The nitrogen-induced recovery of alpha-zein gene expression in in vitro cultured opaque2 maize endosperms depends on the genetic background (original) (raw)
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Physiologia …, 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).
The Plant Journal, 1993
The effect of nitrogen (N) nutrition upon the accumulation of seed storage protein has been studied in wildtype and opaqoe-2 mutant (02) maize endosperms grown in vitro, for 5 days, on a solid medium containing different amounts of N in the form of inorganic or organic compounds. As reference, the accumulation of zein proteins in endosperms grown to maturity in vivo under field conditions was also examined. The developmental stage up to 14-19 days after pollination (DAP) defines, both in vivo and in vitro, a phase of growth during which the major zein classes of 20 and 22 kDa are synthesized both in wild-type and o2endosperms. In vitro, the corresponding transcripts of the genes coding the 20 and 22 kDa zeins, and of the zein synthesis associated gene b-32, are also present. Under in vitro conditions, the level of expression of the zein synthesizing system, as monitored by zein accumulation, is dependent on the level and form of N supplied in the medium. In vivo, at stages of development from 19 DAP to maturity, the synthesis of the 22 kDa zein class and of the b-32 protein is under control of the 02 transcriptional activator. It is hypothesized that at early stages of endosperm development (between 9 and 14-19 DAP) a 'metabolic control', based on N availability, is operative, possibly based on a regulatory factor different from 02.
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.
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.
Coordinated Transcriptional Regulation of Storage Product Genes in the Maize Endosperm
Plant Physiology, 1994
We have demonstrated that expression of genes involved in starch and storage protein synthesis of the maize (Zea mays 1.) endosperm are coordinated. Genetic lesions altering synthetic events in one biosynthetic pathway affect expression of genes in both pathways. Initial studies focused on shrunken2 (sh2) and briffle2 (bf2) mutants because these genes encode subunits of the same enzyme, ADP-glucose pyrophosphorylase. Analysis of various sh2and bf2-mutant alleles showed that the most severe mutations also conditioned the largest increase in transcripts. The analysis was extended by monitoring the transcripts of the genes, shrunken1 (shl, structural gene for Suc synthase), sh2, bf2, waxy1 (wxl, structural gene for starch synthase), and those of the large and small zeins in isogenic maize lines at 14, 22, and 30 d postpollination. Endosperms were wild type for all of these genes or contained shl-, sh2-, btl-, bt2-, opaque2 (02-), or amylose-extender7 (del-) dulll (dul-) wxl-mutations. Transcripts increased continually throughout kernel development in the mutants relative to the standard W64A used. Variation in the amount of SUC entering the developing seed also altered transcript amounts. The results indicate that starch and protein biosynthetic genes act in a concerted manner, and both are sensitive to mutationally induced differences. The maize (Zea mays L.) endosperm is a specialized tissue that provides a source of nutrients to the germinated embryo. The vast majority of carbon used in the early steps of seedling development is derived from starch and the storage proteins, the zeins. These storage products account for approximately 90% of the dry weight of a mature maize endosperm. Historically, mutants have provided the most insight into the pathways of starch and zein synthesis. Many endosperm mutations can be classified as affecting starch or zein biosynthesis. Mutations such as 02-, flou y2-, and 07-decrease zein content, and proteins in the other solubility classes increase in abundance (Mertz et al., 1964; Nelson et al., 1965; Ma and Nelson, 1975; Habben et al., 1993). 02 has been shown to encode a transacting regulatory protein involved in zein transcription (Schmidt et al., 1990). S and L zeins represent the 19-and 22-kD classes of zein polypeptides and an 02mutation affects mainly the production of 22-kD zein proteins (Langridge et al., 1982).
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.
The EMBO journal, 1989
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 ...
The Plant Cell, 1996
Zeins are seed storage proteins that form accretions called protein bodies in the rough endoplasmic reticulum of maize endosperm cells. Four types of zeins, a, p, y , and 6, aggregate in a distinctive spatial pattern within the protein body. We created transgenic tobacco plants expressing a-zein, y-zein, or both to examine the interactions between these proteins leading to the formation of protein bodies in the endosperm. Whereas y-zein accumulated in seeds of these plants, stable accumulation of a-zein required simultaneous synthesis of y-zein. The zein proteins formed accretions in the endoplasmic reticulum similar to those in maize endosperm. Protein bodies were also found in protein storage vacuoles. The accumulation of both types of zeins peaked early in development and declined during maturation. Even in the presente of y-zein, there was a turnover of a-zein, suggesting that the interaction between the two proteins might be transitory. We suggest that y-zein plays an important role in protein body formation and demonstrate the utility of tobacco for studying interactions between different zeins.
The Plant Cell, 1996
Zeins are seed storage proteins that form accretions called protein bodies in the rough endoplasmic reticulum of maize endosperm cells. Four types of zeins, a, p, y , and 6, aggregate in a distinctive spatial pattern within the protein body. We created transgenic tobacco plants expressing a-zein, y-zein, or both to examine the interactions between these proteins leading to the formation of protein bodies in the endosperm. Whereas y-zein accumulated in seeds of these plants, stable accumulation of a-zein required simultaneous synthesis of y-zein. The zein proteins formed accretions in the endoplasmic reticulum similar to those in maize endosperm. Protein bodies were also found in protein storage vacuoles. The accumulation of both types of zeins peaked early in development and declined during maturation. Even in the presente of y-zein, there was a turnover of a-zein, suggesting that the interaction between the two proteins might be transitory. We suggest that y-zein plays an important role in protein body formation and demonstrate the utility of tobacco for studying interactions between different zeins.