Linda A. Franklin

Two components of onset and recovery during photoinhibition of Ulva rotundata

Short-term (up to 5 h) transfers of shade-adapted (100 μmol · m−2 · s−1) clonal tissue of the marine macroalga Ulva rotundata Blid. (Chlorophyta) to higher irradiances (1700, 850, and 350 μmol · m−2 · s−1) led to photoinhibition of room-temperature chlorophyll fluorescence and O2 evolution. The ratio of variable to maximum (Fv/Fm) and variable (Fv) fluorescence, and quantum yield (ϕ) declined with increasing irradiance and duration of exposure. This decline could be resolved into two components, consistent with the separation of photoinhibition into energy-dissipative processes (photoprotection) and damage to photosystem II (PSII) by excess excitation. The first component, a rapid decrease in Fv/Fm and in Fv, corresponds to an increase in initial (Fo) fluorescence and is highly sensitive to 1 mM chloramphenicol. This component is rapidly reversible under dim (40 μmol · m−2 · s−1) light, but is less reversible with increasing duration of exposure, and may reflect damage to PSII. The second (after 1 h exposure) component, a slower decline in Fv/Fm and Fv with declining Fo, appears to be associated with the photoprotective interconversion of violaxanthin to zeaxanthin and is sensitive to dithiothreitol. The accumulation of zeaxanthin in U. rotundata is very slow, and may account for the predominance of increases in Fo at high irradiances.

Natural ultraviolet radiation and photosynthetically active radiation induce formation of mycosporine-like amino acids in the marine macroalga Chondrus crispus (Rhodophyta)

Abstract. The UV-absorbing mycosporine-like amino acids (MAAs) are hypothesized to protect organisms against harmful UV radiation (UVR). Since the physiology and metabolism of these compounds are unknown, the induction and kinetics of MAA biosynthesis by various natural radiation conditions were investigated in the marine red alga Chondrus crispus collected from Helgoland, Germany. Three photosynthetically active radiation (PAR, 400–700 nm) treatments without UVR and three UV-A/B (290–400 nm) treatments without PAR were given. Chondrus crispus collected from 4–6 m depth contained only traces of the MAA palythine. After 24 h exposure to 100% ambient PAR, traces of three additional MAAs, shinorine, palythinol and palythene, were detected, and their concentrations increased strongly during a one-week exposure to all PAR treatments. The concentration of all MAAs varied directly with PAR dose, with palythine and shinorine being four- to sevenfold higher than palythinol and palythene. Likewise, naturally high doses of both UV-A and UV-B resulted in a strong accumulation of all MAAs, in particular shinorine. While shinorine accumulation was much more stimulated by UVR, the content of all other MAAs was more affected by high PAR, indicating an MAA-specific induction triggered by UVR or PAR.

Photoacclimation and photoinhibition in Ulva rotundata as influenced by nitrogen availability

Clonal tissue of the marine chlorophyte macroalga, Ulva rotundata Blid., was transferred from 100 to 1700 μmol photons · m−2 · s−1 under limiting (1.5 μM NH4+maximum, N/P=2) and sufficient (15 μM NH4+maximum, N/P=20) nitrogen supply at 18° C and 11 h light-13 h darkness daily. Photoinhibition was assayed by light-response curves (photosynthetic O2 exchange), and chlorophyll fluorescence at 77 K and room temperature. Daily surface-area growth rate (μSA) in N-sufficient plants increased sixfold over 3 d and was sustained at that level. During this period, respiration (Rd) doubled and light-saturated net photosynthesis capacity (Pm) increased by nearly 50%, indicating acclimation to high light. Quantum yield (ϕ) decreased by 25% on the first day, but recovered completely within one week. The ratio of variable to maximum fluorescence (Fv/Fm) also decreased markedly on the first day, because of an increase in initial fluorescence (Fo) and a decrease in Fm, and partially recovered over several days. Under the added stress of N deficiency, μSA accelerated fivefold over 4 d, despite chronic photoinhibition, then declined along with tissue-N. Respiration doubled, but Pm decreased by 50% over one week, indicating inability to acclimate to high light. Both ϕ and Fv/Fm decreased markedly on the first day and did not significantly recover. Changes in Fo, Fm and xanthophyll-cycle components indicate concurrent photodamage to photosystem II (PSII) and photoprotection by thermal deexcitation in the antenna pigments. Increasing μSA coincided with photoinhibition of PSII. Insufficient diel-carbon balance because of elevated Rd and declining Pm and tissue-N, rather than photochemical damage per se, was the apparent proximate cause of decelerating growth rate and subsequent tissue degeneration under N deficiency in U. rotundata.

Fluorescence quenching during photosynthesis and photoinhibition of Ulva rotundata blid.

The relationships between photoinhibition and photoprotection in high and low-light-grown Ulva were examined by a combination of chlorophyll-fluorescence-monitoring techniques. Tissues were exposed to a computer-controlled sequence of 5-min exposures to red light, followed by 5-min darkness, with stepwise increases in photon flux. Coefficients of chlorophyll fluorescence quenching (1−qP and NPQ) were calculated following a saturating pulse of white light near the end of each 5-min light treatment. Dark-adapted chlorophyll fluorescence parameters (F0 and FV/FM) were calculated from a saturating pulse at the end of each 5-min dark period. Low-light-grown Ulva showed consistently higher 1−qP, i.e. higher reduction status of Q (high primary acceptor of photosystem II), and lower capacity for nonphotochemical quenching (NPQ) at saturating light than did high-light-grown plants. Consequently, low-light plants rapidly displayed photoinhibitory damage (increased F0) at light saturation in seawater. Removal of dissolved inorganic carbon from seawater also led to photoinhibitory damage of high-light-grown Ulva at light saturation, and addition of saturating amounts of dissolved inorganic carbon protected low-light-grown plants against photoinhibitory damage. A large part of NPQ was abolished by treatment with 3 mM dithiothreitol and the processes so inhibited were evidently photoprotective, because dithiothreitol treatment accelerated photoinhibitory damage in both low- and high-light-grown Ulva. The extent of photoinhibitory damage in Ulva was exacerbated by treatment with chloramphenicol (1 mM) without much effect on chlorophyll-quenching parameters, evidently because this inhibitor of chloroplast protein synthesis reduced the rate of repair processes.

BLUE LIGHT AND UV-A RADIATION CONTROL THE SYNTHESIS OF MYCOSPORINE-LIKE AMINO ACIDS IN CHONDRUS CRISPUS (FLORIDEOPHYCEAE)

The induction of UV‐absorbing compounds known as mycosporine‐like amino acids (MAAs) by red, green, blue, and white light (43% ambient radiation greater than 390 nm) was examined in sublittoral Chondrus crispus Stackh. Fresh collections or long‐term cultures of sublittoral thalli, collected from Helgoland, North Sea, Germany, and containing no measurable amounts of MAAs, were exposed to filtered natural radiation for up to 40 days. The MAA palythine (λmax 320 nm) was synthesized in thalli in blue light to the same extent observed in control samples in white light. In contrast, thalli in green or red light contained only trace amounts of MAAs. After the growth and synthesis period, the photosynthetic performance of thalli in each treatment, measured as pulse amplitude modulated chlorophyll fluorescence, was assessed after a defined UV dose in the laboratory. Thalli with MAAs were more resistant to UV than those without, and exposure to UV‐A+B was more damaging than UV‐A in that optimal (Fv/Fm) and effective (φII) quantum yields were lower and a greater proportion of the primary electron acceptor of PSII, Q, became reduced at saturating irradiance. However, blue light‐grown thalli were generally more sensitive than white light control samples to UV‐A despite having similar amounts of MAAs. The most sensitive thalli were those grown in red light, which had significantly greater reductions in Fv/Fm and φII and greater Q reduction. Growth under UV radiation alone had been shown previously to lead to the synthesis of the MAA shinorine (λmax 334 nm) rather than palythine. In further experiments, we found that preexposure to blue light followed by growth in natural UV‐A led to a 7‐fold increase in the synthesis of shinorine, compared with growth in UV‐A or UV‐A+B without blue light pretreatment. We hypothesize that there are two photoreceptors for MAA synthesis in C. crispus, one for blue light and one for UV‐A, which can act synergistically. This system would predispose C. crispus to efficiently synthesize UV protective compounds when radiation levels are rising, for example, on a seasonal basis. However, because the UV‐B increase associated with artificial ozone reduction will not be accompanied by an increase in blue light, this triggering mechanism will have little additional adaptive value in the face of global change unless a global UV‐B increase positively affects water column clarity.

SYNTHESIS OF MYCOSPORINE‐LIKE AMINO ACIDS IN CHONDRUS CRISPUS (FLORIDEOPHYCEAE) AND THE CONSEQUENCES FOR SENSITIVITY TO ULTRAVIOLET B RADIATION

The induction and protective role of the UV‐absorbing compounds known as mycosporine‐like amino acids (MAAs) were examined in sublittoral Chondrus crispus Stackh. transplanted for 2 weeks in the spring and summer to shallow water under three irradiance conditions: PAR (photosynthetically active radiation; 400–700 nm), PAR + UVA (PAR + 320– 400 nm), PAR + UVA + UVB (PAR + UVA + 280– 320 nm). Sublittoral thalli collected around Helgoland, North Sea, Germany, from 6 m below the mean low water of spring tides contained less than 0.1 mg·g−1 dry weight (DW) total MAAs, whereas eulittoral samples contained over 1 mg·g−1 DW. Transplantation to shallow water led to the immediate synthesis of three MAAs in the following temporal order: shinorine (λmax 334 nm), asterina (λmax 330 nm), and palythine (λmax 320 nm), with the shinorine content peaking and then declining after 2 days (exposure to 100 mol photons·m−2). Maximum total MAA content (2 mg·g−1 DW) also occurred after 2 days of induction, exceeding the content normally found in eulittoral samples. Furthermore, the relative proportion of the different MAAs at this time was different than that in eulittoral samples. After 2 days the total content declined to the eulittoral value, with palythine as the principal MAA. Similar data were obtained for all treatments, indicating that MAA synthesis in C. crispus was induced by PAR and not especially stimulated by UV radiation. The ability of photosystem II (PSII) to resist damage by UVB was tested periodically during the acclimation period by exposing samples to a defined UVB dose in the lab. Changes in chlorophyll fluorescence (Fv/Fm and effective quantum yield, φII) indicated that PSII function was inhibited during the initial stage of acclimation but gradually improved with time. No difference among screening treatments was detected except in spring for the samples acclimating to PAR + UVA + UVB. In this treatment Fv/Fm and φII were significantly lower than in the other treatments. During the first week of each experiment, growth rates were also significantly reduced by UVB. The reductions occurred despite maximum MAA content, indicating an incomplete protection of photosynthetic and growth‐related processes.

The effects of temperature acclimation on the photoinhibitory responses of Ulva rotundata Blid.

The effect of acclimation to 25, 18, or 10° C on the relationship between photoprotection and photodamage was tested in low-light-grown (80 μmol · m−2 · s−1) Ulva rotundata Blid. exposed to several higher irradiances at the acclimation temperature. Changes in chlorophyll fluorescence parameters (minimum fluorescence, F0, and the ratio of variable to maximum fluorescence, Fv/Fm, measured after 5 min darkness) were monitored during 5 h transfers to 350, 850, and 1700 μmol · m−2 · s−1, and during recovery after 1- or 5-h treatments. At all temperatures, rate of onset and final extent of photoinhibition, measured by a decrease in Fv/Fm, increased with increasing irradiance. At a given photoinhibitory irradiance, rate of onset was most rapid at 10 ° C, but the extent was temperature-independent. Recovery rates from mild light stress were similar at all temperatures, but recovery from the most extreme photoinhibitory treatment lagged 2 h at 10° C. De-epoxidation of xanthophyll-cycle components proceeded faster and to a lower epoxidation status at 25° C, but there was little difference in the pool size among the three growth conditions. Using chloramphenicol to inhibit chloroplast protein synthesis and dithiothreitol to inhibit violaxanthin de-epoxidation, it was shown that at the lowest light treatment given, the extent of photoinhibition could be attributed both to greater amounts of photodamage and to greater zeaxanthin-related photoprotection at 25 than at 10° C. While these two mechanisms for high-light-induced loss of photosynthetic efficiency were operating at 10° C, there was evidence for a relatively greater proportion of zeaxanthin-unrelated photoprotection at the low temperature. This photoprotective mechanism is related to a rapidly reversible increase in F0 and is insentivite to both chloramphenicol and dithiothreitol.

Natural UV Enhances Chloroplast Protein Damage in Chondrus Crispus, Despite the Presence of UV Screening Pigments

As a result of the daily tidal cycle, eulittoral macroalgae are exposed to high levels of photosynthetically active (PAR, 400–700 nm), UV-A (320–400 nm) and UV-B (280–320 nm) radiation. Exposure to high PAR leads to a decline in photosynthetic efficiency as photoprotective reactions divert excess radiation away from photosystem II (PSII) (1). The decline in efficiency is enhanced in the presence of UV radiation (2), possibly as a result of UV-specific damage to reaction centers which differs from that caused by PAR (3). Sublittoral macroalgae also experience a variable, but less extreme, light climate, and are generally more sensitive to a given light stress than are eulittoral ones (1, 4).