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.

The nucleotide sequence was determined for a 340-kb segment of rice chromosome 2, revealing 56 putative protein-coding genes. This represents a density of one gene per 6.1 kb, which is higher than was reported for a previously sequenced segment of the rice genome. Sixteen of the putative genes were supported by matches to ESTs. The predicted products of 29 of the putative genes showed similarity to known proteins, and a further 17 genes showed similarity only to predicted or hypothetical proteins identified in genome sequence data. The region contains a few transposable elements: one retrotransposon, and one transposon. The segment of the rice genome studied had previously been identified as representing a part of rice chromosome 2 that may be homologous to a segment of Arabidopsis chromosome 4. We confirmed the conservation of gene content and order between the two genome segments. In addition, we identified a further four segments of the Arabidopsis genome that contain conserved gene content and order. In total, 22 of the 56 genes identified in the rice genome segment were represented in this set of Arabidopsis genome segments, with at least five genes present, in conserved order, in each segment. These data are consistent with the hypothesis that theArabidopsis genome has undergone multiple duplication events. Our results demonstrate that conservation of the genome microstructure can be identified even between monocot and dicot species. However, the frequent occurrence of duplication, and subsequent microstructure divergence, within plant genomes may necessitate the integration of subsets of genes present in multiple redundant segments to deduce evolutionary relationships and identify orthologous genes.

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 conserved bifactorial endosperm box found in the promoter of wheat storage protein genes comprises two different cis elements that are thought to be involved in regulating endosperm-specific gene expression. Endosperm nuclear extracts contain binding activities. One is called ESBF-I, which binds to the endosperm motif (EM), and the other is called ESBF-II, which binds to the GCN4-like motif(GLM). Here, we present a functional analysis of the endosperm box of a low-molecular-weight glutenin gene found on the 1D1 chromosome of hexaploid wheat (LMWG-1D1) in transgenic tobacco plants. Our analysis demonstrates the necessity of the EM and GLM for endosperm-specific gene expression and suggests the presence in tobacco of functional counterparts of wheat ESBF-I and ESBF-II. Furthermore, we describe the isolation and characterization of cDNA clones encoding SPA, a seed-specific basic leucine zipper protein from wheat that can activate transcription from the GLMs of the -326-bp LMWG-1D1 promoter in both maize and tobacco leaf protoplasts. This activation is also partially dependent on the presence of functional EMs, suggesting interactions between SPA with ESBF-I-like activities.

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.

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.

We have analyzed the expression of chimeric genes in populations of protoplasts isolated from the photosynthetic and nonphotosynthetic tissues within leaves of transgenic tobacco plants and separated by fluorescence-activated cell sorting. Expression of transcriptional gene fusions controlled by promoters from photosynthesis-associated genes showed a striking dependence on cell type. These patterns of expression were preserved when the gene fusions were transfected into normal (nontransgenic) tobacco leaf protoplasts.

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.

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 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.

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.

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.

The particles of cultures of cucumber mosaic virus (CMV) contain the three genomic species of single-stranded RNA and a sub-genomic species that acts as messenger RNA for CMV particle protein. Some cultures also contain a single-stranded linear RNA molecule that is typically ∼335 nucleotides long1,2. This extra molecule, termed satellite RNA, does not share appreciable nucleotide sequence with CMV genomic RNA3 but replicates only in plants1 or protoplasts4 that are infected with CMV. CMV isolates that do not contain satellite RNA can be cultured repeatedly in plants in a satellite-free state, but when satellite RNA is added to such cultures it is synthesized and persists as a component of the virus isolate5. The effect of satellite RNA on CMV infections depends on the strain of satellite: in many cases the usual symptoms of CMV are suppressed and as a result the infected plants show few symptoms of infection6–8. However, the presence of other strains of satellite RNA leads to the production of severe symptoms that are quite distinct from those of CMV8. Here we describe the transformation of tobacco plants with DNA copies of CMV satellite RNA, the production of satellite RNA transcripts by such plants and the acquisition of satellite RNA by CMV cultures grown in them. These results suggest a means of protecting plants against the effects of CMV and also suggest a method by which satellite RNAs may have evolved.

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.