NADPH-protochlorophyllide oxidoreductase (POR; EC 1.6.99.1) catalyzes the only known light-dependent step in chlorophyll synthesis of higher plants, the reduction of protochlorophyllide (Pchlide) to chlorophyllide. In barley, two distinct immunoreactive POR proteins were identified. In contrast to the light-sensitive POR enzyme studied thus far (POR-A), levels of the second POR protein remained constant in seedlings during the transition from dark growth to the light and in green plants. The existence of a second POR-related protein was verified by isolating and sequencing cDNAs that encode a second POR polypeptide (POR-B) with an amino acid sequence identity of 75% to the POR-A. In the presence of NADPH and Pchlide, the in vitro-synthesized POR-A and POR-B proteins could be reconstituted to ternary enzymatically active complexes that reduced Pchlide to chlorophyllide only after illumination. Even though the in vitro activities of the two enzymes were similar, the expression of their genes during the light-induced transformation of etiolated to green seedlings was distinct. While the POR-A mRNA rapidly declined during illumination of dark-grown seedlings and soon disappeared, POR-B mRNA remained at an approximately constant level in dark-grown and green seedlings. Thus these results suggest that chlorophyll synthesis is controlled by two light-dependent POR enzymes, one that is active only transiently in etiolated seedlings at the beginning of illumination and the other that also operates in green plants.

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letters to nature 80 NATURE | VOL 397 | 7 JANUARY 1999 | www.nature.com (Molecular Probes) was dissolved at 1 mg ml -1 in N-N-dimethylformamide and ionophoretically injected into the neuromuscular cleft. Embryos were placed in 1 3 PBS overnight at 4 8C, mounted in 50% glycerol plus 2% peraformaldehyde, and viewed on a Zeiss confocal microscope. Generation of transformants. Restriction fragments from lim3 genomic phage clones 9 were inserted into P[tau-myc] 4 or HZ50PL lacZ 28 plasmids and transformation was done as described 29 . Three independent lim3A-tau-myc lines and one lim3A-lacZ line were tested. All displayed similar expression patterns. Two of the lim3A-tau-myc insertions, denoted 1.8 and 1.13, were crossed into lim3 mutant backgrounds and gave identical results. UAS-lim3 was made by inserting the lim3 cDNA plus 50 base pairs of the Xenopus laevis b-globin 59 leader from plasmid pNB 25 into plasmid pUAS 18 . For the misexpression studies, several independent transgenic lines, each carrying one or two copies of UAS-lim3, were used. The ftz ng -GAL4.20 driver. To improve the expression levels of the original ftz ng -GAL4 line 19 , we generated 25 new insertion lines by transposase-mediated mobilization. Each line was tested for GAL4 expression by crossing it to¯ies carrying a UAS-tau-myc-GFP reporter 30 , where GFP is green¯uorescent protein. One line, ftz ng -GAL4.20, expressed GAL4 at high levels in most, if not all, motor neurons. As assayed by transactivation of the UAS-tau-myc reporter gene, 89% of hemisegments showed labelling in ISNb and 84% showed labelling in ISNd (n 56).

Plant tissues treated with the naturally occurring cyclopentanone compound methyl jasmonate or exposed to stress causing in panta jasmonate acmulation express disnctive proteins and, concomitantly, reduce the synthesis of most preexsting proteins. One of the recently identified jasmonate-induced proteins, designated JIP6O, in barley is a ribosome-inactivating protein that cleaves polysomes of both animal and plant origin into their ribosomal subunits. By attcing foreign and self ribosomes, respectively, JIP60 appears to be both a defense protein and a potent regulator of protein synthesis in stressed plant tissues.Jasmonates are cyclopentanone derivatives (1, 2) that control plant growth and development in a pleiotropic manner (3). Processes influenced by jasmonates in diverse plant species include embryogenesis and seed germination, leaf senescence and abscission, and stomata closure, for example (for review, see ref.3). Recent results indicate that the methyl ester of (-

Plants are continuously challenged by a variety of abiotic and biotic cues. To deter feeding insects, nematodes and fungal and bacterial pathogens, plants have evolved a plethora of defence strategies. A central player in many of these defence responses is jasmonic acid. It is the aim of this minireview to summarize recent findings that highlight the role of jasmonic acid during programmed cell death, plant defence and leaf senescence.

Chloroplasts synthesize an abundance of different tetrapyrrole compounds. Among them are chlorophyll and its precursor protochlorophyllide (Pchlide), which accumulate in light-and dark-grown plants, respectively. Pchlide is converted to chlorophyllide by virtue of the NADPH:Pchlide oxidoreductase (POR), which, in angiosperms, is the only known light-dependent enzyme of the chlorophyll biosynthetic pathway. In etiolated barley plants, two closely related POR proteins exist termed PORA and PORB, which are nuclear gene products. Here we identified plastid envelope proteins that interact with the cytosolic PORA precursor (pPORA) during its posttranslational chloroplast import. We demonstrate that pPORA interacts with several previously unreported components. Among them is a Pchlide a oxygenase, which provides Pchlide b as import substrate for pPORA, and a tyrosine aminotransferase thought to be involved in the synthesis of photoprotective vitamin E. Two other constituents were found to be orthologs of the GTP-binding proteins Toc33͞34 and of the outer plastid envelope protein Oep16.

The key regulatory enzyme of chlorophyll biosynthesis in higher plants, the light-dependent NADPH:protochlorophyllide oxidoreductase (POR), is a nuclear-encoded plastid protein. Its post-translational transport into plastids is determined by its substrate. The precursor of POR (pPOR) is taken up and processed to mature size by plastids only in the presence of protochlorophyllide (Pchlide). In etioplasts, the endogenous level of Pchlide saturates the demands for pPOR translocation. During the light-induced transformation of etioplasts into chloroplasts, the Pchlide concentration declined drastically, and isolated chloroplasts rapidly lost the ability to import the precursor enzyme. The chloroplasts' import capacity for the pPOR, however, was restored when their intraplastidic level of Pchlide was raised by incubating the organelles in the dark with delta-aminolevulinic acid, a common precursor of tetrapyrroles. Additional evidence for the involvement of intraplastidic Pchlide in regulating the transport of pPOR into plastids was provided by experiments in which barley seedlings were grown under light/dark cycles. The intraplastidic Pchlide concentration in these plants underwent a diurnal fluctuation, with a minimum at the end of the day and a maximum at the end of the night period. Chloroplasts isolated at the end of the night translocated pPOR, whereas those isolated at the end of the day did not. Our results imply that the Pchlide-dependent transport of the pPOR into plastids might be part of a novel regulatory circuit by which greening plants fine tune both the enzyme and pigment levels, thereby avoiding the wasteful degradation of the imported pPOR as well as photodestruction of free Pchlide.