Carnivorous plants acquire significant amounts of nitrogen from insects. The tropical carnivorous plant Nepenthes accumulates acidic fluid containing aspartic proteinase (AP) in its trapping organs (pitchers), suggesting that the plant utilizes insect protein as a nitrogen source. Aspartic proteinases have been purified and characterized from sterile pitcher fluid of several species of Nepenthes; however, there is, as of yet, no information about sequence and expression of Nepenthes AP genes. To identify the pitcher AP, we cloned plant AP homologs from N. alata and examined their expressions. Five AP homologs ( NaAP1-NaAP5) were obtained by reverse transcription-polymerase chain reaction with degenerate primers designed for the conserved sequences of plant APs. Alignment of deduced amino acid sequences with other plant APs demonstrated that NaAP1-NaAP4 contained a plant-specific insert (PSI), a unique sequence of plant AP. However, NaAP5 did not possess the insert, and had a shorter sequence (by >100 amino acids) than the other APs. Northern analysis using a part of the coding region of NaAP1 as a probe showed that bands of approx. 1.8 kb corresponding to the sizes of NaAP1-NaAP4 mRNA were present in roots, stems, leaves, tendrils, and lower part of the pitchers, but a band of approx. 1.3 kb corresponding to the size of NaAP5 mRNA was not observed in any organs. In pitchers, highest expressions of NaAP1-NaAP4 were seen in the lower part of open pitchers containing natural prey, suggesting that the expressions of NaAP1-NaAP4 are coupled with prey capture. Transcripts of NaAP2 and NaAP4 were detected in the digestive glands, where AP secretion may occur. This result suggests that NaAP2 and NaAP4 are the possible APs secreted into the pitcher of N. alata.
Nepenthes is a unique genus of carnivorous plants that can capture insects in trapping organs called pitchers and digest them in pitcher fluid. The pitcher fluid includes digestive enzymes and is strongly acidic. We found that the fluid pH decreased when prey accumulates in the pitcher fluid of Nepenthes alata. The pH decrease may be important for prey digestion and the absorption of prey-derived nutrients. To identify the proton pump involved in the acidification of pitcher fluid, plant proton-pump homologs were cloned and their expressions were examined. In the lower part of pitchers with natural prey, expression of one putative plasma-membrane (PM) H+-ATPase gene, NaPHA3, was considerably higher than that of the putative vacuolar H+-ATPase (subunit A) gene, NaVHA1, or the putative vacuolar H+-pyrophosphatase gene, NaV-HP1. Expression of one PM H+-ATPase gene, Na-PHA1, was detected in the head cells of digestive glands in the lower part of pitchers, where proton extrusion may occur. Involvement of the PM H+-ATPase in the acidification of pitcher fluid was also supported by experiments with proton-pump modulators; vanadate inhibited proton extrusion from the inner surface of pitchers, whereas bafilomycin A1 did not, and fusicoccin induced proton extrusion. These results strongly suggest that the PM H+-ATPase is responsible for acidification of the pitcher fluid of Nepenthes.
Solanesyl diphosphate (SPP) is regarded as the precursor of the side-chains of both plastoquinone and ubiquinone in Arabidopsis thaliana. We previously analyzed A. thaliana SPP synthase (At-SPS1) (Hirooka et al., Biochem. J., 370, 679-686 (2003)). In this study, we cloned a second SPP synthase (At-SPS2) gene from A. thaliana and characterized the recombinant protein.Kinetic analysis indicated that At-SPS2 prefers geranylgeranyl diphosphate to farnesyl diphosphate as the allylic substrate. Several of its features, including the substrate preference, were similar to those of At-SPS1. These data indicate that At-SPS1 and At-SPS2 share their basic catalytic machinery. Moreover, analysis of the subcellular localization by the transient expression of green fluorescent protein-fusion proteins showed that At-SPS2 is transported into chloroplasts, whereas At-SPS1 is likely to be localized in the endoplasmic reticulum in the A. thaliana cells. It is known that the ubiquinone side-chain originates from isopentenyl diphosphate derived from the cytosolic mevalonate pathway, while the plastoquinone side-chain is synthesized from isopentenyl diphosphate derived from the plastidial methylerythritol phosphate pathway. Based on this information, we propose that At-SPS1 contributes to the biosynthesis of the ubiquinone side-chain and that At-SPS2 supplies the precursor of the plastoquinone side-chain in A. thaliana.Key words: isoprenoid; prenyltransferase; nonaprenyl diphosphate; plastoquinone; ubiquinone Plants have two major prenylquinones, plastoquinone and ubiquinone. 1) Although both share the structural feature of a trans-polyprenyl tail attached to the benzoquinone skeleton and have common oxidationreduction properties, their subcellular localization and biochemical roles are distinct. Plastoquinone exists in the thylakoid membrane of the chloroplast and acts as an electron carrier in the photosynthetic electron transfer reaction,2) whereas ubiquinone exists in the inner membrane of the mitochondrion and transfers electrons in the respiratory chain reaction.3)The hydrophobic tails of prenylquinones are C 30 -C 50 in length and serve as membrane anchors. The lengths of the polyprenyl side-chains differ between plastoquinone and ubiquinone, and even among plant species.4) It is thought that these polyprenyl chains are derived from C 30 -C 50 prenyl diphosphates formed by the consecutive condensation of isopentenyl diphosphate (IPP; C 5 ) with allylic diphosphate in the trans-configuration.5) As for the enzymes giving these precursors, several genes isolated from microorganisms have been well characterized, [6][7][8] but information about the plant-origin enzymes has been limited. Recently, we succeeded in the molecular cloning of Arabidopsis thaliana solanesyl diphosphate (SPP; C 45 ) synthase (At-SPS1; formerly designated At-SPS), which catalyzes the trans-type condensation of IPP to yield the C 45 product. 9) Enzymological analysis indicated that At-SPS1 utilizes both farnesyl diphosphate (FPP; C 15 ) and geranylgeranyl di...
Double-stranded RNA (dsRNA) induces sequence-specific gene silencing in eukaryotes through a process known as RNA interference (RNAi). RNAi is now used as a powerful tool for functional genomics in many eukaryotes, including plants. We herein report a dsRNA-mediated transient RNAi assay system using protoplasts from Arabidopsis mesophyll cells and suspension-cultured cells (cell line T87). Introduction of dsRNA into protoplasts led to marked silencing of target transgenes. Our assay system would provide a convenient and efficient way to induce RNAi in protoplasts of the model plant Arabidopsis thaliana.
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