Zein Gene Research Articles

Opaque-2 is a transcriptional activator that recognizes a specific target site in 22-kD zein genes.

opaque-2 (o2) is a regulatory locus in maize that plays an essential role in controlling the expression of genes encoding the 22-kD zein proteins. Through DNase I footprinting and DNA binding analyses, we have identified the binding site for the O2 protein (O2) in the promoter of 22-kD zein genes. The sequence in the 22-kD zein gene promoter that is recognized by O2 is similar to the target site recognized by other "basic/leucine zipper" (bZIP) proteins in that it contains an ACGT core that is necessary for DNA binding. The site is located in the -300 region relative to the translation start and lies about 20 bp downstream of the highly conserved zein gene sequence motif known as the "prolamin box." Employing gel mobility shift assays, we used O2 antibodies and nuclear extracts from an o2 null mutant to demonstrate that the O2 protein in maize endosperm nuclei recognizes the target site in the zein gene promoter. Mobility shift assays using nuclear proteins from an o2 null mutant indicated that other endosperm proteins in addition to O2 can bind the O2 target site and that O2 may be associated with one of these proteins. We also demonstrated that in yeast cells the O2 protein can activate expression of a lacZ gene containing a multimer of the O2 target sequence as part of its promoter, thus confirming its role as a transcriptional activator. A computer-assisted search indicated that the O2 target site is not present in the promoters of zein genes other than those of the 22-kD class. These data suggest a likely explanation at the molecular level for the differential effect of o2 mutations on expression of certain members of the zein gene family.

Mutations of the 22- and 27-kD zein promoters affect transactivation by the Opaque-2 protein.

By utilizing a homologous transient expression system, we have demonstrated that the Opaque-2 (O2) gene product O2 confers positive trans-regulation on a 22-kD zein promoter. This trans-acting function of the O2 protein is mediated by its sequence-specific binding to a cis element (the O2 target site) present in the 22-kD zein promoter. A multimer of a 32-bp promoter fragment containing this O2 target site confers transactivation by O2. A single nucleotide substitution in the O2 target sequence not only abolishes O2 binding in vitro, but also its response to transactivation by O2 in vivo. We have also demonstrated that an amino acid domain including the contiguous basic region and the heptameric leucine repeat is essential for the trans-acting function of the O2 protein. Similar but not identical O2 target sequence motifs can be found in the promoters of zein genes of different molecular weight classes. Conversion of such a motif in the 27-kD zein promoter to an exact O2 target sequence by site-directed mutagenesis was sufficient to increase the binding affinity of the O2 protein in vitro and to confer transactivation by O2 in vivo.

Functional analysis of the regulatory region of a zein gene in transiently transformed protoplasts.

The transcription of zein genes in maize is tissue-specific and developmentally regulated. The 5' regulatory region of many zein genes contains two promoters, P1 and P2, lying approximately 1000 bases apart. The promoter/enhancer activity of various fragments of the two promoter regions of the zein gene E19 have been analysed by means of transient expression experiments. The results indicate that the various regions differentially affect the expression of the GUS reporter gene activity in protoplasts from tobacco leaves, maize immature endosperms and in vitro endosperm cell cultures. In tobacco protoplasts only the proximal promoter region, P2, activates GUS expression, while in endosperm culture cells only the distant promoter, P1, gives significant activity. The P1 region, both in direct and opposite orientation, stimulates a low level of GUS expression in protoplasts from immature endosperms.

Partial Purification and Characterization of Lysine-Ketoglutarate Reductase in Normal and Opaque-2 Maize Endosperms

Lysine-ketoglutarate reductase catalyzes the first step of lysine catabolism in maize (Zea mays L.) endosperm. The enzyme condenses l-lysine and alpha-ketoglutarate into saccharopine using NADPH as cofactor. It is endosperm-specific and has a temporal pattern of activity, increasing with the onset of kernel development, reaching a peak 20 to 25 days after pollination, and there-after decreasing as the kernel approaches maturity. The enzyme was extracted from the developing maize endosperm and partially purified by ammonium-sulfate precipitation, anion-exchange chromatography on DEAE-cellulose, and affinity chromatography on Blue-Sepharose CL-6B. The preparation obtained from affinity chromatography was enriched 275-fold and had a specific activity of 411 nanomoles per minute per milligram protein. The native and denaturated enzyme is a 140 kilodalton protein as determined by polyacrylamide gel electrophoresis. The enzyme showed specificity for its substrates and was not inhibited by either aminoethyl-cysteine or glutamate. Steady-state product-inhibition studies revealed that saccharopine was a noncompetitive inhibitor with respect to alpha-ketoglutarate and a competitive inhibitor with respect to lysine. This is suggestive of a rapid equilibrium-ordered binding mechanism with a binding order of lysine, alpha-ketoglutarate, NADPH. The enzyme activity was investigated in two maize inbred lines with homozygous normal and opaque-2 endosperms. The pattern of lysine-ketoglutarate reductase activity is coordinated with the rate of zein accumulation during endosperm development. A coordinated regulation of enzyme activity and zein accumulation was observed in the opaque-2 endosperm as the activity and zein levels were two to three times lower than in the normal endosperm. Enzyme extracted from L1038 normal and opaque-2 20 days after pollination was partially purified by DEAE-cellulose chromatography. Both genotypes showed a similar elution pattern with a single activity peak eluted at approximately 0.2 molar KCL. The molecular weight and physical properties of the normal and opaque-2 enzymes were essentially the same. We suggest that the Opaque-2 gene, which is a transactivator of the 22 kilodalton zein genes, may be involved in the regulation of the lysine-ketoglutarate reductase gene in maize endosperm. In addition, the decreased reductase activity caused by the opaque-2 mutation may explain, at least in part, the elevated concentration of lysine found in the opaque-2 endosperm.

Functional analysis of the -300 region of maize zein genes.

A 43 bp fragment containing the -300 region upstream control element common to the endosperm expressed zein genes of Zea mays L. has been analyzed by in vivo and in vitro techniques. Transient transformation studies with protoplasts from a maize endosperm culture indicate that the element positively affects CaMV 35S promoter-driven gene expression, and that this effect is both orientation- and position-dependent. Band-shift and Southwestern blotting experiments demonstrate that the element is specifically bound by different sets of DNA-binding proteins from seedling and endosperm nuclei. A 2 bp substitution within the most conserved region of the element both reduces the stimulatory effect on transcription and alters the binding of nuclear proteins from both tissues.

Sequence analysis of linked maize 22 kDa alpha-zein genes.

We have determined the nucleotide sequence of a 7343 bp zein genomic clone (gZ22.8H3) from the maize inbred W64A. Computer-aided analysis of the DNA sequence revealed two contiguous 22 kDa alpha-zein genes. The 5' gene (gZ22.8) encodes a complete polypeptide and contains putative regulatory sequences in both the 5' and 3' flanking regions that are typical of zein genes. In contrast, the 3' gene (psi gZ22.8) appears to be a pseudogene, because it contains numerous insertions and deletions that would prevent translation of the mRNA. Alignment of the 5' and 3' flanking sequences of both genes indicated that they resulted from a 3.3 kb DNA duplication event.

Isolation and characterization of high-mobility-group proteins from maize

Chromosomal nonhistone high-mobility-group (HMG) proteins were purified from nuclei of maize (Zea mays L. cv. A619) endosperm and leaf tissue. Tissuespecific differences were observed in their polypeptide patterns, in in-vitro phosphorylation experiments with a casein-kinase type II, and by Western blot analysis with antisera against different HMG proteins. Gelfiltration chromatography demonstrated that maize HMG proteins occur as monomers. By measuring the capacity of the HMG proteins to bind to the 5' flanking region of a zein gene, the sensitivity of the proteins to different temperatures, salt concentrations and pH values was determined.

A homologous expression system for cloned zein genes

Expression of the genes encoding the 10-, 15-, and 27-kDa zeins is maintained in suspension cultures derived from developing endosperm tissue of maize (Zea mays L.). Although expression of these genes is reduced in endosperm cultures as compared with that in endosperm tissue from developing kernels, it remains specific to the origin of explant, since no transcripts are detected in leaf tissue-derived suspension cultures. Transcript sizes are identical to those in developing seed endosperm tissue. Furthermore, accurate transcription initiation of the 10- and 27-kDa zein genes is observed by S1 nuclease mapping. Protoplasts isolated from endosperm cultures are capable of expressing foreign genes when transfected by electroporation. We demonstrate that the 5' flanking sequences of the 10- and 27-kDa zein genes are capable of promoting chloramphenicol acetyltransferase (CAT) gene expression in these transfected protoplasts. Our observations show that these maize endosperm cultures can be used as an efficient homologous system to study transcriptional regulation of zein genes.

Highly clustered zein gene sequences reveal evolutionary history of the multigene family

We have determined the nucleotide sequences of zein cDNA clones ZG14, ZG15, and ZG35. The three clones have 95 to 98% homology to the previously published sequence of clone A20, and 84% homology to sequences of the zein subfamily A30. Comparison of all sequences of the A30 and A20 subfamilies highlights the following features: the 5′ nontranslated regions are 68 and 57 nucleotides in length for the A20- and A30-like mRNAs, respectively, and contain at least three repeats of the consensus sequence ACGAACAAta gG ; the majority of these genes are highly clustered as judged from pulsed-field gel electrophoresis of high molecular weight maize DNA. Furthermore, we discuss a model for the evolution of the multigene family which stresses the special importance of unequal crossing-over and gene conversion in this system.

Characterization of a maize endosperm culture expressing zein genes and its use in transient transformation assays

An endosperm derived tissue culture of maize (Zea mays L.) variety A636 has been characterised for its ability to synthesize zein protein and respond to a zein gene regulatory element. Western analysis with zein specific antibodies revealed the distinct presence of zein proteins of the 15, 19 and 21 kDa classes in this tissue, in contrast to an embryo-derived Black Mexican Sweet variety tissue culture which exhibited no zein proteins. Transient transformation studies with a cauliflower mosaic virus 35S promoter and luciferase reporter gene construct demonstrate that protoplasts from this tissue culture, but not from the embryo-derived culture, respond positively to the potential enhancer which is located approximately 300 base pairs upstream of the coding region in most zein genes of maize, thus establishing the usefulness of this culture for studies of tissue specific gene regulation.

An arginine to lysine substitution in the bZIP domain of an opaque-2 mutant in maize abolishes specific DNA binding.

The opaque-2 (o2) locus in maize encodes a transcription factor involved in the regulation of zein storage proteins. We have shown previously that the O2 protein contains a leucine zipper domain that binds to promoters of 22-kD zein genes. In this paper we characterize an EMS-induced o2 allele, o2-676, that causes a 50% reduction in zein. We have found that the o2-676 mutant protein does not show specific recognition of zein promoter fragments because of the substitution of a lysine residue for an arginine residue within the bZIP domain of o2-676. This particular arginine is conserved within the bZIP domains of all mammalian, fungal, and plant DNA binding proteins of this class. The correlation between this mutation in o2 and the altered pattern of zein expression strongly suggests that O2 regulates transcription of certain members of the zein multigene family through direct interaction with the zein promoters and not through the transcriptional activation of some other regulator of zein gene expression.

Post-transcriptional regulation of methionine content in maize kernels

Message levels for a methionine-rich 10 kDa zein were determined in three inbred lines of maize and their reciprocal crosses at various stages during endosperm development. Inbred line BSSS-53, which overexpresses the 10 kDa protein in mature kernels, was shown to have higher mRNA levels in developing endosperm, as compared to inbred lines W23 and W64A. Differences in mRNA levels could not be explained by differences in transcription rate of the 10 kDa zein gene, indicating differential post-transcriptional regulation of this storage protein in the different inbred lines analyzed. Among progeny segregating for the BSSS-53 allele of the 10 kDa zein structural gene Zps10/(22), mRNA levels are independent of Zps10/(22) segregation, indicating that post-transcriptional regulation of mRNA levels takes place via a trans-acting mechanism. In the same progeny, mRNA levels are also independent of allelic segregation of the regulatory locus Zpr10/(22). Thus, the trans-acting factor encoded by Zpr10/(22) determines accumulation of 10 kDa zein at a translational or post-translational step. Multiple trans-acting factors are therefore involved in post-transcriptional regulation of the methionine-rich 10 kDa zein.

Monocot regulatory protein Opaque-2 is localized in the nucleus of maize endosperm and transformed tobacco plants.

Protein targeting to the nucleus has been studied extensively in animal and yeast systems; however, nothing is known about nuclear targeting in plants. The Opaque-2 (O2) gene produces a regulatory protein that is responsible for inducing transcription of the alpha-zein class of storage proteins in maize kernels. The cloned O2 gene encodes a protein that contains a leucine zipper DNA binding domain that can interact with zein gene promoters. We have used immunolocalization to show that the O2 protein is present in nuclei in the maize endosperm tissues known to produce alpha-zeins. In addition, neither embryo tissue from wild-type kernels nor endosperm from kernels harboring a null o2 allele contain the O2 protein. Analysis of a transposable, element-induced o2 allele, o2-m20, revealed that sectors of endosperm cells contained the nuclear-localized O2 protein, indicating excision of the transposable element. To study further the nuclear transport of the O2 protein, we have transformed this gene, under the control of a constitutive promoter, into tobacco. Plants were shown to have detectable levels of steady-state O2 mRNA and O2 protein. Immunolocalization of O2 protein in transformed tobacco plants indicated that the O2 protein was transported into tobacco nuclei. Therefore, we have developed a system to study nuclear targeting in plants and have established that the nuclear transport machinery is similar in monocots and dicots.

Normal and lysine-containing zeins are unstable in transgenic tobacco seeds

Chimeric genes composed of the beta-phaseolin promoter, an alpha-zein coding sequence and its modified versions containing lysine codons, and a beta-zein polyadenylation signal were inserted into the genome of tobacco by Agrobacterium-mediated transformation. alpha-Zein mRNA levels in the transgenic tobacco seeds 20 days after self-pollination varied between 1.0% and 2.5% of the total mRNA population. At 25 days after pollination the 19 kDa alpha-zein was immunologically detected with a polyclonal antiserum in protein extracts from the seeds of transgenic plants. The transgenic plant with the highest level of zein gene expression had an alpha-zein content that was approximately 0.003% of the total seed protein. The amount of alpha-zein in other transgenic plants varied between 1 x 10(-4)% and 1 x 10(-5)% of the total seed protein. The differences in the amounts of mRNA and protein did not correlate with the lysine substitutions introduced into the alpha-zein protein. Polysomes translating alpha-zein mRNA isolated from tobacco seeds contained fever ribosomes than those from maize endosperm, but this did not appear to be the cause of the inefficient protein synthesis. In vivo labelling and immunoprecipitation indicated that newly synthesized alpha-zein was degraded in tobacco seeds with a half-life of less than 1 hour.

Analysis of promoter activity from an ?-zein gene 5? flanking sequence in transient expression assays

Three DNA regions required for high levels of transcription were identified by transient gene expression analysis of the 5' flanking region of a 19 kDa alpha-zein gene. For these analyses, the zein promoter region was fused to the beta-glucuronidase (GUS) gene and assayed by transient expression in carrot protoplasts. A 107-bp sequence (-114/-8) containing the TATA box resulted in low levels of GUS activity. Addition of the proximal 75 bp (-189/-114) doubled the level of GUS expression, and a further increase in expression was obtained when additional upstream sequences (-483/-226) were placed 5' of the zein promoters. Zein upstream sequences enhanced transcription independently of the -189/-114 region. Although the -189/-114 region was not essential for transcription, it was important to obtain maximum GUS activity. A 121 bp upstream sequence (-347/-226) that contains the conserved TGTAAAG sequence gave high levels of GUS activity when placed in either orientation 5' of the zein promoter sequences. However, nucleotides -347 to -309, containing the TGTAAAG sequence, could be deleted from this fragment without a significant change in GUS activity. Zein upstream sequences did not promote transcription of the GUS gene in somatic maize protoplasts. The upstream activating sequence from the cauliflower mosaic virus (CaMV) 35S promoter placed 5' of deletion mutants of the zein promoter also failed to produce GUS activity above background.

The maize zein gene zE19 contains two distinct promoters which are independently activated in endosperm and anthers of transgenic Petunia plants

The maize zein gene zE19 contains two distinct promoters which are independently activated in endosperm and anthers of transgenic Petunia plants

The maize zein gene zE19 contains two distinct promoters which are independently activated in endosperm and anthers of transgenic Petunia plants

The activity, tissue specificity and temporal expression of the tandem promoter region preceding a maize zein gene (zE19, encoding a 19 kDa zein protein) were tested in transgenic Petunia plants. To simplify the analysis, the tandem promoter as well as each of the two separate promoter regions were fused to the beta-glucuronidase (GUS) reporter gene. All of the three constructs directed the synthesis of GUS in the endosperm of transformed seeds indicating that both separate promoters are independently activated and show the same tissue and cell type specificity observed for zein genes in maize. The kinetics of accumulation and the localization of GUS activity are not coordinated with those of Petunia endogenous seed storage proteins during the development of transformed seeds. Unexpectedly, we detected high levels of GUS activity in anthers of transformed Petunia plants for all three constructs. This appears to reflect the expression pattern of zein genes in maize, since we detect zein transcripts in anthers. Finally, we discuss the possible origin and function of the tandem promoter arrangement on the basis of these data.

Multiple proteins bind to the P2 promoter region of the zein gene pMS1 of maize

A 216 bp promoter fragment of the 19 kDa protein zein gene pMS1, containing the CCAAT and TATA boxes, was analysed by a variety of techniques for in vitro interactions with nuclear proteins from endosperm tissue. HMG proteins were found to form stable complexes with these A/T-rich promoter sequences and several specific DNA-binding proteins appear to be involved in the formation of DNA-protein complexes with this fragment. A 29 bp region spanning the two CCAAT boxes was protected from DNase I digestion in footprinting experiments.

Deletion analysis of a zein gene promoter in transgenic tobacco plants

A zein gene (Z4) promoter containing 886 bp upstream from the transcription start site has been shown previously to be active specifically in the endosperm of transgenic tobacco seeds. To investigate the region required for this tissue-specific activity, deletions of the Z4 promoter were constructed and placed upstream of the beta-glucuronidase (GUS) reporter gene. When these deletions were tested in transgenic tobacco plants, seed-specific GUS activity, which reached a peak between 15 and 19 DAP, was observed for promoters extending from -886 to -174. Interestingly, the 174 bp promoter lacked the complete 15 bp consensus sequence found in the same position in all zein genes so far sequenced. With the next shorter promoter in the deletion series (79 bp), which just included the CAAT and TATA elements, negligible GUS activity was observed in seeds. The results demonstrated that 174 bp upstream of the transcription start site are sufficient for tissue-specific and temporally regulated activity of the Z4 promoter in tobacco. At most, two-fold enhanced activity was observed with additional 5' sequences up to -886.

Maize high mobility group proteins bind to CCAAT and TATA boxes of a zein gene promoter.

A 216-base pair promoter fragment of the 19-kDa zein storage protein gene pMS1, containing the CCAAT and TATA boxes, was analyzed for its in vitro interactions with purified high mobility group (HMG) proteins from maize endosperm tissue. It was found that the HMG proteins display a preferential binding of A/T-rich DNA regions like their animal counterparts, although the A/T content seems not to be the only determinant for binding specificity. Surprisingly, a specific binding of the HMG proteins occurs at the CCAAT and the TATA boxes as indicated by footprinting experiments.