SummaryPlant isoprenoids represent a heterogeneous group of compounds which play essential roles not only in growth and development, but also in the interaction of plants with their environment. Higher plants contain two pathways for the biosynthesis of isoprenoids: the mevalonate pathway, located in the cytosol/endoplasmic reticulum, and the recently discovered mevalonate-independent pathway (Rohmer pathway), located in the plastids. In order to evaluate the function of the Rohmer pathway in the regulation of the synthesis of plastidial isoprenoids, we have isolated a tomato cDNA encoding 1-deoxy-D-xylulose 5-phosphate synthase (DXS), the ®rst enzyme of the pathway. We demonstrate in vivo activity and plastid targeting of plant DXS. Expression analysis of the tomato DXS gene indicates developmental and organ-speci®c regulation of mRNA accumulation and a strong correlation with carotenoid synthesis during fruit development. 1-Deoxy-D-xylulose feeding experiments, together with expression analysis of DXS and PSY1 (encoding the fruit-speci®c isoform of phytoene synthase) in wildtype and yellow¯esh mutant fruits, indicate that DXS catalyses the ®rst potentially regulatory step in carotenoid biosynthesis during early fruit ripening. Our results change the current view that PSY1 is the only regulatory enzyme in tomato fruit carotenogenesis, and point towards a coordinated role of both DXS and PSY1 in the control of fruit carotenoid synthesis.

SummaryThe recently discovered 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for the biosynthesis of plastid isoprenoids (including carotenoids) is not fully elucidated yet despite its central importance for plant life. It is known, however, that the ®rst reaction completely speci®c to the pathway is the conversion of 1-deoxy-D-xylulose 5-phosphate (DXP) into MEP by the enzyme DXP reductoisomerase (DXR). We have identi®ed a tomato cDNA encoding a protein with homology to DXR and in vivo activity, and show that the levels of the corresponding DXR mRNA and encoded protein in fruit tissues are similar before and during the massive accumulation of carotenoids characteristic of fruit ripening. The results are consistent with a non-limiting role of DXR, and support previous work proposing DXP synthase (DXS) as the ®rst regulatory enzyme for plastid isoprenoid biosynthesis in tomato fruit. Inhibition of DXR activity by fosmidomycin showed that plastid isoprenoid biosynthesis is required for tomato fruit carotenogenesis but not for other ripening processes. In addition, dormancy was reduced in seeds from fosmidomycin-treated fruit but not in seeds from the tomato yellow ripe mutant (defective in phytoene synthase-1, PSY1), suggesting that the isoform PSY2 might channel the production of carotenoids for abscisic acid biosynthesis. Furthermore, the complete arrest of tomato seedling development using fosmidomycin con®rms a key role of the MEP pathway in plant development.

SummaryPlants are unique in the obligatory nature of their exposure to sunlight and consequently to ultraviolet (UV) irradiation. However, our understanding of plant DNA repair processes lags far behind the current knowledge of repair mechanisms in microbes, yeast and mammals, especially concerning the universally conserved and versatile dark repair pathway called nucleotide excision repair (NER). Here we report the isolation and functional characterization of Arabidopsis thaliana AtRAD1, which encodes the plant homologue of Saccharomyces cerevisiae RAD1, Schizosaccharomyces pombe RAD16 and human XPF, endonucleolytic enzymes involved in DNA repair and recombination processes. Our results indicate that AtRAD1 is involved in the excision of UV-induced damages, and allow us to assign, for the ®rst time in plants, the dark repair of such DNA lesions to NER. The low ef®ciency of this repair mechanism, coupled to the fact that AtRAD1 is ubiquitously expressed including tissues that are not accessible to UV light, suggests that plant NER has other roles. Possible`UV-independent' functions of NER are discussed with respect to features that are particular to plants.

Isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) constitute the basic building block of isoprenoids, a family of compounds that is extraordinarily diverse in structure and function. IPP and DMAPP can be synthesized by two independent pathways: the mevalonate pathway and the recently discovered 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. Although the MEP pathway is essential in most eubacteria, algae and plants and has enormous biotechnological interest, only some of its steps have been determined. We devised a system suitable for the genetic analysis of the MEP pathway in Escherichia coli. A synthetic operon coding for yeast 5-diphosphomevalonate decarboxylase, human 5-phosphomevalonate kinase, yeast mevalonate kinase and E. coli isopentenyl diphosphate isomerase was incorporated in the chromosome of this bacterium. The expression of this operon allowed the synthesis of IPP and DMAPP from mevalonate added exogenously and complementation of lethal mutants of the MEP pathway. We used this system to show that the ygbP, ychB and ygbB genes are essential in E. coli and that the steps catalysed by the products of these genes belong to the trunk line of the MEP pathway.

All living organisms have to protect the integrity of their genomes from a wide range of genotoxic stresses to which they are inevitably exposed. However, understanding of DNA repair in plants lags far behind such knowledge in bacteria, yeast, and mammals, partially as a result of the absence of efficient in vitro systems. Here, we report the experimental setup for an Arabidopsis in vitro repair synthesis assay. The repair of plasmid DNA treated with three different DNA-damaging agents, UV light, cisplatin, and methylene blue, after incubation with whole-cell extract was monitored. To validate the reliability of our assay, we analyzed the repair proficiency of plants depleted in AtRAD1 activity. The reduced repair of UV light-and cisplatin-damaged DNA confirmed the deficiency of these plants in nucleotide excision repair. Decreased repair of methylene blue-induced oxidative lesions, which are believed to be processed by the base excision repair machinery in mammalian cells, may indicate a possible involvement of AtRAD1 in the repair of oxidative damage. Differences in sensitivity to DNA polymerase inhibitors (aphidicolin and dideoxy TTP) between plant and human cell extracts were observed with this assay. INTRODUCTIONThe genomes of all living organisms are constantly subjected to a wide range of genotoxic stresses induced by environmental factors (e.g., UV-B irradiation, bacterial and fungal toxins) as well as by the intermediate products of normal cellular metabolism (e.g., alkylating and oxidizing agents). These can lead to the formation of different types of DNA damage, the persistence of which can block DNA replication and transcription or cause cell cycle arrest and apoptosis (Britt, 1999;Lindahl and Wood, 1999). Incorrect repair can result in heritable point mutations or gross rearrangements such as deletions and insertions. To keep the integrity of their genomes, all organisms have evolved protective mechanisms of repair of a broad variety of DNA lesions. According to the mode of action, the substrate specificity, and the size of the excised DNA fragment, these pathways generally have been classified as direct repair, base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (reviewed by Friedberg, 1996;Sancar, 1996;Wood, 1996;Lindahl and Wood, 1999). These mechanisms were first described in bacteria and later characterized extensively in yeast and mammals (Sancar, 1996;Laat et al., 1999;Le Page et al., 2000;Memisoglu and Samson, 2000). Unfortunately, with the exception of light-dependent reversion of UV light-induced pyrimidine dimers by photolyases, very little is known about DNA repair pathways in plants (reviewed by Vonarx et al., 1998; Britt, 1999).To study different types of DNA repair mechanisms in vitro, the formation of pathway-specific DNA lesions is required. For the study of NER, we chose the following DNAdamaging agents: UV-C and cis -diamminedichloroplatinum II (cisplatin). Additionally, methylene blue-treated DNA was used to distinguish the relative contributions of ...