Keywords

19.1 Introduction

Even though light is essential for photosynthesis, when in excess, it may harm the photosynthetic apparatus itself (Li et al. 2018). Not only the amount of light, but also its quality in terms of wavelength, may have adverse effects on photosynthesis. In particular, the ultraviolet-B light component of the electromagnetic spectrum (280–315 nm) has been shown to be very dangerous to photosynthesis, as it affects a number of electron transfer steps and biochemical reactions with a very high quantum yield (Zavafer et al. 2015). These include damage to the quinone acceptors (Jansen et al. 1996), damage to the Mn cluster of the oxygen evolving complex (Barbato et al. 1995, Hakala et al. 2005), and increased degradation of reaction center D1-protein with concomitant inhibition of its synthesis (Barbato et al. 2000).

An increase in the level of ultraviolet-B light reaching the Earth’s surface has been measured as a consequence of stratospheric ozone depletion (Allen et al. 1998). Since its discovery, this fact has been causing much concern as it may have strong and adverse effects on ecosystems (Caldwell et al. 2007) and have to be considered as one of main face of climate global changes. In line with this view, it seems important to understand, at any level, the effect of this light on photosynthesis. In line with this view, here we report the result from a study based on fluorescence induction and fluorescence decay after a single turnover flash, in which the effect of visible, ultraviolet-B (UVB), and a mixture of the two kinds of light (visible/UVB) is noted on the photosynthetic apparatus of Thellungiella salsuginea, a model organism in the field of plant environmental stress physiology. Our results indicated that the effect of ultraviolet-B light is different depending on whether visible light is present or not in the background. We found that ultraviolet-B light affects mainly the donor side of photosystem II and electron carriers beyond QB; however, when it was administrated together with visible light (which, per se, did not have any adverse effect), electron transfer between QA and QB was affected too.

19.2 Materials and Methods

Thellungiella salsuginea was grown as described by Goussi et al. (2018). Leaves from 4-week-old plants were used in all experiments. For irradiation, detached leaves were left to float on water and exposed to different kinds of light for either 1 or 3 h. Control leaves were kept in the dark until the end of experiments. Irradiation conditions were as follows: white light, 400 μmol·m−2·s−1, and UVB light (from a Vilber-Lourmat lamp), 1 μmol·m−2·s−1. Before acquisition of fluorescence transients, plants were dark adapted for at least 30 min. Measurements were carried out as described by Pagliano et al. (2006) and analysis of chlorophyll transients performed as described by Strasser and Srivastava (1995). Statistical analysis was carried out by using the Origin software package, version 9.

19.3 Results

Irradiation of Thellungiella salsuginea leaves under our experimental conditions brought about a different effect depending on the particular light treatment. In terms of FV/FM, the intensity of visible white light used in this study did not significantly affect the maximal efficiency of PSII, whereas the presence of UVB, alone or in combination with white light, caused a marked decrease on FV/FM, depending on irradiation time (Table 19.1). This decrease, similar in both irradiation conditions (UVB, visible/UVB), was due to both an increase in F0 and a decrease in FM (Table 19.1 and Fig. 19.1a,b).

Table 19.1 Absolute (Fo, Fm, Fm, Fk, Fj) and relative (Fv/Fm, Fk/Fj) values of chlorophyll fluorescence from control and light-treated plants
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Fig. 19.1
figure 1

Unnormalized chlorophyll fluorescence transients recorded from plants treated either for 1 h (a) or 3 h (b) with white light (up triangles), UVB (squares), and with W/UVB (diamonds). Transient from dark control (circles) is also reported

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In Fig. 19.1a,b, chlorophyll transients (recorded after 30 min of dark adaptation) from leaves treated with visible, ultraviolet-B (UVB), or ultraviolet-B in combination with visible light (visible/UVB) for 1 h (1A) or 3 h (1B) are shown. The fluorescence Ft of the dark-adapted control leaves showed a typical OJIP transient (Strasser et al. 2004). As stated above, UVB treatment, alone or combined with background white light (visible/UVB), induced an increase in F0; at the same time, a considerable decrease of FM was observed. The effect was slightly more pronounced after 3 h of irradiation. At variance, irradiation with white light did not produce any major adverse effects.

To further investigate this point, transients shown in Fig. 19.1a,b were normalized at F0 and FM and then plotted as relative variable fluorescence, VOP (Fig. 19.2a,b). While visible light did not affect the shape of the transients, UVB light produced a strong effect, which was different depending on the presence or absence of background white light. When administrated alone, ultraviolet-B light produced a general loss fluorescence (decrease of all phases, i.e., OJ, Ji, and IP); at variance, when visible light was present together with UVB light, a strong increase of the OJ and a decrease in the JI and IP phases was observed. This effect was qualitatively similar at 1 and 3 h of irradiation, but after 3 h it was more pronounced. By calculating ΔVOP (Fig. 19.2c,d) it became clear that the presence of visible light (visible/UVB) brought about a strong band peaking at about 2–3 ms, whose intensity increased with irradiation time; the appearance of this band was a specific effect of irradiation with visible/UVB, as it was induced neither by UVB nor by visible light alone. In addition, irrespective of the presence of visible light, UVB light brought about the appearance of two negative bands peaking about at 20 and 100 ms, corresponding to a decrease of phases JI and IP, respectively.

Fig. 19.2
figure 2

Normalized (Fo, Fm) chlorophyll fluorescence transients (VOP) from plants treated either for 1 h (a) or 3 h (b) with white light (up triangles), UVB (squares), and with W/UVB (diamonds). Transient from dark control (circles) is also reported. ΔVOP was calculated as VOP (treated)-VOOP(control); plants were treated either for 1 h (c) or 3 h (d). Triangles, white-light-treated minus dark control; squares, UVB-treated minus dark control; diamonds, white/UVB-treated minus dark control

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To further elucidate, at least semiquantitatively, the observed differences in fluorescence kinetics, main bands occurring from O to P transients were analyzed separately. This was achieved by double normalizing curves between 0 and 300 μs (L band, between O and K steps, Fig. 19.3a,b) and between 0 and 2 ms (K band, between O and J steps, Fig. 19.4a,b). K (Fig. 19.3c,d) and L (Fig. 19.4c,d) bands were obtained, and then plotted, as the difference between treated plants with ultraviolet-B (either in the presence or absence of visible light) and dark control. After 1 h of irradiation, ΔVOK revealed a positive L band, peaking between 100 and 200 μs. The maximum amplitude of the L band was after irradiation with UVB after 3 h, whereas when present together with white light, the effect of UVB was much mitigated, very similar to that observed with just white light. The kinetic difference of VOJ revealed the so-called K band in treated plants, which appeared as a peak between 250 and 300 μs.

Fig. 19.3
figure 3

Normalized (Fo, Fk) chlorophyll fluorescence transients (VOK) from plants treated either for 1 h (a) or 3 h (b) with white light (up triangles), UVB (squares), and with W/UVB (diamonds). Transient from dark control (circles) is also reported. ΔVOk (L band) was calculated as VOK (treated)-VOOK (control); plants were treated either for 1 h (c) or 3 h (d). Triangles, white-light-treated minus dark control; squares, UVB-treated minus dark control; diamonds, white/UVB-treated minus dark control

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Fig. 19.4
figure 4

Normalized (Fo, Fj) chlorophyll fluorescence transients (VOj) from plants treated either for 1 h (a) or 3 h (b) with white light (up triangles), UVB (squares), and with W/UVB (diamonds). Transient from dark control (circles) is also reported. ΔVOI (K band) was calculated as VOI (treated)-VOOI (control); plants were treated either for 1 h (c) or 3 h (d). Triangles, white-light-treated minus dark control; squares, UVB-treated minus dark control; diamonds, white/UVB-treated minus dark control

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Changes in IP phase have been measured by transient normalization to the time range of 30–200 ms and expressed as VIP = (Ft − FI) / (Fm − FI) (Fig. 19.5a,b). Upon treatment with UVB light, a loss of IP phase was observed, bigger when visible light was present. Irradiation with visible light did not produce any adverse effect on this phase of the transient (Fig. 19.5a,b). Finally, fluorescence decay after a single turnover flash (in the μs range) was investigated. Visible light did not affect fluorescence decay, whereas UVB light, particularly when visible light was present in the background, produced a strong slowing down of fluorescence decay (Fig. 19.6).

Fig. 19.5
figure 5

Normalized (Fi, Fp) chlorophyll fluorescence transients (VIP) from plants treated either for 1 h (a) or 3 h (b) with white light (up triangles), UVB (squares), and with W/UVB (diamonds). Transient from dark control (circles) is also reported

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Fig. 19.6
figure 6

Fluorescence decay in leaves treated for either 1 h (a) or 3 h (b) in plants treated with white light (dashed line), UVB light (solid line), or W/UVB light (dash dot line). Fluorescence decay from dark control is also shown (dotted line)

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19.4 Discussion

The effect of UVB on photosynthetic apparatus has widely been investigated. There seems to be a number of different targets in the thylakoid membranes for this kind of radiation. Damage to the Mn cluster (Barbato et al. 1995; Hakala et al. 2005; Vass 2012; Vass et al. 1996) associated with cleavage of the D1 protein originating a 23-kDa C-terminal fragment (Barbato et al. 1995, 2000) is considered as the main target for this radiation, but additional target, such as the QB site (Trebst and Depka 1990; Vass et al. 1996) has been suggested. The effect of irradiation with a mixture of the two lights has been less investigated, but available data (Sicora et al. 2003) indicate that, even though administrated at the same time, the two kinds of radiations might have different and noninteracting targets. Results reported in this study suggest a slightly different view. UVB irradiation, apparently, did not affect QB site, as the rising in fluorescence in the 0–3 ms (OJ phase) is similar to the control. At variance, when analysis is restricted to the first 300 μs or the Fk/Fj ratio is considered (i.e., the time required for water to diffuse to the donor side of the photosystem), UVB adversely affects the chlorophyll transient, confirming the donor side of PSII as a main target for UVB light. However, when white light is superimposed to UVB, the appearance of a strong band in the first 3 ms of the transient suggests that additional targets are hit, most likely the QB site. Accordingly, decay of fluorescence after a single saturating flash supports this possibility. Thus, we suggest that under mixed light conditions (at least when the light intensities here reported are used), electron transfer between QA and QB may be one main target. Clearly, if sensibility to UVB light increases when white light is present, it is likely that the actual target is a reduced or semireduced form of quinones.

The effect of UVB on the second part of the chlorophyll transient (JI, IP) instead seems to be essentially independent on the presence of visible light. In fact, a decrease in both JI and IP phases is observed. These phases are thought to describe electron transfer steps beyond quinone acceptor leading to reduction of PSI acceptors. If so, we have to conclude that cytochrome b6/f and/or PSI could be specifically targeted for damage. This possibility remains to be investigated.