Altered starch structure is associated with endosperm modification in Quality Protein Maize

Abstract

The biochemical basis of modified kernel texture in Quality Protein Maize (QPM) is poorly understood. Proteomic analysis of several QPM lines indicated increased levels of granule-bound starch synthase I in the soluble nonzein protein fraction of these genotypes. Increased extraction of this enzyme reflected a change in starch structure, which was manifested as shorter amylopectin branches and increased starch-granule swelling. In mature kernels, these alterations in starch structure were associated with interconnections between starch granules that resulted in a vitreous kernel phenotype. Understanding the molecular basis for this previously uncharacterized starch structure will accelerate the development of QPM.

Fig. 1.

Phenotypic comparison of CM105⁺, o2, and mo2 kernels. (A) Transmission of light by mature kernels. (B) SDS/PAGE analysis of zein and nonzein proteins, respectively, from equal amounts of endosperm flour of CM105⁺ (lanes 1 and 5), CM105o2 (lanes 2 and 6), and CM105mo2 (lanes 3 and 7). The identity of specific types of zeins is shown on the left, and molecular masses of the size markers in lane 4 are shown on the right.

Fig. 2.

Two-dimensional gel patterns of GBSS I proteins from wild-type, o2, and mo2 genotypes. Nonzein proteins were separated by isoelectric focusing with immobilized pH 4–7 gradient strips and 12.5% SDS/PAGE. Compared with CM105⁺ (A) and CM105o2 (B), nonzeins from CM105mo2 contained larger amounts of a cluster of 56-kDa polypeptides identified as GBSS I. Vitreous mo2 kernels (F) from a segregating F₂ ear of a cross between K0326Y QPM and W64Ao2 contained more GBSS I than opaque kernels from the same ear (E) or the W64Ao2 parent (D). The opaque RI-5 (G) had smaller amounts of GBSS I than the vitreous RI-32 (H).

Fig. 3.

Differential extraction of GBSS I in the presence of SDS from wild-type, o2, and mo2 genotypes. Endosperm flour was extracted without (Upper) or with (Lower) SDS in the extraction buffer, and the resulting starch was solubilized by boiling in SDS/PAGE sample buffer. The total amount of granule-associated GBSS I was similar from all three CM105 lines (lane 1, CM105⁺; lane 2, CM105o2; lane 3, CM105mo2) and from vitreous or opaque kernels from a K0326Y × W64Ao2 cross (lane 4, vitreous kernels; lane 5, opaque kernels). In the presence of SDS, more GBSS I was extracted from starch granules of CM105mo2 (Lower, lane 3, compared with lanes 1 and 2) and vitreous kernels from the K0326Y × W64Ao2 cross (Lower, lane 4, compared with lane 5).

Fig. 4.

Analysis of starch structure in wild-type, o2, and mo2 maize genotypes. (A) Sedimentation assay measuring swelling of starch granules in water at room temperature. (B) Measurement of amylose content by I₂ binding. (C–E) Difference plots contrasting the lengths of α-1,4-linked glucose polymers of debranched amylopectin molecules from mo2 and o2 genotypes. (C) Compared with CM105o2, CM105mo2 has more very short α-1,4-linked glucose chains and a lower abundance of chains with a DP of 25–35. (D) Vitreous mo2 kernels from an F₂ ear of K0326Y QPM by W64Ao2 have a greater proportion of α-1,4-glucose chains with a DP of 12–25 and a decreased proportion of chains with a DP of 27–42, compared with opaque kernels from the same ear. (E) The vitreous RI-32 had a slightly increased proportion of short α-1,4-linked glucose chains and a prominent reduction in chains with a DP of 20–35 compared with the opaque RI-5. (F) Compared with CM105⁺ and o2, wide-angle x-ray scattering of CM105mo2 starch granules showed an increase in the peak at 20.0° 2θ and a small new peak at 13.2° 2θ. (G) The other opaque and vitreous lines tested had x-ray scattering patterns similar to CM105⁺ and CM105o2.

Fig. 5.

Scanning electron microscopy analysis of starch granules in mature endosperms and purified starch. (A) The CM105⁺ kernel contained spherical starch grains embedded in a proteinaceous matrix containing protein bodies. (B) The CM105o2 kernel had smooth, spherical starch grains with very little matrix surrounding them. (C) The CM105mo2 kernel contained starch grains with circular surface depressions at which adjacent starch granules made contact (*). The arrows mark connections between adjacent starch granules. Purified starch from CM105⁺ (D) and CM105o2 (E) consisted of round, smooth granules, whereas that from CM105mo2 (F) consisted of irregularly shaped granules, and there were many points at which adjacent granules form contacts (arrows). Opaque (G) and vitreous (H) kernels from a segregating F₂ ear of K0326Y QPM by W64Ao2 were similar to CM105o2 and CM105mo2, respectively. Likewise, opaque RI-5 (I) and vitreous RI-32 (J) kernels were similar to CM105o2 and CM105mo2, respectively.