We have isolated a cDNA (PAR corresponding to a single nuclear gene that encodes an approximately 30-kD major protein of bell pepper (Capsicum annuum L.) fruit chromoplasts. RNA and protein analyses revealed that, although at a low level, this gene is also expressed in every organ of the plant, the amount of the corresponding transcript and protein dramatically increasing in the latter stages of fruit development. Western-blot and immunocytochemical analyses of purified chloroplasts from leaves and fruits and of chromoplasts from red fruits showed that the encoded protein is the major component of plastoglobules and fibrils and is localized on the outer surface of these lipid structures. Analyses of PAPin plants belonging to different taxa revealed that it is expressed and highly conserved in both monocotyledonous and dicotyledonous plants. The presence of the protein in plastids not differentiating into chromoplasts indicates that PAP is expressed irrespective of the ontogeny of various plastid lines. In light of our results and since the encoded protein, identical to that previously named ChrB or fibrillin, is present in plastoglobules from several species and accumulates in the fibrils of bell pepper chromoplast, we propose to designate it as a plastid-lipid-associated protein.

We have isolated a 454-bp cDNA that encodes a nove1 fruit-specific defensin from bell pepper (Capsicum annuum). The encoded 75-amino-acid polypeptide contains an N-terminal domain characteristic of a signal peptide and a 48-amino-acid mature domain named 11. The mature protein, from which the N-terminal amino acid sequence was determined, contains eight cysteines that form four intramolecular disulfide bridges, suggesting a monomeric form for 11. In healthy fruits J1 is undetectable at the green stage but high levels accumulate during ripening. In wound areas of the green fruit the accumulation of 11 dramatically increased, suggesting a role for Jl in the plant's defense response. Moreover, we have demonstrated that 11 possesses an antifungal activity. We have isolated and characterized the corresponding two homologous genes (jl-1 and jl-2) that exist in the hell pepper genome. Both genes are interrupted hy the insertion, at the same position, of one intron of 853 bp for j7-7 and 4900 bp for j7-2. Northern blot and reverse transcriptase-polymerase chain reaction and restriction fragment length polymorphism analyses revealed that jl-7 transcripts are present only in fruits, only in trace amounts in mature green fruits, and that they accumulate to high levels in fully ripe fruits, whereas no jl-2 transcripts were detected in the samples monitored.

Cell-specific and light-regulated expression of the .8-glucuronidase (GUS) reporter gene from maize cab-ml and rbcS-m3 promoter sequences was studied in maize leaf segments by using an in situ transient expression microprojectile bombardment assay. The cab-ml gene is known to be strongly photoregulated and to be expressed almost exclusively in mesophyll cells (MC) but not in bundle sheath cells (BSC).Expression of GUS from a 1026-base-pair 5' promoter fragment of cab-ml is very low in dark-grown leaves; GUS expression is increased about 10-fold upon illumination ofdark-grown leaves. In illuminated leaves, the ratio of GUS expression in MC vs. BSC is about 10:1. The cab-ml region between 868 and 1026 base pairs 5' to the translation start confers strong MC-preferred expression on the remainder of the chimeric gene in illuminated leaves, but a region between -39 and -359 from the translation start is required for photoregulated expression. Transcripts of rbcS-m3 are found in BSC but not in MC and are about double in BSC of greening dark-grown seedlings. In contrast to the behavior of the cab-ml-GUS construct, GUS expression driven by 2.1 kilobase pairs of the rbcS-m3 5' region was about twice as high in MC as in BSC of unilluminated dark-grown maize leaves. The number of BSC, but not MC, expressing GUS nearly doubled upon greening of bombarded etiolated leaves. These data suggest that the 5' region ofrbcS-m3 used here could be responsible for most ofthe light-dependent increase in rbcS-m3 transcripts observed in BSC of greening leaves and that transcriptional or posttranscriptional mechanisms are responsible for the lack of rbcS-m3 transcripts in MC. Although the recent generation of transgenic maize (11) carrying 8-glucuronidase (GUS) reporter gene constructs makes this possible in principle, maize transformation and regeneration procedures are lengthy and thus are not convenient for the analysis of numerous modified forms of genes.We have found that DNA precipitated on tungsten microprojectiles can be delivered into MC and BSC in situ in maize leaf segments and that MC-specific and light-induced GUS expression from the cab-ml promoter1 requires two widely separated sequences 5' to the translation start site. In contrast, GUS expression from the rbcS-m3 promoter is about the same in MC and BSC of 24-hr greened leaves, but expression of the rbcS chimeric gene is promoted by light in BSC. MATERIALS AND METHODSPlant Material. Seeds of maize (Zea mays; FR9cms x FR37; Illinois Foundation Seeds, Champaign, IL) were sown in damp Vermiculite and grown at 300C in darkness. Ten-dayold seedlings were harvested under a dim-green safelight, and their second leaves were used in the transient in situ expression assay described below.Transient in Situ Expression Assay. Four 3.5-cm-long segments of the upper halves of the second leaves of 10-day-old dark-grown maize seedlings, a total area of about 12 cm2, were flattened on 0.8% agar Murashige and Skoog medium (GIBCO) in a 5-cm Petri plate with the lower epidermis facing ...

Geranylgeranyl pyrophosphate synthase is a key enzyme in plant terpenoid biosynthesis. Using specific antibodies, a cDNA encoding geranylgeranyl pyrophosphate synthase has been isolated from bell pepper (Capsicum annuum) ripening fruit. The cloned cDNA codes for a high molecular weight precursor of 369 amino acids which contains a transit peptide of approximately 60 amino acids. In-situ immunolocalization experiments have demonstrated that geranylgeranyl pyrophosphate synthase is located exclusively in the plastids. Expression of the cloned cDNA in E. coli has unambiguously demonstrated that the encoded polypeptide catalyzes the synthesis of geranylgeranyl pyrophosphate by the addition of isopentenyl pyrophosphate to an allylic pyrophosphate. Peptide sequence comparisons revealed significant similarity between the sequences of the C. annuum geranylgeranyl pyrophosphate synthase and those deduced from carotenoid biosynthesis (crtE) genes from photosynthetic and non-photosynthetic bacteria. In addition, four highly conserved regions, which are found in various prenyltransferases, were identified. Furthermore, evidence is provided suggesting that conserved and exposed carboxylates are directly involved in the catalytic mechanism. Finally, the expression of the geranylgeranyl pyrophosphate synthase gene is demonstrated to be strongly induced during the chloroplast to chromoplast transition which occurs in ripening fruits, and is correlated with an increase in enzyme activity.