Hans Hartings

The maize regulatory locus Opaque-2 encodes a DNA-binding protein which activates the transcription of the b-32 gene.

The maize locus, Opaque‐2, controls the expression in developing endosperm of structural genes encoding a family of storage proteins, the 22 kd zeins, and an abundant albumin, termed b‐32. It is shown that the promoter of the b‐32 gene is activated in vivo in the presence of the O2 gene product and that the information necessary for this activation resides in a 440 bp DNA fragment containing five O2 binding sites (GATGAPyPuTGPu). Two of these sites are embedded in copies of the ‘endosperm box’, a motif thought to be involved in endosperm‐specific expression, which is also represented in 22 kd zein promoters. The O2 protein is also shown to be capable of binding in vitro and activating in vivo, its own promoter.

The O2 gene which regulates zein deposition in maize endosperm encodes a protein with structural homologies to transcriptional activators

The structure of the zein regulatory gene Opaque 2 of Zea mays has been determined by sequence analysis of genomic and cDNA clones. The size of O2 mRNA is 1751 bp [poly(A) tail not included] containing a major open reading frame (ORF) of 1380 bp preceded by three short ORFs of 3, 21 and 20 amino acid residues. The main ORF comprises 1362 bp and is composed of six exons ranging in size from 465 to 61 bp and five introns of 678 bp to 83 bp. A putative protein 454 amino acids long was derived by the theoretical translation of the genomic sequences corresponding to exons. The opaque 2 protein contains a domain similar to the leucine zipper motif identified in DNA binding proteins of animal protooncogenes such as fos, jun and myc, and in the transcriptional activators GCN4 and C/EBP. The region of 30 amino acid residues next to the leucine repeats towards the N terminus is rich in basic amino acids and is also homologous to a domain present in fos, jun and GCN4. Moreover, in the carboxy terminal region an amino acid motif closely resembling a metal binding domain is present.

Maize Histone Deacetylase hda101 Is Involved in Plant Development, Gene Transcription, and Sequence-Specific Modulation of Histone Modification of Genes and Repeats

Enzymes catalyzing histone acetylation and deacetylation contribute to the modulation of chromatin structure, thus playing an important role in regulating gene and genome activity. We showed that downregulation and overexpression of the maize (Zea mays) Rpd3-type hda101 histone deacetylase gene induced morphological and developmental defects. Total levels of acetylated histones and histone acetylation of both repetitive and nonrepetitive sequences were affected in hda101 transgenic mutants. However, only transcript levels of genes but not repeats were altered. In particular, hda101 transgenic mutants showed differential expression of genes involved in vegetative-to-reproductive transition, such as liguleless2 and knotted-like genes and their repressor rough sheath2, which are required for meristem initiation and maintenance. Perturbation of hda101 expression also affected histone modifications other than acetylation, including histone H3 dimethylation at Lys-4 and Lys-9 and phosphorylation at Ser-10. Our results indicate that hda101 affects gene transcription and provide evidence of its involvement in setting the histone code, thus mediating developmental programs. Possible functional differences between maize hda101 and its Arabidopsis thaliana ortholog HDA19 are discussed.

Identification and characterisation of an RPD3 homologue from maize (Zea mays L.) that is able to complement an rpd3 null mutant of Saccharomycescerevisiae

Abstract In mammals, yeast and Drosophila, the histone deacetylase RPD3 proteins can alter the expression of genes involved in fundamental biological processes by affecting the degree of acetylation of histones and changing chromatin structure. Here we report the isolation of a cDNA sequence encoding an RPD3 homologue from maize, which is able to complement the phenotype of an rpd3 null mutant of the yeast Saccharomyces cerevisiae. The expression of the corresponding gene(s) was assessed in different maize tissues. The number of homologous loci was estimated by Southern hybridisation to be in the range of two to three, and the chromosomal location of one of these loci was determined. Phylogenetic analysis and tests for relative divergence rates, using related RPD3 sequences from different species, were performed, and suggest that different polymorphic forms of RPD3-like proteins that evolve at distinct rates are present in the species considered.

The b-32 protein from maize endosperm: characterization of genomic sequences encoding two alternative central domains.

As derived from a cDNA clone, the structure of the b-32 protein ofZea mays, a putative regulatory factor of zein expression, has a central acidic region separated by two domains covered by secondary structure motifs. In this work, three b-32 genomic clones were selected from two genomic libraries obtained from the maize inbred lines W64A and A69Y. The nucleotide sequences of the complete coding region of eachb-32 gene, as well as long stretches of their 5′ and 3′ flanking regions, were determined. Introns are not present in the b-32 genomic sequences. Minor variations among the three genes and an earlier reported b-32 cDNA indicates that they constitute a gene family showing a characteristic polymorphism. Such a polymorphism is highly evident in large segments of the upstream regulatory sequences. Interestingly, when compared with cDNA (W64A) or with geneb-32.120 (W64A), the genesb-32.129 (W64A) andb-32.152 (A69Y) show three jumps of the reading frame in the central part of the coding region, resulting in a completely different sequence of the b-32 protein central domain. In all cases, variations in the N- and C-terminal domains account only for microheterogeneity.

The b-32 protein from maize endosperm, an albumin regulated by the O2 locus: nucleic acid (cDNA) and amino acid sequences.

SummaryThe cDNA coding for the b-32 protein, an albumin expressed in maize endosperm cells under the control of the O2 and O6 loci, has been cloned and the complete amino acid sequence of the protein derived. A lambda gt11 cDNA library from mRNA of immature maize endosperm was screened for the expression of the b-32 protein using antibodies against the purified protein. One of the positive clones obtained was used to isolate a full-length cDNA clone. By Northern analysis, the size of the b-32 mRNA was estimated to be 1.2 kb. Hybrid-selected translation assays show that the message codes for a protein with an apparent molecular weight of 30–35 kDa. The nucleotide sequence shows that several internal repeats are present. The protein has a length of 303 amino acid residues (mol. wt. 32430 dalton) and its sequence shows the following features: no signal peptide is observable; it contains seven tryptophan residues, an amino acid absent in maize storage proteins; polar and hydrophobic residues are spread along the sequence; several pairs of basic residues are present in the N-terminal region; the secondary structure allows the prediction of two structural domains for the b-32 protein that would fold up giving rise to a globular shape. The cloning of this gene may help in understanding the role of the O2 and O6 loci in regulating the deposition of zein, the major storage protein of maize endosperm.

The maize WD-repeat gene ZmRbAp1 encodes a member of the MSI/RbAp sub-family and is differentially expressed during endosperm development.

Abstract. Members of the MSI/RbAp sub-family of WD-repeat proteins are widespread in eukaryotic organisms and form part of multiprotein complexes that are involved in various biological pathways, including chromatin assembly, regulation of gene transcription, and cell division. In this study we report the isolation and characterization of a cDNA sequence from Zea mays, which encodes an RbAp-like protein (ZmRbAp1) that binds acetylated histones H3 and H4 and suppresses mutations that have a negative effect on the Ras/cAMP pathway in yeast. The ZmRbAp genes form a gene family and are expressed in different tissues of Z. mays L. plants. Determination of its expression pattern during maize seed development revealed that ZmRbAp transcripts are abundant during the initial stages of endosperm formation. In addition, the transcripts are specifically localized in shoot apical meristem and leaf primordia of the embryo. A possible role for the ZmRbAp genes in early endosperm differentiation and plant development is discussed.

Structural and functional analysis of an Opaque-2-related gene from sorghum.

The Opaque-2 (O2) gene from maize encodes a transcriptional activator of the b-ZIP class. We have isolated and characterized a gene from sorghum, related in sequence to the O2 gene from maize. A single copy of the gene is present in sorghum. Both genomic and cDNA sequences of the O2-related sorghum gene were determined. The sequence is highly homologous to maize O2 both in the promoter and in the coding region. The most closely related sequences contain the b-ZIP domain with only 11 amino acid substitutions in a total of 122 residues. In transient expression assays, the sorghum O2-related coding sequence, expressed from a CaMV 35S promoter, activates expression from the maize b-32 promoter as effectively as that obtained with the maize O2 sequence.

Molecular analysis of opaque -2 alleles from Zea mays L. reveals the nature of mutational events and the presence of a hypervariable region in the 5′ part of the gene

Ten recessive Opaque-2 (O2) alleles of independent origin were characterized at the molecular level. The results revealed a high level of polymorphism at the O2 locus. In addition, our data suggest the possible cause for the recessive character of some of the alleles investigated, and allow us to infer some conclusions concerning the degree of relationship between the o2 mutations. Comparison of genomic sequences spanning the first exon and obtained from a series of wild-type and recessive alleles revealed the presence of a hypervariable region, involving different dipeptides, in the N-terminal part of the O2 protein.

Molecular analysis of theBg-rbg transposable element system ofZea mays L.

SummaryThe two components of theBg-rbg transposable element system of maize have been cloned. TheBg element, isolated from the mutable allelewx-m32 :: Bg is inserted in the intron of theWaxy (Wx) gene between exons 12 and 13. The length of the element is of 4869 bp.Bg has 5 by terminal inverted repeats, and generates upon insertion an 8 by direct duplication of the target sequence. Both ends of theBg element contain a 76 by direct repeat adjacent to the terminal inverted repeats. The hexamer motif TATCGkCG is here repeated several times in direct or inverse orientation. Therbg element was isolated from the mutable alleleo2m(r) where it is located in the promoter region of theOpaque-2 (O2) gene.rbg is approximately 4.5 kb in length, has terminal inverted repeats identical to those of theBg element, and is also flanked by an 8 by direct duplication at the target site. LikeBg, rbg carries the 76 by direct repeats. Restriction enzyme analysis reveals that, compared toBg, the receptor element is distinguishable by small deletion and insertion events. Sequence data indicate that not more than 75% homology exists at the DNA level between therbg element and the autonomousBg element.