The regulation of zein biosynthesis in maize endosperm.

KEY MESSAGE: We review the current knowledge regarding the regulation of zein storage proteins biosynthesis and protein body formation, which are crucial processes for the successful accumulation of nutrients in maize kernels. Storage proteins in the seeds of crops in the grass family (Poaceae) are a major source of dietary protein for humans. In maize (Zea mays), proteins are the second largest nutrient component in the kernels, accounting for ~ 10% of the kernel weight. Over half of the storage proteins in maize kernels are zeins, which lack two essential amino acids, lysine and tryptophan. This deficiency limits the use of maize proteins in the food and feed industries. Zeins are encoded by a large super-gene family. During endosperm development, zeins accumulate in protein bodies, which are derived from the rough endoplasmic reticulum. In recent years, our knowledge of the pathways of zein biosynthesis and their deposition within the endosperm has been greatly expanded. In this review, we summarize the current understanding of zeins, including the genes encoding these proteins, their expression patterns and transcriptional regulation, the process of protein body formation, and other biological processes affecting zein accumulation.

Seed-Specific Expression of the Arabidopsis AtMAP18 Gene Increases both Lysine and Total Protein Content in Maize.

Lysine is the most limiting essential amino acid for animal nutrition in maize grains. Expression of naturally lysine-rich protein genes can increase the lysine and protein contents in maize seeds. AtMAP18 from Arabidopsis thaliana encoding a microtubule-associated protein with high-lysine content was introduced into the maize genome with the seed-specific promoter F128. The protein and lysine contents of different transgenic offspring were increased prominently in the six continuous generations investigated. Expression of AtMAP18 increased both zein and non-zein protein in the transgenic endosperm. Compared with the wild type, more protein bodies were observed in the endosperm of transgenic maize. These results implied that, as a cytoskeleton binding protein, AtMAP18 facilitated the formation of protein bodies, which led to accumulation of both zein and non-zein proteins in the transgenic maize grains. Furthermore, F1 hybrid lines with high lysine, high protein and excellent agronomic traits were obtained by hybridizing T6 transgenic offspring with other wild type inbred lines. This article provides evidence supporting the use of cytoskeleton-associated proteins to improve the nutritional value of maize.

Comparison of membrane targeting strategies for the accumulation of the human immunodeficiency virus p24 protein in transgenic tobacco.

Membrane anchorage was tested as a strategy to accumulate recombinant proteins in transgenic plants. Transmembrane domains of different lengths and topology were fused to the cytosolic HIV antigen p24, to promote endoplasmic reticulum (ER) residence or traffic to distal compartments of the secretory pathway in transgenic tobacco. Fusions to a domain of the maize seed storage protein γ-zein were also expressed, as a reference strategy that leads to very high stability via the formation of large polymers in the ER lumen. Although all the membrane anchored constructs were less stable compared to the zein fusions, residence at the ER membrane either as a type I fusion (where the p24 sequence is luminal) or a tail-anchored fusion (where the p24 sequence is cytosolic) resulted in much higher stability than delivery to the plasma membrane or intermediate traffic compartments. Delivery to the tonoplast was never observed. The inclusion of a thrombin cleavage site allowed for the quantitative in vitro recovery of p24 from all constructs. These results point to the ER as suitable compartment for the accumulation of membrane-anchored recombinant proteins in plants.

Kernel lysine content does not increase in some maize opaque2 mutants.

The recessive mutant allele of the opaque2 gene (o2) alters the endosperm protein pattern and increases the kernel lysine content of maize (Zea mays L.). In this study, sequencing results showed that the o2 mutant was successfully introgressed into 12 elite normal maize inbred lines by marker assisted selection (MAS). The average genetic similarity between these normal inbred lines and their o2 near-isogenic lines (NILs) was more than 95%. Kernel lysine content increased significantly in most of o2 NILs lines relative to normal elite inbreds, but remained unchanged in the genetic backgrounds Dan598o2 and Liao2345o2. Moreover, the kernel characteristics of these two o2 NILs did not differ from the other inbred lines. The results of lysine content analysis in the F1 hybrids between Liao2345o2 and Dan598o2 and other o2 NILs demonstrated that gene(s) other than opaque2 may control kernel lysine content in these two o2 NILs. The results of zein analysis showed that 22-kD α-zein synthesis was reduced or absent, and the 19-kD α-zein synthesis was greatly reduced compared with the recurrent parents in most o2 NILs except for Dan598o2 and Liao2345o2. Our results indicate that gene(s) other than opaque2 may play more important roles in zein synthesis and kernel lysine content in some maize genetic backgrounds.

Protein body-inducing fusions for high-level production and purification of recombinant proteins in plants.

For the past two decades, therapeutic and industrially important proteins have been expressed in plants with varying levels of success. The two major challenges hindering the economical production of plant-made recombinant proteins include inadequate accumulation levels and the lack of efficient purification methods. To address these limitations, several fusion protein strategies have been recently developed to significantly enhance the production yield of plant-made recombinant proteins, while simultaneously assisting in their subsequent purification. Elastin-like polypeptides are thermally responsive biopolymers composed of a repeating pentapeptide 'VPGXG' sequence that are valuable for the purification of recombinant proteins. Hydrophobins are small fungal proteins capable of altering the hydrophobicity of their respective fusion partner, thus enabling efficient purification by surfactant-based aqueous two-phase systems. Zera, a domain of the maize seed storage protein γ-zein, can induce the formation of protein storage bodies, thus facilitating the recovery of fused proteins using density-based separation methods. These three novel protein fusion systems have also been shown to enhance the accumulation of a range of different recombinant proteins, while concurrently inducing the formation of protein bodies. The packing of these fusion proteins into protein bodies may exclude the recombinant protein from normal physiological turnover. Furthermore, these systems allow for quick, simple and inexpensive nonchromatographic purification of the recombinant protein, which can be scaled up to industrial levels of protein production. This review will focus on the similarities and differences of these artificial storage organelles, their biogenesis and their implication for the production of recombinant proteins in plants and their subsequent purification.

The accumulation of alpha-zein in transgenic tobacco endosperm is stabilized by co-expression of beta-zein.

The cysteine-poor alpha-zein is the major prolamin storage protein fraction in maize endosperm and is localized in the interior of protein bodies with delta-zein, whereas the hydrophobic cysteine-rich beta- and gamma-zein are found on the exterior of the PB. In transgenic tobacco endosperm expressing zein genes, alpha-zein was unstable unless co-expressed with gamma-zein. Here we showed that alpha-zein was also stabilized by beta-zein. Small accretions of alpha- and beta-zeins, similar in appearance to maize protein bodies, were localized to the endoplasmic reticulum within tobacco endosperm cells. The zein proteins were also localized to protein storage vacuoles in a more dispersed pattern, suggesting that they were transported there after they were post-translationally sequestered into the ER.

Maize opaque endosperm mutations create extensive changes in patterns of gene expression.

Maize starchy endosperm mutants have kernel phenotypes that include a brittle texture, susceptibility to insect pests, and inferior functional characteristics of products made from their flour. At least 18 such mutants have been identified, but only in the cases of opaque2 (o2) and floury2 (fl2), which affect different aspects of storage protein synthesis, is the molecular basis of the mutation known. To better understand the relationship between the phenotypes of these mutants and their biochemical bases, we characterized the protein and amino acid composition, as well as the mRNA transcript profiles, of nearly isogenic inbred lines of W64A o1, o2, o5, o9, o11, Mucuronate (Mc), Defective endosperm B30 (DeB30), and fl2. The largest reductions in zein protein synthesis occur in the W64A o2, DeB30, and fl2 mutants, which have approximately 35 to 55% of the wild-type level of storage proteins. Zeins in W64A o5, o9, o11, and Mc are within 80 to 90% of the amount found in the wild type. Only in the cases of o5 and Mc were significant qualitative changes in zein synthesis observed. The pattern of gene expression in normal and mutant genotypes was assayed by profiling endosperm mRNA transcripts at 18 days after pollination with an Affymetrix GeneChip containing >1400 selected maize gene sequences. Compared with W64A sugary1, a mutant defective in starch synthesis, alterations in the gene expression patterns of the opaque mutants are very pleiotropic. Increased expression of genes associated with physiological stress, and the unfolded protein response, are common features of the opaque mutants. Based on global patterns of gene expression, these mutants were categorized in four phenotypic groups as follows: W64A+ and o1; o2; o5/o9/o11; and Mc and fl2.

Sequence, regulation, and evolution of the maize 22-kD alpha zein gene family.

We have isolated and sequenced all 23 members of the 22-kD alpha zein (z1C) gene family of maize. This is one of the largest plant gene families that has been sequenced from a single genetic background and includes the largest contiguous genomic DNA from maize with 346,292 bp to date. Twenty-two of the z1C members are found in a roughly tandem array on chromosome 4S forming a dense gene cluster 168,489-bp long. The twenty-third copy of the gene family is also located on chromosome 4S at a site approximately 20 cM closer to the centromere and appears to be the wild-type allele of the floury-2 (fl2) mutation. On the basis of an analysis of maize cDNA databases, only seven of these genes appear to be expressed including the fl2 allele. The expressed genes in the cluster are interspersed with nonexpressed genes. Interestingly, some of the expressed genes differ in their transcriptional regulation. Gene amplification appears to be in blocks of genes explaining the rapid and compact expansion of the cluster during the evolution of maize.

Nitrogen and hormonal responsiveness of the 22 kDa alpha-zein and b-32 genes in maize endosperm is displayed in the absence of the transcriptional regulator Opaque-2.

The maize (Zea mays L.) b-ZIP transcriptional activator Opaque-2(O2) regulates the synthesis of major endosperm proteins. In the o2 homozygote, 22 kDa zein prolamins and the b-32 ribosome-inactivating protein are greatly reduced in level. An in vitro endosperm culture system has been studied in which o2 endosperm synthesizes 22 kDa zein and b-32 in response to nitrogen supplements. An increase in 22 kDa zein mRNA concentration is also seen, implying an effect at the level of transcription or differential RNA turnover. The nitrogen-dependent induction of 22 kDa zein synthesis in cultured o2 endosperm was further investigated by analysing transient expression of reporter constructs. The highest response to nitrogen was exhibited by the intact 22 kDa zein promoter. Removal of individual O2 binding sites either reduced or increased overall promoter activity, but always decreased the nitrogen-dependent stimulation of activity. This effect was observed equally in wild-type and o2 mutant endosperm. It is concluded that a factor other then O2 is responsible for activating the 22 kDa zein promoter under high-nitrogen culture conditions. Despite its occurrence in the absence of O2 protein, the nitrogen response is mediated through binding at O2 binding sites. An induction of 22 kDa zein and b-32 synthesis in cultured o2 endosperm could also be achieved on nitrogen-free media by the addition of abscisic acid or methyl jasmonate.

Differential stability of zein mRNA in developing corn kernel.

The lifetime of the zein mRNA in a developing corn (Zea mays L.) kernel under genome transcription blockade with actinomycin D (in vivo) and in a cell-free system (in vitro) was studied. After a 10 h blockade of gene transcription with actinomycin D, only 55% of 19 kDa zein mRNA and 40% of 22 kDa mRNA were detected in a developing kernel of normal corn. In that of the opaque-2 mutant 80% of 19 kDa zein mRNA remained. To examine the relative stability of poly(A)-containing mRNA, cell-free systems from rabbit reticulocyte lysate and wheat-germ extract were used. In both cases only 40% of 19 kDa zein mRNA and 60% of 22 kDa zein mRNA decayed during a 30 min incubation. Differential mRNA degradation of poly(A)-containing zein mRNA was observed on affinity chromatography; poly(A)-containing 19 kDa zein mRNA from normal corn partially decayed by elution from poly(U)-Sepharose whereas that from opaque-2 remained stable. These data suggest that differential mRNA stability is an important factor in the regulation of the zein gene expression in a developing corn kernel.

Cysteine tRNAs of plant origin as novel UGA suppressors.

We have isolated and sequenced chloroplast (chl) and cytoplasmic (cyt) cysteine tRNAs from Nicotiana rustica. Both tRNAs carry a GCA anticodon but beyond that differ considerably in their nucleotide sequences. One obvious distinction resides in the presence of N6-isopentenyladenosine (i6A) and 1-methylguanosine (m1G) at position 37 in chl and cyt tRNA(Cys) respectively. In order to study the potential suppressor activity of tRNAs(Cys) we used in vitro synthesized zein mRNA transcripts in which an internal UGA stop codon had been placed in either the tobacco rattle virus (TRV)- or tobacco mosaic virus (TMV)-specific codon context. In vitro translation was carried out in a messenger- and tRNA-dependent wheat germ extract. Both tRNA(Cys) isoacceptors stimulate read-through over the UGA stop codon, however, chl tRNA(GCA)Cys is more efficient than the cytoplasmic counterpart. The UGA in the two viral codon contexts is suppressed to about the same extent by either of the two tRNAs(Cys), whereas UGA in the beta-globin context is not recognized at all. The interaction of tRNA(GCA)Cys with UGA requires an unconventional G:A base pair in the wobble position, as postulated earlier for plant tRNA(G psi A)Tyr misreading the UAA stop codon. This is the first case that a cysteine-accepting tRNA has been characterized as a natural UGA suppressor.

Maternal-specific demethylation and expression of specific alleles of zein genes in the endosperm of Zea mays L.

Zeins constitute 60-70% of maize endosperm protein. Zein genes are specifically transcribed in the endosperm, and a correlation has been established between tissue-specific expression and demethylation. Three inbred lines and their reciprocal crosses were analysed to assess for allele-specific differences in methylation, transcription and translation. DNAs from endosperm, embryo and seedling tissues analysed by cleavage with methylation-sensitive restriction enzymes and Southern blot hybridization with zein cDNA and genomic sequences show that specific demethylation of zein sequences occurs only in endosperm and is restricted to the maternal complements. Steady-state transcript accumulation of zein mRNA assessed by RNase protection assay reveals qualitative and quantitative differences among endosperm RNAs of the inbreds and of their reciprocal hybrids. Moreover, two-dimensional gel electrophoresis of zein proteins identified polypeptides that are maternally imprinted in reciprocal crosses. These results indicate that endosperm-specific expression of specific zein alleles may occur via parental imprinting and disclose a possible role of methylation in regulating the expression of genes differently contributed in the endosperm by the maternal and paternal genomes.

Determinants of the high-methionine trait in wild and exotic germplasm may have escaped selection during early cultivation of maize.

The 18 kDa high-methionine delta-class zein gene from maize has been cloned, and its regulation, structure, and map position studied. These studies have shown that (i) zein genes may also contain tryptophan and lysine codons, (ii) the 18 kDa and the related 10 kDa zein gene are coordinately regulated, but their products accumulate to different levels in a genotype-dependent manner, (iii) the duplication of delta-zein genes probably involved unequal crossing over, (iv) no copy correction in either direction has occurred from teosinte to modern corn, and (v) the duplication of of the 18 kDa zein gene probably occurred before the tetraploidization of a progenitor chromosome. The work shows that important nutritional quality determinants like the high-methionine seed proteins are abundant in several exotic and wild corn varieties and low in most of the inbreds screened. The lack of a selectable phenotype for such quality traits during initial domestication and breeding of corn would have eliminated cis and trans regulatory determinants from the germplasm used in modern corn breeding. Examples of the high-methionine delta-class zeins shown here may be generally applicable in explaining the low nutritional quality of most present-day corn grown.

RFLP mapping of the maize dzr1 locus, which regulates methionine-rich 10 kDa zein accumulation.

The dzrl locus in maize posttranscriptionally regulates the accumulation of methionine-rich 10 kDa zein in the endosperm. An allele of this locus present in the inbred line BSSS53, dzrl + BSSS53, conditions several-fold higher accumulation of the 10 kDa zein in comparison with standard inbred lines, leading to enrichment of methionine content in BSSS53 by 30%. In a population segregating for high and low 10 kDa zein, dzr1 + BSSS53 was found tightly to cosegregate with a 22 kDa zein gene cluster, belonging to the Z1C subfamily of alpha-zeins that is located on chromosome 4S. One member of this gene cluster, azs22/6, was estimated to be located less than 0.4 cM from dzr1 + BSSS53, while three other 22 kDa zein genes mapped 3.4 cM away. Restriction fragment length polymorphism (RFLP) mapping of dzr1 was conducted using additional maize DNA markers and orthologous rice DNA markers. One maize marker, php20725, was identified that mapped 1.1 cM from dzr1, proximal to the centromere. Another marker derived from rice, rz329, mapped 6.6 cM distal to dzr1. Pulsed-field gel electrophoresis (PFGE) of the 22 kDa zein cluster showed that probably all copies of the 22 kDa zein genes are present within a 200 kb SalI fragment. The recombination frequency within this cluster was estimated to be 20-fold higher than that predicted for the maize genome.

opaque-15, a maize mutation with properties of a defective opaque-2 modifier.

An opaque mutation was identified that reduces gamma-zein synthesis in maize endosperm. The mutation, opaque-15, causes a 2- to 3-fold reduction in gamma-zein mRNA and protein synthesis and reduces the proportion of the 27-kDa gamma-zein A gene transcript. Although the protein bodies in opaque-15 are similar in size and morphology compared to wild type, there are fewer of them in developing endosperm cells. The opaque-15 mutation maps near the telomere of chromosome 7L, coincident with an opaque-2 modifier locus. Based on its phenotype, opaque-15 appears to be a mutation of an opaque-2 modifier gene.

Identification of a transcriptional activator-binding element in the 27-kilodalton zein promoter, the -300 element.

By utilizing a homologous transient-expression system, we have shown that a 58-bp sequence from the gamma-class 27-kDa zein promoter, spanning from -307 to -250 relative to the transcription start site, confers a high level of transcriptional activity on a truncated plant viral promoter. The transcriptional activity mediated by the 58-bp sequence is orientation independent, and it is further enhanced as a result of its multimerization. A similarly high level of transcriptional activity was also observed in protoplasts isolated from leaf tissue-derived maize suspension cells. In vitro binding and DNase I footprinting assays with nuclear protein prepared from cultured endosperm cells revealed the sequence-specific binding of a nuclear factor(s) to a 16-nucleotide sequence present in the 58-bp region. The nuclear factor binding sequence includes the -300 element, a cis-acting element highly conserved among different zein genes and many other cereal storage protein genes. A 23-bp oligonucleotide sequence containing the nuclear factor binding site is sufficient for binding the nuclear factor in vitro. It also confers a high level of transcriptional activity in vivo, but in an orientation-dependent manner. Four nucleotide substitutions in the -300 element drastically reduced binding and transcriptional activation by the nuclear factor. The same nuclear factor is abundant in the developing kernel endosperm and binds to the -300 element region of the 27-kDa or the alpha-class zein promoter. These results suggest that the highly conserved -300 element is involved in the common regulatory mechanisms mediating the coordinated expression of the zein genes.

Three Tetrahymena tRNA(Gln) isoacceptors as tools for studying unorthodox codon recognition and codon context effects during protein synthesis in vitro.

Three glutamine tRNA isoacceptors are known in Tetrahymena thermophila. One of these has the anticodon UmUG which reads the two normal glutamine codons CAA and CAG, whereas the two others with CUA and UmUA anticodons recognize UAG and UAA, respectively, which serve as termination codons in other organisms. We have employed these tRNA(Gln)-isoacceptors as tools for studying unconventional base interactions in a mRNA- and tRNA-dependent wheat germ extract. We demonstrate here (i) that tRNA(Gln)UmUG suppresses the UAA as well as the UAG stop codon, involving a single G:U wobble pair at the third anticodon position and two simultaneous wobble base pairings at the first and third position, respectively, and (ii) that tRNA(Gln)CUA, in addition to its cognate codon UAG, reads the UAA stop codon which necessitates a C:A mispairing in the first anticodon position. These unorthodox base interactions take place in a codon context which favours readthrough in tobacco mosaic virus (TMV) or tobacco rattle virus (TRV) RNA, but are not observed in a context that terminates zein and globin protein synthesis. Furthermore, our data reveal that wobble or mispairing in the middle position of anticodon-codon interactions is precluded in either context. The suppressor activities of tRNAs(Gln) are compared with those of other known naturally occurring suppressor tRNAs, i.e., tRNA(Tyr)G psi A and tRNA(Trp)CmCA. Our results indicate that a 'leaky' context is neither restricted to a single stop codon nor to a distinct tRNA species.

Allele-specific parental imprinting of dzr1, a posttranscriptional regulator of zein accumulation.

Parental imprinting describes the phenomenon of unequivalent gene function based on transmission from the female or male parent. We have discovered parental imprinting of an allele of the dzr1 locus that posttranscriptionally regulates the accumulation of 10-kDa zein in the maize endosperm. The imprinted allele of MO17 inbred origin, dzr1 + MO17, conditions low accumulation of the 10-kDa zein and is dominant when transmitted through the female but recessive when transmitted through the male. Analyzing endosperms with equal parental contributions of dzr1 + MO17 ruled out the possibility that the unequivalent phenotype of dzr1 + MO17 was due to parental dosage imbalance in the triploid endosperm. Second-generation studies show that the dominant or recessive phenotype of dzr1 + MO17 is determined at every generation based on immediate parental origin with no grandparental effect.