Tse-Min Lee

Nitric Oxide Up-regulates the Expression of Methionine Sulfoxide Reductase Genes in the Intertidal Macroalga Ulva fasciata for High Light Acclimation

Nitric oxide (NO) has emerged as a fundamental signal molecule involved in the responses of plant to stress. A role for NO in the regulation of methionine sulfoxide reductase (MSR) mRNA expression and high light acclimation was studied in a green macroalga Ulva fasciata Delile. Transfer from darkness to high light (≥1,200 μmol photons m(-2) s(-1)) inhibited photosynthesis and growth but increased NO production and UfMSRA and UfMSRB transcripts. Treatment with an NO scavenger, 2-(4-carboxy- phenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (cPTIO), at 1,200 μmol photons m(-2) s(-1) caused a further growth inhibition accompanied by an inhibition of the increase of UfMSRA and UfMSRB transcripts by high light, while treatment with an NO generator, sodium nitroprusside (SNP), alleviated the growth inhibition and enhanced UfMSRA and UfMSRB expression. Exposure to moderate light (300 μmol photons m(-2) s(-1)) conditions also increased UfMSRA and UfMSRB transcripts, which were not affected by cPTIO treatment but were enhanced by SNP treatment. So, NO does not mediate the up-regulation of UfMSR genes by transfer to moderate light possibly as a precautionary mechanism in the sense of increasing light intensities in the daytime. In conclusion, NO production can be induced in U. fasciata upon exposure to high light for up-regulation of UfMSRA and UfMSRB expression but the level of NO production is not sufficient for acquisition of full tolerance to high light stress. Enhanced NO production by an exogenously applied NO generator can effectively trigger the high light acclimation process, including UfMSRA and UfMSRB expression.

Hydrogen peroxide production protects Chlamydomonas reinhardtii against light-induced cell death by preventing singlet oxygen accumulation through enhanced carotenoid synthesis.

The effect of hydrogen peroxide (H₂O₂) on carotenoid synthesis in Chlamydomonas reinhardtii under light-induced stress at 3000 μmol m⁻² s⁻¹ has been investigated. This very high light (VHL) illumination triggered a transient increase in H₂O₂ production during the initial 30 min of light stress, followed by singlet oxygen (¹O₂) production, growth inhibition and necrotic cell death. The carotenoid content was slightly reduced during the first 30 min of VHL illumination and strongly diminished after 60 min, while the expression of the transcripts of enzymes involved in carotenoid biosynthesis, including phytoene synthase (PSY), phytoene desaturase (PDS), and lycopene ɛ-cyclase (LCYE), initially increased and then decreased. Lycopene β-cyclase (LCYB) transcripts did not change. Treatment with dimethylthiourea, a H₂O₂ scavenger, under VHL conditions reduced H₂O₂ production and PSY and PDS transcript levels and accelerated the reduction of carotenoids, the production of ¹O₂, viability loss and necrotic cell death. Pretreatment with 0.1 μM methyl viologen or 0.2 mM H₂O₂ under 50 μmol m⁻² s⁻¹ low light for 60 min increased VHL tolerance, carotenoid content, and PSY and PDS transcripts, while LCYB and LCYE transcripts were not affected. These results suggest that H₂O₂, produced under VHL stress, ameliorates the ¹O₂-mediated oxidative damage to C. reinhardtii through a reduction in the degree of carotenoid breakdown by activation of de novo carotenoid synthesis.

Modulation of gene expression of carotene biosynthesis-related protein by photosynthetic electron transport for the acclimation of intertidal macroalga Ulva fasciata to hypersalinity and excess light

A gene (UfCBR) encoding carotene biosynthesis-related (CBR) protein that potentially functions for the dissipation of excessive energy has been cloned from the intertidal green macroalga Ulva fasciata Delile. Hypersalinity and high light ≥300 µmol m(-2) s(-1) increased both UfCBR mRNA level and non-photochemical quenching (NPQ). The increase of UfCBR mRNA level and NPQ by high light was inhibited by treatment of photosynthetic electron transport inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea or 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, but not by stigmatellin, an inhibitor that blocks electron transfer from quinol oxidase to iron-sulfur protein in cytochrome b(6) f complex. Treatment of dimethylthiourea, an H(2) O(2) scavenger, under 1200 µmol m(-2) s(-1) condition inhibited H(2) O(2) accumulation but did not affect UfCBR mRNA level, while treatment of H(2) O(2) in 150 µmol m(-2) s(-1) condition decreased UfCBR mRNA level. Thus, an reactive oxygen species-independent redox control via a more reduced state downstream the cytochrome b(6) f complex is involved in high light up-regulation of UfCBR expression in U. fasciata. The expression of UfCBR in U. fasciata against oxidative stress occurring in high light or high salinity in relation to NPQ is discussed.

Reactive oxygen species modulate the differential expression of methionine sulfoxide reductase genes in Chlamydomonas reinhardtii under high light illumination.

Illumination of Chlamydomonas reinhardtii cells at 1000 (high light, HL) or 3000 (very high light, VHL) µmol photons m(-2)  s(-1) intensity increased superoxide anion radical (O(2)(•-)) and hydrogen peroxide (H(2)O(2)) production, and VHL illumination also increased the singlet oxygen ((1)O(2)) level. HL and VHL illumination decreased methionine sulfoxide reductase A4 (CrMSRA4) transcript levels but increased CrMSRA3, CrMSRA5 and CrMSRB2.1 transcripts levels. CrMSRB2.2 transcript levels increased only under VHL conditions. The role of reactive oxygen species (ROS) on CrMSR expression was studied using ROS scavengers and generators. Treatment with dimethylthiourea (DMTU), a H(2)O(2) scavenger, suppressed HL- and VHL-induced CrMSRA3, CrMSRA5 and CrMSRB2.1 expression, whereas H(2)O(2) treatment stimulated the expression of these genes under 50 µmol photons m(-2)  s(-1) conditions (low light, LL). Treatment with diphenylamine (DPA), a (1)O(2) quencher, reduced VHL-induced CrMSRA3, CrMSRA5 and CrMSRB2.2 expression and deuterium oxide, which delays (1)O(2) decay, enhanced these gene expression, whereas treatment with (1)O(2) (rose bengal, methylene blue and neutral red) or O(2)(•-) (menadione and methyl viologen) generators under LL conditions induced their expression. DPA treatment inhibited the VHL-induced decrease in CrMSRA4 expression, but other ROS scavengers and ROS generators did not affect its expression under LL or HL conditions. These results demonstrate that the differential expression of CrMSRs under HL illumination can be attributed to different types of ROS. H(2)O(2), O(2) (•-) and (1)O(2) modulate CrMSRA3 and CrMSRA5 expression, whereas H(2)O(2) and O(2)(•-) regulate CrMSRB2.1 and CrMSRB2.2 expression, respectively. (1)O(2) mediates the decrease of CrMSRA4 expression by VHL illumination, but ROS do not modulate its decrease under HL conditions.

Polyamine effects on protein disulfide isomerase expression and implications for hypersalinity stress in the marine alga Ulva lactuca Linnaeus(1).

Full‐length protein disulfide isomerase (UfPDI) cDNA was cloned from the intertidal macroalga Ulva lactuca Linnaeus. Modulation of UfPDI expression by stresses and polyamines (PA) was studied. UfPDI transcription and enzyme activity were increased by hypersalinity (90) or high light illumination (1,200 μmol photons · m−2 · s−1), decreased by the addition of 100 μM CuSO4. An exposure to a salinity of 90 decreased PA contents. Treating with PA biosynthetic inhibitors, D‐arginine (D‐Arg) or α‐methyl ornithine (α‐MO), led to a further decrease and also inhibited UfPDI expression and recovery of the growth rate. These results suggest that PAs are required to activate UfPDI expression with hypersalinity, even PA contents are decreased at a salinity of 90. The induction of UfPDI expression by hypersalinity of 90 and tolerance to hypersalinity could be enhanced if internal PA contents rise. Sung et al. (2011b) showed that PA contents could be increased by pretreating with putrescine (Put, 1 mM), spermidine (Spd, 1 mM), or spermine (Spm, 1 mM) at a salinity of 30. Therefore, PA pretreatment effect on UfPDI expression was examined. Pretreatment with Spd and Spm, but not with Put, enhanced UfPDI expression after transferred to a salinity of 90 and restored the growth rate. In conclusion, induction of UfPDI expression by Spd or Spm before exposure to hypersaline conditions and continuous up‐regulation after hypersalinity exposure are required for the acquisition of hypersalinity tolerance in the intertidal green macroalga U. lactuca.

A role for glutathione reductase and glutathione in the tolerance of Chlamydomonas reinhardtii to photo-oxidative stress

The role of glutathione reductase (GR; EC 1.6.4.2) in the tolerance of Chlamydomonas reinhardtii P.A. Dangeard to high-intensity light stress (HL, 1400 μmol m-2  s-1 ) was examined. Cells survived under high light (HL) stress, although their growth was inhibited after long-term treatment (9-24 h). GR activity increased 1 h after HL treatment. The contents of total glutathione, reduced glutathione (GSH) and glutathione disulfide (GSSG) increased 1-3 h after HL treatment and then decreased after 24 h, while the GSH:GSSG ratio (glutathione redox potential) decreased after 3-9 h and recovered after 24 h. The transcript abundance of GR, CrGR1 (Cre06.g262100) and CrGR2 (Cre09.g396252) as well as glutathione synthesis-related genes, CrGSH1 (Cre02g077100.t1.1) and CrGSH2 (Cre17.g70800.t1.1), increased with a peak near 1 h after HL treatment. Except for enhanced glutathione synthesis, the GR-mediated glutathione redox machinery is also critical for the tolerance of C. reinhardtii cells to HL stress. Therefore, GR was downregulated or upregulated to investigate the importance of GR in HL tolerance. The CrGR1 knockdown amiRNA line exhibited low GR transcript abundance, GR activity and GSH:GSSG ratio and could not survive under HL conditions. Over-expression of CrGR1 or CrGR2 driven by a HSP70A:RBCS2 fusion promoter resulted in a higher GR transcript abundance, GR activity and GSH:GSSG ratio and led to cell survival when exposed to high-intensity illumination, i.e. 1800 μmol m-2  s-1 . In conclusion, GR-mediated modulation of the glutathione redox potential plays a role in the tolerance of Chlamydomonas cells to photo-oxidative stress.