Molecular basis of the waxy endosperm starch phenotype in broomcorn millet (Panicum miliaceum L.)

Abstract

Waxy varieties of the tetraploid cereal broomcorn millet (Panicum miliaceum L.) have endosperm starch granules lacking detectable amylose. This study investigated the basis of this phenotype using molecular and biochemical methods. Iodine staining of starch granules in 72 plants from 38 landrace accessions found 58 nonwaxy and 14 waxy phenotype plants. All waxy types were in plants from Chinese and Korean accessions, a distribution similar to that of the waxy phenotype in other cereals. Granule-bound starch synthase I (GBSSI) protein was present in the endosperm of both nonwaxy and waxy individuals, but waxy types had little or no granule-bound starch synthase activity compared with the wild types. Sequencing of the GBSSI (Waxy) gene showed that this gene is present in two different forms (L and S) in P. miliaceum, which probably represent homeologues derived from two distinct diploid ancestors. Protein products of both these forms are present in starch granules. We identified three polymorphisms in the exon sequence coding for mature GBSSI peptides. A 15-bp deletion has occurred in the S type GBSSI, resulting in the loss of five amino acids from glucosyl transferase domain 1 (GTD1). The second GBSSI type (L) shows two sequence polymorphisms. One is the insertion of an adenine residue that causes a reading frameshift, and the second causes a cysteine-tyrosine amino acid polymorphism. These mutations appear to have occurred in parallel from the ancestral allele, resulting in three GBSSI-L alleles in total. Five of the six possible genotype combinations of the S and L alleles were observed. The deletion in the GBSSI-S gene causes loss of protein activity, and there was 100% correspondence between this deletion and the waxy phenotype. The frameshift mutation in the L gene results in the loss of L-type protein from starch granules. The L isoform with the tyrosine residue is present in starch granules but is nonfunctional. This loss of function may result from the substitution of tyrosine for cysteine, although it could not be determined whether the cysteine isoform of L represents the functional type. This is the first characterization of mutations that occur in combination in a functionally polyploid species to give a fully waxy phenotype.

FIG. 1.

Structure of the GBSSI sequence analyzed in Panicum miliaceum showing the L and S gene types (putative homeologues) and the relative sizes of the exons (wide bars, numbered analogously to Setaria italica) and introns. Approximate positions and 5′ to 3′ direction of primers used for PCR are marked with arrows (primers not to scale). The mutations identified in the L and S loci are identified.

FIG. 2.

Starch granules of Panicum miliaceum stained with 0.01× Lugol′s solution. (a) Nonwaxy type, stained blue–black (accession 58 plant 1). (b) Waxy type, stained red–brown (accession 85 plant 1).

FIG. 3.

(a) Western blot of soluble (S) and insoluble (I) protein fractions from endosperm starch, developed with antibarley GBSSI antibody and IgA antirabbit phosphatase conjugate. The band at ∼52 kDa represents the GBSSI protein. The larger ∼80-kDa band most likely represents a second isoform of GBSS in broomcorn millet. (b) Detail of selected samples (insoluble protein fractions only) from a parallel blot (using different plants of the same genotypes) to that in figure 5(a). The S₋₁₅/L_f genotype, lacking five amino acids, produces a slightly smaller GBSSI band than the S₀/L_f genotype. A double band can be seen in the S₋₁₅/L_Y genotype, with a smaller band of the same size as in the S₋₁₅/L_f sample and a larger band comparable in size with the S₀ GBSSI. The larger band probably represents the (inactive) L_Y protein.

FIG. 4.

Maximum likelihood tree of GBSS amino acid sequences, using the JTT + I + G model of protein evolution with 1,000 bootstrap replicates. Percentage bootstrap support values are shown. GenBank accession numbers for the GBSS proteins: Arabidopsis thaliana GBSSI NP_174566; Austrostipa aristiglumis GBSSI ABU98330.1; Elymus scaber GBSSI ABU98331.1; Hordeum vulgare GBSSI CAA30755, GBSSIb AAM74054; Manihot esculenta GBSSI CAA52273; Microlaena stipoides GBSSI ABU98332.1; Oryza australiensis GBSSI ABU98325.1; Oryza glaberrima GBSSI BAA01272; Oryza rufipogon GBSSI ABU98326.1; Oryza sativa GBSSI CAA46294, GBSSII AAL58572; Panicum miliaceum GBSSI-L type ADA61154, GBSSI S-type ADA61162 (this study); Pisum sativum GBSSI AAB26591; Setaria italica GBSSI BAC06486; Solanum tuberosum GBSSI CAA41359; Sorghum bicolor GBSSI Q43134; Sorghum leiocladum GBSSI ABU98327.1; Triticum aestivum GBSSI AAB26860; and Zea mays GBSSI P04713.

FIG. 5.

Three-dimensional structure of the Agrobacterium tumefaciens glycogen synthase protein (Buschiazzo et al. 2004). The amino acids ALNKKAV that correspond to the ALNKEAL motif in the Panicum miliaceum GBSSI-S–type protein are highlighted and indicated with the arrow. A color version of this figure is available as supplementary figure S2, Supplementary Material online.

FIG. 6.

Ms/ms fragmentation of [D489-P531 + 4H]⁴⁺ ion 1,114.5m/z. A series of b³⁺and b⁴⁺ ions can be seen which correspond to those expected from the peptide, confirming the sequence of the C-terminal section. Zoomscan of selected peaks confirmed 3+ charge state.