Molecular structure of three mutations at the maize sugary1 locus and their allele-specific phenotypic effects

Abstract

Starch production in all plants examined is altered by mutations of isoamylase-type starch-debranching enzymes (DBE), although how these proteins affect glucan polymer assembly is not understood. Various allelic mutations in the maize (Zea mays) gene sugary1 (su1), which codes for an isoamylase-type DBE, condition distinct kernel phenotypes. This study characterized the recessive mutations su1-Ref, su1-R4582::Mu1, and su1-st, regarding their molecular basis, chemical phenotypes, and effects on starch metabolizing enzymes. The su1-Ref allele results in two specific amino acid substitutions without affecting the Su1 mRNA level. The su1-R4582::Mu1 mutation is a null allele that abolishes transcript accumulation. The su1-st mutation results from insertion of a novel transposon-like sequence, designated Toad, which causes alternative pre-mRNA splicing. Three su1-st mutant transcripts are produced, one that is nonfunctional and two that code for modified SU1 polypeptides. The su1-st mutation is dominant to the null allele su1-R4582::Mu1, but recessive to su1-Ref, suggestive of complex effects involving quaternary structure of the SU1 enzyme. All three su1- alleles severely reduce or eliminate isoamylase-type DBE activity, although su1-st kernels accumulate less phytoglycogen and Suc than su1-Ref or su1-R4582::Mu1 mutants. The chain length distribution of residual amylopectin is significantly altered by su1-Ref and su1-R4582::Mu1, whereas su1-st has modest effects. These results, together with su1 allele-specific effects on other starch- metabolizing enzymes detected in zymograms, suggest that total DBE catalytic activity is the not the sole determinant of Su1 function and that specific interactions between SU1 and other components of the starch biosynthetic system are required.

Figure 1

Kernel phenotypes of su1-mutants. A, The su1-Ref mutant phenotype. The ear shown was obtained by self-pollination of an su1-Ref/Su1 heterozygote. B, The su1-st mutant phenotype. The ear shown was obtained by self-pollination of an su1-st/Su1 heterozygote. Mutant kernels (examples indicated by the arrows) and wild-type kernels segregated at a frequency of approximately 1:3, respectively. C, Phenotype of su1-Ref/su1-st kernels. The ear shown is the result of a cross of an su1-st/su1-st plant as the male parent to an su1-Ref/su1-Ref plant as the female parent. D, Phenotype of su1-R4582::Mu1/su1-st kernels. The ear shown is the result of a cross of an su1-R4582::Mu1/su1-R4582::Mu1 plant as the male parent to an su1-st/su1-st plant as the female parent.

Figure 2

RNA gel-blot analysis. Total RNA was extracted from endosperm of kernels harvested at 20 DAP, fractionated on a 1% (w/v) agarose-formaldehyde gel, and hybridized with Su1 cDNA antisense RNA. RNA concentration variations can be visualized by the ethidium bromide-stained rRNA, shown in the lower panel. The lanes shown were run simultaneously in the same gel.

Figure 3

PCR and RT-PCR analyses. A, Relative positions of oligonucleotide primers within the Su1 cDNA. Exons comprising the Su1 cDNA are represented by numbered boxes. Primers used for the detection of alternatively spliced transcripts are indicated by arrows above and below the boxes (depicting sense and antisense sequences, respectively). B, RT-PCR amplifications. DNA was reverse-transcribed from purified mRNA isolated from 20 DAP whole kernels homozygous for Su1 (lanes 1 and 3) and su1-st  (lanes 2 and 4). Primer pair 797/JD02 was used for the amplifications in lanes 1 and 2, and primer pair JD03/JD18 was used for the amplifications in lanes 3 and 4. C, PCR amplification of genomic DNA from seedling tissue homozygous for Su1 (lane 1) and su1-st (lane 2) with primer pair JD19/JD20 to amplify the region of the gene containing the Toad element.

Figure 4

Nucleotide sequence analysis of the su1-st locus. The su1-st locus contains an insertion of the 638-bp Toad sequence (shown in bold) in exon 10. Intron sequence flanking exon 10 is designated by lowercase letters, and exon 10 sequence is designated by uppercase letters. Nucleotides underlined with a dotted line indicate the 10-bp target site duplication of the host sequence. Regions underlined with a solid line indicate 138-bp TIRs within the Toad element. The wild-type acceptor site for the end of intron 9 is designated A+. The intron formed for the creation of the type-II transcript is made by joining cryptic acceptor sites D*1 to A*. This results in a deletion of 18 nt of Su1 sequence from the mature transcript and complete removal of the Toad sequence. The intron formed for the type-III transcript is produced by the use of D*2 as the donor site and A* as the acceptor site, removing most of the Toad sequence as an intron, but leaving an insertion of 30 bp in the mature mRNA. The wild-type donor site for the start of intron 10 is indicated as D+. The highly conserved GT and AG dinucleotides found at the termini of most plant introns are highlighted at the indicated splice donor/acceptor sites. The nucleotides indicated correspond to the wild-type Su1 genomic sequence (GenBank accession no. AF030882).

Figure 5

HPAEC-PAD analysis of chain length distributions. A, Amylopectin from Su1, su1-R4582::Mu1, su1-Ref, and su1-st homozygous kernels harvested 30 DAP was debranched with Pseudomonas isoamylase. Chain length distributions were determined using HPAEC-PAD and normalized to the total peak area. Differences in chain lengths of su1-R4582::Mu1, su1-Ref, and su1-st amylopectin-like molecules relative to Su1 amylopectin are shown in difference plots below the respective mutant profiles. B, WSP from su1-Ref and su1-st kernels harvested 30 DAP was debranched and separated, as described for A. A difference plot comparing the WSP chain distributions is below the su1-st profile.

Figure 6

Native PAGE/activity gels (zymograms) of starch modifying activities. A, Twenty-five micrograms of total protein from Su1 and su1-R4582::Mu1 homozygous kernels harvested at 20 DAP was separated on a 5-cm native polyacrylamide gel, then electroblotted to a starch-containing gel. Starch modifying enzyme activities were visualized by staining with I₂/KI. B, Fifty or 100 μg total protein from Su1, su1-R4582::Mu1, su1-st, and su1-Ref homozygous kernels harvested at 20 DAP was analyzed as described for A on 15-cm native PAGE/activity gels. Arrows A through G indicate starch modifying activities that exhibit allele-specific variation.