The O2 gene which regulates zein deposition in maize endosperm encodes a protein with structural homologies to transcriptional activators (original) (raw)
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PLANT PHYSIOLOGY, 2000
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.
The Plant Cell, 1993
The opaque2 (o2) modifier genes convert the soft endosperm of an o2 mutant to a hard, vitreous phenotype. The primary biochemical change associated with the expression of these genes is a two- to threefold increase in synthesis of the 27-kD gamma-zein storage protein. To investigate the mechanism of modifier gene activity, we examined the level of gamma-zein mRNA and protein synthesis during the early stages of endosperm development in normal, o2, and modified o2 geno-types. Although the o2 mutation was found to reduce expression of the 27-kD gamma-zein genes, the activity of o2 modifier genes dramatically increased the level of both gamma-zein protein and mRNAs as early as 16 days after pollination. At this stage, transcription of gamma-zein genes is reduced by approximately 50% in both o2 and modified o2 genotypes compared to wild type. Thus, it appears that the modifiers regulate gamma-zein synthesis through a post-transcriptional mechanism. Analysis of transcripts from the two nearly identical genes (A and B) encoding the 27-kD gamma-zein protein showed differences in the mRNA ratios in different genotypes. In modified o2 mutants, accumulation of A over B transcript was greatly enhanced during endosperm development. Somatic recombination at this locus was found to reduce the number of B genes in the endosperm, but this could not account for the preferential accumulation of the A transcript. Our results suggest that a product of the o2 modifier genes increases the translation or stability of the A gene mRNA, leading to enhanced synthesis of 27-kD gamma-zein protein.
Proceedings of the National Academy of Sciences, 1997
The prolamin box (P-box) is a highly conserved 7-bp sequence element (5-TGTAAAG-3) found in the promoters of many cereal seed storage protein genes. Nuclear factors from maize endosperm specifically interact with the P-box present in maize prolamin genes (zeins). The presence of the P-box in all zein gene promoters suggests that interactions between endosperm DNA binding proteins and the P-box may play an important role in the coordinate activation of zein gene expression during endosperm development. We have cloned an endosperm-specific maize cDNA, named prolaminbox binding factor (PBF), that encodes a member of the recently described Dof class of plant Cys 2 -Cys 2 zinc-finger DNA binding proteins. When tested in gel shift assays, PBF exhibits the same sequence-specific binding to the P-box as factors present in maize endosperm nuclei. Additionally, PBF interacts in vitro with the basic leucine zipper protein Opaque2, a known transcriptional activator of zein gene expression whose target site lies 20 bp downstream of the P-box in the 22-kDa zein gene promoter. The isolation of the PBF gene provides an essential tool to further investigate the functional role of the highly conserved P-box in regulating cereal storage protein gene expression.
Characterization of Polypeptides Corresponding to Clones of Maize Zein mRNAS
PLANT PHYSIOLOGY, 1987
Zeins, the storage proteins of maize (Zea mays) are a complex group of polypeptides encoded by a large multigene family. The a-zein proteins, which account for about 70% of the total, show both size and charge heterogeneity. Although clones corresponding to several different alpha zeins have been characterized, it has not been possible to correlate these sequences with individual zein polypeptides. By translating in Xenopus oocytes RNAs transcribed in vitro from cloned zein mRNAs, we were able to identify the encoded proteins among native zeins or zeins synthesized in oocytes with total zein mRNA. There was no correlation between the isoelectric points of these proteins and the homology of their oding DNA sequences, as the proteins encoded by two closely homologous cDNAs migrated with greater charge heterogeneity than those encoded by less homologous clones. In addition, the size of the proteins as determined by SDS polyacrylamide gel electrophoresis did not always correlate with the length of the protein deduced from the DNA sequence. The ability to match cloned zein sequences to individual native proteins wifl enable the genetic mapping of cloned genes as well as the analysis of their translational regulation.
Solution conformational analysis of the α-zein proteins of maize. J. Biol. Chem 268:26253-26259
Journal of Biological Chemistry
Small angle x-ray scattering and viscometric analyses of the a-zeins of maize in solution indicated that the molecules were asymmetric. Structure predictions of consensus sequences for the two classes of a-zeins, 219 and 222, were in good agreement with the ahelical contents determined by circular dichroism. Dimensions determined by small angle x-ray scattering and viscometry indicated a predominantly a-helical conformation. The data are discussed in relation to models for the solution conformation and to earlier models for a-zeins structure. The alcohol soluble zein storage proteins (prolamins) of maize (Zea mays) are a mixture of proteins and constitute about 50-60% of total endosperm protein. Their only known function is to act as a store of nitrogen for the developing seed. The zeins are rich in glutamine, proline, and unlike other cereal prolamins from wheat, barley and rye, in leucine and alanine. The zeins are separated into four classes, a-, p-, y-, and 6-, on the basis of differences in solubility and sequence (1). The a-zeins constitute 75-85% of the total fraction and comprise two protein groups, the Z19 and 222 zeins, with molecular masses by SDS-PAGE' of about 19 and 22 kDa, respectively. Analysis by isoelectric focusing indicates the azeins to be a heterogeneous mixture of up to 15 proteins (2). A number of complete a-zeins sequences have been deduced from cloned cDNAs and genes (3, 4), showing that the Z19 and 222 components consist of about 210 and 245 residues, with true molecular weights of about 23,000-24,000 and 26,000-27,000, respectively. The sequences show unique Nand C-terminal domains of about 36-37 and 10 residues, respectively, separated by a repetitive domain consisting of blocks of between about 14 and 25 residues (with an average length of 20 residues). The size difference between the Z19 and 222 zeins results from the insertion of an additional repeat unit in the C-terminal end of the protein. This insertion gives a total of 10 repeats in the 222 zeins compared with 9 in the 219 zeins. The p-, y-and 6-zeins show no sequence homology with the a-zeins. Although the pand 6-zeins do not contain repeated sequence motifs, which is unusual for
The Plant Cell, 1993
In maize endosperm, genes encoding the 22-kD zein class of storage proteins are regulated by the OPAQUE2 locus. The Opaque2 (02) protein shares homology with the basic domainlleucine zipper class of transcriptional activators. Using microprojectile bombardment, we have shown that 0 2 is capable of transactivating a 22-kD zein promoter in maize endosperm suspension cultures and in longitudinal sections of intact endosperm. Two mutant forms of the 02 gene were constructed by deleting regions that encode either the basic domain or the first 175 N-terminal residues.of the 0 2 protein.
THE PLANT CELL ONLINE, 2001
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 ␣ -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 ␥ -and ␦ -zeins, and members of these gene families, especially the ␥ -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 ␣ -, ␥ -, and ␦ -zein gene families, which provides evidence that ␥ -zeins are synthesized throughout the endosperm before ␣ -and ␦ -zeins. This observation is consistent with earlier studies that suggested that ␥ -zeins play an important role in prolamin protein body assembly. Analysis of endosperm cDNAs also revealed several previously unidentified proteins, including a 50-kD ␥ -zein, an 18-kD ␣ -globulin, and a legumin-related protein. Immunolocalization of the 50-kD ␥ -zein showed this protein to be located at the surface of prolamin-containing protein bodies, similar to other ␥ -zeins. The 18-kD ␣ -globulin, however, is deposited in novel, vacuole-like organelles that were not described previously in maize endosperm.
Mucronate, Mc, a dominant gene of maize which interacts with opaque-2 to suppress zein synthesis
Theoretical and Applied Genetics, 1983
This paper describes a new dominant mutation of maize, Mc, which interferes in the endosperm with the synthesis of storage proteins. The mutant is characterized by an opaque phenotype; it reduces the deposition of zein and it increases the level of methionine. The mutation is specifically related to storage protein synthesis since soluble and insoluble carbohydrates are present at normal levels. The main interest of this mutant lies in its synergistic interaction with oFaque-2 in repressing zein synthesis. In the double mutant o2Mr the accumulation of zein is reduced to less than 10% of that of the normal endosperm. The control on zein synthesis exerted by the double mutant is at the level of production or stability of translatable zein mRNAs. The double mutant o2Mc germinates well offering the opportunity of using it in biochemical and molecular studies related to storage protein synthesis; the reduced endosperm weight of o2Mc negates its practical utilization in breeding maize for quality.
Physiologia Plantarum, 2001
The effect of nitrogen nutrition on the accumulation of seed In these growth conditions, fresh and dry weights increased in storage proteins has been studied in vitro by cultivating on agar both wild-type and o2 endosperms, irrespective of the genetic media maize (Zea mays L.) endosperm explants from seeds at background. In 4 out of the 5 o2 mutant genotypes analysed we 10 days after pollination. The experiments were performed on detected an accumulation of the zHs similar to the corresponding wild-type explants or seeds. However, in one of these various genetic backgrounds bearing different opaque2 (o2) mutants, Mo17o2R, the addition of amino acids to the culture mutant alleles and on the corresponding wild-type lines. In the seed of the o2 genotypes the high molecular weight-zein media had no effect on the zH accumulation. We showed that the Mo17o2R behaviour is not due to a negative regulation but polypeptides (zHs), whose transcription is Opaque2 (O2) reguto the absence of putative transcription factor(s) able to regulate lated, are absent or extremely reduced. The endosperms were incubated on basal agar medium with amino acid supply. the zH transcription occurring in the other o2 mutants. specific 2D pattern and are characterised by the presence or the absence of some of these polypeptides, or by differences in their relative abundance (Lund et al. 1995, Ciceri et al. 2000). During seed maturation, the expression of the different subsets of zein genes is under the control of a few regulatory loci. The best characterised among them is Opaque2 (O2) which codes for a basic domain/leucine zipper transcriptional factor that activates the expression of most of the zH1 and zH2 genes (for review see Schmidt 1993). In some o2 mutant endosperms, the zH1 and zH2 polypeptides are severely reduced in their relative amounts compared with the other size classes (Ciceri et al. 2000). The synthesis of storage proteins in the seed is also under nutritional control and is quantitatively depending on nitrogen availability in the ear (Singletary and Below 1989, 1990). Dry weight, starch and protein content of in vitro grown kernels are enhanced by amino acid addition up to 14 mM (Singletary et al. 1990).