Functional analysis of cis-regulatory elements within the promoter of the tobacco late pollen gene g10.

The tobacco gene g10 is preferentially and maximally expressed in mature pollen, shows homology to pectate lyases, and is the putative homologue of the tomato gene lat56. Analysis of regulatory elements within the g10 promoter was carried out to verify the importance of putative regulatory sequence motifs. Analysis of transgenic plants showed that 1190 bp of g10 5' sequence directed preferential expression of GUS in pollen, with bimodal peaks of expression just before and during pollen mitosis I, and in mature anthers. This was confirmed by northern analysis of native g10 transcripts in isolated spores. Transient expression analysis defined the minimal g10 promoter region capable of directing expression in pollen as -86 to +217. Three upstream regions within -427 bp modulate the expression from g10. Gain-of-function analyses showed that the region from -106 to -53 could enhance pollen-specific expression of a minimal CaMV 35S promoter. These analyses further showed that sequences upstream of -86 modulate expression in pollen, but are not essential for preferential pollen expression. The function of a conserved GTGA motif shared between the tobacco g10 and tomato lat56 promoters was demonstrated in g10. Thus, further functional evidence is provided for the conservation of mechanisms for the regulation of late pollen genes across species.

Introns and their positions affect the translational activity of mRNA in plant cells.

In an attempt to further increase transgene expression levels in plants over and above the enhancement obtained with a 5' untranslated leader intron, three different maize introns were inserted at three different positions within the coding sequence of the luciferase reporter gene. Constructs were transformed into maize (Black Mexican Sweet) cells and protoplasts, and their activity determined. Although all introns tested were correctly spliced, only one of them in a particular position was able to enhance gene expression. Correct splicing sites were used for intron removal and the quantity of luciferase mRNA produced did not differ significantly. These data indicate that both the position and the sequence of an intron have marked effects on expression levels, suggesting that nuclear processing of the pre-mRNA determines final expression levels through the structure of the mRNP.

Isolation and characterization of the wheat prolyl isomerase FK506-binding protein (FKBP) 73 promoter.

The wheat FK506-binding protein (FKBP) 73 is a member of the peptidyl prolyl cis-trans isomerase gene family, which catalyses the interconversion between the cis and trans forms of the peptide bond preceding proline residues in proteins. A 3.5 kb sequence 5' upstream of the ATG codon of the wheat FKBP73 was isolated from a wheat genomic library, and characterized by deletion analysis and transient expression in wheat embryos. The 1517 bp fragment is referred to as the full promoter due to the maximal activity of the fused luciferase reporter gene. Sequence analysis revealed the presence of three abscisic acid (ABA)-responsive elements (ABREs) proximal to coupling elements (CE1-like), a putative lectin box, two putative binding sites for the myb transcription factor and a 36 bp fragment which exhibits 100% identity to the pSau3A9 clone located in the centromeric region of wheat chromosomes. In a transient expression assay the promoter preserved the tissue specificity described in vivo, namely it is expressed only in germinating embryos and young shoots. The promoter was induced 1.9-fold by ABA, the minimal promoter was designated at -221 and the TATA box located at -137. The inducibility by ABA and the expression during germination may indicate that FKBP73 belongs to the group of genes induced by ABA upon germination.

Characterization of a gene encoding a single-subunit bacteriophage-type RNA polymerase from maize which is alternatively spliced.

Single-subunit RNA polymerases belonging to the T3/T7 bacteriophage family are thought to be common throughout eukaryotes. We report the isolation and characterization of a nucleus-encoded single-subunit RNA polymerase gene from maize. This gene is highly homologous to other single-subunit RNA polymerase genes from Arabidopsis, Chenopodium. yeast and Neurospora crassa involved in organellar transcription. Genomic Southern analysis reveals 10 to 15 hybridising fragments, suggesting that maize contains a small gene family. The isolated gene contains 19 exons and its genomic structure is highly conserved when compared to the three Arabidopsis homologues. Unlike the case in Arabidopsis, intron-12 of the maize bacteriophage-type RNA polymerase gene is alternatively spliced. Quantitative RT-PCR revealed that the resultant alternatively spliced transcript represents approximately 21 to 26% of the total polymerase mRNA in maize coleoptiles. The orthologous wheat bacteriophage-type RNA polymerase is also alternatively spliced and the intron exhibits 78% identity to maize intron-12. The conservation in alternative splicing between wheat and maize and its absence from Arabidopsis suggest a functional requirement for the alternatively spliced product.

The effect of altered codon usage on luciferase activity in tobacco, maize and wheat.

A comparison of the wild-type firefly luciferase reporter gene to a codon-modified gene, available from Promega, demonstrates that in tobacco cell cultures, an increase in G+C content of 1.8%, as a consequence of 36 A/T→G/C synonymous codon alterations and removal of the lysosomal targeting sequence, has no significant effect on expression. In maize Black Mexican Sweet cells and wheat scutellum, increases in activity of 14- to 23-fold and 53- to 59-fold, respectively, are obtained using the codon-modified luciferase with the UBI1 promoter and its leader intron. The observed increase in luc+ expression is most likely a consequence of differences in codon usage reflecting tRNA abundance rather than an increase in the efficiency of intron splicing resulting from the small increase in the G+C content of the coding sequence. This difference in light emission between the wild-type and codon-modified luciferases can be clearly visualised in a low-light imaging camera, making the latter a much more sensitive and useful reporter gene for detecting luciferase activity in vivo.

An analysis of the relative activities of a number of promoter constructs from genes which are expressed during late pollen development as determined by particle bombardment.

The promoters of a tobacco actin gene, a tobacco pectate lyase, a tobacco and maize polygalacturonase and aBrassica S-locus related gene have been fused to theß-glucuronidase reporter gene and their activities determined by biolistic transient assay in tobacco pollen. In stably transformed tobacco all the transgenes with the exception of Cauliflower Mosaic Virus-35S-ß-glucuronidase appear to express efficiently in maturing pollen. Transient assay analysis showed that the tobacco pectate lyase and the polygalacturonase constructs were 8x more active than the tobacco actin construct, and that the tobacco polygalacturonase construct was some 33x more active than the maize polygalacturonase construct. Constructional manipulations that altered the lengths of the 5'-untranslated leaders including one which resulted in the removal of a 490 bp leader intron had little effect on the observed level of expression. However, the alteration of the context of the ATG from A/TnnATGG to CnnATGT resulting in a 70% reduction in the observed levels of activity, was obtained with the pectate lyase and polygalacturonase promoters. An identical reductional was also observed in transgenic plant populations transformed with the polygalacturonase transgenes.

Characterization of a tobacco gene encoding a pollen-specific polygalacturonase.

We report here the isolation and characterization of a gene which is specifically expressed during late pollen development in Nicotiana tabacum L. cv. Havana and which exhibits homology to bacterial, fungal and plant polygalacturonases. This gene is ca. 4.3 kb, from the transcription start-site to the 3' polyadenylation-site sequences. It contains three introns of 620, 706 and 1400 bp and encodes a 1.5 kb message that contains an A-rich 5'-untranslated-leader sequence of 81 bases and a variable-length 3'-untranslated sequence of between 180 and 320 bases. Located within intron 3 is a 414 bp sequence which exhibits 79% homology to a sequence within the endochitinase gene; both sequences share the same internal repeat structure and exhibit features consistent with them being defective transposable elements. The predicted protein sequence coded for by Npg1 shows, in addition to a number of highly conserved cysteines, four conserved domains with the bacterial and fungal polygalacturonase genes. The pollen-specific polygalacturonases as a group can be distinguished from the fruit-ripening polygalacturonases by a number of criteria. It is suggested that these differences reflect the functional differences between plant endo- and exo-polygalacturonases. Npg1 is one of a two-member gene family expressed predominantly in the male gametophyte upon first microspore mitosis. From expression studies of promoter::GUS transgenes it is clear that the -744 bp to +74/+85 bp of Npg1 sequence (with respect to the transcription start site) is sufficient to drive the expression of the GUS reporter gene in a manner that reflects the spatial and temporal expression of Npg1 as determined by dot-blot and northern analysis.

Identification of a defective transposable element in tobacco.

A putative defective transposable element has been identified in tobacco. This element has been found and characterised in two separate parts of the tobacco genome, specifically within the 3rd intron of the pollen-specific polygalacturonase gene (Npg1) and upstream of the endochitinase gene (Chn50). The element is ca. 0.4 kb in length and is bounded by conserved inverted repeats and putative target site duplications. It appears to fall into the category of non-autonomous transposable elements.

Partial characterization of the Nicotiana tabacum actin gene family: evidence for pollen-specific expression of one of the gene family members.

The actin gene family of Nicotiana tabacum has been partially characterised by Southern hybridisation and by isolating lambda EMBL4 recombinants from a genomic library having homology to the soybean actin gene, Sac3. The number of actin genes with homology to Sac3 is estimated at between 20 to 30, based on Southern hybridisation and library screening, though the total gene family may be larger. Twenty-four recombinant lambda clones were isolated, 18 had unique restriction profiles and from these, 2 clones, Tac9 and Tac25, were selected for further study. The region of Tac25 hybridizing to Sac3 was sequenced and shown to contain an open reading frame (ORF) with homology to actin. Partial sequencing of Tac9 revealed a sequence with homology to the third exon of Tac25 and Sac3. The two tobacco actin sequences were compared to other reported actin gene sequences; Tac25 was closely related to the allelic potato actins, Pac58 and Pac85, while Tac9 was more related to Pac79 than to other plant actins. Northern hybridisation analysis showed that while Tac9 detected actin transcripts in RNA from root, leaf, stigma and pollen, Tac25 transcripts were only detected in pollen RNA.

Molecular characterization of one of the maize polygalacturonase gene family members which are expressed during late pollen development.

A gene exhibiting homology to the polygalacturonases of several species, including tomato and Oenothera, has been shown by RNA dot-blot analysis and in situ hybridization experiments to be expressed post-first microspore mitosis in maize. A 2.87 kbp section of the promoter fused to E. coli beta-glucuronidase (uidA) coding sequence conferred the correct spatial and temporal expression in transgenic tobacco plants. However, low levels of expression were detected in other tissues, and in particular in the tissues surrounding the vascular branch points of leaf nodes. The maize polygalacturonase gene is one member of a highly conserved gene family. The lack of detectable expression in sporophytic tissues and the isolation of a number of related cDNAs from maize suggests that all expressed members of this family show the same spatial and temporal regulation.

Isolation and characterization of a tobacco gene with homology to pectate lyase which is specifically expressed during microsporogenesis.

A genomic clone has been isolated which contains an open reading frame of 1191 bp interrupted by two small introns. The ORF has been sequenced and the transcriptional start determined. The predicted amino acid sequence shows homology to the deduced amino acid sequences of two pollen-specific pectate lyase genes identified in tomato. The genomic clone was isolated using a partial cDNA clone, TP10, which had been isolated from a Nicotiana tabacum pollen cDNA library by means of differential screening. TP10 has been fully sequenced and contains an open reading frame of 792 bp which shows 96% homology to the ORF in the genomic clone. The transcript corresponding to TP10 is maximally expressed late in pollen development, and has not been detected in vegetative tissues.

Transfer of methomyl and HmT-toxin sensitivity from T-cytoplasm maize to tobacco.

The mitochondrial gene, T-urf13, which is unique to the T-cytoplasm of maize, has been expressed in tobacco plants using the Cauliflower Mosaic Virus 35S promoter. Tobacco plants expressing T-urf13 exhibit a variety of responses to methomyl. Leaf discs and petiole sections bleach when exposed to methomyl or HmT-toxin; this effect increases with the age of the tissue. The bleaching effect is not however observed when light is excluded. Plants homozygous for T-urf13 exhibit extreme sensitivity when sprayed with methomyl. The growth of seedling which are either homozygous or heterozygous for T-urf13 is inhibited by methomyl and by kanamycin, whereas seedlings from untransformed tobacco or tobacco which has lost the T-urf13 gene through segregation are sensitive to kanamycin but develop normally when exposed to methomyl. The results demonstrate that T-URF13 need not be specifically targeted to the mitochondrion for it to induce methomyl or HmT-toxin sensitivity in tobacco.

Pollen specific cDNA clones from Zea mays.

We have cloned and sequenced four pollen-specific cDNAs. None of the clones are complete at their 5' ends. One of the clones shows significant homology to the tomato fruit-ripening polygalacturonase and to a pollen-specific polygalacturonase from Oenothera. The other three clones have no significant homologies to any reported sequence.

Application of the beta-glucuronidase gene fusion system to Saccharomyces cerevisiae.

Bacterial beta-glucuronidase (GUS) has been described as a useful reporter enzyme for gene fusion studies in bacteria and plants. Here we report the expression of GUS in yeast to illustrate further applications of this enzyme as a quantitative tool for measuring gene activity, as a colour selection marker and as a versatile system for protein targeting studies. There is no intrinsic GUS activity in any yeast strain tested. GUS was expressed in transgenic yeast on a multiple-copy vector under the control of the alcohol dehydrogenase 1 (ADH1) promoter. The enzyme is stable in yeast and its activity may be monitored by very sensitive colorimetric or fluorometric methods in extracts, or by the histochemical reagent 5-bromo-4-chloro-3-indolylglucuronide (X-Gluc) on plates. To test the efficacy of GUS as a reporter for targeting proteins into different subcellular compartments in vivo, we fused the presequence of the mitochondrial tryptophanyl-tRNA-synthetase gene (MSW) to the amino terminus of GUS. The activity of the fusion protein is not substantially impaired and it is imported efficiently into yeast mitochondria.

A yeast mitochondrial presequence functions as a signal for targeting to plant mitochondria in vivo.

To date, the presequence of the mitochondrial beta-subunit of ATPase from tobacco is the only signal sequence that has been shown to target a foreign protein into plant mitochondria in vivo. Here we report that the presequence of a yeast mitochondrial protein directs bacterial beta-glucuronidase (GUS) specifically into the mitochondrial compartment of transgenic tobacco plants. Fusions between the presequence of the mitochondrial tryptophanyl-tRNA-synthetase gene from yeast and the GUS gene have been introduced into tobacco plants and yeast cells. In both systems, proteins containing the complete yeast mitochondrial presequence are efficiently imported in the mitochondria. Measurements of GUS activity in different subcellular fractions indicate that there is no substantial misrouting of the chimeric proteins in plant cells. In vitro synthesized GUS fusion proteins have a higher molecular weight than those found inside yeast and tobacco mitochondria, suggesting a processing of the precursors during import. Interestingly, fusion proteins translocated across the mitochondrial membranes of tobacco have the same size as those that are imported into yeast mitochondria. We conclude that the processing enzyme in plant mitochondria may recognize a proximate or even the same cleavage site within the mitochondrial tryptophanyl-tRNA-synthetase presequence as the matrix protease from yeast.

A specific rearrangement of mitochondrial DNA induced by tissue culture.

The induction, growth and regeneration of sugar beet callus to whole plants were all found to be highly genotype-specific. Regenerants of one line (of sterile cytoplasm) were obtained and a study of the chloroplast and mitochondrial DNA in these somaclones was undertaken by gel electrophoresis and cosmid hybridization. In one somaclone a rearrangement in the mitochondrial genome was observed; the novel arrangement of this part of the genome was identical to the corresponding area of the genome of the normal cytoplasm though it was otherwise of sterile type. This suggests that mitochondrial DNA may have a propensity to undergo certain types of rearrangement.

Location and nucleotide sequence of two tRNA genes and a tRNA pseudo-gene in the maize mitochondrial genome: evidence for the transcription of a chloroplast gene in mitochondria.

We report the nucleotide sequence of three tRNA genes from maize mitochondria. The genes are located in two BamHI fragments, 3.55 and 5.7 kb long, adjacent to the S2 sequence in the maize mitochondrial genome. On the 3.55 kb BamHI fragment, we have characterized a tRNA(Cys)(GCA) gene. A strong sequence homology of this tRNA(Cys)(GCA) gene with its chloroplast counterpart in wheat suggests that it may be part of a chloroplast DNA insertion into the mitochondrial genome. This gene has been found to be transcribed in the mitochondrion. Two tRNA genes are located on the 5.7 kb BamHI fragment, separated from each other by 250 bp. One is a mitochondrial tRNA(Ser)(GCU) gene. The other, a non-transcribed tRNA(Phe)-like gene, is interrupted by a 49 base-pair inserted DNA sequence in the variable loop and has a Leu (UAA) anticodon.