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At the time the programme began, two loci had already been identified that affected seed shape through an influence on embryo development (as opposed to testa development). Kooistra described two mutant lines that had wrinkled seeds. The dry seed of both mutants had a starch content reduced by about 40% (on a dry weight basis). One was identified as being at the original rugosus (r) locus (rugosus meaning wrinkled) which had been described by Gregor Mendel in the 19^th century and given the gene symbol, r, by White. The other represented a new locus, so Kooistra renamed Mendel's locus (incorrectly as it happens) the r_a locus, and he termed the second r_b. Both loci were subsequently renamed correctly as r and rb by Blixt. Over 30 wrinkled-seeded mutants were isolated. Complementation testing indicated that there were 5 wrinkled-seeded loci that affected the starch content of the seed, the original two and 3 new ones named, rug3, rug4 and rug5. The locus that influenced starch, but not seed shape was named low amylose (lam).

  
Mutations at the r locus, which encodes a starch-branching enzyme (SBEI or SBEA), exhibit considerable pleiotropy. The lack of enzyme activity in the mutant brings about a decrease in the amylopectin content of the starch considerably. Wild-type pea starch usually contains about 30% amylose. In the r mutant, this increases to 70%. The consequences of the decrease in starch are increases in the lipid content of the embryo, the sugar content, the osmotic pressure and the water uptake. There is also a change in the composition of the storage protein. These events have been linked using a 'Jigsaw' model which holds that the change in the osmotic environment is the key event in the pleiotropy of the locus. Essentially the model pertains to all loci that bring about a decrease in the starch content of the embryo. The r locus was one of the loci investigated by Gregor Mendel in his work on peas.

  
The rb mutants decrease the activity of the enzyme ADPglucose pyrophosphorylase and increase its sensitivity to allosteric regulation. All show a decrease in the starch content to about 30% of the dry weight - a very similar amount to those at the r locus. The effect on starch composition is quite different, however, in that the amylose content is lowered to ca. 20% of the starch.

Mutants at the rug3 locus have a severe effect on the starch content of the seed, lowering it to between 1 and 12% of the dry weight (depending on the allele). Similar mutants have been isolated in Nicotiana sylvestris and Arabidopsis thaliana. In both these species, mutants have been isolated whose leaves have been shown to lack starch. In rug3 mutants, however, the starchless phenotype has been demonstrated in leaves, roots and seeds. The evidence from biochemical assays and linkage studies indicated that the pea mutant alleles decrease the activity of plastidial phosphoglucomutase which, in pea, is the minor form of the activity. This indicates that the hypothesis of Hill and Smith is correct in that glucose-6-phosphate must be the imported substrate for starch synthesis in peas. Furthermore, the rug3 mutants demonstrate unequivocally that starchless mutants can exist in starch-accumulating crop plants and support the view held by Porter that starch is "the product of excess assimilation" rather than "a purposive reserve for future metabolic events" since the plants are completely viable.

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The starch content of rug4 embryos is only 10% less than the wild type and consequently the seed are mildly wrinkled. Investigations into the rug4 locus indicate that it affects the activity of sucrose synthase, embryos from the mutants having only 5% the activity of wild type. In leaves the reduction is about 50%, whereas in nodules activity is reduced such that the nodules cannot function correctly and the plants consequently are nitrogen deficient and derive little nitrogen from fixation by Rhizobium. In the field the plants grow very poorly without additional nitrogen. Recent studies have shown that the locus encodes one isoform of sucrose synthase.

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In the biosynthetic pathway to starch, there are a number of isoforms of starch synthases. Mutants affecting the major granule bound starch synthase do not affect the amount of starch significantly (see lam below). In pea, there is an active starch synthase isoform of 77 kDa (SSII). Mutants at the rug5 locus lack this enzyme activity and the starch content of their seed is decreased to the same degree as in r and rb mutants. The amylose content,however, is increased to ca. 50% of the starch. Structural studies indicate that the starch from rug5 is very different from that of the wild type in that it contains more short chains (DP15 and less), more long chains (DP>1000), but fewer intermediate chains (DP15-45) in the amylopectin. The physicochemical properties of the starch are very different from those of the wild type and any other pea mutant as are the starch granules. A mutant (sta3) with similar effects on starch structure has also been isolated from Chlamydomonas. This mutant affects the activity of one of the soluble starch synthases in this organism and is the only mutant similar to rug5 in plants

  
Mutants that do not decrease the starch content of the seed significantly, but affect its composition should not possess wrinkled seeds. Such mutants are usually of the 'waxy' maize type. In potato, the equivalent mutant (amf) was isolated on the basis of iodine staining. The amf mutant and other such 'waxy'-like mutants contain essentially amylopectin. Amylopectin has different iodine affinity and staining properties to amylose, producing a red colour rather than the usual indigo blue of normal starches. A screen of the original mutagenised population based on the colouration of starch grains transferred from dry seeds to filter paper led to the isolation of 5 alleles at a locus named lam (low amylose). These mutants had round seeds, as predicted, and were similar to others in their class in that they produced no, or very low amounts of, amylose. The mutants were shown to lack a 59kDa granule bound starch synthase.

Key references:

Ball SG, Guan H-P, James M, Myers A, Keeling p, Mouille G, Buleon A, Colonna P, Preiss J. 1996. From glycogen to amylopectin: a model for the biogenesis of the plant starch granule. Cell 86, 349-52.

Bhattacharyya MK, Smith AM, Ellis THN, Hedley C, Martin C. 1990. The wrinkled-seed character of peas described by Mendel is caused by a transposon-like insertion in a gene encoding starch branching enzyme. Cell 60, 115-22.

Blixt S. 1972. Mutation genetics in Pisum. Agri Hortique Genetica 30, 1-293.

Blixt S. 1977. Gene symbols of Pisum. Pisum Newsletter 9, supplement.

Casey R, Domoney C, Smith AM. 1993. Biochemistry and molecular biology of seed products. In: Casey R, Davies DR, eds. Peas: genetics, molecular biology and biotechnology. Wallingford: CAB International, 121-63.

Craig J, Lloyd JR, Tomlinson K, Barber L, Edwards A, Wang TL, Martin C, Hedley CL, Smith AM. 1998. Mutations in the gene encoding starch synthase II profoundly alter amylopectin structure in pea embryos. Plant Cell 10, 413-426.

Craig J, Barratt P, Tatge H, Déjardin A, Handley L, Gardner CD, Barber L, Wang TL, Hedley CL, Martin C, Smith AM. Mutations at the rug4 locus alter the carbon and nitrogen metabolism of pea plants through an effect on sucrose synthase.  Plant Journal 17, 353-362, 1999.

Creech R. 1965. Genetic control of carbohydrate synthesis in maize. Genetics 52, 1175-86.

Denyer K, Barber LM, Burton R, Hedley CL, Hylton CM, Johnson S, Jones DA, Marshall J, Tatge H, Tomlinson K, Wang TL. 1995. The isolation and characterisation of novel low-amylose mutants of Pisum sativum L. Plant, Cell and Environment 18, 1019-26.

Harrison CJ, Hedley CL and Wang TL. 1998. Evidence that the rug3 locus of pea Pisum sativum L. encodes plastidial phosphoglucomutase confirms that the imported substrate for starch synthesis in pea amyloplasts is glucose-6-phosphate. The Plant Journal, 13, 753-62.

Hill LM, Smith AM. 1991. Evidence that glucose-6-phosphate is imported as the substrate for starch synthesis by the plastids of developing pea embryos. Planta 185, 91-6.

Hylton C and Smith AM. 1992. The rb mutation of peas causes structural and regulatory changes in ADP glucose pyrophosphorylase from developing embryos. Plant Physiology 99, 1626-34.

Kooistra E. 1962. On the differences between smooth and three types of wrinkled peas. Euphytica 11, 357-73.

Martin C, Smith A. 1995. Starch Biosynthesis. The Plant Cell 7, 971-85.

Mendel G. 1865. Versuche über pflanzen-hybriden. Verhandlungen des naturforshenden Vereins in Brünn 4, 3-47.

Nelson O, Pan D. 1995. Starch synthesis in maize endosperms. Annual Review of Plant Physiology and Plant Molecular Biology 46, 475-96.

Turner S, Barratt DHP, Casey R. 1990. The effect of different alleles at the r locus on the synthesis of seed storage proteins in Pisum sativum. Plant Molecular Biology 14, 793-803.

Wang TL, Hadavizideh A, Harwood A, Welham TJ, Harwood WA, Faulks R, Hedley CL. 1990. An analysis of seed development in Pisum sativum. XIII. The chemical induction of storage product mutants. Plant Breeding 105, 311-20.

Wang TL, Hedley CL. 1991. Seed development in peas: Knowing your three 'r's' (or four, or five). Seed Science Research 1, 3-14.

Wang TL, Hedley CL. 1993.Genetic and developmental analysis of the seed. In: Peas: Genetics, Molecular Biology and Biotechnology (Eds. Casey R, Davies DR). Wallingford, CAB International, pp. 83-120.

Wang TL, Bogracheva TYa, Hedley CL. 1998. Starch: as simple as A, B, C? Journal of Experimental Botany 49, 481-502, 1998.

White OE. 1917. Studies of inheritance in Pisum. II. The present state of knowledge of heredity and variation in peas. Proceedings of the American Philosophical Society 56, 487-588.