Modes of hydrocarbon oil biosynthesis revealed by comparative gene expression analysis for race A and race B strains of Botryococcus braunii.

To clarify the oil biosynthetic routes of the oil-producing green alga Botryococcus braunii, here the race-specific gene expression patterns were examined using representative strains of race A and race B producing fatty acid- and triterpene-derived hydrocarbon oils, respectively. The strain-specific gene expression patterns in the BOT-88-2 strain (race A) and the BOT-22 strain (race B) were revealed by transcriptome comparison and real-time PCR quantification. For race A, it was inferred from the gene expression patterns that the fatty acid elongation in the acyl-carrier-protein (acp)-bound form followed by further elongation in the coenzyme A (CoA)-bound form is the major route of oil biosynthesis. The fatty acids may be desaturated in both acp- and CoA-bound forms and once metabolized into glycerolipids prior to further elongation. For race B, relatively direct entry of photosynthetic products from the reductive pentose phosphate cycle into the mevalonate-independent triterpene biosynthesis was implicated.

Transcriptome analysis of an oil-rich race B strain of Botryococcus braunii (BOT-70) by de novo assembly of 5'-end sequences of full-length cDNA clones.

Here the transcriptome of an oil-rich race B strain of Botryococcus braunii (BOT-70) was analyzed to mine genetic information useful in biofuel development. A full-length-enriched cDNA library was constructed via the oligo-capping method and the 5' ends of 11,904 randomly chosen cDNA clones were sequenced. Homology search using BLASTX identified candidate BOT-70 genes for majority of the reactions required for biosynthesis of botryococcenes through the mevalonate-independent pathway. The sequence retrieval from the transcriptome dataset implicated that an alternative entry route into the mevalonate-independent pathway via xylulose-5-phosphate, rather than the conventional entry route via 1-deoxy-d-xylulose-5-phosphate, is predominantly active. Analysis of N-terminal sequences of the retrieved genes indicated that the final reactions of botryococcene biosynthesis are likely to take place outside of chloroplasts. The transcriptome dataset has been deposited in the GenBank/EMBL/DDBJ database.

Transcriptome analysis of an oil-rich race B strain of Botryococcus braunii (BOT-22) by de novo assembly of pyrosequencing cDNA reads.

To gain genetic insights into the biosynthesis of botryococcene oils in Botryococcus braunii race B, a transcriptome dataset of the BOT-22 strain containing 27,427 non-redundant sequences assembled from 209,429 complementary DNA reads was obtained via high-throughput 454 sequencing. Relatively reliable prediction of the gene product was feasible for 725 non-redundant sequences based on homology to previously characterized database sequences. Regarding the botryococcene oil biosynthesis, genes putatively associated with the mevalonate-independent isoprenoid biosynthesis pathway were retrieved, while no genes were found for the mevalonate pathway, suggesting that botryococcenes are biosynthesized through the mevalonate-independent pathway in B. braunii. All transcriptome sequences have been deposited in the GenBank/EMBL/DDBJ database.

Transcriptome analysis of an oil-rich race A strain of Botryococcus braunii (BOT-88-2) by de novo assembly of pyrosequencing cDNA reads.

To gain genetic information of oil-producing algae Botryococcus braunii, a novel dataset of 185,936 complementary DNA (cDNA) reads was obtained via pyrosequencing for the representative race A strain (strain BOT-88-2) exhibiting high oil productivity. The cDNA reads were assembled to retrieve 29,038 non-redundant sequences and 964 of them were successfully annotated based on similarity to database sequences. The transcriptome data embraced candidate genes for majority of enzymes involved in the biosynthesis of unsaturated very long-chain fatty acids. The transcriptome dataset has been deposited in the GenBank/EMBL/DDBJ database.

Ethylene and salicylic acid control glutathione biosynthesis in ozone-exposed Arabidopsis thaliana.

Ozone produces reactive oxygen species and induces the synthesis of phytohormones, including ethylene and salicylic acid. These phytohormones act as signal molecules that enhance cell death in response to ozone exposure. However, some studies have shown that ethylene and salicylic acid can instead decrease the magnitude of ozone-induced cell death. Therefore, we studied the defensive roles of ethylene and salicylic acid against ozone. Unlike the wild-type, Col-0, Arabidopsis mutants deficient in ethylene signaling (ein2) or salicylic acid biosynthesis (sid2) generated high levels of superoxide and exhibited visible leaf injury, indicating that ethylene and salicylic acid can reduce ozone damage. Macroarray analysis suggested that the ethylene and salicylic acid defects influenced glutathione (GSH) metabolism. Increases in the reduced form of GSH occurred in Col-0 6 h after ozone exposure, but little GSH was detected in ein2 and sid2 mutants, suggesting that GSH levels were affected by ethylene or salicylic acid signaling. We performed gene expression analysis by real-time polymerase chain reaction using genes involved in GSH metabolism. Induction of gamma-glutamylcysteine synthetase (GSH1), glutathione synthetase (GSH2), and glutathione reductase 1 (GR1) expression occurred normally in Col-0, but at much lower levels in ein2 and sid2. Enzymatic activities of GSH1 and GSH2 in ein2 and sid2 were significantly lower than in Col-0. Moreover, ozone-induced leaf damage observed in ein2 and sid2 was mitigated by artificial elevation of GSH content. Our results suggest that ethylene and salicylic acid protect against ozone-induced leaf injury by increasing de novo biosynthesis of GSH.

Cytosolic dehydroascorbate reductase is important for ozone tolerance in Arabidopsis thaliana.

Dehydroascorbate reductase (DHAR) is a key component of the ascorbate recycling system. Three functional DHAR genes are encoded in the Arabidopsis genome. Ozone exposure increased the expression of the cytosolic DHAR (cytDHAR) gene alone. We characterized an Arabidopsis mutant with a deficient cytDHAR. The mutant completely lacked cytDHAR activity and was highly ozone sensitive. The amounts of total ascorbate and glutathione were similar in both lines, but the amount of apoplastic ascorbate in the mutant was 61.5% lower. These results indicate that the apoplastic ascorbate, which is generated through the reduction of DHA by cytDHAR, is important for ozone tolerance.

Salicylic acid accumulation under O3 exposure is regulated by ethylene in tobacco plants.

Ozone (O3), a major photochemical oxidant, induces leaf injury concomitant with salicylic acid (SA) synthesis. In pathogen-infected leaves, SA is synthesized via two pathways, involving phenylalanine or isochorismate. SA biosynthesis under O3 fumigation is not well understood. When we applied 14C-labeled benzoic acid (a precursor of SA in the pathway via phenylalanine) to O3-exposed tobacco leaves, it was effectively metabolized to SA. However, the activity and mRNA level of isochorismate synthase (ICS) were not increased. In contrast, ICS activity was increased in O3-exposed Arabidopsis thaliana L. These results suggest that SA is synthesized via benzoic acid from phenylalanine in O3-exposed tobacco leaves but via isochorismate in Arabidopsis. Ethylene is a plant hormone that promotes leaf damage in O3-exposed plants. During O3 exposure, transgenic plants with a phenotype of reduced O3-induced ethylene production accumulated less SA than did wild-type plants. O3 increased the activity of phenylalanine ammonia-lyase (PAL) and the transcript levels of the chorismate mutase (CM) and PAL genes in wild-type tobacco, but their induction was suppressed in the transgenic plants. These results indicate that ethylene promotes SA accumulation by regulating the expression of the CM and PAL genes in O3-exposed tobacco.