Homology-dependent gene silencing phenomena in plants have received considerable attention, especially when it was discovered that the presence of homologous sequences not only affected the stability of transgene expression, but that the activity of endogenous genes could be altered after insertion of homologous transgenes into the genome. Homology-mediated inactivation most likely comprises at least two different molecular mechanisms that induce gene silencing at the transcriptional or posttranscriptional level, respectively. In this review we discuss different mechanistic models for plant-specific inactivation mechanisms and their relationship with repeat-specific silencing phenomena in other species.

Homology-dependent gene silencing in plants

We analyzed cDNA libraries from developing endosperm of the B73 maize inbred line to evaluate the expression of storage protein genes. This study showed that zeins are by far the most highly expressed genes in the endosperm, but we found an inverse relationship between the number of zein genes and the relative amount of specific mRNAs. Although alpha-zeins are encoded by large multigene families, only a few of these genes are transcribed at high or detectable levels. In contrast, relatively small gene families encode the gamma- and delta-zeins, and members of these gene families, especially the gamma-zeins, are highly expressed. Knowledge of expressed storage protein genes allowed the development of DNA and antibody probes that distinguish between closely related gene family members. Using in situ hybridization, we found differences in the temporal and spatial expression of the alpha-, gamma-, and delta-zein gene families, which provides evidence that gamma-zeins are synthesized throughout the endosperm before alpha- and delta-zeins. This observation is consistent with earlier studies that suggested that gamma-zeins play an important role in prolamin protein body assembly. Analysis of endosperm cDNAs also revealed several previously unidentified proteins, including a 50-kD gamma-zein, an 18-kD alpha-globulin, and a legumin-related protein. Immunolocalization of the 50-kD gamma-zein showed this protein to be located at the surface of prolamin-containing protein bodies, similar to other gamma-zeins. The 18-kD alpha-globulin, however, is deposited in novel, vacuole-like organelles that were not described previously in maize endosperm.

Genomics Analysis of Genes Expressed in Maize Endosperm Identifies Novel Seed Proteins and Clarifies Patterns of Zein Gene Expression

The wx-K mutation results from the insertion of a copia-like retrotransposon into exon 12 of the maize waxy gene. This retrotransposon, named Hopscotch, has one long open reading frame encoding all of the domains required for transposition. Computer-assisted database searches using Hopscotch and other plant copia-like retroelements as query sequences have revealed that ancient, degenerate retrotransposon insertions are found in close proximity to 21 previously sequenced plant genes. The data suggest that these elements may be involved in gene duplication and the regulation of gene expression. Similar searches using the Drosophila retrotransposon copia did not reveal any retrotransposon-like sequences in the flanking regions of animal genes. These results, together with the recent finding that reverse-transcriptase sequences characteristic of copia-like elements are ubiquitous and diverse in plants, suggest that copia-like retrotransposons are an ancient component of plant genomes.

https://www.pnas.org/content/pnas/91/25/11792.full.pdf

Retrotransposons in the flanking regions of normal plant genes: a role for copia-like elements in the evolution of gene structure and expression

The B-hordeins are the major group of prolamin storage proteins in barley (Hordeum vulgare L.) and they are encoded by a small multigene family that is expressed specifically in the developing endosperm. We report the complete nucleotide sequence of a clone of one B-hordein gene (pBHR184). The cloned gene contains no introns and belongs to the B1 sub-family of B-hordein genes. Comparison of the 5'-flanking sequences of pBHR184 with those of related S-rich prolamin genes from wheat shows that several short sequences within 600 bp upstream of the translation initiation codon are strongly conserved. A sequence that is conserved at around -300 bp in the S-rich prolamins is also conserved at similar locations in genes encoding the two major classes of maize prolamin (the Z19 and Z21 zeins) and appears to be unique to prolamin genes. We discuss the possible role of this '-300 element' in the control of gene expression in the developing cereal endosperm.

Nucleotide sequence of a B1 hordein gene and the identification of possible upstream regulatory elements in endosperm storage protein genes from barley, wheat and maize

GENE EXPRESSION DURING PLANT DEVELOPMENT 439 Phenotypic Variation of Plant Development 439 Seed Development 440 ZEIN PROTEINS AND GENES 441 The Prolamin Fraction of Maize Storage Proteins 441 The Zein Protein F amities. . . . . . . . . . . . . . . . . .. . .... . . .. . .. .... ... . . . . . . . . . . . . . . . . . . . . . . .. . . ... . . . . 441 The Primary Structure of the Z1 Protein Family 442 Zein Subfamilies 443 Control of Zein Synthesis 445 Structure and Regulation of Zein Genes in Plants 446 Storage Proteins in Other Plants 450 REGULATORY SEQUENCES ON PLANT mRNAs 45 1 Information Derived from Multigene Families 45 1 The Initiator Codon AUG in Plant mRNAs 45 1 The Polyadenylation Signal Sequence . . . .. . 46 1

Angelo Spena

Papers

Two adjacent genomic zein sequences: structure, organization and tissue-specific restriction pattern