compared with single transitions for A- and B-type starches. It was shown that B polymorphs gave a transition with a lower peak temperature than A. The disruption of crystallinity during gelatinization began from the hilum area and was propagated along the granule, accompanied by swelling of disrupted areas. It is proposed that the swelling of disrupted parts of the granule decreases the melting temperature of the neighboring crystallites resulting in the progressive disruption of crystalline areas. The gelatinization process is dependent on the arrangement of A and B polymorphs within the granule.

An analysis of seed development in Pisum sativum L.

SummaryPea embryos have been treated with xenobiotic substances to manipulate specific cellular events. Taxol was able to induce condensation of chromatin in endoreduplicated nuclei, resulting in cells with either normal or increased numbers of chromosomes at all stages of the mitotic cycle. Application of the synthetic auxin, 2,4-D allowed a proportion of dividing cells to be retained throughout the culture period. However, this did not alter the range of different nuclear DNA levels when compared with embryos grown in the absence of the auxin. The effect of applying aphidicolin produced the most dramatic alteration in cellular behavior by limiting the increase in DNA levels. This resulted in some cells of 4 C DNA level containing vicilin. These results are discussed in the light of previous findings, when it was suggested that the onset of storage protein deposition is linked to the cessation of mitosis and subsequent cell expansion rather than to elevated cellular DNA levels as once thought.

The effect of water content on the ordered/disordered structures in starches.

(13)C cross-polarization magic angle spinning NMR has been used to study the ordered and disordered structures of starches with different water contents. The amorphous regions of starch have been shown to produce NMR patterns only if they are in a glassy state, the widths, positions, and areas of the peaks to some extent being dependent on the temperature and the water content of the starch. In the amorphous region, the peaks were all Gaussian in shape, while the peaks in the ordered regions had Lorentz profiles. Water contents in the range 10-50% did not influence the proportion of double helices in the starch. Decreasing the water content to 1-3%, however, resulted in a significant decrease in the proportion of double helices, the effect being greater in B- than in A-type starches. It is suggested that short-range order structures in starches (double helices) are stabilized by becoming part of long-range order structures (crystallites).

Determination of the polymorphic composition of smooth pea starch

Abstract A method for estimating the proportions of ‘A’ and ‘B’ polymorphs comprising a sample of ‘C’ type starch is proposed which uses established experimental techniques with commercially available spreadsheet and X-ray analysis software. Waxy maize, potato and smooth pea starches were used to provide X-ray diffraction patterns characteristic of the ‘A’, ‘B’ and ‘C’ starch polymorphs. Samples of amorphous starches were also prepared. The method initially involved subtraction of the amorphous phase and instrumental background from the X-ray diffraction patterns of each starch sample using the spreadsheet program, Lotus 1-2-3. The remainder of the pattern, representing the crystalline portion of the starch sample, was then analysed by profile fitting to elucidate the positions and areas of individual diffraction peaks. The ratio of the total peak area to the areas under peaks characteristic of ‘A’ and ‘B’ type starches, respectively, were used to calculate the relative proportions of these polymorphs in smooth pea starch. These proportions were found to be 56±3% ‘A’ polymorph to 44±3% ‘B’ polymorph. A ‘C’ type pattern was constructed by using Lotus 1-2-3 to combine diffraction patterns from the crystalline portions of ‘A’ and ‘B’ type starches in the proportions given above. Polymorph patterns were obtained by manipulation of the diffraction patterns from the crystalline portions of starches using Lotus 1-2-3. An ‘A’ type pattern was obtained by subtraction of a ‘B’ type pattern from that of a ‘C’ type. Similarly, a ‘B’ type pattern was obtained by subtraction of an ‘A’ type pattern from that of a ‘C’ type.

The effect of mutant genes at the r, rb, rug3, rug4, rug5 and lam loci on the granular structure and physico-chemical properties of pea seed starch

The granular structure and gelatinisation properties of starches from a range of pea seed mutants were studied. Genes which affect the supply of substrate during starch synthesis (rb, rug3, rug4) affected the total crystallinity and possibly increased the content of A polymorphs in the starch. Conversely, genes directly affecting the synthesis of starch polymers (r, rug5, lam) increased the content of B polymorphs, but had a minimal effect on total crystallinity. During gelatinisation, starches from the rb, rug3, rug4 and lam mutants had narrow endothermic peaks which were similar to starch from the wild-type, although all the starches had different peak temperatures and enthalpy changes. Starches from r and rug5 mutants were very different to all other starches, having a very wide transition during gelatinisation. In addition, the amylopectin in starch from these mutants had altered chain lengths for those parts of the polymer which form the ordered structures in the granule.

Atomic force microscopy of pea starch granules: granule architecture of wild-type parent, r and rb single mutants, and the rrb double mutant

AFM studies have been made of the internal structure of pea starch granules. The data obtained provides support for the blocklet model of starch granule structure (Carbohydr. Polym. 32 (1997) 177-191). The granules consist of hard blocklets dispersed in a softer matrix material. High-resolution images have yielded new insights into the detailed structure of growth rings within the granules. The blocklet structure is continuous throughout the granule and the growth rings originate from localised defects in blocklet production distributed around the surface of spheroidal shells within the granules. A mutation at the rb locus did not lead to significant changes in granule architecture. However, a mutation at the r locus led to loss of growth rings and changed blocklet structure. For this mutant the blocklets were distributed within a harder matrix material. This novel composite arrangement was used to explain why the granules had internal fissures and also changes in gelatinisation behaviour. It is suggested that the matrix material is the amylose component of the granule and that both amylose and amylopectin are present within the r mutant starch granules in a partially-crystalline form. Intermediate changes in granule architecture have been observed for the double mutant rrb.

Seed development in peas: knowing your three ‘r's’ (or four, or five)

The value of the garden pea (Pisum sativum) both as an experimental tool and food crop has led to an accumulated wealth of genetic variation. This paper describes how some of the variation may be harnessed for studying seed development. One can identify genotypic differences for maternal, embryonic and cellular components and the interaction between them. The impact of these components on processes such as cell division, partitioning and the deposition of storage products can then be assessed by utilizing new techniques such as immunocytochemistry and in situ hybridization.The rugosus loci, which induce wrinkling of the seed, are examples of how major genes can be exploited to dissect seed development. Alleles at one of these loci, r, have been defined at both the molecular and biochemical levels. A much clearer picture can now be drawn of the pleiotropic nature of the genes, from their effects on starch composition to those on cellular development, which has implications for seed development in many species. The rugosus loci are the only ones known to affect seed development in peas. By analogy with cereal seeds, however, one would anticipate other mutants in the starch pathway which also affect the shape of the seed. Here, we report the induction of new rugosus-like mutants, compare them to starch mutants in other species and examine the ways in which they may help our understanding of starch biosynthesis and the regulation of seed development.

Determination of the effect of r and rb mutations on the structure of amylose and amylopectin in pea (Pisum sativum L.)

The structure of starch from pea lines near-isogenic except for mutation at the r and rb loci was compared with that of the wild-type. The isolated starch polysaccharides were fractionated by size exclusion chromatography, and the constituent chain profile of the branched amylopectin components examined. A mutation at the rb locus had little effect on starch structure and composition. A mutation at the r locus (a genetic lesion affecting the activity of an isoform of starch branching enzyme) resulted in an increase in the amylose content of the starch and a change in the structure of amylopectin.