Specific combinations of zein genes and genetic backgrounds influence the transcription of the heavy-chain zein genes in maize opaque-2 endosperms

Abstract

The transcript levels of heavy-chain zein genes (zH1 and zH2) and the occurrence of the zH polypeptides in different opaque-2 (o2) lines were investigated by RNA-blot analyses and by sodium dodecylsulfate-polyacrylamide gel electrophoresis or two-dimensional gel electrophoresis protein fractionations. Four mutant alleles o2R, o2T, o2It, and o2-676 introgressed into different genetic backgrounds (GBs) were considered. The mono-dimensional gel electrophoresis zein pattern can be either conserved or different among the various GBs carrying the same o2 allele. Likewise, in the identical GB carrying different o2 alleles, the zein pattern can be either conserved or differentially affected by the different mutant allele. Zein protein analysis of reciprocal crosses between lines with different o2 alleles or the same o2 showed in some case a more than additive zH pattern in respect to the o2 parent lines. Electrophoretic mobility shift assay approaches, with O2-binding oligonucleotide and endosperm extracts from the above o2 lines, failed to reveal o2-specific retarded band in any of the o2 extracts. The results suggest that the promoter of some zH1 and zH2 contains motif(s) that can respond to factors other than O2.

Figure 1

SDS-PAGE analysis of zein polypeptides from wild-type and o2 inbreds. Approximately 15 μg of zein protein extracts was loaded in each lane of A and B. Genotypes are reported at the top (see also Table I). For a proper comparison, wild-type (+) and opaque-2 (o2) in a given GB, abbreviated as in Table I, are also indicated at the bottom of each panel. A, Electrophoretic mobility of zein polypeptides extracted from mature seeds. The relative mobility of the H1 and H2 heavy chains and the L1, L2, and L3 light chains are indicated at the left. Bracket indicates the mobility of the sulfur-rich γ-zein polypeptides. The R802 O2w1 line was not inserted in this analysis; it shows, however, a pattern identical to the one of the Oh43 O2w1. B, Electrophoretic mobility of zeins extracted from seeds at maturity (M) or at different DAP as indicated at the top of each lane.

Figure 2

Two-dimensional (2D) gel analysis of zein protein extracts. Approximately 100 μg of zein protein extracts was loaded for each genotype. In the first dimension (IEF), the equilibrium pH gradient from left to right was between 6.5 and 9.0 as reported at the top and this applies for all the panels. Genotypes, abbreviated as reported in Table I, are indicated at the left or at the right of each panel. Dots on the left identify the five major size classes of zeins.

Figure 3

Southern analysis of genomic DNA from different maize lines digested with EcoRI and probed with O2 cDNA (A) and with the heavy chain zein cDNA M1 (B). Maize lines, abbreviated as in Table I, are reported on the top and apply for both panels. Molecular mass markers in kb are on the left. Approximately 10 μg of DNA was digested to completion as described by Bianchi and Viotti (1988) and applied in each lane.

Figure 4

A, Slot-blot analysis of total RNAs of maize genotypes as reported on the right. RNA was isolated from endosperms at 20 and 25 DAP, as indicated at the bottom. For each genotype and DAP, 1 and 2 μg loads were analyzed as indicated on the right. The total RNA at 20 DAP from the Xo2R line was not available in our stock of frozen seeds. Lane L contains total RNA from maize leaf in 1- and 2-μg horizontal loads from left to right, respectively. Probes are reported at the top of each strip. The filter was subjected to a series of sequential hybridization-stripping cycles using the probes indicated from left to right. B, Northern analysis of total RNA from endosperms of maize lines as indicated at the top. The wild-type RNA from endosperms at 20 and 25 DAP from left to right was 5 μg for each load and the o2 RNAs (20 DAP) were 10 μg for each load. Probes are indicated at the right of each panel. M1′ shows a longer exposure than M1. The filter was subjected to a series of sequential hybridizations to the four probes (from top to bottom) and stripping. Exposure time, at −80°C with intensifying screens, was 6 d for O2 and 5 h for zein probes. rDNA blots were exposed for 1 h at room temperature.

Figure 5

A, 2D analysis of zein extracts from the Yo2It and Oh676 lines and from their reciprocal crosses as indicated at the side of each panel. The pH gradient (from 6.5–9.0) is indicated at the top and applies for all the panels. Dots indicate the five major size classes. Arrow indicates the zein spot expressed in the reciprocals. B, EMSAs of double strand oligonucleotide containing the O2-binding site. Protein extracts from endosperms and bacterial protein extracts from E. coli, expressing (O2be) or nonexpressing (be) the O2 polypeptide, were used in this analysis. Wild-type and o2 maize lines or o2 hybrids are indicated at the top of each lane. Addition (+) of O2-antiserum (O2-Ab) is indicated. Open arrow indicates the migration of the oligonucleotide bands shifted by O2 protein in O2be and Y+. Closed arrow indicates the super-shift determined by the addition of O2-antiserum to the mixture.

Figure 6

SDS-PAGE analysis of zein polypeptides from o2 genotypes and their reciprocal crosses. Zein protein extracts were obtained from mature seeds and loaded in the amounts indicated at the bottom. Dots on the left side identify the five major size classes of zeins.