Data_Sheet_1_Biosynthesis of Ascorbic Acid as a Glucose-Induced Photoprotective Process in the Extremophilic Red Alga Galdieria partita.pdf

The extremophilic red alga Galdieria partita is a facultative heterotroph that occupies mostly low-light microhabitats. However, the exceptional detection of abundant populations of G. partita in sunlight-exposed soil raises the possibility that exogenous organic carbon sources protect cells from photo-oxidative damage. The present study aimed to identify the photoprotective process activated by exogenous glucose under photo-oxidative stress. We demonstrated that exogenous glucose mitigated the photo-oxidative damage of cells exposed to 300 μmol photons m–2 s–1 photosynthetic active radiation. Photosynthesis carbon assimilation scarcely contributed to the cell growth in the presence of glucose, but the photosynthetic apparatus was nevertheless maintained and protected by glucose in a concentration-dependent manner. Supplementation of glucose increased expression of the L-gulonolactone oxidase gene essential for ascorbic acid biosynthesis, whereas no enhanced expression of the genes involved in carotenoid or tocopherol biosynthesis was observed. Under the photo-oxidative stress condition, the ascorbic acid content was strongly enhanced by exogenous glucose. We propose that the biosynthesis of ascorbic acid is one of the major photoprotective processes induced by exogenous glucose. The elucidation of how ascorbic acid is involved in scavenging reactive oxygen species provides key insights into the photoprotective mechanism in red algae.</p

Data_Sheet_4_Biosynthesis of Ascorbic Acid as a Glucose-Induced Photoprotective Process in the Extremophilic Red Alga Galdieria partita.xlsx

The extremophilic red alga Galdieria partita is a facultative heterotroph that occupies mostly low-light microhabitats. However, the exceptional detection of abundant populations of G. partita in sunlight-exposed soil raises the possibility that exogenous organic carbon sources protect cells from photo-oxidative damage. The present study aimed to identify the photoprotective process activated by exogenous glucose under photo-oxidative stress. We demonstrated that exogenous glucose mitigated the photo-oxidative damage of cells exposed to 300 μmol photons m–2 s–1 photosynthetic active radiation. Photosynthesis carbon assimilation scarcely contributed to the cell growth in the presence of glucose, but the photosynthetic apparatus was nevertheless maintained and protected by glucose in a concentration-dependent manner. Supplementation of glucose increased expression of the L-gulonolactone oxidase gene essential for ascorbic acid biosynthesis, whereas no enhanced expression of the genes involved in carotenoid or tocopherol biosynthesis was observed. Under the photo-oxidative stress condition, the ascorbic acid content was strongly enhanced by exogenous glucose. We propose that the biosynthesis of ascorbic acid is one of the major photoprotective processes induced by exogenous glucose. The elucidation of how ascorbic acid is involved in scavenging reactive oxygen species provides key insights into the photoprotective mechanism in red algae.</p

Data_Sheet_2_Biosynthesis of Ascorbic Acid as a Glucose-Induced Photoprotective Process in the Extremophilic Red Alga Galdieria partita.xlsx

The extremophilic red alga Galdieria partita is a facultative heterotroph that occupies mostly low-light microhabitats. However, the exceptional detection of abundant populations of G. partita in sunlight-exposed soil raises the possibility that exogenous organic carbon sources protect cells from photo-oxidative damage. The present study aimed to identify the photoprotective process activated by exogenous glucose under photo-oxidative stress. We demonstrated that exogenous glucose mitigated the photo-oxidative damage of cells exposed to 300 μmol photons m–2 s–1 photosynthetic active radiation. Photosynthesis carbon assimilation scarcely contributed to the cell growth in the presence of glucose, but the photosynthetic apparatus was nevertheless maintained and protected by glucose in a concentration-dependent manner. Supplementation of glucose increased expression of the L-gulonolactone oxidase gene essential for ascorbic acid biosynthesis, whereas no enhanced expression of the genes involved in carotenoid or tocopherol biosynthesis was observed. Under the photo-oxidative stress condition, the ascorbic acid content was strongly enhanced by exogenous glucose. We propose that the biosynthesis of ascorbic acid is one of the major photoprotective processes induced by exogenous glucose. The elucidation of how ascorbic acid is involved in scavenging reactive oxygen species provides key insights into the photoprotective mechanism in red algae.</p

Data_Sheet_3_Biosynthesis of Ascorbic Acid as a Glucose-Induced Photoprotective Process in the Extremophilic Red Alga Galdieria partita.xlsx

The extremophilic red alga Galdieria partita is a facultative heterotroph that occupies mostly low-light microhabitats. However, the exceptional detection of abundant populations of G. partita in sunlight-exposed soil raises the possibility that exogenous organic carbon sources protect cells from photo-oxidative damage. The present study aimed to identify the photoprotective process activated by exogenous glucose under photo-oxidative stress. We demonstrated that exogenous glucose mitigated the photo-oxidative damage of cells exposed to 300 μmol photons m–2 s–1 photosynthetic active radiation. Photosynthesis carbon assimilation scarcely contributed to the cell growth in the presence of glucose, but the photosynthetic apparatus was nevertheless maintained and protected by glucose in a concentration-dependent manner. Supplementation of glucose increased expression of the L-gulonolactone oxidase gene essential for ascorbic acid biosynthesis, whereas no enhanced expression of the genes involved in carotenoid or tocopherol biosynthesis was observed. Under the photo-oxidative stress condition, the ascorbic acid content was strongly enhanced by exogenous glucose. We propose that the biosynthesis of ascorbic acid is one of the major photoprotective processes induced by exogenous glucose. The elucidation of how ascorbic acid is involved in scavenging reactive oxygen species provides key insights into the photoprotective mechanism in red algae.</p

One out of Four: HspL but No Other Small Heat Shock Protein of <em>Agrobacterium tumefaciens</em> Acts as Efficient Virulence-Promoting VirB8 Chaperone

<div><p>Alpha-crystallin-type small heat shock proteins (sHsps) are ubiquitously distributed in most eukaryotes and prokaryotes. Four sHsp genes named <em>hspL</em>, <em>hspC</em>, <em>hspAT1</em>, and <em>hspAT2</em> were identified in <em>Agrobacterium tumefaciens</em>, a plant pathogenic bacterium capable of unique interkingdom DNA transfer via type IV secretion system (T4SS). HspL is highly expressed in virulence-induced growth condition and functions as a VirB8 chaperone to promote T4SS-mediated DNA transfer. Here, we used genetic and biochemical approaches to investigate the involvement of the other three sHsps in T4SS and discovered the molecular basis underlying the dominant function of HspL in promoting T4SS function. While single deletion of <em>hspL</em> but no other sHsp gene reduced T4SS-mediated DNA transfer and tumorigenesis efficiency, additional deletion of other sHsp genes in the <em>hspL</em> deletion background caused synergistic effects in the virulence phenotypes. This is correlated with the high induction of <em>hspL</em> and only modest increase of <em>hspC</em>, <em>hspAT1</em>, and <em>hspAT2</em> at their mRNA and protein abundance in virulence-induced growth condition. Interestingly, overexpression of any single sHsp gene alone in the quadruple mutant caused increased T4SS-mediated DNA transfer and tumorigenesis. Thermal aggregation protecting assays <em>in vitro</em> indicated that all four sHsps exhibit chaperone activity for the model substrate citrate synthase but only HspL functions as efficient chaperone for VirB8. The higher VirB8 chaperone activity of HspL was also demonstrated <em>in vivo,</em> in which lower amounts of HspL than other sHsps were sufficient in maintaining VirB8 homeostasis in <em>A. tumefaciens.</em> Domain swapping between HspL and HspAT2 indicated that N-terminal, central alpha-crystallin, and C-terminal domains of HspL all contribute to HspL function as an efficient VirB8 chaperone. Taken together, we suggest that the dominant role of HspL in promoting T4SS function is based on its higher expression in virulence-induced condition and its more efficient VirB8 chaperone activity as compared to other sHsps.</p> </div

AS-induced mRNA and protein expression of four sHsp genes in <i>A. tumefaciens</i>.

<p>(A) Quantitative RT-PCR analysis of sHsp mRNA levels in <i>A. tumefaciens</i> wild type (WT) strain NT1RE(pJK270) and the <i>ΔhspL</i> mutant. Relative expression level is normalized by 16S rRNA as an internal control and the mean values of fold-change relative to the DMSO control of WT. Data are mean with standard deviation (SD) of 2 biological replicates, each of which contains 3 technical replicates. (B) Each of the sHsp genes expressing proteins tagged with HA and driven by its upstream promoter region on plasmid was expressed in wild type (WT) strain NT1RE(pJK270) and the <i>ΔhspL</i> mutant. The agrobacterial cells were grown in ABMES medium (pH5.5) at 25°C in the absence (−, DMSO control) or the presence of AS for 16 hrs. The total cell lysates were detected by western blot analysis with specific antibody against HA, VirB2, or RpoA. Numbers on the left are molecular masses of reference proteins in kDa.</p

Mobilization efficiency of RSF1010 derivative PML122<i>Δkm</i>::Tc^R between <i>Agrobacterium</i> strains.

<p>Mobilization efficiency of RSF1010 derivative PML122<i>Δkm</i>::Tc^R between <i>Agrobacterium</i> strains.</p

Effects of IPTG-induced His-tagged HspL, HspC, HspAT1 or HspAT2 on VirB8 accumulation.

<p>(A) <i>ΔhspL</i>(pTrc-HspL-His), <i>ΔhspL</i>(pTrc-HspC-His), <i>ΔhspL</i>(pTrc-HspAT1-His), and <i>ΔhspL</i>(pTrc-HspAT2-His) were grown in AB-MES (pH 5.5) containing 200 µM AS at 28°C with or without 0.4 mM IPTG for 24 h. The total cell lysates were detected by western blot analysis with specific antiserum. (B) <i>ΔhspL</i>(pTrc-HspL-His) and<i>ΔhspL</i>(pTrc-HspAT2-His) were grown in AB-MES (pH 5.5) containing 200 µM AS at 28°C with different concentrations of IPTG (0∼80 µM) for 24 h. The signal of VirB8 or sHsp-His was determined by western blot using anti-VirB8 or anti-His antiserium and the intensity of signal was quantified. The signal of VirB8 or sHsp-His was normalized by the signal of RpoA (loading control). The regression curve was created using the increase of signal intensity of VirB8 as Y-axis and sHsp-His as X-axis. The lineal range of signals was selected to calculate regression curve, the increase of signal intensity of VirB8 as Y-axis and sHsp-His as X-axis.</p

Tumorigenesis and RSF1010 transfer efficiency assays of single and multiple sHsp deletion mutants.

<p>(A) Tumorigenesis efficiency is presented as number of tumor per disc averaged from more than 60 potato discs, with standard errors. Three independent experiments were carried out with similar results with the representative result shown. (B) The RSF1010 transfer efficiency was evaluated as number of transconjugants per input donor. Average values for relative RSF1010 transfer efficiency from three independent experiments are shown with standard errors, in which the efficiency of wild type strain NT1RE(pJK270) (WT) was set at 100% and that of other strains is shown relative to that of NT1RE(pJK270). Means annotated with the same letter (a-d) are not significantly different; those with different letters are significantly different (<i>P</i><0.05) according to Duncan’s multiple range test. The absolute RSF1010 transfer efficiency and the numbers of transconjugants and input donors obtained from three independent experiments were shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049685#pone-0049685-t001" target="_blank">Table 1</a>.</p

Chaperone activity assay of sHsps.

<p>(A) Thermal aggregation protection assays using model substrate CS (600 nM) was carried out at 43°C in the absence (□) or presence of HspL-His₆ (♦), HspC-His₆ (•), HspAT1-His₆ (▴) or HspAT2-His₆ (▾) at concentration of 1.2 µM respectively. (B) 1 µM GST-VirB8 was used as substrate and was carried out at 50°C in the absence (□) or presence of HspL-His₆ (♦), HspC-His₆ (•), HspAT1-His₆ (▾) or HspAT2-His₆ (▴) at concentration of 2 µM respectively. (C) 1 µM GST-VirB8 was used as substrate and was carried out at 50°C in the absence (□) or presence of HspAT2-His₆ with concentration at 1 µM(•), 2 µM(▾), 4 µM(▴), 8 µM(▪) and 16 µM (<>\raster="rg1"<>), or of 2 µM HspL-His₆ (♦). Aggregation was monitored in absorbance at 360 nm and presented as a function of time.</p