Transgenic tobacco (Nicotiana tabacum L. cv. Samsun) plants with reduced levels of the Calvin cycle enzyme sedoheptulose-1,7-bisphosphatase (SBPase; EC 3.1.3.37) were produced using an antisense construct in which the expression of a tobacco SBPase cDNA clone was driven by the cauli¯ower mosaic virus (CaMV) promoter. The reduction in SBPase protein levels observed in the primary transformants correlated with the presence of the antisense construct and lower levels of the endogenous SBPase mRNA. No changes in the amounts of other Calvin cycle enzymes were detected using Western blot analysis. The SBPase antisense plants with less than 20% of wild-type SBPase activity were observed to display a range of phenotypes, including chlorosis and reduced growth rates. Measurements of photosynthesis, using both light-dosage response and CO 2 response curves, of T1 plants revealed a reduction in carbon assimilation rates, which was apparent in plants retaining 57% of wild-type SBPase activity. Reductions were also observed in the quantum eciency of photosystem II. This decrease in photosynthetic capacity was re¯ected in a reduction in the carbohydrate content of leaves. Analysis of carbohydrate status in fully expanded source leaves showed a shift in carbon allocation away from starch, whilst sucrose levels were maintained in all but the most severely aected plants. Plants with less than 15% of wild-type SBPase activity were found to contain less than 5% of wild-type starch levels. The results of this preliminary analysis indicate that SBPase activity may limit the rate of carbon assimilation.Abbreviations: A = rate of CO 2 assimilation; CaMV = cauliower mosaic virus; C i = intercellular CO 2 concentration; FBPase = fructose-1,6-bisphosphatase; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; LSU = large subunit; PRKase = phosphoribulokinase; RuBP = ribulose-1,5-bisphosphate; SBP = sedoheptulose-1,7-bisphosphate; SBPase = sedoheptulose-1,7-bisphosphatase Correspondence to: C.A. Raines;

Protein phosphorylation represents one of the major mechanisms for transcription factor activation. Here we demonstrate a molecular mechanism by which phosphorylation by mitogen‐activated protein (MAP) kinases leads to changes in transcription factor activity. MAP kinases stimulate DNA binding and transcriptional activation mediated by the mammalian ETS‐domain transcription factor Elk‐1. Phosphorylation of the C‐terminal transcriptional activation domain induces a conformational change in Elk‐1, which accompanies the stimulation of DNA binding. C‐terminal phosphorylation is coupled to activation of DNA binding by the N‐terminal DNA‐binding domain via an additional intermediary domain. Activation of DNA binding is mediated by an allosteric mechanism involving the key phosphoacceptor residues. Together, these results provide a molecular model for how phosphorylation induces changes in Elk‐1 activity.

The nucleotide sequence encoding the chloroplast enzyme, sedoheptulose-l,7-bisphosphatase [Sed(l,7)P2ase], was obtained from wheat cDNA and genomic clones. The transcribed region of the Sed(1 ,7)P2ase gene has eight exons (72 -507 bp) and seven introns (85 -626 bp) and encodes a precursor polypeptide of 393 amino acids. Comparison of the deduced amino acid sequence of Sed(1 ,7)P2ase with those of fructose-l,6-bisphosphatase [Fru(l ,6)P2ase] enzymes from a variety of sources reveals 19% identity, rising to 42% if conservative changes are considered. Most importantly, the amino acid residues which form the active site of Fru(l,6)P2ase are highly conserved in the Sed(1 ,7)P2ase molecule, indicating a common catalytic mechanism. Interestingly, although the activities of both Sed(1 ,7)P2ase and chloroplast Fru(1 ,6)P2ase are modulated by light via the thioredoxin system, the amino acid sequence motif identified as having a role in this regulation in chloroplast Fru(1 ,6)P2ase is not found in the Sed(l,7)P2ase enzyme.The photosynthetic carbon-reduction cycle is the primary pathway of carbon fixation which in higher plants takes place in the stroma of chloroplasts. This cycle is autocatalytic; in addition to producing substrates for starch and sucrose biosynthesis, it must regenerate the carbon dioxide acceptor, ribulose 1,s-bisphosphate. A balance between the regenerative and export functions is critical, and this is believed to be achieved by mechanisms which regulate the catalytic activity of a number of key enzymes of the cycle. In addition to the constraints imposed by ribulose-1 ,5-bisphosphate carboxylase on the overall rate of carbon flow, three further enzymes, sedoheptulose-1,7-bisphosphatase [Sed(l,7)P2ase], fructose-1,6-bisphosphatase [Fru(l ,6)P2ase] and phosphoribulokinase, have been proposed to have prominent roles in regulating the flow of intermediates (Woodrow and Berry, 1988). The reactions catalysed by these enzymes are essentially irreversible and their activities are stimulated significantly by light (Wirtz et al., 1982).Sed(1 ,7)P2ase catalyses the dephosphorylation of Sed( 1 ,7)Pz, forming sedoheptulose 7-phosphate (Sed7P) and inorganic phosphate, and this is an essentially irreversible reaction which commits intermediates to the regenerative part of the photosynthetic carbon-reduction cycle. The activity of Abbreviotions. Sed(1 ,7)P2ase, sedoheptulose-1.7-bisphosphatase; Fru(l,6)Pzase. fructose-l,6-bisphosphatase; Sed7P, sedoheptulose 7-phosphate; Fru(2,6)P2, fructose 2,6-bisphosphate; Fru6P. fructose 6-phosphate.Enzymes. Sedoheptulose-l,7-bisphosphatase (EC 3.1.3.37); chloroplast fructose-l,6-bisphosphatase (EC 3.1.311 1). sity of Essex,

The control of DNA binding by eukaryotic transcription factors represents an important regulatory mechanism. Many transcription factors are controlled by cisacting autoinhibitory modules that are thought to act by blocking promiscuous DNA binding in the absence of appropriate regulatory cues. Here, we have investigated the determinants and regulation of the autoinhibitory mechanism employed by the ETS-domain transcription factor, PEA3. DNA binding is inhibited by a module composed of a combination of two short motifs located on either side of the ETS DNA-binding domain. A second type of protein, Ids, can act in trans to mimic the effect of these cis-acting inhibitory motifs and reduce DNA binding by PEA3. By using a one-hybrid screen, we identified the basic helix-loop-helix-leucine zipper transcription factor USF-1 as an interaction partner for PEA3. PEA3 and USF-1 form DNA complexes in a cooperative manner. Moreover, the formation of ternary PEA3⅐USF-1⅐DNA complexes requires parts of the same motifs in PEA3 that form the autoinhibitory module. Thus the binding of USF-1 to PEA3 acts as a switch that modifies the autoinhibitory motifs in PEA3 to first relieve their inhibitory action, and second, promote ternary nucleoprotein complex assembly.

We report here the isolation and nucleotide sequence of genomic clones encoding the chloroplast enzyme sedoheptulose-1,7-bisphosphatase (SBPase) from Arabidopsis thaliana. The coding region of this gene contains eight exons (72-76 bp) and seven introns (75-91 bp) and encodes a polypeptide of 393 amino acids. Unusually, the 5' non-coding region contains two additional AUG codons upstream of the translation initiation codon. A comparison of the deduced Arabidopsis and wheat SBPase polypeptide sequences reveals 78.6%, identity. Expression studies showed that the level of SBPase mRNA in Arabidopsis and wheat is regulated in a light-dependent manner and is also influenced by the developmental stage of the leaf. Although the Arabidopsis SBPase gene is present in a single copy, two hybridizing transcripts were detected in some tissues, suggesting the presence of alternate transcription start sites in the upstream region.