Delayed Leaf Senescence in Tobacco Plants Transformed with tmr, a Gene for Cytokinin Production in Agrobacterium.

The aim of this study was to investigate whether enhanced levels of endogenous cytokinins could influence plant development, particularly leaf senescence. Tobacco plants were transformed with the Agrobacterium tumefaciens gene tmr, under the control of the soybean heat shock promoter HS6871. This gene encodes the enzyme isopentenyl transferase, which catalyzes the initial step in cytokinin biosynthesis. After heat shock, the cytokinin level increased greatly and the level of tmr mRNA, undetectable at 20[deg]C, rose and remained high for up to 8 hours. The levels of cytokinin and tmr mRNA were substantially lower by 24 hours. Transformed plants grown at 20[deg]C were shorter, had larger side shoots, and remained green for longer than untransformed plants. The differences were more pronounced after several heat shocks of whole plants or defined areas of leaves. Our results demonstrated that plant morphology and leaf senescence can be manipulated by changing the endogenous level of cytokinins.

Characterisation of the 5'-leader sequence of tobacco mosaic virus RNA as a general enhancer of translation in vitro.

Uncapped messenger RNAs (mRNAs) encoding calf preprochymosin, chicken prelysozyme, or Escherichia coli beta-glucuronidase (GUS) were synthesized in vitro, with or without a 5'-terminal 67-nucleotide sequence (omega') derived from the untranslated 5'-leader (omega) of tobacco mosaic virus (TMV) RNA. Messenger RNAs were translated in vitro, in messenger-dependent systems derived from rabbit reticulocytes (MDL), wheat-germ (WG) or E. coli (EC). The omega' sequence enhanced expression of each mRNA in almost every translation system. While MDL was the least responsive to omega', this sequence proved particularly efficient in permitting translation of the eukaryotic mRNAs in EC, despite the absence of a consensus Shine-Dalgarno sequence in either the mRNAs or omega'. The local context of the initiation codon (AUG) in two GUS mRNA constructs did not influence the relative enhancement caused by the omega' sequence. These findings extend the utility of omega' as a general enhancer of translation for both prokaryotic and eukaryotic mRNAs in either 80S- or 70S-ribosome-based systems.

A rapid transformation method for Solanum tuberosum using binary Agrobacterium tumefaciens vectors.

A tuber disc transformation and regeneration system was devised for potato (Solanum tuberosum). Tuber discs were found to be the most morphogenetic organ on a medium previously optimised for tomato regeneration. Shoot regeneration from tuber discs was rapid and transformed as shown by nopaline assays and Southern blot analysis. The ease and speed of the tuber disc method will allow for the increased use of this commercially important plant in transformation studies.

T-DNA of the Agrobacterium Ti and Ri plasmids.

The study of T-DNA transmission from Agrobacterium Ti or Ri plasmid into the genomes of higher plant cells has revealed much about the consequences of transformation. It is now clear that the transformed phenotype is caused by hormonal changes produced directly or indirectly by T-DNA genes. The opine synthases are enzymes encoded in T-DNA that function in the plant cell. Our level of understanding of T-DNA-encoded functions is already sufficient to reveal clear and feasible ways to exploit T-DNA as a gene vector. What remains to challenge the crown gall investigator are many questions of fundamental importance: What is the mechanism of the seemingly illegitimate recombination between T-DNA and plant DNA, and is this process catalyzed by bacterial or host plant enzymes, or both? Do T-DNA genes encode enzymes that catalyze biosynthesis of auxin- and cytokinin-active substances? What gene in T-DNA confers immunity to A. tumefaciens, and what is its mode of action? Does T-DNA insert into random or specific sites in the host plant genome? Did T-DNA derive from plant genetic information or has prokaryotic DNA arrived at functional eukaryotic gene structure by convergent evolution? Although there is keen interest in T-DNA as a vector for genetic engineering, it holds equal interest as a unique interface between the biology of prokaryotes and eukaryotes.

Multiple transcripts of T-DNA detected in nopaline crown gall tumors.

Crown gall plant tumors contain neoplastic cells transformed by incorporation of a foreign DNA element, T-DNA, derived from a large tumor-inducing plasmid in the inciting Agrobacterium strain. T-DNA is covalently joined to the nuclear DNA of the tumor cell, and RNA transcripts from T-DNA are present in polyadenylated form on polysomes. This paper presents a detailed analysis of those parts of T-DNA transcribed in a nopaline-type tobacco teratoma, BT37, whose T-DNA has been mapped and cloned. Northern blots of polyA+ RNA were probed with 21 different nick-translated T-DNA fragments, and at least 13 well-defined transcripts were visualized.

Targeting a foreign protein to chloroplasts using fusions to the transit peptide of a chlorophyll a/b protein.

We have constructed chimaeric genes consisting of sequences encoding the transit peptide and 4, 16, 24, 53 or 126 amino-terminal residues of the mature chlorophyll a/b binding (Cab) apoprotein fused to the Escherichia coli gene encoding beta-glucuronidase (GUS). These genes were introduced into tobacco plants and the fate of the fusion proteins they encode was analysed. Less than 1% of the total activity of fusion proteins containing the transit peptide and 4 (FP4) or 16 (FP16) amino-terminal amino acids of the mature Cab protein was associated with chloroplasts. Moreover, FP4 appears to be unprocessed. This is in striking contrast to fusion proteins containing the transit peptide and 24 (FP24), 53 (FP53) or 126 (FP126) amino-terminal residues of the mature Cab polypeptide. Approximately 98%, 96% or 75%, respectively, of the total activity of these fusion proteins was associated with purified intact chloroplasts, and protease protection experiments showed that of this, approximately 98%, 87% or 50%, respectively, was located within this organelle. Furthermore, both FP24 and FP53 appear to be processed. However, less than 10% of the activity of those fusion proteins translocated into chloroplasts was associated with thylakoid membranes.

Expression of tobacco mosaic virus coat protein by a cauliflower mosaic virus promoter in plants transformed by Agrobacterium.

The 35S transcript promoter of cauliflower mosaic virus directs the synthesis of an abundant RNA in infected leaf tissue that is both a template for reverse transcription during virus replication and also a polycistronic mRNA. The 35S promoter was isolated and linked to a cDNA of the 3' end of tobacco mosaic virus that encodes the coat protein of this single-stranded RNA virus. After transformation of this gene construction into tobacco plants using a disarmed Agrobacterium binary vector, viral coat protein synthesis was detected in transformed leaf tissue. A chimaeric 35S coat protein RNA was detected in transformed callus.

Spatial and temporal patterns of transcription of a wound-induced gene in potato.

Transcriptional fusions between the gene encoding win2 from potato and the reporter gene encoding beta-glucuronidase (GUS) have been used to study the spatial and temporal patterns of wound induced gene activity in transgenic potato and tobacco plants. Gene fusions containing a full length win2 promoter were found to be correctly regulated in response to mechanical wounding in transgenic potato, but not in the heterologous host, tobacco. Sequences greater than 560 bp upstream of the transcription start site of win2 were shown to be important for wound inducibility. The dramatic induction of GUS activity detected using fluorometric assays of extracts of wounded and aged leaves of several independent win2--GUS transformants was consistent with the kinetics of win2 mRNA accumulation. Histochemical analysis of wounded leaves showed that transcription first occurred in cells immediately adjacent to the wound, and was then progressively induced in cells associated with the vascular system at a distance from the wound site. In tubers, a localized response to wounding was observed, and this only spread to other parts of the tuber if it had started to sprout. It was concluded that active vascular transport was necessary for the spread of wound response. Win2--GUS fusions were also expressed as part of normal plant development, as GUS activity was detected in the developing buds and in a layer of cells associated with the lenticels of unwounded tubers.

GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.

We have used the Escherichia coli beta-glucuronidase gene (GUS) as a gene fusion marker for analysis of gene expression in transformed plants. Higher plants tested lack intrinsic beta-glucuronidase activity, thus enhancing the sensitivity with which measurements can be made. We have constructed gene fusions using the cauliflower mosaic virus (CaMV) 35S promoter or the promoter from a gene encoding the small subunit of ribulose bisphosphate carboxylase (rbcS) to direct the expression of beta-glucuronidase in transformed plants. Expression of GUS can be measured accurately using fluorometric assays of very small amounts of transformed plant tissue. Plants expressing GUS are normal, healthy and fertile. GUS is very stable, and tissue extracts continue to show high levels of GUS activity after prolonged storage. Histochemical analysis has been used to demonstrate the localization of gene activity in cells and tissues of transformed plants.

Endoplasmic reticulum targeting and glycosylation of hybrid proteins in transgenic tobacco.

The correct compartmentation of proteins to the endomembrane system, mitochondria, or chloroplasts requires an amino-terminal signal peptide. The major tuber protein of potato, patatin, has a signal peptide in common with many other plant storage proteins. When the putative signal peptide of patatin was fused to the bacterial reporter protein beta-glucuronidase, the fusion proteins were translocated to the endoplasmic reticulum in planta and in vitro. In addition, translocated beta-glucuronidase was modified by glycosylation, and the signal peptide was correctly processed. In the presence of an inhibitor of glycosylation, tunicamycin, the enzymatically active form of beta-glucuronidase was assembled in the endoplasmic reticulum. This is the first report of targeting a cytoplasmic protein to the endoplasmic reticulum of plants using a signal peptide.

The eli1 mutation reveals a link between cell expansion and secondary cell wall formation in Arabidopsis thaliana.

Mutants with altered patterns of lignification have been identified in a population of mutagenised Arabidopsis seedlings. One of the mutants exhibited ectopic lignification (eli) of cells throughout the plant that never normally lignify. The reduced expansion of eli1 cells resulted in a stunted phenotype, and xylem cells were misshapen and failed to differentiate into continuous strands, causing a disorganized xylem. Analysis of phenotypes associated with double mutants of eli1 lit (lion's tail), a cell expansion mutant, indicated that the primary defect in eli1 plants may be inappropriate initiation of secondary wall formation and subsequent aberrant lignification of cells caused by altered cell expansion. Related ectopic lignification phenotypes were also observed in other cell expansion mutants, suggesting a mechanism that senses cell size and controls subsequent secondary wall formation. Interactions between eli1 and wol (woodenleg), a mutant altering xylem cell specification, revealed a role for ELI1 in promoting formation of continuous xylem strands, and demonstrated that ELI1 functions during cell elongation zone in the primary root and other tissues.

PLANT TRANSCRIPTION FACTOR STUDIES.

Major advances have been made in understanding the role of transcription factors in gene expression in yeast, Drosophila, and man. Transcription factor modification, synergistic events, protein-protein interactions, and chromatin structure have been successfully integrated into transcription factor studies in these organisms. While many putative transcription factors have been isolated from plants, most of them are only poorly characterized. This review summarizes examples where molecular biological techniques have been successfully employed to study plant transcription factors. The functional analysis of transcription factors is described as well as techniques for studying the interactions of transcription factors with other proteins and with DNA.

The activities of acidic and glutamine-rich transcriptional activation domains in plant cells: design of modular transcription factors for high-level expression.

The aim of this work was to design strong transcriptional activators that can be used to regulate plant gene expression. The contribution of different components in a transcription factor and target gene system was assayed by measuring transcriptional activation. Each component was optimised to achieve maximal reporter gene expression in transient protoplast transformation assays. The DNA-binding domain of the yeast transcriptional activator GAL4 was studied in the context of fusion proteins with activation domains of the herpes simplex virus protein VP16 or the tomato Myb-like activator THM18. Multimerisation of the activation domain and insertion of a homopolymeric glutamine stretch was used to increase transcription factor potency. Evidence is presented that these modifications can result in even more active transcription factors when they are combined. Finally, it was demonstrated using competition experiments that transcription factors with acidic activation domains can mutually suppress their activation potentials when expressed at high levels.

Localization of sequences in wheat endosperm protein genes which confer tissue-specific expression in tobacco.

The developing cereal grain accumulates large quantities of proteins which are unique to the endosperm tissue. The DNA sequences which determine their endosperm-specific expression have not yet been identified. In the absence of a suitable transformation-regeneration system for cereals, we have investigated whether chimaeric genes consisting of low mol. wt (LMW) and high mol. wt (HMW) glutenin gene upstream sequences coupled to the coding region of the bacterial chloramphenicol acetyl transferase (CAT) gene could be specifically expressed in transgenic tobacco. The fusions, made in a Ti-derived binary vector, were introduced into tobacco via Agrobacterium tumefaciens-mediated transformation and their activity assayed. Both the LMW and HMW glutenin chimaeric genes exhibited endosperm-specific CAT activity in the transformed plants. In addition, a deletion series of the LMW glutenin sequence indicated that sequences present between 326 bp and 160 bp upstream of the transcription start point are necessary to confer endosperm-specific CAT activity.

Impaired sucrose-induction mutants reveal the modulation of sugar-induced starch biosynthetic gene expression by abscisic acid signalling.

Plants both produce and utilize carbohydrates and have developed mechanisms to regulate their sugar status and co-ordinate carbohydrate partitioning. High sugar levels result in a feedback inhibition of photosynthesis and an induction of storage processes. We used a genetic approach to isolate components of the signalling pathway regulating the induction of starch biosynthesis. The regulatory sequences of the sugar inducible ADP-glucose pyrophosphorylase subunit ApL3 were fused to a negative selection marker. Of the four impaired sucrose induction (isi) mutants described here, two (isi1 and isi2) were specific to this screen. The other two mutants (isi3 and isi4) showed additional phenotypes associated with sugar-sensing screens that select for seedling establishment on high-sugar media. The isi3 and isi4 mutants were found to be involved in the abscisic acid signalling pathway. isi3 is allelic to abscisic acid insensitive4 (abi4), a gene encoding an Apetala2-type transcription factor; isi4 was found to be allelic to glucose insensitive1 (gin1) previously reported to reveal cross-talk between ethylene and glucose signalling. Here we present an alternative interpretation of gin1 as an allele of the ABA-deficient mutant aba2. Expression analysis showed that ABA is unable to induce ApL3 gene expression by itself, but greatly enhances ApL3 induction by sugar. Our data suggest a major role for ABA in relation to sugar-signalling pathways, in that it enhances the ability of tissues to respond to subsequent sugar signals.

MYB61 is required for mucilage deposition and extrusion in the Arabidopsis seed coat.

We have undertaken a systematic reverse genetic approach to understand R2R3-MYB gene function in Arabidopsis. Here, we report the functional characterization of MYB61 based on the phenotype of three independent insertion alleles. Wide-ranging phenotype screens indicated that MYB61 mutants were deficient in seed mucilage extrusion upon imbibition. This phenotype was expressed in the sporophytic tissues of the seed. Deposition and extrusion of the principal component of the mucilage, a relatively unbranched rhamnogalacturonan, were reduced in the MYB61 mutant seed coats. Additional defects in the maturation of the testa epidermal cells suggested a potential deficiency in extracellular secretion in myb61 lines. Consistent with a proposed role in testa development, reverse transcription-polymerase chain reaction analysis showed the highest MYB61 expression in siliques, which was localized to the seed coat by a beta-glucuronidase (GUS) reporter gene fusion. Lower levels of GUS expression were detected in developing vascular tissue. Parallel analysis of the ttg1-1 mutant phenotype indicated that this mutant showed more severe developmental defects than myb61 and suggested that MYB61 may function in a genetic pathway distinct from that of TTG1. The transient nature of seed epidermal characteristics in the ttg1-1 mutant suggested that TTG1 was required for maintenance rather than initiation of testa epidermal differentiation. Germination and seedling establishment were compromised in the myb61 and ttg1-1 mutants under conditions of reduced water potential, suggesting a function for Arabidopsis seed mucilage during germination in dry conditions.

The complete nucleotide sequence of the potexvirus white clover mosaic virus.

The complete nucleotide sequence (5845 nucleotides) of the genomic RNA of the potexvirus white clover mosaic virus (WC1MV) has been determined from a set of overlapping cDNA clones. Forty of the most 5'-terminal nucleotides of WC1MV showed homology to the 5' sequences of other potexviruses. The genome contained five open reading frames which coded for proteins of Mr 147, 417, Mr 26,356, Mr 12,989, Mr 7,219 and Mr 20,684 (the coat protein). The Mr 147,417 protein had domains of amino acid sequence homology with putative polymerases of other RNA viruses. The Mr 26,356 and Mr 12,989 proteins had homology with proteins of the hordeivirus barley stripe mosaic virus RNA beta and the furovirus beet necrotic yellow vein virus (BNYVV) RNA-2. A portion of the Mr 26,356 protein was also conserved in the cylindrical inclusion proteins of two potyviruses. The Mr 7,219 protein had homology with the 25K putative fungal transmission factor of BNYVV RNA-3.

In vivo footprinting of a low molecular weight glutenin gene (LMWG-1D1) in wheat endosperm.

The quality of the wheat grain is determined by the quantity and composition of storage proteins (prolamins) which are synthesized exclusively in endosperm tissue. We are investigating the mechanisms underlying the regulation of expression of a prolamin gene, the low molecular weight glutenin gene LMWG-1D1. The LMWG-1D1 promoter contains the endosperm box, a sequence motif highly conserved in the promoter region of a large number of storage protein genes, which is thought to confer endosperm-specific expression of prolamin genes. Here we show by in vivo DMS footprinting of wheat endosperm tissue that the endosperm box becomes occupied by putative trans-acting factors during grain ripening. During early stages of development the endosperm motif within the 5' half of the endosperm box becomes occupied first, followed by binding of a second activity to a GCN4/jun-like motif in the 3' half just prior to the stage of maximum gene expression. Occupancy of the endosperm box is highly tissue-specific: no protection was observed in husk and leaf tissues. Several binding activities were identified in vitro from nuclear protein extracts of wheat endosperm which bind specifically to the endosperm and GCN4/jun motifs identified by in vivo footprinting.

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.