The impact of GBSSI inactivation on starch structure and functionality in EMS-induced mutant lines of wheat

Abstract

Background: Starch, a major component of wheat (Triticum aestivum L.) grain, plays a crucial role in determining processing quality. Granule-bound starch synthase I (GBSSI), the enzyme primarily responsible for elongating α-1,4-glucan chains into linear amylose molecules, is a key determinant of starch quality. In this study, a mutant population of the wheat cultivar SM126, a high-quality variety form Sichuan, China, was generated using ethyl methanesulfonate (EMS) mutagenesis. This research investigates the effects of GBSSI inactivation on starch structure and functionality.

Results: A waxy mutant (Wx-Abd) was identified by screening an M4 seed library with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of grain endosperm flour. DNA sequencing revealed a single nucleotide polymorphism (SNP) in the fourth exon, causing a premature stop codon and inactivation of the Wx-Abd allele. In previous work, the Wx-abD mutant was identified in the M2 generation, and crossing the M2-31 line with the M4-6165 line produced four distinct Wx protein subunits in the SM126 background. Comparisons between the Wx-abd line and the wild-type SM126 (Wx-AbD) showed significant differences in starch properties. The Wx-abd line exhibited reduced Wx gene expression, a distinct surface depression on starch granules, and a higher proportion of B-type starch granules. Notably, it exhibited significantly lower amylose content (7.02%) compared to SM126 (22.32%), along with a reduction in total starch content. Additionally, the Wx-abd line showed a higher gelatinization temperature.

Conclusion: Inactivation of GBSSI in the Wx-abd line resulted in altered starch structure, particularly a decrease in amylose content and changes in granule morphology. These findings suggest that the Wx-abd line represents a valuable genetic resource for wheat breeding programs focused on improving starch quality for food production, with its high agronomic performance making it suitable for further breeding applications.

Keywords: GBSSI; Gene expression; Starch properties; Thermodynamic properties; Waxy wheat.

Fig. 1

SDS-PAGE analysis of waxy proteins in SM126 and its derivatives. (a) Identification of Wx-D1 protein subunit deletion mutant. White arrows indicate the present and absence of waxy proteins in the M4-6165 mutant. (M: Protein Marker; WT: Wild-type (SM126); Lanes 1–3: M4-6475 line; Lanes 4–6: M4-6165 line; Lanes 7–9: M4-6436 line; Lanes 10–12: M2-6166 line). (b) Hybridization of the F1 generation from the M4-6165 × M2-31 cross. (M: Protein Marker; WT: Wild-type (SM126); CS: Chinese Spring; Lanes 1–10: F1 hybrids of M4-6165 × M2-31). (c) Detection of different Wx protein subunits in SM126. (CS: Chinese Spring; Lanes 1–3: Lack of Wx-D1 protein (Wx-abD); Lanes 4–6: Lack of Wx-A1 protein (Wx-Abd); Lanes 7–9: Lack of Wx-B1 protein (Wx-AbD); Lanes 10–12: Lack of all Wx protein subunits (Wx-abd)

Fig. 2

Analysis of Wx-D1 gene sequences in wheat parental SM126 and mutant M4-6165 lines. The black boxes represent exon regions, and the lines represent introns. The regions targeted by the PCR primers are indicated by black arrows. The first amplified region spans from exon 1 to intron 5, and the second region spans from exon 5 to exon 11. The SNP in exon 4 and the associated amino acid variation are highlighted in red

Fig. 3

Identification of a KASP (Kompetitive Allele-Specific PCR) marker for four different Wx protein subunits in SM126.Genotyping of the M6 population, M4-6165, and M2-31 lines was performed using fluorescence signals for discrimination. The two clusters represent the genotypes of homozygous M4-6165 mutants and homozygous SM126 wild-type. Mutants M2-31 and M4-6165 (HEX-tagged, Wx-A1/Wx-D1 silenced) formed the heterozygous group

Fig. 4

Analysis of the expression levels (a-c) and (d) GBSSI enzyme activity of wheat Wx protein subunits in SM126 lines (Wx-abD, Wx-Abd, and Wx-AbD). GBSSI activity and expression levels were measured in endosperms 15 days after anthesis (DAA). Asterisks indicate statistically significant differences between the Wx-AbD and mutant lines, as determined by Student’s t-test (**P < 0.01)

Fig. 5

Investigation of the effects of different Wx protein subunits on grain morphology. Grain length and width (a, b) were measured in four SM126 lines. For each line, 50 randomly selected seeds were measured three times. Statistical significance was determined using a threshold of P < 0.01

Fig. 6

Analysis of total starch (a), amylose content (b), protein content (c), and granule types (d) in the four different Wx protein subunits of SM126 lines. Significant differences were observed among the various Wx proteins (P < 0.01), except for protein content, where no significant variations were found. The data presented are the means of three replicates

Fig. 7

Starch granule structure in the four different Wx protein subunits of SM126 lines analyzed using Scanning Electron Microscopy (SEM). The red arrow indicates A-type starch granules, and the blue arrow indicates B-type starch granules