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PubMed1992

Import and processing of the precursor of the Rieske FeS protein of tobacco chloroplasts.

cDNA clones encoding the precursor of the Rieske FeS protein of tobacco chloroplasts have been characterised and shown to derive from two different genes. The 5' ends of the corresponding transcripts have been cloned using primer extension and PCR. The nucleotide sequences of the cDNAs (and their 5' extensions) predict precursors for the tobacco proteins which differ in 4 amino acid residues out of a total of 228 residues and show high homology with the pea and spinach precursors. The tobacco precursor proteins contain N-terminal presequences of 49 amino acid residues which lack 17 amino acid residues present at the N-terminus of the spinach presequence. The 26 kDa precursor obtained by transcription and translation of one of these cDNAs in vitro was efficiently imported and correctly processed to the mature 20 kDa protein by isolated pea or tobacco chloroplasts. The precursor was also processed to its mature size by a peptidase present in the stroma of chloroplasts.

Amino Acid Sequence↗

PubMed1996

Antimicrobial peptides from Mirabilis jalapa and Amaranthus caudatus: expression, processing, localization and biological activity in transgenic tobacco.

The cDNAs encoding the seed antimicrobial peptides (AMPs) from Mirabilis jalapa (Mj-AMP2) and Amaranthus caudatus (Ac-AMP2) have previously been characterized and it was found that Mj-AMP2 and Ac-AMP2 are processed from a precursor preprotein and preproprotein, respectively [De Bolle et al., Plant Mol Biol 28:713-721 (1995) and 22:1187-1190 (1993), respectively]. In order to study the processing, sorting and biological activity of these antimicrobial peptides in transgenic tobacco, four different gene constructs were made: a Mj-AMP2 wild-type gene construct, a Mj-AMP2 mutant gene construct which was extended by a sequence encoding the barley lectin carboxyl-terminal propeptide, a known vacuolar targeting signal [Bednarek and Raikhel, Plant Cell 3: 1195-1206 (1991)]; an Ac-AMP2 wild-type gene construct; and finally, an Ac-AMP2 mutant gene construct which was truncated in order to delete the sequence encoding the genuine carboxyl-terminal propeptide. Processing and localization analysis indicated that an isoform of Ac-AMP2 with a cleaved-off carboxyl-terminal arginine was localized in the intercellular fluid fraction of plants expressing either wild-type or mutant gene constructs. Mj-AMP2 was recovered extracellularly in plants transformed with Mj-AMP2 wild-type gene construct, whereas an Mj-AMP2 isoform with a cleaved-off carboxyl-terminal arginine accumulated intracellularly in plants expressing the mutant precursor protein with the barley lectin propeptide. The in vitro antifungal activity of the AMPs purified from transgenic tobacco expressing any of the four different precursor proteins was similar to that of the authentic proteins. However, none of the transgenic plants showed enhanced resistance against infection with either Botrytis cinerea or Alternaria longipes.

Amino Acid Sequence↗

PubMed1993

Immunodetection of the plum pox virus helper component in infected plants and expression of its gene in transgenic plants.

Tobacco plants (Nicotiana tabacum cv. Xanthi) have been transformed via Agrobacterium tumefaciens vectors, with cDNAs corresponding to the plum pox virus (PPV) cistron 2 encoding helper component (HC-Pro) and with the first two and half cistrons of the PPV genome. Presence of the HC-Pro in PPV-infected plants and transgenic plants transformed with the gene coding for this protein was investigated using specific polyclonal antibodies produced against the PPV HC-Pro. The results suggest that two proteases are involved in the processing of the PPV N-terminal polyprotein to yield a protein of 48 k (HC-Pro). HC-Pro autolytically cleaves at its carboxyl-terminus and a proteolytic activity, probably associated with the protein (P1) encoded by the cistron 1, is required for the cleavage in planta between the proteins derived from cistrons 1 and 2.

Agrobacterium tumefaciens↗

PubMed2000

Cross-talk between posttranscriptionally silenced neomycin phosphotransferase II transgenes.

Tobacco plants containing a transgene locus with two chimeric neomycin phosphotransferase II (nptII) genes in tail-to-tail orientation (locus 1) show posttranscriptional gene silencing. The silenced nptII transgenes of locus 1 can downregulate the expression of homologous nptII transgenes in hybrid plants. The 3' region of the silenced nptII genes located in the center of the inverted repeat locus 1 is extensively methylated. Moreover, 3' segments of in trans-inactivated transgenes also become methylated, suggesting cross-talk between homologous posttranscriptionally silenced genes. Our results are in accordance with the hypothesis that this cross-talk can be mediated by specially featured RNAs.

DNA Methylation↗

PubMed1999

The arcelin-5 gene of Phaseolus vulgaris directs high seed-specific expression in transgenic Phaseolus acutifolius and Arabidopsis plants.

The regulatory sequences of many genes encoding seed storage proteins have been used to drive seed-specific expression of a variety of proteins in transgenic plants. Because the levels at which these transgene-derived proteins accumulate are generally quite low, we investigated the utility of the arcelin-5 regulatory sequences in obtaining high seed-specific expression in transgenic plants. Arcelin-5 is an abundant seed protein found in some wild common bean (Phaseolus vulgaris L.) genotypes. Seeds of Arabidopsis and Tepary bean (Phaseolus acutifolius A. Gray) plants transformed with arcelin-5 gene constructs synthesized arcelin-5 to levels of 15% and 25% of the total protein content, respectively. To our knowledge, such high expression levels directed by a transgene have not been reported before. The transgenic plants also showed low plant-to-plant variation in arcelin expression. Complex transgene integration patterns, which often result in gene silencing effects, were not associated with reduced arcelin-5 expression. High transgene expression was the result of high mRNA steady-state levels and was restricted to seeds. This indicates that all requirements for high seed-specific expression are cis elements present in the cloned genomic arcelin-5 sequence and trans-acting factors that are available in Arabidopsis and Phaseolus spp., and thus probably in most dicotyledonous plants.

Arabidopsis↗

PubMed1990

Spacing between GT-1 binding sites within a light-responsive element is critical for transcriptional activity.

Dissection of the light-responsive element (LRE) located between -166 and -50 of rbcS-3A from pea has revealed critical spacing requirements between the two GT-1 binding sites for light-responsive transcription. An increase in spacing between the two sites by as little as 2 bp reduces dramatically the rbcS-3A transcript levels in vivo. Mutation of the 10 bp between the binding sites leads to slightly lower transcript levels, as do deletions of either 3 bp or 8 bp. Deletions of 5 bp or 7 bp from between the GT-1 binding sites do not affect rbcS-3A transcript levels; however, a deletion of 10 bp virtually abolishes the activity of this element. These spacing changes within the light-responsive element similarly affect transcription of a divergently oriented and truncated nopaline synthase promoter. Most spacing changes between the two GT-1 binding sites, however, do not impair the binding of GT-1 to this element in vitro. Together with previous observations, these results suggest that the nuclear factor GT-1 may interact with the binding sites in either a productive or nonproductive manner and that GT-1 binding is necessary but not sufficient for light-responsive transcription. We also discuss our results in relation to the observed spacing of similar sequence elements present within other light-responsive promoters.

Amino Acid Oxidoreductases↗

PubMed1994

Hypocotyl expression and light downregulation of the soybean tubulin gene, tubB1.

The tubB1 beta-tubulin gene of Glycine max (previously named s beta 1) is highly expressed only in rapidly elongating regions of etiolated seedling hypocotyls and this expression is strongly downregulated when the seedlings are exposed to light. Primer extension demonstrated that the gene was transcribed in these tissues and contained two sites of transcriptional initiation. To determine the mechanism regulating tubB1 expression, a chimeric reporter gene was constructed by fusing 5' upstream regions of tubB1 to a promoterless beta-glucuronidase (GUS) gene and these constructs were introduced into protoplasts by electroporation. Strong transient expression of the reporter gene was obtained after electroporation of chimeric constructs containing 1 kb of tubB1 5' upstream sequence into tobacco protoplasts. Deletion of the distal most 300 bp from the 5' sequence of tubB1 enhanced expression, suggesting the possibility of a negative transcriptional regulator in this region. Additional deletions of the 5' sequence reduced expression substantially. Constructs containing a tubB1 3' terminus were expressed at much lower levels than those containing a nopaline synthase (NOS) 3' terminus. The tubB1-GUS chimeric gene also was introduced into tobacco by Agrobacterium-mediated Ti plasmid transformation and the organ-specific expression pattern of the chimeric gene was determined in seedlings of the transgenic plants. Hypocotyls exhibited strong GUS activity when the seedlings were germinated in darkness, but lacked the GUS enzyme when the seedlings were germinated in the light.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1992

Vicilin with carboxy-terminal KDEL is retained in the endoplasmic reticulum and accumulates to high levels in the leaves of transgenic plants.

Gene constructs were designed to test the effect of the endoplasmic reticulum (ER)-targeting signal, KDEL, on the level of accumulation of a foreign protein in transgenic plants. The gene for the pea seed protein vicilin was modified by the addition of a sequence coding for this tetrapeptide at its carboxyl terminus. The altered gene was placed under the control of a CaMV 35S promoter and its expression in the leaves of both tobacco and lucerne (alfalfa) was compared with that of an equivalent vicilin construct lacking the KDEL-coding sequence. The presence of the ER-targeting signal led to a greatly enhanced accumulation of the heterologous protein. In lucerne and tobacco leaves, the level of vicilin-KDEL protein was 20 and 100 times greater than that of the unmodified vicilin, respectively. These differences in expression level could not be explained by corresponding differences in the steady-state levels or the translatability of the mRNAs. However, when the stability of vicilin and vicilin-KDEL proteins was compared in their respective transgenic hosts, unmodified vicilin was found to be degraded with a half-life of 4.5 h while vicilin-KDEL was much more stable with a half-life of more than 48 h. Immunogold labelling of leaf tissues from transgenic lucerne and tobacco showed the presence of vicilin associated with large aggregates within the ER lumen of vicilin-KDEL plants. No such aggregates were detected in transgenic plants expressing wild-type vicilin. It is concluded that the carboxy-terminal KDEL caused the retention of the modified vicilin in the ER, and that this retention led to the increased stability and higher level of accumulation of vicilin-KDEL in leaves of transgenic plants.

Amino Acid Sequence↗

PubMed2000

Plants rendered herbicide-susceptible by cauliflower mosaic virus-elicited suppression of a 35S promoter-regulated transgene.

Crop plants genetically modified for herbicide tolerance were some of the first to be released into the environment. Frequently, the cauliflower mosaic virus (CaMV) 35S promoter is used to drive expression of the herbicide tolerance transgene. We analyzed the response to CaMV infection of a transgenic oilseed rape line containing the bialaphos tolerance gene (BAR) from Streptomyces hygroscopicus, regulated by the 35S promoter. Oilseed rape is susceptible to CaMV, but plants recover from infection. CaMV infection altered the expression of the herbicide tolerance gene such that plants became susceptible to the herbicide. The effect on transgene expression differed in infections with viral pathogenic variants typical of those found in natural situations worldwide. Susceptibility to the herbicide was most likely a result of transcriptional gene silencing of the transgene. Our results show that transgene phenotypes can be modified by pathogen invasion.

Brassica↗

PubMed1992

Light regulatory sequences are located within the 5' portion of the Fed-1 message sequence.

We have previously shown that element(s) mediating a light-induced increase in the abundance of Fed-1 mRNA in the leaves of transgenic tobacco plants are located within the transcribed portion of the gene. As part of an effort to define the mechanism of this effect, we report here that cis-acting elements capable of mediating a 5-fold light-induced increase in the abundance of this mRNA are located within a region comprising the 5' leader and first third of the Fed-1 coding sequence. No activity was detected in the 3' untranslated region of the gene. In a gain-of-function assay, the 5' region was found to be capable of conferring light responsiveness on three different reporter sequences, although experiments with the gusA reporter were complicated by an apparent negative light effect on the stability of this mRNA. Deletion experiments show that at least one essential light regulatory element is located in the 5' untranslated region of Fed-1 between nucleotides +19 and +57. Additional Fed-1 sequences, including a portion of the protein coding region, are required to confer positive responsiveness on the gusA reporter. These additional sequences may include specific light regulatory elements or simply provide an environment in which the leader element can function normally.

Chromosome Deletion↗

PubMed1995

RNA-mediated transposition of the tobacco retrotransposon Tnt1 in Arabidopsis thaliana.

The tobacco (Nicotiana tabacum) retrotransposon Tnt1 was introduced into Arabidopsis thaliana. In this heterologous host plant species, Tnt1 undergoes an RNA-mediated transposition and creates a 5 bp duplication at the insertion sites. This is the first report of transposition of a retrotransposon after introduction into a heterologous host species. Tnt1 transposed during in vitro regeneration of transformed A.thaliana, but no transposition event was detected as happening in T2 and T3 generation plants. Newly synthesized copies of Tnt1 can integrate into coding regions of the host DNA. Our results open up the possibility of using Tnt1 as a new tool for insertional mutagenesis and functional analysis of plant genomes, in addition to the strategies of T-DNA and transposon tagging.

Amino Acid Sequence↗

PubMed1995

Monocotyledonous C4 NADP(+)-malate dehydrogenase is efficiently synthesized, targeted to chloroplasts and processed to an active form in transgenic plants of the C3 dicotyledon tobacco.

Chloroplastic NADP(+)-malate dehydrogenase (cpMDH, EC 1.1.1.82) is a key enzyme in the carbon-fixation pathway of some C4 plants such as the monocotyledons maize or Sorghum. We have expressed cpMDH from Sorghum vulgare Pers. in transgenic tobacco (Nicotiana tabacum L.) (a dicotyledonous C3 plant) by using a gene composed of the Sorghum cpMDH cDNA under the control of cauliflower mosaic virus 35S promoter. High steady-state levels of cpMDH mRNA were observed in isogenic dihaploid transgenic tobacco lines. Sorghum cpMDH protein was detected in transgenic leaf extracts, where a threefold higher cpMDH activity could be measured, compared with control tobacco leaves. The recombinant protein was identical in molecular mass and in N-terminal sequence to Sorghum cpMDH. The tobacco cpMDH protein which has a distinct N-terminal sequence, could not be detected in transgenic plants. Immunocytochemical analyses showed that Sorghum cpMDH was specifically localized in transgenic tobacco chloroplasts. These data indicate that Sorghum cpMDH preprotein was efficiently synthesized, transported into and processed in tobacco chloroplasts. Thus, C3-C4 photosynthesis specialization or monocotyledon-dicotyledon evolution did not affect the chloroplastic protein-import machinery. The higher levels of cpMDH in transgenic leaves resulted in an increase of L-malate content, suggesting that carbon metabolism was altered by the expression of the Sorghum enzyme.

Amino Acid Sequence↗

PubMed1997

Targeted deletion of sprA from the tobacco plastid genome indicates that the encoded small RNA is not essential for pre-16S rRNA maturation in plastids.

The small plastid RNA (spRNA) which includes a segment that is complementary to the pre-16S rRNA has been suggested to facilitate maturation of pre-16S rRNA in tobacco. To investigate the function of spRNA, the gene encoding it (sprA) was removed from the plastid genome using targeted gene deletion. We report here that deletion of sprA does not significantly affect pre-16S rRNA maturation, nor does it cause any obvious phenotype. Although the spRNA still may be involved in rRNA maturation, it is non-essential under normal growth conditions.

Gene Deletion↗

PubMed1999

Plastome engineering of ribulose-1,5-bisphosphate carboxylase/oxygenase in tobacco to form a sunflower large subunit and tobacco small subunit hybrid.

Targeted gene replacement in plastids was used to explore whether the rbcL gene that codes for the large subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase, the key enzyme of photosynthetic CO2 fixation, might be replaced with altered forms of the gene. Tobacco (Nicotiana tabacum) plants were transformed with plastid DNA that contained the rbcL gene from either sunflower (Helianthus annuus) or the cyanobacterium Synechococcus PCC6301, along with a selectable marker. Three stable lines of transformants were regenerated that had altered rbcL genes. Those containing the rbcL gene for cyanobacterial ribulose-1,5-bisphosphate carboxylase/oxygenase produced mRNA but no large subunit protein or enzyme activity. Those tobacco plants expressing the sunflower large subunit synthesized a catalytically active hybrid form of the enzyme composed of sunflower large subunits and tobacco small subunits. A third line expressed a chimeric sunflower/tobacco large subunit arising from homologous recombination within the rbcL gene that had properties similar to the hybrid enzyme. This study demonstrated the feasibility of using a binary system in which different forms of the rbcL gene are constructed in a bacterial host and then introduced into a vector for homologous recombination in transformed chloroplasts to produce an active, chimeric enzyme in vivo.

Amino Acid Sequence↗

PubMed2000

Post-transcriptional regulation of phenylalanine ammonia-lyase expression in tobacco following recovery from gene silencing.

Introduction of a bean phenylalanine ammonia-lyase (PAL) transgene into tobacco plants results in epigenetic post-transcriptional gene silencing which is unstable, such that after self-pollination first generation progeny may become PAL over-expressors. The change from gene silencing to PAL over-expression is accompanied by a loss of cytosine methylation of the PAL transgene and reduced methylation of the endogenous tobacco PAL2 gene, but not the PAL1 gene. These changes are associated with the appearance of high levels of bean PAL and tobacco PAL2 transcripts in the total RNA fraction from PAL over-expressing plants. However, tobacco PAL2 transcripts are inefficiently recruited into polysomes, and tobacco PAL2 protein is not detected in leaves of PAL over-expressing or wild-type lines. Thus, in spite of the post-transcriptionally controlled increase in tobacco PAL2 transcripts in PAL over-expressors, the increased PAL activity is primarily the result of the increase in bean PAL transcripts and corresponding enzymatic activity. These results reveal a complex cross-talk between expression of the PAL transgene and the corresponding endogenous PAL genes at the levels of transcription, transcript stability and polysomal recruitment during sense transgene-mediated silencing and subsequent over-expresson of PAL in tobacco.

DNA Methylation↗

PubMed2000

Characterization of the potato MADS-box gene STMADS16 and expression analysis in tobacco transgenic plants.

A new MADS-box gene, STMADS16, has been cloned in Solanum tuberosum L. that is expressed in all vegetative tissues of the plant, mainly in the stem, but not in flower organs. STMADS16 expression is established early during vegetative development and is not regulated by light. Sequence similarity besides the spatial and temporal expression patterns allow to define a novel MADS-box subfamily comprising STMADS16 and the gene STMADS11. Expression of the STMADS16 sense cDNA under the control of the 35S cauliflower mosaic virus promoter modifies the inflorescence structure by increasing both internode length and flower proliferation of the inflorescence meristems, and confers vegetative features to the flower. Moreover, STMADS16 ectopic expression overcomes the increase in flowering time and node number produced under short-day photoperiod, while the flowering time is not affected in long-day conditions. These results are discussed in terms of a possible role for STMADS16 in promoting vegetative development.

Amino Acid Sequence↗

PubMed1999

Processing of preproricin in transgenic tobacco.

The plant protein toxin ricin has found widespread application as a potential therapeutic agent for many human diseases and in disease-model systems such as those involving apoptosis. Genetic engineering and expression of the complete two-polypeptide chain toxin have only been possible in plants, specifically in transgenic tobacco carrying the preproricin gene under the control the cauliflower mosaic virus 35S promoter. Production of modified ricin for altered controllable activity and/or fusion therapeutics to target delivery requires knowledge of the heterologous processing that occurs when preproricin is expressed in tobacco. Here, recombinant ricin from transgenic tobacco was purified using lectin affinity chromatography and characterized using various biochemical and biophysical techniques. Coomassie blue staining of an SDS-PAGE gel of lactose-agarose purified material identified predominant proteins of 30 and 35 kDa molecular weight. Western analysis using anti-ricin a- and b-chain antibodies confirmed the expression and purification of recombinant ricin, with identical protein banding profiles to that of authentic castor-bean-derived ricin. High-resolution gel filtration chromatography characterized the lactose binding complex as a 66-kDa native molecular weight protein which could be separated into 30- and 35-kDa proteins upon incubation with the reducing agent dithiothreitol. N-terminal sequencing of the recombinant ricin a-chain revealed that an equimolar ratio of two alternately processed peptides was present, which varied by an additional amino acid derived from the signal peptide. Similar analysis of ricin b-chain again identified two forms of this polypeptide as well; however, full-length ricin b-chain and b-chain missing the first alanine residue were present at 11:1 molar ratios. Transgenic tobacco plants expressing ricin were used to develop a stable cell suspension culture system from callus induced with the growth regulators 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine. Double sandwich enzyme-linked immunosorbent assay using anti-ricin b-chain antibodies and Western analysis identified soluble ricin in the media of the cultures, indicating that cell cultures provide a safe and simple means to produce properly processed recombinant ricin.

Amino Acid Sequence↗

PubMed1998

Isolation and characterization of a rice homebox gene, OSH15.

In many eukaryotic organisms including plants, homeobox genes are thought to be master regulators that establish the cellular or regional identities and specify the fundamental body plan. We isolated and characterized a cDNA designated OSH15 (Oryza sativa homeobox 15) that encodes a KNOTTED-type homeodomain protein. Transgenic tobacco plants overexpressing the OSH15 cDNA showed a dramatically altered morphological phenotype caused by disturbance of specific aspects of tobacco development, thereby indicating the involvement of OSH15 in plant development. We analyzed the in situ mRNA localization of OSH15 through the whole plant life cycle, comparing the expression pattern with that of another rice homeobox gene, OSH1. In early embryogenesis, both genes were expressed as the same pattern at a region where the shoot apical meristem would develop later. In late embryogenesis, the expression pattern of the two genes became different. Whereas the expression of OSH1 continued within the shoot apical meristem, OSH15 expression within the shoot apical meristem ceased but became observable in a ring shaped pattern at the boundaries of some embryonic organs. This pattern of expression was similar to that observed around vegetative or reproductive shoots, or the floral meristem in mature plants. RNA in situ localization data suggest that OSH15 may play roles in the shoot organization during early embryogenesis and thereafter, OSH15 may be involved in morphogenetic events around the shoot apical meristem.

Amino Acid Sequence↗