Constitutive or Inducible Protective Mechanisms against UV-B Radiation in the Brown Alga Fucus vesiculosus? A Study of Gene Expression and Phlorotannin Content Responses

Abstract

A role as UV sunscreens has been suggested for phlorotannins, the phenolic compounds that accumulate in brown algae in response to a number of external stimuli and take part in cell wall structure. After exposure of the intertidal brown alga Fucus vesiculosus to artificial UV-B radiation, we examined its physiological responses by following the transcript level of the pksIII gene encoding a phloroglucinol synthase, likely to be involved in the first step of phlorotannins biosynthesis. We also monitored the expression of three targeted genes, encoding a heat shock protein (hsp70), which is involved in global stress responses, an aryl sulfotransferase (ast), which could be involved in the sulfation of phlorotannins, and a vanadium bromoperoxidase (vbpo), which can potentially participate in the scavenging of Reactive Oxygen Species (ROS) and in the cross-linking and condensation of phlorotannins. We investigated whether transcriptional regulation of these genes is correlated with an induction of phlorotannin accumulation by establishing metabolite profiling of purified fractions of low molecular weight phlorotannins. Our findings demonstrated that a high dose of UV-B radiation induced a significant overexpression of hsp70 after 12 and 24 hours following the exposure to the UV-B treatment, compared to control treatment. The physiological performance of algae quantified by the photosynthetic efficiency (Fv/Fm) was slightly reduced. However UV-B treatment did not induce the accumulation of soluble phlorotannins in F. vesiculosus during the kinetics of four weeks, a result that may be related to the lack of induction of the pksIII gene expression. Taken together these results suggest a constitutive accumulation of phlorotannins occurring during the development of F.vesiculosus, rather than inducible processes. Gene expression studies and phlorotannin profiling provide here complementary approaches to global quantifications currently used in studies of phenolic compounds in brown algae.

Fig 1. Photosynthesis efficiency of Fucus vesiculosus under chronic exposure to UV-B radiation during 4 weeks.

The optimal quantum yield (Fv/Fm) of apical parts of F.vesiculosus thalli measured through imaging PAM fluorimetry during exposure to PAR (controls, white losanges) and UV-B condition (black squares).Values represent means of three independent replicates and bars represent the SE. Statistically significant differences (LSMEANS 0.05) between treatments are indicated by stars (*).

Fig 2. Relative gene expression in Fucus vesiculosus in controlled condition (white square) and exposed to UV-B (black square) during 72 hours are presented for pksIII (a), ast6 (b), hsp70 (c), and vbpo (d).

The expression of a gene is normalized to the geometric mean of the expression of 2 reference genes (ef1α, tua) in the same algal sample and to the mean of its expression in the three control algae at each time point. Values represent means of three independent replicates and bars represent the SE. Letters indicate significant difference (LSMEANS 0.05).

Fig 3. Quantification of total soluble phenol contents (mg equivalent phloroglucinol.g^-1 DW) before purification in controlled condition (white square) and exposed to UV-B (black square).

Values represent means of three independent replicates and bars represent the SD.

Fig 4. Quantification of soluble phenol contents in semi-purified phlorotannins fractions (mg equivalent phloroglucinol.g^-1 DW) in controlled condition (white square) and exposed to UV-B (black square).

Values represent means of three independent replicates and bars represent the SD. Mean with different letters indicate significant difference between all samples along the kinetic (LSMEANS 0.05). Stars (*) indicate significant difference between control and UV-B treatment (LSMEANS 0.05).

Fig 5. Ultra-HPLC-ESI-MS chromatogram of a precursor ion at m/z 621.0886 corresponding to a DP5 oligomer of phlorotannin in semi-purified fractions extracted from control (a) and UV-B treated algae (b) after 2 weeks.

Mass spectrum from the chromatogram identified as DP5 (c). The theorical mass spectrum of DP5: C₃₀H₂₁O₅ in the negative ion mode ([M-H]- = 621.0886 m/z) (d).