Globulin-1 gene expression in regenerable Zea mays (maize) callus.

Since maize callus cultures regenerate plants via somatic embryogenesis, one might expect to find similar proteins in both zygotic embryos and tissue cultures. The 63-kD globulin protein designated GLB1, the expression of which is regulated by abscisic acid (ABA), is one such protein. When maize Type I regenerable callus was exposed for 24 h to 0.1 m M ABA or a water stress induced by 0.53 M mannitol, GLB1 was produced as determined by Western analysis. This protein was not detected in ABA or mannitol-treated regenerable cultured tissue of a null genotype or in tissues not exposed to ABA or water stress. Exposure to ABA in the culture medium increased the callus ABA levels greatly but a mannitol-induced water stress had only a small effect on ABA levels. Regenerable callus exposed to 0.1 m M ABA also produced mRNA that hybridized on a Northern blot with a globulin- 1 gene ( Glb1) probe. When both Type I and Type II regenerable cultured tissues were exposed to regeneration medium without ABA or mannitol, several GLB1 antibody immunoreactive proteins were produced. These proteins were not detected in regenerated plants nor in non-regenerable callus treated with ABA. These results suggest that: (1) at least for expression of Glb1, somatic embryogenesis is similar to zygotic embryogenesis, (2) there may be a regulatory role for auxin in the processing of Glb1-encoded polypeptides since fewer are seen when dicamba is present in the medium, (3) ABA has a role in somatic embryogenesis, and (4) regenerability of a maize callus culture may be assessed by treating the cultured tissue with 0.1 m M ABA to determine if GLB1 proteins are induced.

Nucleotide sequence analysis of a novel globulin1 null allele from the Illinois high protein strain of maize.

The highly polymorphic maize globulin1 (glb1) gene encodes an abundant embryo storage protein. The present study extends the analysis of glb1 variants to further explore the nature of polymorphism at this locus. The null allele Glb1-N1Hb, derived from the Illinois High Protein (IHP) strain of maize was characterized at the molecular level by nucleotide sequence analysis. Among other differences, a single-base insertion leading to a premature termination codon in the carboxyl-terminal half of the otherwise normal protein was observed. The likely reasons for the absence of GLB1 protein accumulation in the IHP strain of maize are discussed.

Evidence for the thiamine biosynthetic pathway in higher-plant plastids and its developmental regulation.

Thiamine or vitamin B-1, is an essential constituent of all cells since it is a cofactor for two enzyme complexes involved in the citric acid cycle, pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase. Thiamine is synthesized by plants, but it is a dietary requirement for humans and other animals. The biosynthetic pathway for thiamine in plants has not been well characterized and none of the enzymes involved have been isolated. Here we report the cloning and characterization of two cDNAs representing members of the maize thi1 gene family encoding an enzyme of the thiamine biosynthetic pathway. This assignment was made based on sequence homology to a yeast thiamine biosynthetic gene and by functional complementation of a yeast strain in which the endogenous gene was inactivated. Using immunoblot analysis, the thi1 gene product was found to be located in a plastid membrane fraction. RNA gel blot analysis of various tissues and developmental stages indicated thi1 expression was differentially regulated in a manner consistent with what is known about thiamine synthesis in plants. This is the first report of cDNAs encoding proteins involved in thiamine biosynthesis for any plant species.

Identification of cDNA clones corresponding to two inducible nitrate reductase genes in soybean: analysis in wild-type and nr1 mutant.

Among higher plants, soybean is unique in that biochemically it has been characterized as having two constitutive nitrate reductase (cNR) isoforms and one substrate-inducible nitrate reductase (iNR) isoform in leaves. All three NR isoforms are expressed in cv. Williams 82 while the nr1 mutant expresses only the iNR isoform. The genetic and molecular mechanisms for regulation of these isoforms have not been elucidated. We describe here the isolation, by reverse transcription-polymerase chain reaction (RT-PCR), of two cDNA clones encoding soybean NR. They were designated as iNR1 and iNR2, respectively, since both were inducible by nitrate. The iNR1 and iNR2 cDNAs cover total encoding regions of 2661 and 2673 nucleotides, respectively. The iNR1 clone shows a 12 bp deletion at the 5' end, relative to iNR2. They show overall similarity of 89% at the nucleotide level, and 87% at the amino acid level. Like all plant NRs cloned so far, deduced amino acid sequences between iNR1 and iNR2 show greatest variation at the N-terminal region while no difference was observed at the C-terminus. Soybean iNR mRNAs were found to be different from those of maize and tobacco in response to tungsten inhibitor treatment, since the inhibitor decreased the steady-state levels of mRNA for soybean iNR and for NiR. Using the same 5' regions of both cDNAs as the probes, Southern blot analysis of genomic DNA revealed differences in organization between iNR1 and iNR2. The genomic DNA from wild-type Williams 82 soybean was shown to have three Eco RI fragments while the nr1 mutant lacked an 8 kb fragment when probed with iNR1 cDNA. Likewise, the nr1 mutant lacked three Hae III restriction fragments when probed with iNR1 cDNA. When probed with iNR2, both wildtype and nr1 mutant showed one identical Eco RI band and two identical Hae III bands. In northern blots, the steady-state level of iNR1 mRNA was similar for the nr1 mutant and the wild-type parent after 20 to 48 h induction by nitrate. Based on the Eco RI and Hae III restriction enzyme digestion patterns observed in Southern blot analysis of soybean DNA, it is concluded that in soybean iNR1 is encoded by a small multiple gene family and iNR2 is a single gene.

Molecular analysis of two cDNA clones encoding acidic class I chitinase in maize.

The cloning and analysis of two different cDNA clones encoding putative maize (Zea mays L.) chitinases obtained by polymerase chain reaction (PCR) and cDNA library screening is described. The cDNA library was made from poly(A)+ RNA from leaves challenged with mercuric chloride for 2 d. The two clones, pCh2 and pCh11, appear to encode class I chitinase isoforms with cysteine-rich domains (not found in pCh11 due to the incomplete sequence) and proline-/glycine-rich or proline-rich hinge domains, respectively. The pCh11 clone resembles a previously reported maize seed chitinase; however, the deduced proteins were found to have acidic isoelectric points. Analysis of all monocot chitinase sequences available to date shows that not all class I chitinases possess the basic isoelectric points usually found in dicotyledonous plants and that monocot class II chitinases do not necessarily exhibit acidic isoelectric points. Based on sequence analysis, the pCh2 protein is apparently synthesized as a precursor polypeptide with a signal peptide. Although these two clones belong to class I chitinases, they share only about 70% amino acid homology in the catalytic domain region. Southern blot analysis showed that pCh2 may be encoded by a small gene family, whereas pCh11 was single copy. Northern blot analysis demonstrated that these genes are differentially regulated by mercuric chloride treatment. Mercuric chloride treatment caused rapid induction of pCh2 from 6 to 48 h, whereas pCh11 responded only slightly to the same treatment. During seed germination, embryos constitutively expressed both chitinase genes and the phytohormone abscisic acid had no effect on the expression. The fungus Aspergillus flavus was able to induce both genes to comparable levels in aleurone layers and embryos but not in endosperm tissue. Maize callus growth on the same plate with A. flavus for 1 week showed induction of the transcripts corresponding to pCh2 but not to pCh11. These studies indicate that the different chitinase isoforms in maize might have different functions in the plant, since they show differential expression patterns under different conditions.

Structure of the amplified 5-enolpyruvylshikimate-3-phosphate synthase gene in glyphosate-resistant carrot cells.

The structure of amplified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) DNA of carrot suspension-cultured cell lines selected for glyphosate resistance was analyzed to determine the mechanism of gene amplification in this plant system. Southern hybridization of the amplified DNA digested with several restriction enzymes probed with a petunia EPSPS cDNA clone showed that there were differences in fragment sizes in the amplified DNA from one highly resistant cell line in comparison with the parental line. Cloning of the EPSPS gene and 5' flanking sequences was carried out and two different DNA structures were revealed. A 13 kb clone contained only one copy of the EPSPS gene while a 16 kb clone contained an inverted duplication of the gene. Southern blot analysis with a carrot DNA probe showed that only the uninverted repeated DNA structure was present in all of the cell lines during the selection process and the inverted repeat (IR) was present only in highly amplified DNA. The two structures were present in about equal amounts in the highly amplified line, TC 35G, where the EPSPS gene was amplified about 25-fold. The presence of the inverted repeat (IR) was further verified by resistance to S1 nuclease hydrolysis after denaturation and rapid renaturation, showing foldback DNA with the IR length being 9.5 kb. The junction was also sequenced. Mapping of the clones showed that the size of the amplified carrot EPSPS gene itself is about 3.5 kb. This is the first report of an IR in amplified DNA of a target enzyme gene in selected plant cells.

Regeneration of Transgenic Soybean (Glycine max) Plants from Electroporated Protoplasts.

Transgenic soybean (Glycine max [L.] Merr.) plants were regenerated from calli derived from protoplasts electroporated with plasmid DNA-carrying genes for a selectable marker, neomycin phosphotransferase (NPTII), under the control of the cauliflower mosaic virus 35-Svedberg unit promoter, linked with a nonselectable mannityl opine synthesis marker. Following electroporation and culture, the protoplast-derived colonies were subjected to kanamycin selection (50 micrograms per milliliter) beginning on day 15 for 6 weeks. Approximately, 370 to 460 resistant colonies were recovered from 1 x 10(6) electroporated protoplasts, giving an absolute transformation frequency of 3.7 to 4.6 x 10(-4). More than 80% of the kanamycin-resistant colonies showed NPTII activity, and about 90% of these also synthesized opines. This indicates that the linked marker genes were co-introduced and co-expressed at a very high frequency. Plants were regenerated from the transformed cell lines. Southern blot analysis of the transformed callus and leaf DNA demonstrated the integration of both genes. Single-plant assays performed with different plant parts showed that both shoot and root tissues express NPTII activity and accumulate opines. Experiments with NPTII and mannityl opine synthesis marker genes on separate plasmids resulted in a co-expression rate of 66%. These results indicate that electroporation can be used to introduce both linked and unlinked genes into the soybean to produce transformed plants.

Molecular basis for allelic polymorphism of the maize Globulin-1 gene.

An abundant protein in maize (Zea mays L.) embryos is a storage globulin encoded by the polymorphic Glb1 gene. Several Glb1 protein size alleles and a null allele have been described. Here we report the isolation and nucleotide sequence analysis of genomic clones corresponding to two Glb1 size alleles (Glb1-L and Glb1-S) and to the Glb1-0 null allele. The Glb1-L and Glb1-0 alleles differ from Glb1-S by the presence of small nucleotide insertions which are imperfect or perfect duplications, respectively, of adjacent sequences. In the case of Glb1-L, the insertion is in-frame and results in a protein larger than that encoded by Glb1-S, whereas in Glb1-0 the insertion causes a translational frameshift which introduces a premature termination codon. Although steady-state levels of Glb1-0 transcripts are extremely low in Glb1-0/0 embryos, nuclear transcription assays indicate that the Glb1-0 gene is transcribed at a level comparable to that of Glb1-L. This suggests that the low amounts of Glb1-0 transcripts in the cytoplasm may be due to mRNA instability.

Characterization of the maize Globulin-2 gene and analysis of two null alleles.

The most abundant proteins present in maize (Zea mays L.) embryos are saline-soluble globulins. A Mr 45,000 globulin component, designated GLB2, is encoded by the Glb2 gene. A cDNA clone corresponding to Glb2 was used as radiolabeled probe to examine the expression of Glb2 in developing embryos and other maize tissues. Glb2 transcripts accumulate during embryo development and are not detectable in germinating kernels. Glb2 transcripts are found only in the developing embryo, and not in endosperm, seedling, or unfertilized ears. Analysis of globulin profiles in embryos homozygous for either a previously described null allele, Glb-2-0, or a novel null allele, Glb2-N1, revealed that these embryos lack not only the GLB2 protein but also globulins of lower molecular mass which may represent processed forms of GLB2. Southern blot analysis of DNA from Glb2-0/0 and Glb2-N1/N1 plants in which a Glb2-specific clone is used as probe indicates that the two null alleles are genetically distinct.

Molecular characterization of the major maize embryo globulin encoded by the glb1 gene.

One of the most abundant proteins in maize (Zea mays L.) embryos is the molecular weight 63,000 globulin encoded by the Glb1 gene. To obtain DNA clones corresponding to Glb1, a cDNA library corresponding to RNA from developing maize embryos was constructed in a lambda expression vector and screened with antibodies specific for Glb1-encoded proteins. Here we report the complete nucleotide sequence, as determined from two overlapping clones, of pcGlb 1S, a 2009 base pair clone containing the entire translated region of Glb1. The deduced amino acid sequence of pcGlb 1S shows similarities to 7S-type seed storage proteins of wheat and legumes. Southern blot analysis of maize DNA confirms previous genetic studies which had indicated the presence of a single copy of Glb1 per haploid genome. Northern blot analysis indicates that Glb1 transcripts are present throughout most of embryo development and that expression of this gene is limited to seed tissues. Embryos homozygous for a Glb1 null allele, in which Glb1-encoded proteins are not detectable, contain low levels of Glb1 transcripts which are a different size from those encoded by functional alleles. This suggests that the defect in the null allele is at the level of gene transcription or RNA processing.

Characterization of embryo globulins encoded by the maize Glb genes.

Two of the most abundant proteins in maize embryos are saline-soluble, water-insoluble globulins. One is a Mr 63,000 protein encoded by the Glb1 gene and the other is a Mr 45,000 component encoded by the Glb2 gene. Both proteins accumulate to high levels during embryo development and are rapidly degraded during the early stages of seed germination. Amino acid composition analysis indicates that these proteins may serve as storage reserves to provide sources of nitrogen and carbon to the germinating embryo. Amino-terminal sequence analysis of the final Glb1 gene product, GLB1, and its immediate precursor, GLB1', indicates that the latter is proteolytically cleaved near the amino terminus to form GLB1. In addition to these biochemical studies, we describe the identification of a novel maize variant which lacks the protein product of the Glb2 gene.

Structural and transcriptional analysis of DNA sequences flanking genes that encode 19 kilodalton zeins.

The nucleotide sequence of gz19ab11, a clone that corresponds to the coding and flanking sequences of an Mr 19,000 alpha zein, was determined. Comparison of the DNA sequences flanking this gene with those of other members of the gene subfamily showed that sequence conservation extends 820 nucleotides into the 5' flanking region and 130 nucleotides into the 3' flanking region. Southern blot analysis of maize DNA indicated that highly repetitive sequences are located within 950 bp 5' and 300 bp 3' to the protein coding region of these genes. The coding region of gz19ab11 is similar to but not identical with cDNA clones corresponding to Mr 19,000 zeins, and analysis of zein transcripts indicated that this gene is expressed exclusively in endosperm tissue. RNAs which correspond to transcripts originating 60 nucleotides, and more than 800 nucleotides, upstream of the initiation codon were detected for this and a related gene. However, the concentration of the large RNA species was several orders of magnitude less than that of the shorter RNAs. The functional significance of these large RNA transcripts in zein gene expression is unclear.

Synthesis of globulins in maize embryos.

The two major components of the globulin fraction in Zea mays embryos are specified by the Prot gene. Pulse-chase analysis of protein synthesis in cultured, immature embryos indicates that the smaller Prot-specific polypeptide, PROT, is derived from the larger polypeptide, PROT'. These experiments also demonstrate that PROT' is derived from a short-lived precursor polypeptide, prePROT'. The primary Prot-specific translation product, as detected by in vitro translation of immature embryo RNA, is of a lower apparent molecular weight than pre-PROT', suggesting the involvement of co- and/or post-translational modification in the production of prePROT'.