Quantification of dominance for proteins pleiotropically affected by opaque-2 in maize

Abstract

The maize opaque-2 (o2) mutation is known to have numerous pleiotropic effects. Using two different genetic backgrounds, endosperm proteins from isogenic lines of the o2 locus and their reciprocal hybrids were compared by high-resolution two-dimensional electrophoresis (2-D PAGE), and the 2-D gels stained with Coomassie blue or silver were analysed using a computerassisted system for quantification of polypeptide spots. The aim of this work was (i) to identify polypeptides, the amount of which is affected by the single gene substitution, and (ii) to assess the dominance relations for every affected polypeptide. The triploid nature of the endosperm allowed the effects of 0, 1, 2 and3 doses of the wild-type 02 allele to be examined. When using a basic pH range in the first dimension (IEF), we observed the known effects of o2 on prolamin storage proteins, mostly being 22 kDa, zeins. In a pH 5-7 IEF range, we detected 14 polypeptides whose amount is affected by the o2 mutation in both backgrounds. Eight of them were more abundant in the wild type than in the mutant and six were more abundant in the mutant than in the wild type. Dominance of the wild type allele was found for the amounts of 10 polypeptides, while the amounts of two of them displayed additive inheritance. This is an example where pleiotropically related characters display different inheritance, which could generate heterozygous phenotypes with original properties as compared to their homozygous parents. Because 02 belongs to the basic region-leucine zipper family of transcription activators, our results show that 2-D PAGE of isogenic lines represents a straightforward method to identify gene products affected by a transcription factor, whether they are products of its target genes, or products indirectly affected by far-reaching effects. This is a prerequisite to understanding the multiple molecular and physiological consequences of the action of such factors.Keywords: dominance, isogenic lines, maize, opaque-2, plelotropy. IntroductionNumerous single mutations are known that have multiple phenotypic consequences (Caspari, 1952;Wright, 1968). Various mechanisms can be put forward to explain pleiotropy. For example, a mutation of a gene coding for a trans-acting transcription factor binding to the promoter regions of different genes could result in variation of the characters they control (Hope & Struhl, 1985;Sassone-Corsi, 1985). At the post-translational level, it has been shown that differences in the degree of sialylation of multiple hydrolases could be due to the mutation of the gene coding for neuraminidase (Womack et at., 1981). Keightley & Kacser (1987) investigated a branched metabolic pathway where the two outputs represent two characters whose variation is affected by changes in any of the genetically specified enzymes in the system. However, characterization of the mutation may not be sufficient 38 to predict or understand all the diverse pleiotropic effects. Recent cloning of the r (rugosus) gene in pea revealed that it encodes a starch-br...

Maize Opaque Endosperm Mutations Create Extensive Changes in Patterns of Gene Expression[W]

“…Among these are a large number of genes associated functionally with stress, cellular perturbation, and pathogen responses (Table 3). This major shift in expression from zein to nonzein genes (Table 1) is consistent with changes in patterns of protein synthesis in the endosperm that were described previously in this mutant (Damerval and De Vienne, 1993;Habben et al, 1993;Damerval and Le Guilloux, 1998).…”

“…Only in the case of o2 and fl2 is the molecular basis for the mutation understood. o2 encodes a transcription factor that regulates genes encoding the 22-kD ␣ -zeins as well as several other proteins (Damerval and De Vienne, 1993;Habben et al, 1993). fl2 corresponds to a point mutation in the signal peptide of a 22-kD ␣ -zein protein (Coleman et al, 1997b).…”

Retracted: Opaque7 Encodes an Acyl-Activating Enzyme-Like Protein That Affects Storage Protein Synthesis in Maize Endosperm

“…The cloning and characterization of some of the opaque mutants has revealed important regulatory mechanisms for zeins accumulation in maize endosperm. The O2 gene, which encodes a defective basic-domain-leucine-zipper transcription factor, regulates several endosperm-expressed genes, in particular the 22-kDa a-zeins (Schmidt et al 1987(Schmidt et al , 1990Damerval and De Vienne …”

Assessing factors for reliable quantitative proteomics based on two‐dimensional gel electrophoresis

“…The staining procedure was modified from [30] as described in [38], except that the gels were stained to equilibrium with 0.1% w/v CBB in 17% ethanol for 72 h at room temperature. Detections with 35 S and colloidal Coomassie blue were compared using four replicates of 30 mL.…”