SummaryThe Arabidopsis bZIP transcription factor gene ATB2 has been shown previously to be expressed in a light-regulated and tissue-specific way. Here we describe the precise localization of ATB2 expression, using transgenic lines containing an ATB2 promoter-GUS reporter gene construct. The observed expression pattern suggests a role for ATB2 in the control of processes associated with the transport or utilization of metabolites. Remarkably, expression of the ATB2-GUS reporter gene construct was specifically repressed by sucrose. Other sugars, such as glucose and fructose, alone or in combination, were ineffective. Repression was observed at external sucrose concentrations exceeding 25 mM. Transcript levels of both the endogenous ATB2 gene and the ATB2-GUS reporter gene were not repressed by sucrose, suggesting that sucrose affects mRNA translation. This translational regulation involves the ATB2 leader sequence because deletion of the leader resulted in loss of sucrose repression. Our results provide evidence for a sucrose-specific sugar sensing and signalling system in plants.

The question whether sucrose (Suc) is present inside plastids has been long debated. Low Suc levels were reported to be present inside isolated chloroplasts, but these were argued to be artifacts of the isolation procedures used. We have introduced Suc-metabolizing enzymes in plastids and our experiments suggest substantial Suc entry into plastids. The enzyme levansucrase from Bacillus subtilis efficiently synthesizes fructan from Suc. Targeting of this enzyme to the plastids of tobacco (Nicotiana tabacum) and potato (Solanum tuberosum) plants leads to high-level fructan accumulation in chloroplasts and amyloplasts, respectively. Moreover, introduction of this enzyme in amyloplasts leads to an altered starch structure. Expression of the yeast invertase in potato tuber amyloplasts results in an 80% reduction of total Suc content, showing efficient hydrolysis of Suc by the plastidic invertase. These observations suggest that Suc can enter plastids efficiently and they raise questions as to its function and metabolism in this organelle.Plastids are of tremendous metabolic importance. Next to photosynthesis they are involved in the synthesis of fatty acids, amino acids, starch, and many compounds of secondary metabolism. This diverse metabolic capacity of plastids requires an extensive array of selective transporting systems for interaction with other cellular compartments. Plastids are surrounded by two membranes, the inner and the outer membrane. In the inner membrane of the plastid envelope, many metabolite specific transporters are present, whereas the outer membrane contains nonspecific porin-like channels. The envelope outer membrane was proposed to be non-selective and permeable for many small molecules (Heldt and Sauer, 1971). However, recent data suggest that outer membranes can also act as selective and regulated molecular sieves (Flü gge, 2000; Neuhaus and Wagner, 2000; Soll et al., 2000).Several metabolite transporters in plastids have now been identified (Emes and Neuhaus, 1997; Flü gge, 1998; Neuhaus and Wagner, 2000). The wellknown triose phosphate/phosphate translocator exports the triose phosphates generated by photosynthetic CO 2 fixation into the cytosol. The phosphoenolpyruvate/phosphate translocator is responsible for the import of phosphoenolpyruvate into plastids for several plastidic metabolic pathways, like the shikimate pathway or amino acid synthesis (Streatfield et al., 1999). Another phosphate antiporter is the Glc-6-P/phosphate translocator (Naeem et al., 1997;Wischmann et al., 1999). The imported Glc-6-P in amyloplasts can be used for starch biosynthesis or in the oxidative pentose phosphate pathway (Naeem et al., 1997). Next to sugar-phosphates, unphosphorylated carbohydrates like Glc and maltose can be transported (Schleucher et al., 1998) and recently a gene encoding plastidic Glc translocator was identified (Weber et al., 2000). Furthermore, plastids contain transporters involved in ammonia and nitrogen assimilation, transporting Glu, Gln, and oxaloacetate in exchange for malat...

Es:sr.herichia coli outer-membrane phospholipase A (OMPLA) is thought to be a tnember of the class of serine hydrolases, having a classical Asp-His-Ser catalytic triad [Horrevoets, A. J. G., Verheij, k1. M. & de Haas, G. H. (199'1) Eicr: .I. Biocherrr. 198, 247-2531. To idenlify the histidinc residue that is important for catalytic activity, the four histidine residues in E. c d i OMPLA that arc conserved in other enterobacterial OMPLA enzymes werc replaced by cysteine residues using PCR-directed, site-specific mutagenesis. The resulting mutant proteins were all well cxpressed and displayed heat modifiability, indicating that they were properly folded. Enzyme assays showed that only the Hisl42Cys mutant protein was lacking enzymatic activity. In addition, a His142Gly mutant protein appeared to he inactive. These result? show that His142 is important for the enzymatic activity of OMPLA.Keqrwords: outer-membrane protein; phospholipase A; active site; site-directed mutagenesis; Escherichiu coli.Outer-membrane phospholipase A (OMPLA) of Es:Jheric.hiti coli is a 30-kDa protein that is eiicodcd by the p1dA gene. It is widely disseminated among members of the Enterobacteriaceae family (Brok et al., 1994), but its physiological role is still unknown. It has been shown that R. coli OMPLA is required for efficient secretion of bacteriocins (Pugsley and Schwiutn, 1984; Luirink et al., 1986), but it is unlikely that this is the primary [unction of the protein. OMPLA activity must be well regulated since otherwise the enzyme would degrade the cell envelope. In normally growing cells, OMPLA appears to be dormant (A~idet et al., 1974). However, high OMPLA activity can be induced by damaging the membrane, e.g. by phage-induced lysis (Cronan and Wulff, 1960) or by temperalure shock (de Geus et al., 19x3). The 1~ldA genes of E. coli (Homtnii et al., 1984), Snlm!pplonell~i typhimurium, Klebsiellci pneurnoniuf,, and Proteus vulgnris (Rrok et al., 1094) have been cloned and sequenced, and they show high mutual sequence similarity.OMPLA has several enzymatic activities, i.e. those of phospholipases A, and A,, 1 -acyl-and 2-acyl-lysophospholipase, and diacylglycerol lipase, with (he phospholipase A, activity being six times higher thnn the phospholipase A, activity (Horrevoets et nl., 1989). It has bcen suggested (Horrevoets et al., 1991) that OMPLA belongs 10 thc class of serine hydrolases whose members catalyse cster hydrolysis via an ucyl cnzyme interniediate. However, in contrast to serine proteases (Kraut, 1977), OMPI>A is not inactivated by water-solublc serine hydrolase inhibitors like diisopropyltluorophosphate and phcnylmethylsulfotiyl fluoride. Only water-insoluble inhibitors, like n-hexadecylsulfonyl C~f~rrt..s~~o~~rluncr to H. M. Verheij,

SummaryThe consumption of fructans as a low caloric food ingredient or dietary fibre is rapidly increasing due to health benefits. Presently, the most important fructan source is chicory, but these fructans have a simple linear structure and are prone to degradation. Additional sources of high-quality tailor-made fructans would provide novel opportunities for their use as food ingredients. Sugar beet is a highly productive crop that does not normally synthesize fructans. We have introduced specific onion fructosyltransferases into sugar beet. This resulted in an efficient conversion of sucrose into complex, onion-type fructans, without the loss of storage carbohydrate content.

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