Many transgenic plant studies use constitutive promoters to express transgenes. For certain genes, deleterious effects arise from constant expression in all tissues throughout development. We describe a chemically inducible plant gene expression system, with negligible background activity, that obviates this problem. We demonstrate its potential by showing inducible manipulation of carbon metabolism in transgenic plants. Upon rapid induction of yeast cytosolic invertase, a marked phenotype appears in developing leaves that is absent from leaves that developed before induction or after it has ceased.

SummarySucrose produced in source leaves is the predominant carbon source for developing sink tissues in most higher plants. Consequently the rate of sucrose synthesis is likely to be important for sink development and final crop yield. Two sucrose biosynthetic enzymes are believed to possess regulatory properties with respect to the rate of sucrose synthesis: (i) cytosolic FBPase and (ii) sucrose phosphate synthase. To study the impact of reduced photosynthetic sucrose biosynthesis on plant growth and crop yield a cDNA clone encoding cytosolic FBPase was isolated from a potato leaf cDNA library and used for antisansa experiments in transgenic potato plants. The cDNA clone cy-F1, containing an open reading frame of 1020 bp highly homologous (85%) to other known sequences of plant cytosolic FBPases, was cloned in reversed orientation between the 35S CaMV promoter and the octopine synthase polyadenylation signal. Out of 75 independent transformants five transgenic lines having 9 to 55 % of the wild-type FBPase activity were chosen for further analysis.A 45% reduction of the cytosolic FBPasa activity did not cause any measurable change in metabolite concentrations, growth behaviour or photosynthetic parameters of the transgenic plants. Inhibition of cytosolic FBPase activity below 20% of the wild-type activity led to an accumulation of 3-PGA, triose-phosphates and fructose-l,6-bisphosphete in source leaves. This resulted in a reduced light-saturated rate of assimilation measured via gas exchange and a decreased photosynthetic rate under conditions of the leaf disc electrode with saturating light and CO2. Measuring photosynthetic carbon fluxes by labelling leaf discs with 14C02 revealed a 53-65% reduction of sucrose synthesis whereas starch synthesis decreased only by 18-24%. The flux into the anionic and cationic

Antibodies raised against a peptide fragment (residues 60-456) of potato sucrose phosphate synthase (SPS) were used to investigate whether potato plants contain multiple forms of SPS. When a partially purified preparation of SPS from cold-stored potato tubers was separated on 5 % polyacrylamide gel electrophoresis (PAGE), four immunopositive bands were found with estimated molecular weights of 125, 127, 135 and 145 kDa. These bands were also found in rapidly prepared extracts and were termed SPS-la, SPS-lb, SPS-2 and SPS-3, respectively. Direct evidence that SPS-la and SPS-lb represent active SPS was provided by the finding that both are greatly reduced in plants expressing an antisense sequence derived from the potato leaf SPS gene. SPS-2 was not decreased in the antisense plants, indicating that it has a significantly different sequence. Evidence that SPS-2 represents active SPS was obtained by showing that the amount of SPS-la and SPS-lb protein remaining in the leaves and tubers of antisense potato plants was too low to account for the remaining SPS activity. The four immunopositive SPS forms had different tissue distributions. SPS-la was the major form in all tissues except petals, sepals and stamens. SPS-lb and SPS-2 were absent in very young growing tissues hut were present as minor forms in source leaves and sprouting tubers. The SPS-lh level was especially high in petals and sepals, and the SPS-

SummaryElucidating the role of viral genes in transgenic plants revealed that the movement protein (MP| from tobacco mosaic virus is responsible for altered carbohydrate allocation in tobacco and potato plants. To study whether this is a general feature of viral MPs, the movement protein MP17 of potato leafroll virus (PLRV), a phloem-restricted luteovirus, was constitutively expressed in tobacco plants. Transgenic lines were strongly reduced in height and developed bleached and sometimes even necrotic areas on their source leaves. Levels of soluble sugars and starch were significantly increased in source leaves. Yet, in leaf laminae the hexose-phosphate content was unaltered and ATP reduced to only a small extent, indicating that these leaves were able to maintain homeostatic conditions by co~npartmentalization of soluble sugars, probably in the vacuole. On the contrary, midribs contained lower levels of soluble sugars, ATP, hexose-phosphates and UDP-glucose supporting the concept of limited uptake and catabolism of sucrose in the phloem. The accumulation of carbohydrates led to a decreased photosynthetic capacity and carboxylation efficiency of ribulose-l,5-bisphosphate carboxylase/oxygenase (rubisco) probably owing to decreased expression of photosynthetic proteins. In parallel, levels of pathogenesis-related proteins were elevated which may be the reason for the obtained limited resistance against the unrelated potato virus Y (PVY) N in the transgenic tobacco plants. Ultrathin sections of affected leaves harvested from 2-week-old plants revealed plasmodesmal alterations in the phloem tissue while plasmodesmata between mesophyll cells were indistinguishable from wildtype. These data favour the phloem tissue to be the primary site of PLRV MP17 action in altering carbohydrate metabolism.

Sucrose-phosphate synthase (SPS) from leaves of spinach (Spinacia oleracea L.) has been purified to homogeneity by a procedure involving precipitation with polyethylene glycol and chromatography over diethylaminoethylcellulose, omega-aminohexyl-agarose, Mono Q and Blue Affinity columns. The purification factor was 838 and the final specific activity was 1.3 nkat.(mg protein)-1. On denaturing gels the major polypeptide was 120 kDa but there was also a variable amount of smaller polypeptides in the range of 90 to 110 kDa. A new activity stain was developed to allow visualization of SPS in gels. The holoenzyme had a molecular weight of about 240 and 480 kDa in native gels and Sepharose, respectively. A high-titre polyclonal antibody was obtained which reacted with SPS from other species including wheat, potato, banana and maize. Screening of a spinach-leaf cDNA-expression library with the antibody allowed the isolation of a full-length clone. Sequencing revealed a predicted molecular weight of 117649 Da, and considerable homology with the recently published sequence for maize leaf (Worrell et al. 1991, Plant Cell 3, 1121-1130). Expression of the spinach-leaf SPS gene in Escherichia coli resulted in biological activity, revealed by the presence of SPS activity in extracts and the accumulation of sucrose-6-phosphate and sucrose in the bacteria.