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Glycerol-3-phosphate dehydrogenase (GPDH) catalyzes the conversion of dihydroxyacetone phosphate (DHAP) and NADH to glycerol-3-phosphate (G3P) and NAD(+). G3P is important as a precursor for glycerol and glycerolipid synthesis in microalgae. A GPDH enzyme has been previously purified from the green microalga Chlamydomonas reinhardtii, however, no genes coding for GPDH have been characterized before. In this study, we report the in silico characterization of three putative GPDH genes from C. reinhardtii: CrGPDH1, CrGPDH2, and CrGPDH3. These sequences showed a significant similarity to characterized GPDH genes from the microalgae Dunaliella salina and Dunaliella viridis. The prediction of the three-dimensional structure of the proteins showed the characteristic fold topology of GPDH enzymes. Furthermore, the phylogenetic analysis showed that the three CrGPDHs share the same clade with characterized GPDHs from Dunaliella suggesting a common evolutionary origin and a similar catalytic function. In addition, the K(a)/K(s) ratios of these sequences suggested that they are under purifying selection. Moreover, the expression analysis showed a constitutive expression of CrGPDH1, while CrGPDH2 and CrGPDH3 were induced in response to osmotic stress, suggesting a possible role for these two sequences in the synthesis of glycerol as a compatible solute in osmoregulation, and perhaps also in lipid synthesis in C. reinhardtii. This study has provided a foundation for further biochemical and genetic studies of the GPDH family in this model microalga, and also opportunities to assess the potential of these genes to enhance the synthesis of TAGs for biodiesel production.

The aim of this study was to investigate the potential of the green microalga Chlorella saccharophila as a source of oil for biodiesel production. We evaluated for the first time, the effect of salinity and/or nitrogen depletion (ND) on cell growth, lipid accumulation and lipid profile in this microalga. The fatty acid methyl esters (FAME) identified for C. saccharophila in this study consisted of C-16:0, C-18:0, C-18:1 cis, and C-18:1 trans. Among these, C-18:1 (indicator of biodiesel quality) was the main FAME found, representing approximately 76 and 80% of total FAME under normal and ND growing conditions, respectively. Under a normal growing condition this microalga showed 154.63 mg l(-1) d(-1), 63.33 mg l(-1) d(-1), and 103.73 mg l(-1) of biomass productivity, lipid productivity, and FAME yield, respectively. The higher biomass productivity (159.58 mg l(-1) d(-1)), lipid productivity (99.33 mg l(-1) d(-1)), and FAME yield (315.53 mg l(-1)) were obtained under the ND treatment. In comparison to other related studies, our results suggest that C. saccharophila can be considered as a suitable source of oil for biodiesel production.

Glycerol-3-phosphate dehydrogenase (GPDH) catalyzes the conversion of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P) and plays a central role in the synthesis of glycerol and triacylglycerides (TAGs). Osmotic stress has been shown to induce the accumulation of glycerol and TAGs in the green microalga Chlamydomonas reinhardtii. In a previous study, we identified three GPDH homologs (CrGPDH1, CrGPDH2, and CrGPDH3) in this microalga. We found that CrGPDH2 and CrGPDH3 were expressed in response to 200 mM NaCl treatment, suggesting that these two genes play roles in glycerol and TAGs synthesis and in osmotic stress tolerance. In this study, we report on the functional characterization of CrGPDH2 and CrGPDH3. A concentration of NaCl as low as 5 mM for 5 min was sufficient to induce the expressions of both genes. We mapped the cDNA ends of CrGPDH2 and CrGPDH3 using RLM-RACE and cloned their full-length cDNAs. The expression of these two cDNAs in the Saccharomyces cerevisiae gpd1Δgpd2Δ double mutant confirmed that both CrGPDH2 and CrGPDH3 have GPDH activity. The genetic complementation analysis revealed that CrGPDH2 and CrGPDH3 were able to restore glycerol production and rescue the salt sensitivity of this mutant. Compared with CrGPDH3, CrGPDH2 conferred higher glycerol production and greater salt tolerance when expressed in the gpd1Δgpd2Δ double mutant. Together, these findings show that CrGPDH2 and CrGPDH3 encode functional homologs of the S. cerevisiae GPD1 gene that is involved in glycerol synthesis and osmotic stress tolerance.

Banana bunchy top virus (BBTV) has a multi-component genome of circular, single-stranded DNA. BBTV replicates via a rolling-circle mechanism, probably involving sequence-specific interaction of the replication initiation protein (Rep) with iterated sequences (iterons) within the viral genome. Three putative iterons (designated F1, F2 and R), with the sequence GGGAC, have been identified in the intergenic region of each BBTV component. To investigate their role in replication, each of the iterons was mutated, singularly and in tandem, in a BBTV DNA-N 1?1mer and the ability of these molecules to be replicated by the BBTV 'master' Rep was evaluated in banana cells using transient biolistic assays. All iteron mutants were replicated less efficiently than the native DNA-N. Mutation of the F1 and R iterons caused a 42 and 62 % reduction in DNA-N replication, respectively, whereas mutation of the F2 and combined F1F2 iteron virtually abolished DNA-N replication.Banana bunchy top virus (BBTV) is the type member of the genus Babuvirus in the family Nanoviridae and has a genome comprising at least six circular, single-stranded DNA components (referred to as DNA-R, -S, -C, -M, -N and -U3; Vetten et al., 2005), each of approximately 1 kb (Burns et al., 1995). Based on similarities among nanovirus DNAs and those of the geminiviruses, it has generally been accepted that BBTV replication occurs by a rolling-circle-type mechanism (Stenger et al., 1991). In geminiviruses, iterated DNA sequences (iterons) play an important role in the replication process by acting as recognition sites for sequencespecific binding of their cognate replication initiation proteins (Reps). Mutation of these sites can affect Rep binding negatively in vitro and replication in vivo (Chatterji et al., 2000;Choi & Stenger, 1996; Fontes et al., 1994a, b;Orozco et al., 1998). Although putative iteron sequences have been identified in the non-coding regions of some nanovirus DNAs, including Faba bean necrotic yellows virus (FBNYV), Milk vetch dwarf virus (MDV) and Subterranean clover stunt virus (SCSV) , their exact role in replication has not been demonstrated experimentally.Analysis of the intergenic regions of BBTV DNA-R, -S, -C, -M, -N and -U3 (Horser, 2000) has identified a putative iteron sequence (GGGAC) occurring as a tandem repeat (designated iterons F1 and F2, respectively) on the virion-sense strand, 39 of the stem-loop, and as a single iteron (designated iteron R) on the complementary strand, 59 of the stem-loop. The direct repeat iterons, F1 and F2, were located 2 nt 39 of the stem-loop in DNA-R, -S, -C, -M and -N, whereas in DNA-U3, they commenced 1 nt 39 of the stem-loop. However, the location of iteron R varied, being 10 (DNA-N), 19 (DNA-R, -S, -C and -M) and 90 (DNA-U3) nt upstream of the 59 base of the stem-loop.We investigated the role of the F1, F2 and R iterons in BBTV replication by assessing the ability of the BBTV 'master' Rep (M-Rep) (encoded by DNA-R) to replicate native and iteron mutants of DNA-N in banana embryogenic cells. DNA-N...