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.

Kinesin-4 Functions in Vesicular Transport on Cortical Microtubules and Regulates Cell Wall Mechanics during Cell Elongation in Plants.

In plants, anisotropic cell expansion depends on cortical microtubules that serve as tracks along which macromolecules and vesicles are transported by the motor kinesins of unknown identities. We used cotton (Gossypium hirsutum) fibers that underwent robust elongation to discover kinesins that are involved in cell elongation and found Gh KINESIN-4A expressed abundantly. The motor was detected by immunofluorescence on vesicle-like structures that were associated with cortical microtubules. In Arabidopsis thaliana, the orthologous motor At KINESIN-4A/FRA1, previously implicated in cellulose deposition during secondary growth in fiber cells, was examined by live-cell imaging in cells expressing the fluorescently tagged functional protein. The motor decorated vesicle-like particles that exhibit a linear movement along cortical microtubules with an average velocity of 0.89 µm/min, which was significantly different from those linked to cellulose biosynthesis. We also discovered that At KINESIN-4A/FRA1 and the related At KINESIN-4C play redundant roles in cell wall mechanics, cell elongation, and the axial growth of various vegetative and reproductive organs, as the loss of At KINESIN-4C greatly enhanced the defects caused by a null mutation at the KINESIN-4A/FRA1 locus. The double mutant displayed a lack of cell wall softening at normal stages of rapid cell elongation. Furthermore, enhanced deposition of arabinose-containing carbohydrate was detected in the kinesin-4 mutants. Our findings established a connection between the Kinesin-4-based transport of cargoes containing non-cellulosic components along cortical microtubules and cell wall mechanics and cell elongation in flowering plants.

Ozone-Induced Rice Grain Yield Loss Is Triggered via a Change in Panicle Morphology That Is Controlled by ABERRANT PANICLE ORGANIZATION 1 Gene.

Rice grain yield is predicted to decrease in the future because of an increase in tropospheric ozone concentration. However, the underlying mechanisms are unclear. Here, we investigated the responses to ozone of two rice (Oryza Sativa L.) cultivars, Sasanishiki and Habataki. Sasanishiki showed ozone-induced leaf injury, but no grain yield loss. By contrast, Habataki showed grain yield loss with minimal leaf injury. A QTL associated with grain yield loss caused by ozone was identified in Sasanishiki/Habataki chromosome segment substitution lines and included the ABERRANT PANICLE ORGANIZATION 1 (APO1) gene. The Habataki allele of the APO1 locus in a near-isogenic line also resulted in grain yield loss upon ozone exposure, suggesting APO1 involvement in ozone-induced yield loss. Only a few differences in the APO1 amino acid sequences were detected between the cultivars, but the APO1 transcript level was oppositely regulated by ozone exposure: i.e., it increased in Sasanishiki and decreased in Habataki. Interestingly, the levels of some phytohormones (jasmonic acid, jasmonoyl-L-isoleucine, and abscisic acid) known to be involved in attenuation of ozone-induced leaf injury tended to decrease in Sasanishiki but to increase in Habataki upon ozone exposure. These data indicate that ozone-induced grain yield loss in Habataki is caused by a reduction in the APO1 transcript level through an increase in the levels of phytohormones that reduce leaf damage.

Genomic structure of the cucumber CPD photolyase gene.

Light-dependent transcriptional activation of the photolyase gene imparts UVB tolerance to a plant. In the present study, the cucumber CPD photolyase gene (CsPHR) was isolated from a genomic DNA library and its genomic structure was scrutinized. As a result, putative light-responsive cis-acting elements were found in the CsPHR promoter.

Desiccation tolerance of Botryococcus braunii (Trebouxiophyceae, Chlorophyta) and extreme temperature tolerance of dehydrated cells.

Botryococcus braunii Kützing, a green colonial microalga, occurs worldwide in both freshwater and brackish water environments. Despite considerable attention to B. braunii as a potential source of renewable fuel, many ecophysiological properties of this alga remain unknown. Here, we examined the desiccation and temperature tolerances of B. braunii using two newly isolated strains BOD-NG17 and BOD-GJ2. Both strains survived through 6- and 8-month desiccation treatments but not through a 12-month treatment. Interestingly, the desiccation-treated cells of B. braunii gained tolerance to extreme temperature shifts, i.e., high temperature (40 °C) and freezing (-20 °C). Both strains survived for at least 4 and 10 days at 40 and -20 °C, respectively, while the untreated cells barely survived at these temperatures. These traits would enable long-distance dispersal of B. braunii cells and may account for the worldwide distribution of this algal species. Extracellular substances such as polysaccharides and hydrocarbons seem to confer the desiccation tolerance.

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.

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.

Isolation of herbicide-resistant mutants of Botryococcus braunii.

Aiming at herbicide-assisted cultivation of Botryococcus braunii for prevention of algal contamination, herbicide-tolerant mutant lines of B. braunii were established for two widely used herbicides, methyl viologen and glufosinate. Some established mutant lines exhibited vigorous oil production and growth in herbicide-containing media. Because the two herbicides were effective in controlling the growth of the algal competitors of B. braunii, these mutants can be directly used in industrial attempts for cost-effective oil production in herbicide-assisted non-axenic systems. This is the first report of mutagenesis of B. braunii.

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.

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.

An unidentified ultraviolet-B-specific photoreceptor mediates transcriptional activation of the cyclobutane pyrimidine dimer photolyase gene in plants.

Cyclobutane pyrimidine dimers (CPDs) constitute a majority of DNA lesions caused by ultraviolet-B (UVB). CPD photolyase, which rapidly repairs CPDs, is essential for plant survival under sunlight containing UVB. Our earlier results that the transcription of the cucumber CPD photolyase gene (CsPHR) was activated by light have prompted us to propose that this light-driven transcriptional activation would allow plants to meet the need of the photolyase activity upon challenges of UVB from sunlight. However, molecular mechanisms underlying the light-dependent transcriptional activation of CsPHR were unknown. In order to understand spectroscopic aspects of the plant response, we investigated the wavelength-dependence (action spectra) of the light-dependent transcriptional activation of CsPHR. In both cucumber seedlings and transgenic Arabidopsis seedlings expressing reporter genes under the control of the CsPHR promoter, the action spectra exhibited the most predominant peak in the long-wavelength UVB waveband (around 310 nm). In addition, a 95-bp cis-acting region in the CsPHR promoter was identified to be essential for the UVB-driven transcriptional activation of CsPHR. Thus, we concluded that the photoperception of long-wavelength UVB by UVB photoreceptor(s) led to the induction of the CsPHR transcription via a conserved cis-acting element.

Induction and inhibition of cyclobutane pyrimidine dimer photolyase in etiolated cucumber (Cucumis sativus) cotyledons after ultraviolet irradiation depends on wavelength.

Under polychromatic ultraviolet (UV) irradiation (maximum energy at 327 nm) the activity of DNA photolyase specific to cyclobutane pyrimidine dimers (CPDs), CPD photolyase, increased by an amount which depended on UV irradiance, and the level of CPD photolyase gene (CsPHR) transcripts temporarily increased before the activity reached a constant level. UV light (>320 nm) was more effective than visible light at increasing CPD photolyase activity. In contrast, monochromatic UV irradiation at wavelengths <300 nm increased the level of CsPHR transcripts similarly to irradiation at wavelengths >320 nm, but reduced CPD photolyase activity compared with the dark control. Exposure of a CPD photolyase solution to UV-C (254 nm) reduced enzyme activity and induced accumulation of H(2)O(2). Addition of H(2)O(2) to the enzyme solution also inactivated CPD photolyase activity. These results suggest the possibility that reactive oxygen species participate in the inactivation of CPD photolyase in cotyledons exposed to UV irradiation of <300 nm.

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.

Isolation of an ozone-sensitive and jasmonate-semi-insensitive Arabidopsis mutant (oji1).

A novel ozone-sensitive mutant was isolated from Arabidopsis T-DNA tagging lines. This mutant revealed severe foliar injury and higher ethylene emission than the wild type under ozone exposure. The ozone-induced injury and ethylene emission were suppressed by pretreatment with aminoethoxyvinyl glycine, an inhibitor of ethylene biosynthesis, both in this mutant and wild-type plants. Pretreatment with methyl-jasmonate (MeJA) at 10 micro M, however, suppressed the ozone-induced ethylene emission and foliar injury only in the wild-type plants. This mutant was less sensitive to jasmonate than the wild type, estimated by the MeJA-induced inhibition of root elongation and ozone-induced expression of AtVSP1, a jasmonate-inducible gene. Thus, this mutant was named oji1 (ozone-sensitive and jasmonate-insensitive 1). These results suggest that the ozone sensitivity of oji1 is caused by the increase in ozone-induced emission of ethylene as a result of low sensitivity to jasmonate, which plays defensive roles under stress conditions.