Subcellular compartmentalization of maize storage proteins in Xenopus oocytes injected with zein messenger RNAs.

Maize storage proteins synthesized in oocytes were compartmentalized in membrane vesicles because they were resistant to hydrolysis by protease, unless detergent was present. The site of storage protein deposition within the oocyte was determined by subcellular fractionation. Optimal separation of oocyte membranes and organelles was obtained when EDTA and high concentrations of NaCl were included in the homogenization and gradient buffers. Under these conditions, fractions in sucrose gradients containing a heterogeneous mixture of smooth membranes (presumably endoplasmic reticulum, Golgi apparatus, and plasma membrane, density = 1.10-1.12 g/cm3), mitochondria (densities = 1.14 and 1.16 g/cm3), yolk platelets (density = 1.21 g/cm3), and a dense matrix material (density = 1.22 g/cm3) could be separated. Some zein proteins were recovered in the mixed membrane fraction, but the majority occurred in vesicles sedimenting with yolk platelets and granular material at a density of approximately 1.22 g/cm3. When metrizamide was included in the gradient to increase the density, little of the dense matrix material was isolated, and vesicles containing zein proteins were separated from other oocyte components. These vesicles were similar to protein bodies in maize endosperm because they were of identical density and contained the same group of polypeptides.

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.

Aggregation of lysine-containing zeins into protein bodies in Xenopus oocytes.

Zeins, the storage proteins of maize, are totally lacking in the essential amino acids lysine and tryptophan. Lysine codons and lysine- and tryptophan-encoding oligonucleotides were introduced at several positions into a 19-kilodalton zein complementary DNA by oligonucleotide-mediated mutagenesis. A 450-base pair open reading frame from a simian virus 40 (SV40) coat protein was also engineered into the zein coding region. Messenger RNAs for the modified zeins were synthesized in vitro with an SP6 RNA polymerase system and injected into Xenopus laevis oocytes. The modifications did not affect the translation, signal peptide cleavage, or stability of the zeins. The ability of the modified zeins to assemble into structures similar to maize protein bodies was assayed by two criteria: assembly into membrane-bound vesicles resistant to exogenously added protease, and ability to self-aggregate into dense structures. All of the modified zeins were membrane-bound; only the one containing a 17-kilodalton SV40 protein fragment was unable to aggregate. These findings suggest that it may be possible to create high-lysine corn by genetic engineering.

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.

Genetic modification of seed proteins.

Knowledge concerning the genetic modification of seed proteins to improve their nutritional quality has advanced significantly over the past two years. Research in this area has focused almost exclusively on model systems, rather than on agronomically important plants. The extent to which genetic engineering of seed-protein genes will improve crop seed nutritional quality remains to be determined.

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.

Modification of maize-seed-protein quality.

The storage proteins of maize are a group of alcohol-soluble polypeptides called zeins. These proteins are synthesized in the developing endosperm, where they form protein bodies within the rough endoplasmic reticulum. Because they account for more than half of the total seed protein, zeins are the primary determinants of the amino acid composition of the seed. All of the zeins are devoid of lysine an essential amino acid for monogastric animals. We have modified the genes encoding zeins so that they encode proteins that contain lysine and tryptophan. Analysis of the synthesis and processing of these modified zein proteins indicates that the addition of lysine and tryptophan does not interfere with their association into protein bodies.

Oat seed globulin: subunit characterization and demonstration of its synthesis as a precursor.

The predominant storage protein of oat (Avena sativa L.) seeds is a saline-soluble globulin with a mol wt of 320,000 which is composed of six large (M(r) = 35,000 to 40,000) and six small (M(r) = 20,000 to 25,000) subunits. Experiments were conducted to further describe the subunit polypeptides and to identify the initial translation products of globulin mRNAs. Approximately 20 large subunits and 10 small subunits were resolved by two-dimensional gel analysis. The large and small subunits had acidic and basic isoelectric points, respectively. Disulfide-linked complexes of one large and one small subunit were isolated by extraction in buffer lacking a reducing agent. The NH(2)-terminal sequence of the small subunits was homologous to a small subunit of soybean glycinin. Immunoprecipitation of in vitro translation products of poly(A)(+) RNA with anti-oat globulin sera yielded M(r) = 60,000 to 68,000 polypeptides. In vivo labeling of spikelets with radioactive amino acids resulted in high amounts of incorporation into polypeptides with M(r) = 65,000 to 68,000 which were immunoprecipitated with anti-globulin sera. These two results suggest oat globulin is synthesized as a higher mol wt precursor which is subsequently processed to yield the large and small subunit polypeptides.

Zein gene organization in maize and related grasses.

Zein cDNA clones were used to study the organization of zein genes within the genome of the inbred maize W64A. When individual clones for the two larger molecular-weight classes of zein proteins (Mr = 22,000; Mr = 19,000) were used as probes for Southern blot hybridizations of genomic DNA, multiple restriction fragments were found to hybridize. Reconstruction analyses using moderately stringent criteria were used to estimate a total of 70-80 zein sequences within the genome of this inbred maize. The hybridization patterns suggest that zein sequences are clustered within the same restriction fragment. When criteria permitting less cross-hybridization of homologous sequences (Tm - 10 degrees C) were used, the banding pattern changed, with some of the bands being reduced in intensity or eliminated entirely. Therefore, by control of hybridization criteria, particular zein genes may be more readily distinguished in a Southern blot analysis. The Southern blot hybridization pattern for the Mr = 15,000 zein was less complex. Only a single major band was found, with sufficient hybridization intensity for two or three genes. Genomic Southern analyses of other inbred maizes and related grasses showed similarly complex hybridization patterns with cDNA probes for the 19,000- and 22,000-molecular-weight zeins, suggesting that these sequences have been conserved over evolutionary time. The zein multigene family may therefore have arisen by gene duplication before divergence of the maize, teosinte, and Tripsacum species from a common ancestor.

Influence of Ionic Strength, pH, and Chelation of Divalent Metals on Isolation of Polyribosomes from Tobacco Leaves.

A procedure was developed for extracting polysomes from tobacco (Nicotiana sp) leaves. Unexpanded leaves ground in a medium consisting of 200 mm tris-HCl, pH 9, 400 mm KCl, 200 mm sucrose, and 35 mm MgCl(2) yielded larger amounts of polysomes with less degradation than polysomes from leaves extracted with buffers of lower ionic strength or pH. Extraction of polysomes from expanded leaves required the inclusion of ethyleneglycol-bis(2-aminoethyl ether)tetraacetic acid (EGTA, a divalent cation chelator with a high affinity for Ca(2+), Cu(2+), and Zn(2+)). EGTA also improved isolation of polysomes from unexpanded leaves. Addition of 25 mm Ca(2+), Cu(2+), or Zn(2+) to extracts from young leaves precipitated polysomes, and density gradient profiles of polysome preparations from the cation treatments mimicked profiles from expanded leaves which were extracted without EGTA. Polysome precipitation by Ca(2+) was prevented by EGTA. Endogenous Ca(2+) was present in unexpanded leaves in sufficient concentrations (25 mm) to cause some precipitation of polysomes during extraction, and this cation increased by 60% in expanded leaves. Cu(2+) and Zn(2+) were not present in amounts sufficient to cause polysome precipitation. The results show that recovery of polyribosomes may be reduced by divalent cations in leaf tissue, and this can be overcome by chelation of these ions with EGTA.

Polyribosomes from Peas: V. An Attempt to Characterize the Total Free and Membrane-bound Polysomal Population.

Attempts were made to isolate and characterize the total population of free and membrane-bound polysomes from the elongating region of dark-grown pea stems (Pisum sativum L.). Partial separation of free from membrane-bound polysomes was achieved by relatively low speed centrifugation of the homogenate. Complete separation was not achieved. Based on analysis of the rRNA content of various subcellular fractions, fractionated tissue yielded greater than 95% of the rRNA found in whole tissue. Approximately 45% of the ribosomal material was membrane-bound (released by detergent) and was found in the "wall" (13%), the "nuclear" pellet (2%), and the "mitochondrial" pellet (29%). The remaining 55%, consisting primarily of free polysomes, could be recovered free from membranous material by sedimentation through a dense (700 mg/ml) sucrose pad for 90 hours. The advantages and disadvantages of using sucrose pads for the separation of free and membrane-bound polysomes are discussed.

Binding of an endosperm-specific nuclear protein to a maize beta-zein gene correlates with zein transcriptional activity.

Promoter regions of alpha- and beta-zein genes were analyzed for binding of nuclear proteins from developing endosperm and seedling tissue of maize. Using a band-shift assay, we identified two distinct protein factors, alpha-1 and beta-1, that interacted specifically with alpha- and beta-zein gene promoter regions, respectively. Alpha-1 was present in nuclei from both endosperm and seedling tissue, whereas beta-1 was found only in nuclei from developing endosperm tissue. Mixing of nuclear extracts demonstrated that seedling tissue contained undetectable amounts of beta-1, rather than having an inhibitor for formation of the beta-1/DNA complex. Chemical footprinting analysis localized the beta-1 recognition site to a 22 bp sequence flanked by CCAT and TATA boxes. The apparent molecular mass of beta-1 was determined to be 29 kDa by southwestern blotting. Based on in vitro binding assays, the greatest concentration of the beta-1 in endosperm nuclei is at 16 days after pollination, which coincides with the time of highest transcriptional activity of the beta-zein gene. These results suggest that beta-1 may act as a tissue-specific, trans-acting regulator of the expression of the beta-zein gene in developing maize endosperm.

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.

Polyribosomes from Peas: II. Polyribosome Metabolism during Normal and Hormone-induced Growth.

Polyribosomes as large as 10-mers (strands of messenger RNA bearing 10 ribosomes) were isolated from etiolated pea (Pisum sativum L. var. Alaska) stem tissue during all stages of development when methods were used which essentially eliminated ribonuclease activity during extraction. Actively growing tissue, harvested from the apical 10 mm, yielded many large polyribosomes and a low (<20%) proportion of monosomes. Similar tissue, allowed to age by applying lanolin to decapitated apices, showed a progressive decrease in number of larger polyribosomes and an increase in the proportion of monosomes. Hormone treatments, which prolonged growth and delayed aging, delayed the loss in large polyribosomes and the increase in proportion of monosomes. Growth-stimulating hormones, added to previously aged tissue, stimulated the production of many large polyribosomes in pre-existing cells.It is suggested that (a) large polyribosomes occur in all regions of the pea stem, (b) changes in polyribosome distribution appear to precede changes in growth rate, (c) loss of larger polyribosomes is closely related to a decrease in mRNA templates followed more gradually by loss of ribosomes, (d) hormone-stimulated continuation of growth is accomplished through maintenance of available mRNA.Methods are described, involving detailed analysis of polysome distribution, which, although they cannot be used to measure changes in initiation of ribosomes on to mRNA, do permit measurement of the amount of polysomal-associated mRNA present in tissues at different stages of growth. These analyses lead to the further suggestion that hormone stimulation of growth of previously nongrowing tissue is accomplished primarily through an increase in available mRNA prior to synthesis of ribosomes.

Polyribosomes from Peas: III. Stimulation of Polysome Degradation by Exogenous and Endogenous Calcium.

Attempts were made to isolate microsomes from Pisum sativum L. var. Alaska by low speed centrifugation of a postmitochondrial supernatant made 8 mm in Ca(2+). However, the addition of Ca(2+) in concentrations as low as 1 mm to the postmitochondrial supernatant resulted in extensive polysome degradation. Degradation was dependent on both Ca(2+) concentration and the duration of incubation. Resuspension of isolated polysomes in Ca(2+)-containing buffer did not result in degradation, whereas resuspension in Ca(2+)-containing postpolysomal supernatant did. Both Ca(2+) and a heat-labile factor in the supernatant were required for polysome degradation. The degradation in the homogenate with or without added Ca(2+) could be reduced by (a) dilution with larger volumes of grinding buffer, (b) increasing the concentration of tris-HCl in the grinding buffer, (c) adding diethylpyrocarbonate or ethyleneglycol-bis (2-aminoethylether) tetraacetic acid (a specific calcium chelator) prior to homogenization or immediately after the addition of Ca(2+). Endogenous Ca(2+) can increase the destruction of polysomes during their isolation in this tissue, presumably by activating a ribonuclease. Addition of Ca(2+) is not a useful technique for separating undegraded free and membrane-bound polyribosomes.

Synthesis and deposition of zein in protein bodies of maize endosperm.

The origin of protein bodies in maize (Zea mays L.) endosperm was investigated to determine whether they are formed as highly differentiated organelles or as protein deposits within the rough endoplasmic reticulum. Electron microscopy of developing maize endosperm cells showed that membranes surrounding protein bodies were continuous with rough endoplasmic reticulum membranes. Membranes of protein bodies and rough endoplasmic reticulum both contained cytochrome c reductase activity indicating a similarity between these membranes. Furthermore, the proportion of alcohol-soluble protein synthesized by polyribosomes isolated from protein body or rough endoplasmic reticulum membranes was similar, and the alcohol-soluble or -insoluble proteins showed identical [(14)C]leucine labeling. These results demonstrated that protein bodies form simply as deposits within the rough endoplasmic reticulum.Messenger RNA that directed synthesis of only the smaller molecular weight zein subunit was separated from mRNA that synthesized both subunits by sucrose gradient centrifugation. This result demonstrated that separate but similar sized mRNAs synthesize the major zein components. In vitro translation products of purified mRNAs or polyribosomes were approximately 2,000 daltons larger than native zein proteins, suggesting that the proteins are synthesized as zein precursors. When intact rough endoplasmic reticulum was placed in the in vitro protein synthesis system, proteins corresponding in molecular weight to the native zein proteins were obtained.

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.