Identification of Mutator insertional mutants of starch-branching enzyme 2a in corn

Abstract

Starch-branching enzymes (SBE) break the alpha-1,4 linkage of starch, re-attaching the chain to a glucan chain by an alpha-1,6 bond, altering starch structure. SBEs also facilitate starch accumulation by increasing the number of non-reducing ends on the growing chain. In maize (Zea mays), three isoforms of SBE have been identified. To examine the function of the SBEIIa isoform, a reverse genetics polymerase chain reaction-based screen was used to identify a mutant line segregating for a Mutator transposon within Sbe2a. To locate the insertion within the second exon of Sbe2a, the genomic sequence of Sbe2a containing the promoter and 5' end was isolated and sequenced. Plants homozygous for sbe2a::Mu have undetectable levels of Sbe2a transcripts and SBEIIa in their leaves. Characterization of leaf starch from sbe2a::Mu mutants shows reduced branching similar to yet more extreme than that seen in kernels lacking SBEIIb activity. Characterization of endosperm starch from sbe2a::Mu mutants shows branching that is indistinguishable from wild-type controls. These mutant plants have a visible phenotype resembling accelerated senescence, which was correlated with the Mutator insertion within Sbe2a. This correlation suggests a specific role for SBEIIa in leaves, which may be necessary for normal plant development.

Figure 1

Identification of sbe::Mu mutants and expression of Sbe2a in individuals from two F₂ populations (98023-1 through 5 and 98025-1 through 6). A, Detection of sbe2a::Mu alleles in two independent F₂ populations via PCR using primers 2A2 and MuTIR. Gel electrophoresis of amplification products (top) and hybridization of amplification products to Sbe2a cDNA clone (bottom). B, Detection of wild-type Sbe2a alleles via PCR using primers 2A1 and 2A2. Presence of Mutator insertion interferes with PCR resulting in no band in samples from homozygous plants. Combined with part A identifies homozygous wild-type individuals (98023-2, 3, 5), heterozygotes (98023-1, 4, and 98025-4, 5, 6), and homozygous mutants (98025-1, 2, 3). C, Northern blot using seedling tissue hybridized with Sbe2a cDNA clone (top). Gel electrophoresis of RNA showing sample loading (bottom).

Figure 2

Structure of the genomic sequence from the isolated clone (0–4.71 kb) and PCR products (4.71–5.08 kb). The thin line indicates the region 5′ of the known cDNA sequence. Shaded boxes indicate exons and open boxes indicate introns. Lower diagram includes Mutator insertion sites for lines 98023 (approximately position 4.135) and 98025 (between 4.507/4.508) as determined via size and sequence of PCR products. Diagram also indicates positions of Sbe2a-specific primers (2A1, 2A32, 2A31, and 2A2). Corresponding locations from the Sbe2a cDNA sequence are noted (cDNA bp).

Figure 3

Leaf phenotypes of sbe2a::Mu mutant and their controls. A, Leaf phenotypes of sbe2a::Mu mutant and wild-type controls from line 98025. Leaves at node 5 from line 98025 were photographed from a homozygous sbe2a::Mu mutant and its full-sibling wild-type control at 30 DAE. B, Leaf phenotypes of plants with the reduced senescence phenotype and sbe2a::Mu mutant and wild-type controls. Leaves at node 7 were photographed from a homozygous sbe2a::Mu mutant (sbe2a::Mu) and its full-sibling wild-type control (Wildtype) alone with the two plants displaying reduced senescence (#18 and #39) at 50 DAE.

Figure 4

Molecular analysis of sbe2a::Mu mutants. A through C, Selection of homozygous sbe2a::Mu mutants (lanes 3–5) and wild-type controls (lanes 1 and 2) and the expression patterns of SBEs as compared with inbred line W64A 20 DAP endosperm (En). D, Western analysis of mutant and control genotypes in kernels. A, Identification of sbe2a::Mu mutants and their full-sibling wild-type controls from a BC₁F₂ population. PCR was used to detect the wild-type allele (primers 2A1/2A2) and the mutant allele (primers 2A2/MuTIR) using DNA extracted from seedlings. Gel electrophoresis of PCR products (top) and hybridization to Sbe2a cDNA clone (bottom). B, RT-PCR analysis of homozygous sbe2a::Mu mutants and their full-sibling wild-type controls to detect transcripts in 30 DAE leaf tissue using primers 2A2 and 2A32 and Sbe2b primers 2B1 and 2B2. C, Western analysis of homozygous sbe2a::Mu mutants and their full-sibling wild-type controls to detect the presence of SBEI, SBEIIa, and SBEIIb in 30 DAE leaf tissue using the non-specific SBEI antibody (top). The high M_r proteins cross-reacting with this antibody are unknown. Identity of SBEIIa and SBEIIb were verified using antibodies to SBEIIa (middle) and SBEIIb (bottom). D, Western analysis of homozygous sbe2a::Mu mutants and controls to detect the presence of SBEIIa in dry kernel tissue using the SBEIIa antibody. Endosperm protein extracts are from fresh sweet corn (lane 1) and from dry kernels of an unrelated Mu mutant wild type for Sbe2a (lane 2), sbe2a:Mu(lane 3), ae-ref (lane 4), and W64A wild type (lane 5). Lane 6 contains protein extract from leaves of an sbe2a::Mu/Sbe2a heterozygote.

Figure 5

Analysis of starch samples from sbe2a::Mu 30 DAE leaves and endosperm. A, SEC of whole starches from leaves of homozygous sbe2a::Mu mutants (right) and their full-sibling wild-type controls (left). Mutants and wild-type controls were selected from a BC₁F₂ population. B, HPSEC of debranched starches from leaves of homozygous sbe2a::Mu mutant (right) and their full-sibling wild-type controls (left). Mutant and wild-type controls were selected from a BC₁F₂ population. C, HPSEC of debranched endosperm starches from kernels of homozygous sbe2a::Mu mutant (right) and W64A wild-type controls (left). The mutant was selected from a BC₃F₃ population. D, HPSEC of debranched starch from endosperm of homozygous amylose extender mutant (ae-ref).

Figure 6

Segregation of senescence phenotype and the wild-type Sbe2a allele in a BC₁F₂ population. The wild-type Sbe2a allele was amplified from DNA extracted from each of 52 plants at 32 DAE using primers 2A2 and 2A32. The molecular mass of two of the bands within the 1-kb ladder (L) are shown to the left. The phenotype observed for each plant is indicated above each lane as normal (N) or senescent (S). Plants 18 and 39 displayed senescence at 10 to 32 DAE, and reduced senescence after 50 DAE.

Figure 7

Western detection of SBEIIa in plants with the reduced senescence phenotype and sbe2a::Mu mutant and wild-type controls. Total protein was extracted and blotted using leaves at node 9 from homozygous sbe2a::Mu mutant and its full-sibling wild-type control along with the two plants displaying reduced senescence (nos. 18 and 39) at 55 DAE. The western blot was probed using the SBEIIa specific antibody.

Figure 8

Pedigree of populations used in this study. Independent F₁ plants were identified by Pioneer personnel (Trait Utility System for Corn F₁). Self progeny of these plants were crossed to inbred line W64A to create BC₁F₂ and BC₃F₃ populations for analysis. Plant 98025-1 was identified as homozygous for sbe2a::Mu (see “Results”). Plants 98133-2, 98133-3, 98159, 99050, and 99052 were determined to be heterozygous for sbe2a::Mu via PCR genotyping (data not shown). Plant 00096 was similarly identified via PCR to be homozygous for sbe2a:Mu (data not shown).