Ascorbate (AsA), the most abundant water-soluble redox compound in plants and eukaryotic algae, has multiple functions. There is compelling genetic evidence that the biosynthesis of AsA proceeds via a D-mannose/L-galactose pathway and is the most significant source of AsA in plants. AsA plays important roles in antioxidative defense, particularly via the AsA/glutathione cycle. AsA peroxidase (APX) plays a central role in the cycle and is emerging as a key enzyme in cellular H(2)O(2) metabolism. Plants possess diverse APX isoenzymes in cellular compartments, including the chloroplast, cytosol, and microbody. In algae, however, the number and distribution of APX proteins are quite limited. Recent progress in molecular biological analysis of APX isoenzymes has revealed elaborate mechanisms for the tissue-dependent regulation of two chloroplastic APX isoenzymes by alternative splicing, and for redox regulation of cytosolic APX gene expression in response to light stress. Furthermore, transgenic plants overexpressing a chloroplastic APX isoenzyme enable us to evaluate the behavior of the enzyme under conditions of photo-oxidative stress. Molecular physiological analysis has revealed that cytosolic APX is part of the system modulating the cellular H(2)O(2) level in redox signaling.

The D-mannose/L-galactose pathway for the biosynthesis of vitamin C (L-ascorbic acid; AsA) has greatly improved the understanding of this indispensable compound in plants, where it plays multifunctional roles. However, it is yet to be proven whether the same pathway holds for all the different organs of plants, especially the fruit-bearing plants, at different stages of development. Micro-Tom was used here to elucidate the mechanisms of AsA accumulation and regulation in tomato fruits. The mRNA expression of the genes in the D-mannose/L-galactose pathway were inversely correlated with increasing AsA content of Micro-Tom fruits during ripening. Feeding L-[6-¹⁴C]AsA to Micro-Tom plants revealed that the bulk of the label from AsA accumulated in the source leaf was transported to the immature green fruits, and the rate of translocation decreased as ripening progressed. L-Galactose feeding, but neither D-galacturonate nor L-gulono-1,4-lactone, enhanced the content of AsA in immature green fruit. On the other hand, L-galactose and D-galacturonate, but not L-gulono-1,4-lactone, resulted in an increase in the AsA content of red ripened fruits. Crude extract prepared from insoluble fractions of green and red fruits showed D-galacturonate reductase- and aldonolactonase-specific activities, the antepenultimate and penultimate enzymes, respectively, in the D-galacturonate pathway, in both fruits. Taken together, the present findings demonstrated that tomato fruits could switch between different sources for AsA supply depending on their ripening stages. The translocation from source leaves and biosynthesis via the D-mannose/L-galactose pathway are dominant sources in immature fruits, while the alternative D-galacturonate pathway contributes to AsA accumulation in ripened Micro-Tom fruits.

Microalgae have recently been attracting attention as a potential platform for the production of biofuels. Euglena gracilis, a unicellular phytoflagellate, has been proposed as an attractive feedstock to produce biodiesel because it can produce large amounts of wax esters, consisting of medium-chain fatty acids and alcohols with 14:0 carbon chains. E. gracilis cells highly accumulate a storage polysaccharide, a β-1,3-glucan known as paramylon, under aerobic conditions. When grown aerobically and then transferred into anaerobic conditions, E. gracilis cells degrade paramylon to actively synthesize and accumulate wax esters. Thus, the enhanced accumulation of paramylon through the genetic engineering of photosynthesis should increase the capacity for wax ester production. We herein generated transgenic Euglena (EpFS) cells expressing the cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphosphatase (FBP/SBPase), which is involved in the Calvin cycle, to enhance its photosynthetic activity. FBP/SBPase was successfully expressed within Euglena chloroplasts. The cell volume of the EpFS4 cell line was significantly larger than that of wild-type cells under normal growth conditions. The photosynthetic activity of EpFS4 cells was significantly higher than that of wild type under high light and high CO2, resulting in enhanced biomass production, and the accumulation of paramylon was increased in transgenic cell lines than in wild-type cells. Furthermore, when EpFS cell lines grown under high light and high CO2 were placed on anaerobiosis, the productivity of wax esters was approximately 13- to 100-fold higher in EpFS cell lines than in wild-type cells. Our results obtained here indicate that the efficiency of biomass production in E. gracilis can be improved by genetically modulating photosynthetic capacity, resulting in the enhanced production of wax esters. This is the first step toward the utilization of E. gracilis as a sustainable source for biofuel production under photoautotrophic cultivation.

The phytoflagellated protozoan, Euglena gracilis, has been proposed as an attractive feedstock for the accumulation of valuable compounds such as β-1,3-glucan, also known as paramylon, and wax esters. The production of wax esters proceeds under anaerobic conditions, designated as wax ester fermentation. In spite of the importance and usefulness of Euglena, the genome and transcriptome data are currently unavailable, though another research group has recently published E.gracilis transcriptome study during our submission. We herein performed an RNA-Seq analysis to provide a comprehensive sequence resource and some insights into the regulation of genes including wax ester metabolism by comparative transcriptome analysis of E.gracilis under aerobic and anaerobic conditions. The E.gracilis transcriptome analysis was performed using the Illumina platform and yielded 90.3 million reads after the filtering steps. A total of 49,826 components were assembled and identified as a reference sequence of E.gracilis, of which 26,479 sequences were considered to be potentially expressed (having FPKM value of greater than 1). Approximately half of all components were estimated to be regulated in a trans-splicing manner, with the addition of protruding spliced leader sequences. Nearly 40 % of 26,479 sequences were annotated by similarity to Swiss-Prot database using the BLASTX program. A total of 2080 transcripts were identified as differentially expressed genes (DEGs) in response to anaerobic treatment for 24 h. A comprehensive pathway enrichment analysis using the KEGG pathway revealed that the majority of DEGs were involved in photosynthesis, nucleotide metabolism, oxidative phosphorylation, fatty acid metabolism. We successfully identified a candidate gene set of paramylon and wax esters, including novel β-1,3-glucan and wax ester synthases. A comparative expression analysis of aerobic- and anaerobic-treated E.gracilis cells indicated that gene expression changes in these components were not extensive or dynamic during the anaerobic treatment. The RNA-Seq analysis provided comprehensive transcriptome information on E.gracilis for the first time, and this information will advance our understanding of this unique organism. The comprehensive analysis indicated that paramylon and wax ester metabolic pathways are regulated at post-transcriptional rather than the transcriptional level in response to anaerobic conditions.

Reactive oxygen species (ROS) have dual functions in plant cells as cytotoxic molecules and emergency signals. The balance between the production and scavenging of these molecules in chloroplasts, major sites for the production of ROS, is one of the key determinants for plant acclimation to stress conditions. The water-water cycle is a crucial regulator of ROS levels in chloroplasts. In this cycle, the stromal and thylakoid membrane-attached isoforms of ascorbate peroxidase (sAPX and tAPX, respectively) are involved in the metabolism of H₂O₂. Current genome and phylogenetic analyses suggest that the first mono-functional APX was generated as sAPX in unicellular green algae, and that tAPX occurred in multicellular charophytes during plant evolution. Chloroplastic APXs, especially tAPX, have been considered to be the source of a bottleneck in the water-water cycle, at least in higher plants, because of their high susceptibility to H₂O₂. A number of studies have succeeded in improving plant stress resistance by reinforcing the fragile characteristics of the enzymes. However, researchers have unexpectedly failed to find a ‘stress-sensitive phenotype’ among loss-of-function mutants at least in the laboratory conditions. Interestingly, the susceptibility of enzymes to H₂O₂ may have been acquired during plant evolution, thereby allowing for the flexible use of H₂O₂ as a signaling molecule in plants, and this is supported by growing lines of evidence for the physiological significance of chloroplastic H₂O₂ as a retrograde signal in plant stress responses. By overviewing historical, biochemical, physiological, and genetic studies, we herein discuss the diverse functions of chloroplastic APXs as antioxidant enzymes and signaling modulators.