Genomics analysis of genes expressed in maize endosperm identifies novel seed proteins and clarifies patterns of zein gene expression.

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 alpha-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 gamma- and delta-zeins, and members of these gene families, especially the gamma-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 alpha-, gamma-, and delta-zein gene families, which provides evidence that gamma-zeins are synthesized throughout the endosperm before alpha- and delta-zeins. This observation is consistent with earlier studies that suggested that gamma-zeins play an important role in prolamin protein body assembly. Analysis of endosperm cDNAs also revealed several previously unidentified proteins, including a 50-kD gamma-zein, an 18-kD alpha-globulin, and a legumin-related protein. Immunolocalization of the 50-kD gamma-zein showed this protein to be located at the surface of prolamin-containing protein bodies, similar to other gamma-zeins. The 18-kD alpha-globulin, however, is deposited in novel, vacuole-like organelles that were not described previously in maize endosperm.

Investigating the hows and whys of DNA endoreduplication.

Endoreduplication is a form of nuclear polyploidization that results in multiple, uniform copies of chromosomes. This process is common in plants and animals, especially in tissues with high metabolic activity, and it generally occurs in cells that are terminally differentiated. In plants, endoreduplication is well documented in the endosperm and cotyledons of developing seeds, but it also occurs in many tissues throughout the plant. It is thought that endoreduplication provides a mechanism to increase the level of gene expression, but the function of this process has not been thoroughly investigated. Numerous observations have been made of endoreduplication, or at least extra cycles of S-phase, as a consequence of mutations in genes controlling several aspects of cell cycle regulation. However, until recently there were few studies directed at the molecular mechanisms responsible for this specialized cell cycle. It is suggested that endoreduplication requires nothing more elaborate than a loss of M-phase cyclin-dependent kinase activity and oscillations in the activity of S-phase cyclin-dependent kinase.

Genetic analysis of amino acid accumulation in opaque-2 maize endosperm.

The opaque-2 mutation in maize (Zea mays) is associated with an increased level of free amino acids (FAA) in the mature endosperm. In particular, there is a high concentration of lysine, the most limiting essential amino acid. To investigate the basis for the high-FAA phenotype of opaque-2 maize, we characterized amino acid accumulation during endosperm development of several wild-type and opaque-2 inbreds. Oh545o2 was found to have an exceptionally high level of FAA, in particular those derived from aspartate (Asp) and intermediates of glycolysis. The FAA content in Oh545o2 is 12 times greater than its wild-type counterpart, and three and 10 times greater than in Oh51Ao2 and W64Ao2, respectively. We crossed Oh545o2 to Oh51Ao2 and analyzed the F(2:3) progeny to identify genetic loci linked with the high FAA level in these mutants. Quantitative trait locus mapping identified four significant loci that account for about 46% of the phenotypic variance. One locus on the long arm of chromosome 2 is coincident with genes encoding a monofunctional Asp kinase 2 and a bifunctional Asp kinase-homo-Ser dehydrogenase-2, whereas another locus on the short arm of chromosome 3 is linked with a cytosolic triose phosphate isomerase 4. The results suggest an alternation of amino acid and carbon metabolism leads to overproduction and accumulation of FAA in opaque-2 mutants.

Aspartate kinase 2. A candidate gene of a quantitative trait locus influencing free amino acid content in maize endosperm.

The maize (Zea mays) Oh545o2 inbred accumulates an exceptionally high level of free amino acids, especially lysine (Lys), threonine (Thr), methionine, and iso-leucine. In a cross between Oh545o2 and Oh51Ao2, we identified several quantitative trait loci linked with this phenotype. One of these is on the long arm of chromosome 2 and is linked with loci encoding aspartate (Asp) kinase 2 and Asp kinase (AK)-homoserine dehydrogenase (HSDH) 2. To investigate whether these enzymes can contribute to the high levels of Asp family amino acids, we measured their specific activity and feedback inhibition properties, as well as activities of several other key enzymes involved in Lys metabolism. We did not find a significant difference in total activity of dihydrodipicolinate synthase, HSDH, and Lys ketoglutarate reductase between these inbreds, and the feedback inhibition properties of HSDH and dihyrodipicolinate synthase by Lys and/or Thr were similar. The most significant difference we found between Oh545o2 and Oh51Ao2 is feedback inhibition of AK by Lys but not Thr. AK activity in Oh545o2 is less sensitive to Lys inhibition than that in Oh51Ao2, with a Lys I50 twice that of Oh51Ao2. AK activity in Oh545o2 endosperm is also higher than in Oh51Ao2 at 15 d after pollination, but not 20 d after pollination. The results indicate that the Lys-sensitive Asp kinase 2, rather than the Thr-sensitive AK-HSDH2, is the best candidate gene for the quantitative trait locus affecting free amino acid content in Oh545o2.

Quantitative trait locus mapping of loci influencing elongation factor 1alpha content in maize endosperm.

The nutritional value of maize (Zea mays) seed is most limited by its protein quality because its storage proteins are devoid of the essential amino acid lysine (Lys). The Lys content of the kernel can be significantly increased by the opaque-2 mutation, which reduces zein synthesis and increases accumulation of proteins that contain Lys. Elongation factor 1alpha (eEF1A) is one of these proteins, and its concentration is highly correlated with the Lys content of the endosperm. We investigated the genetic regulation of eEF1A and the basis for its relationship with other Lys-containing proteins by analyzing the progeny of a cross between a high (Oh51Ao2) and a low (Oh545o2) eEF1A maize inbred. We identified 83 simple sequence repeat loci that are polymorphic between these inbreds; the markers are broadly distributed over the genome (1,402 cM) with an average interval of 17 cM. Genotypic analysis of the F(2) progeny revealed two significant quantitative trait loci that account for 25% of the variance for eEF1A content. One of these is on the short arm of chromosome 4 and is linked with a cluster of 22-kD alpha-zein coding sequences; the other quantitative trait locus is on the long arm of chromosome 7. The content of alpha-zein and gamma-zein was measured in pools of high- and low-eEF1A individuals obtained from this cross, and a higher level of alpha-zein was found to cosegregate with high eEF1A content. Allelic variation at the 22-kD alpha-zein locus may contribute to the difference of eEF1A content between Oh51Ao2 and Oh545o2 by increasing the surface area of protein bodies in the endosperm and creating a more extensive network of cytoskeletal proteins.

Opaque2 modifiers alter transcription of the 27-kDa gamma-zein genes in maize.

Opaque2 modifier genes cause a two- to three-fold increase in the amount of gamma-zein RNA and protein in maize kernels, and can convert the soft, starchy endosperm of an opaque2 mutant to a hard, vitreous phenotype. We analyzed several aspects of transcriptional and post-transcriptional regulation of gamma-zein gene expression in wild-type, opaque2 and modified opaque2 genotypes to investigate the molecular mechanisms by which opaque2 modifiers influence the expression of gamma-zein genes. We found that the poly(A) tails of the gamma-zein RNAs A and B were of similar length in normal, opaque2 and modified opaque2 genotypes. Multiple poly(A) addition sites were detected for the gamma-zein A and B RNAs, but no evidence was obtained that o2 modifiers influence the selection of these sites. Nucleotide sequence analysis of gamma-zein A and B cDNAs derived from 18-DAP endosperm from normal, opaque2, and modified opaque2 kernels confirmed the use of eight different poly(A) addition sites for gamma-zein A transcripts and six different sites for the gamma-zein B transcripts. It also revealed that the A/B gamma-zein RNA ratio in modified opaque2 was at least 40:1, compared to 1:1 in wild type and 3:1 in opaque2. Nuclear run-on transcription assays showed a dramatic shift in the transcription rate of the gamma-zein A gene relative to the B gene in the modified opaque2 genotype. These results are consistent with a model in which the two opaque2 modifier loci influence gamma-zein gene expression through different mechanisms: one affects transcription of the gamma-zein locus and the other influences the steady state level of gamma-zein RNA.

Characterization of maize (Zea mays L.) Wee1 and its activity in developing endosperm.

We report the characterization of a maize Wee1 homologue and its expression in developing endosperm. Using a 0.8-kb cDNA from an expressed sequence tag project, we isolated a 1.6-kb cDNA (ZmWee1), which encodes a protein of 403 aa with a calculated molecular size of 45.6 kDa. The deduced amino acid sequence shows 50% identity to the protein kinase domain of human Wee1. Overexpression of ZmWee1 in Schizosaccharomyces pombe inhibited cell division and caused the cells to enlarge significantly. Recombinant ZmWee1 obtained from Escherichia coli is able to inhibit the activity of p13(suc1)-adsorbed cyclin-dependent kinase from maize. ZmWee1 is encoded by a single gene at a locus on the long arm of chromosome 4. RNA gel blots showed the ZmWee1 transcript is about 2.4 kb in length and that its abundance reaches a maximum 15 days after pollination in endosperm tissue. High levels of expression of ZmWee1 at this stage of endosperm development imply that ZmWee1 plays a role in endoreduplication. Our results show that control of cyclin-dependent kinase activity by Wee1 is conserved among eukaryotes, from fungi to animals and plants.

The eEFIA gene family is differentially expressed in maize endosperm.

eEF1A appears to be a multifunctional protein in eukaryotes, where it serves as a protein synthesis factor as well as a cytoskeletal protein. In maize endosperm, the eEF1A concentration is highly correlated with lysine content, and eEF1A synthesis is increased in opaque2 mutants compared to wild type. To investigate the basis for the increased synthesis of eEF1A in opaque2, we characterized the genes encoding this protein and measured their relative level of expression in endosperm and other tissues. Maize contains 10 to 15 eEF1A genes that are nearly identical in nucleotide and amino acid sequences. However, these genes can be distinguished based on their 3' non-coding sequences, which are less conserved. By screening endosperm and seedling cDNA libraries, we show that most of the maize eEF1A genes are expressed, and the relative level of their transcripts varies in different tissues. At least five genes are transcribed in the endosperm, and two account for ca. 80% of the RNA transcripts. The expression of several genes is enhanced in opaque2 endosperm, although the significance of this is unclear.

Characterization of maize elongation factor 1A and its relationship to protein quality in the endosperm.

The protein synthesis elongation factor 1A (eEF1A) is a multifunctional protein in eukaryotic cells. In maize (Zea mays L.) endosperm eEF1A co-localizes with actin around protein bodies, and its accumulation is highly correlated with the protein-bound lysine (Lys) content. We purified eEF1A from maize kernels by ammonium sulfate precipitation, ion-exchange, and chromatofocusing. The identify of the purified protein was confirmed by microsequencing of an endoproteinase glutamic acid-C fragment and by its ability to bundle actin. Using purified eEF1A as a standard, we found that this protein contributes 0.4% of the total protein in W64A+ endosperm and approximately 1% of the protein in W64Ao2. Because eEF1A contains 10% Lys, it accounts for 2.2% of the total Lys in W64A+ and 2.3% of the Lys in W64Ao2. However, its concentration predicts 90% of the Lys found in endosperm proteins of both genotypes, indicating that eEF1A is a key component of the group of proteins that determines the nutritional quality of the grain. This notion is further supported by the fact that in floury2, another high-Lys mutant, the content of eEF1A increases with the dosage of the floury2 gene. These data provide the biochemical basis for further investigation of the relationship between eEF1A content and the nutritional quality of cereals.

Expression of a mutant alpha-zein creates the floury2 phenotype in transgenic maize.

The maize floury2 mutation results in the formation of a soft, starchy endosperm with a reduced amount of prolamin (zein) proteins and twice the lysine content of the wild type. The mutation is semidominant and is associated with small, irregularly shaped protein bodies, elevated levels of a 70-kDa chaperone in the endoplasmic reticulum, and a novel 24-kDa polypeptide in the zein fraction. The 24-kDa polypeptide is a precursor of a 22-kDa alpha-zein protein that is not properly processed. The defect is due to an alanine-to-valine substitution at the C-terminal position of the signal peptide, which causes the protein to be anchored to the endoplasmic reticulum. We postulated that the phenotype associated with the floury2 mutation is caused by the accumulation of the 24-kDa alpha-zein protein. To test this hypothesis, we created transgenic maize plants that produce the mutant protein. We found that endosperm in seeds of these plants manifests the floury2 phenotype, thereby confirming that the mutant alpha-zein is the molecular basis of this mutation.

A defective signal peptide tethers the floury-2 zein to the endoplasmic reticulum membrane.

The maize (Zea mays L.) floury-2 (fl2) mutation is associated with a general decrease in storage protein synthesis, altered protein body morphology, and the synthesis of a novel 24-kD alpha-zein storage protein. Unlike storage proteins in normal kernels and the majority of storage proteins in fl2 kernels, the 24-kD alpha-zein contains a signal peptide that would normally be removed during protein synthesis and processing. The expected processing site of this alpha-zein reveals a putative mutation alanine-->valine (Ala-->Val) that is not found at other junctions between signal sequences and mature proteins. To investigate the impact of such a mutation on signal peptide cleavage, we have assayed the 24-kD fl2 alpha-zein in a co-translational processing system in vitro. Translation of RNA from fl2 kernels or synthetic RNA encoding the fl2 alpha-zein in the presence of microsomes yielded a 24-kD polypeptide. A normal signal peptide sequence, generated by site-directed mutagenesis, restored the capacity of the RNA to direct synthesis of a properly processed protein in a cell-free system. Both the fl2 alpha-zein and the fl2 alpha-zein (Val-->Ala) were translocated into the lumen of the endoplasmic reticulum. The processed fl2 alpha-zein (Val-->Ala) was localized in the soluble portion of the microsomes, whereas the fl2 alpha-zein co-fractionated with the microsomal membranes. By remaining anchored to protein body membranes during endosperm maturation, the fl2 zein may thus constrain storage protein packing and perturb protein body morphology.

The maize gamma-zein sequesters alpha-zein and stabilizes its accumulation in protein bodies of transgenic tobacco endosperm.

Zeins are seed storage proteins that form accretions called protein bodies in the rough endoplasmic reticulum of maize endosperm cells. Four types of zeins, alpha, beta, gamma, and delta, aggregate in a distinctive spatial pattern within the protein body. We created transgenic tobacco plants expressing alpha-zein, gamma-zein, or both to examine the interactions between these proteins leading to the formation of protein bodies in the endosperm. Whereas gamma-zein accumulated in seeds of these plants, stable accumulation of alpha-zein required simultaneous synthesis of gamma-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 presence of gamma-zein, there was a turnover of alpha-zein, suggesting that the interaction between the two proteins might be transitory. We suggest that gamma-zein plays an important role in protein body formation and demonstrate the utility of tobacco for studying interactions between different zeins.

EF-1[alpha] Is Associated with a Cytoskeletal Network Surrounding Protein Bodies in Maize Endosperm Cells.

By using indirect immunofluorescence and confocal microscopy, we documented changes in the distribution of elongation factor-1[alpha] (EF-1[alpha]), actin, and microtubules during the development of maize endosperm cells. In older interphase cells actively forming starch grains and protein bodies, the protein bodies are enmeshed in EF-1[alpha] and actin and are found juxtaposed with a multidirectional array of microtubules. Actin and EF-1[alpha] appear to exist in a complex, because we observed that the two are colocalized, and treatment with cytochalasin D resulted in the redistribution of EF-1[alpa]. These data suggest that EF-1[alpha] and actin are associated in maize endosperm cells and may help to explain the basis of the correlation we found between the concentration of EF-1[alpha] and lysine content. The data also support the hypothesis that the cytoskeleton plays a role in storage protein deposition. The distributions of EF-1[alpha] actin, and microtubules change during development. We observed that in young cells before the accumulation of starch and storage protein, EF-1[alpha], actin, and microtubules are found mainly in the cell cortex or in association with nuclei.

A maize cDNA encoding a member of the retinoblastoma protein family: involvement in endoreduplication.

Retinoblastoma (RB-1) is a tumor suppressor gene that encodes a 105-kDa nuclear phosphoprotein. To date, RB genes have been isolated only from metazoans. We have isolated a cDNA from maize endosperm whose predicted protein product (ZmRb) shows homology to the "pocket" A and B domains of the Rb protein family. We found ZmRb behaves as a pocket protein based on its ability to specifically interact with oncoproteins encoded by DNA tumor viruses (E7, T-Ag, E1A). ZmRb can interact in vitro and in vivo with the replication-associated protein, RepA, encoded by the wheat dwarf virus. The maize Rb-related protein undergoes changes in level and phosphorylation state concomitant with endoreduplication, and it is phosphorylated in vitro by an S-phase kinase from endoreduplicating endosperm cells. Together, our results suggest that ZmRb is a representative of the pocket protein family and may play a role in cell cycle progression. Moreover, certain plant monopartite geminiviruses may operate similarly to mammalian DNA viruses, by targeting and inactivating the retinoblastoma protein, which otherwise induces G1 arrest.

Identification of a maize endosperm-specific cDNA encoding farnesyl pyrophosphate synthetase.

Farnesyl pyrophosphate synthetase (FPS; EC 2.5.1.10) produces the 15-carbon farnesyl pyrophosphate which is utilized in the synthesis of sterols, carotenoids, dolichols, coenzyme Q, heme a and farnesylated proteins. We have cloned this mRNA sequence from a maize endosperm cDNA library and determined the 1378-nucleotide (nt) sequence of the DNA fragment. This sequence specifies an open reading frame of 1050 nt encoding FPS. The deduced amino acid sequence shows a high degree of similarity to FPS from a wide range of organisms. Southern blot analysis indicated that there are at least two FPS gene copies in the maize genome. The cloned FPS is expressed preferentially in maize endosperm and is up-regulated in the endosperm mutants, o2 and fl2.

Expression of protein disulfide isomerase is elevated in the endosperm of the maize floury-2 mutant.

A maize protein disulfide isomerase (PDI, EC 5.3.4.1) cDNA clone was isolated and characterized. The deduced amino acid sequence contains two regions characteristic of the active sites for PDI and a carboxyl-terminal endoplasmic reticulum (ER) retention sequence, Lys-Asp-Glu-Leu. Southern blot analysis indicated the maize PDI is encoded by a single gene that maps to the short arm of chromosome 4. When isolated from the cisternal and protein body ER, the PDI protein resolves into a fast and a slow form on SDS-PAGE. During endosperm development, the PDI RNA level increases between 10 and 14 days after pollination. In floury-2 (fl2) endosperm, which contains an abnormally processed alpha-zein protein, PDI expression is significantly increased, and the level of PDI protein and RNA is positively correlated with the dosage of fl2 alleles. The increase of PDI in fl2 occurs mainly in the cisternal ER fraction, whereas the most dramatic increase of binding protein (BiP) is in the protein body ER. We propose that the induction of PDI in the fl2 mutant reflects its role as a molecular chaperone, and that PDI functions in concert with BiP at different stages of zein processing and assembly into protein bodies.

Activity of single-stranded DNA endonucleases in mung bean is associated with cell division.

A single-strand-specific endonuclease from mung bean sprouts is widely used in molecular biology. However, the biological role of this enzyme is unknown. We studied the spatial and temporal activity of single-stranded DNA endonucleases in mung bean seedling by following enzyme activity that linearizes supercoiled plasmid DNA, a characteristic of this type of enzyme. The formation of a linear molecule from supercoiled DNA was found to occur in two distinguishable steps. The first, which involves introducing a nick into the supercoiled DNA and relaxing it, is very rapid and complete within a few seconds. The second step of cleaving the opposite strand to generate a unit-length linear duplex DNA is a relatively slow process. Analysis of the DNA cleavage sites showed the nuclease preferentially cuts supercoiled DNA at an AT-rich region. Varying levels of nuclease activity could be detected in different tissues of the mung bean seedling. The highest activity was in the root tip and was correlated with histone H1 kinase activity. This implies a link between nuclease activity and cell division. Induction of cell division in mung bean hypocotyls with auxin promoted formation of root primordia and considerably increased the activity of single-stranded DNA endonucleases. The nuclease activity and histone H1 kinase activity were reduced in mung bean cuttings treated with hydroxyurea, but not in cuttings treated with oryzalin. The potential function of single-stranded DNA endonucleases is discussed.

Endoreduplication in maize endosperm: involvement of m phase--promoting factor inhibition and induction of s phase--related kinases.

Endoreduplication is an endonuclear chromosome duplication that occurs in the absence of mitosis and in Zea mays (L.) is required for endosperm development. Induction of DNA synthesis during early stages of endosperm development is maintained by increasing the amount and activity of S phase-related protein kinases, which was demonstrated here by their ability to interact with human E2F or with the adenovirus E1A proteins. In addition it was shown that endoreduplicated endosperm cells contain an inhibitor that suppresses the activity of the M phase-promoting factor (MPF). These results demonstrate that in maize endosperm, endoreduplication proceeds as a result of two events, inhibition of MPF and induction of S phase-related protein kinases.

Elongation factor 1 alpha concentration is highly correlated with the lysine content of maize endosperm.

Lysine is the most limiting essential amino acid in cereals, and for many years plant breeders have attempted to increase its concentration to improve the nutritional quality of these grains. The opaque2 mutation in maize doubles the lysine content in the endosperm, but the mechanism by which this occurs is unknown. We show that elongation factor 1 alpha (EF-1 alpha) is overexpressed in opaque2 endosperm compared with its normal counterpart and that there is a highly significant correlation between EF-1 alpha concentration and the total lysine content of the endosperm. This relationship is also true for two other cereals, sorghum and barley. It appears that genetic selection for genotypes with a high concentration of EF-1 alpha can significantly improve the nutritional quality of maize and other cereals.