Cis-acting Elements and Trans-acting Factors

Regulatory Genes As Trans-Acting Factors

Tissue-Specific Binding Of Trans-Acting Factors

Plant Regulatory Genes Can Function As Trans-Acting Factors

The last topic that we will discuss is molecular biology of regulatory genes. The specific gene that we will discuss is opaque 2 of corn. This gene regulates the expression of the corn storage protein zein. Furthermore, it regulates a specific class of zeins. Four types of seed storage proteins have been identified in seeds and they are classified according to the solution in which they are soluble.

Protein Solube in- Example

Globulins Salt Solution Phaseolin, glycinin

Alcohol-soluble Alcohol Zein, gliadins

Albumin Water

Insouluble None of the above

The following list give the speciific characteristics of the zein proteins of maize.

major storage proteins of corn; alcohol soluble proteins
synthesized 12-50 DAP
5 classes as defined by SDS-PAGE
- 27kd - one species
- 22kd - five species
- 19kd - four to six species
- 15kd - one species
- 10kd - one species
these proteins account for 50-60% of the protein in a normal seed
these proteins are stored in protein bodies
different number of genes encode the various classes
- 22kd - about 25 genes
- 19kd - about 50 genes
- 15kd - 1-2 genes
genes are located on chromosomes 4, 7 and 10

Zein Mutants

A number of mutants have been detected in corn which affect the synthesis of the zein genes. These fall into two classes, those which give an opaque phenotype and those which produce a floury phenotype. Each of the mutants have a unique affect on zein synthesis and they exhibit different interactions in combination with other mutants.

Mutant Gene symbol Gene Action Effect on zein synthesis

Opaque 2 op2 Recessive reduces 21kd>>19kd

Opaque 7 op7 Recessive reduces 19kd>>21kd

op2/op7 Additive reduces 21kd and 19kd equally

Floury2 fl2 Dose dependent reduces 21kd and 19kd equally

fl2/op2 fl2 hypostatic reduces 21kd>>19 kd

fl2/op7 fl2 hypostatic reduces 19kd>>21kd

De*-B30 De*-30 Dominant reduces only 22kd

Mucronate mc Synergistic reduces all zeins to<10% of wild type

The primary interest in these mutants is their increased nutritional quality. The zeins are low in lysine and tryptophan. In the zein mutants, the globulins are increased which makes for a more nutritious seed protein mixture. Some difficulties are encountered though. For example, the op2/op7 double mutant seldom germinates so this mutant cannot provide the dramatic increase in nutritional quality you might expect. Surprisingly, though, the mc/op2 mutant germinates normally even though zein synthesis is nearly shut off.

One of the goals in plant molecular biology has been to clone these regulatory genes. The accumulated evidence strongly suggested that these mutants were regulatory mutants because those zeins that were produced were not structurally altered. This posed a problem for cloning these genes. Whereas it was clear that the gene product of the zein genes were the zein proteins, it could only be hypothesized what was the gene products of the opaque and floury genes. Fortunately, transposon mutants for opaque-2 existed. Therefore, this gene could be cloned by transposon tagging.

The result of this cloning experiment determined that the opaque 2 locus encoded a a protein with leucine zipper domain. Furthermore, its expression was related to that of the 22 kd class of zeins. It was shown that the protein product was able to bind to the promoter of a 22 kd zein gene promoter. Recent analyzes of mutants of the opaque 2 locus determined that the hinge arginine residue had been changed and that protein did not have the ability to bind to the promoter. When this site was changed to arginine, then the binding capability was restored to wild type levels.