Photosystem II Is More Sensitive than Photosystem I to Al3+ Induced Phytotoxicity

Aluminium (Al) the most abundant metal in the earth’s crust is toxic in acid soils (pH < 5.5) mainly in the ionic form of Al3+ species. The ability of crops to overcome Al toxicity varies among crop species and cultivars. Here, we report for a first time the simultaneous responses of photosystem II (PSII) and photosystem I (PSI) to Al3+ phytotoxicity. The responses of PSII and PSI in the durum wheat (Triticum turgidum L. cv. ‘Appulo E’) and the triticale (X Triticosecale Witmark cv. ‘Dada’) were evaluated by chlorophyll fluorescence quenching analysis and reflection spectroscopy respectively, under control (−Al, pH 6.5) and 148 μM Al (+Al, pH 4.5) conditions. During control growth conditions the high activity of PSII in ‘Appulo E’ led to a rather higher electron flow to PSI, which induced a higher PSI excitation pressure in ‘Appulo E’ than in ‘Dada’ that presented a lower PSII activity. However, under 148 μM Al the triticale ‘Dada’ presented a lower PSII and PSI excitation pressure than ‘Appulo E’. In conclusion, both photosystems of ‘Dada’ displayed a superior performance than ‘Appulo E’ under Al exposure, while in both cultivars PSII was more affected than PSI from Al3+ phytotoxicity.

In the form of the trivalent cation Al 3+ , that is toxic to most plants at relatively low concentrations, it is the main limiting factor in the world's arable non-irrigated crop production to over 40% [26,27].

Measurements of Chlorophyll a Fluorescence
Chlorophyll a fluorescence was measured in dark-adapted (20 min) leaf samples, using a pulse amplitude modulation fluorometer (PAM, Walz, Effeltrich, Germany), as described before [35,43]. First, minimal chlorophyll a fluorescence (F o ) was measured by application of a weak modulated light beam (L 1 ) followed by a saturating light pulse (L 2 ) to measure maximal chlorophyll a fluorescence (F m ) in the dark adapted (20 min) samples. Then, by application of the actinic light (L A ) and saturating light pulses, maximum chlorophyll a fluorescence in the light (F m ) was measured, while to assess steady-state photosynthesis (F s ) values, the actinic light (L A ) alone was applied. Minimum chlorophyll a fluorescence in the light (F o ) was measured immediately after turning off the actinic light (L A ) ( Figure 1). The calculated chlorophyll fluorescence parameters with their definitions are given in Table 1.

Plant Material and Growth Conditions
Durum wheat (Triticum turgidum L. cv. 'Appulo E') and the triticale (X Triticosecale Witmark cv. 'Dada') were used to compare the tolerance of the two photosystems to Al toxicity. Seeds obtained from the Institute of Plant Breeding and Genetic Resources, Thermi, Greece, germinated at 22 ± 1 °C for 2 d in the dark. The germinated seeds were transferred in a growth chamber and mounted on nylon-mesh floats on plastic vessels filled with nutrient solution at pH 6.5 [23]. The seedlings were grown in hydroponic culture at controlled environmental conditions as described previously [43].

Al Treatment
Al was supplied at 148 μM as KAl(SO4)212H2O for 14 days. Al-containing pots (nutrient solution plus 148 μM Al) were acidified initially to pH 4.5 with 1N HCl [25], while growth solutions of control plants (nutrient solution only) were adjusted to pH 6.5. According to the GEOCHEM-EZ speciation programme [59] the free Al 3+ activities were calculated to be 16.8 μM [43].

Lipid Peroxidation Measurements
The level of lipid peroxidation of controls and 14-days Al 3+ treated plants was measured as malondialdehyde (MDA) content, as described previously [60], according to the method of Heath and Packer [61]. The concentration of MDA was calculated from the difference of the absorbance at 532 and 600 nm and expressed as nmol (MDA) g −1 fresh weight.

Measurements of Chlorophyll a Fluorescence
Chlorophyll a fluorescence was measured in dark-adapted (20 min) leaf samples, using a pulse amplitude modulation fluorometer (PAM, Walz, Effeltrich, Germany), as described before [35,43]. First, minimal chlorophyll a fluorescence (Fo) was measured by application of a weak modulated light beam (L1) followed by a saturating light pulse (L2) to measure maximal chlorophyll a fluorescence (Fm) in the dark adapted (20 min) samples. Then, by application of the actinic light (LA) and saturating light pulses, maximum chlorophyll a fluorescence in the light (Fm′) was measured, while to assess steady-state photosynthesis (Fs) values, the actinic light (LA) alone was applied. Minimum chlorophyll a fluorescence in the light (Fo′) was measured immediately after turning off the actinic light (LA) (Figure 1). The calculated chlorophyll fluorescence parameters with their definitions are given in Table 1.
The PSII maximum efficiency is an estimate of the maximum efficiency of PSII photochemistry at a given PPFD (photosynthetic photon flux density) The effective quantum yield of photochemical energy conversion in PSII estimating the efficiency at which light absorbed by PSII is used for photochemistry, that means is used for reduction of the primary acceptor of PSII quinone A (QA) The photochemical quenching is a measure of the fraction of open PSII reaction centers, that is the redox state of QA The non-photochemical quenching that reflects heat dissipation of excitation energy Calculated as (F m − F m ')/F m '

ETR
The relative PSII electron transport rate where c is 0.5 since the absorbed light energy is assumed to be equally distributed between PSII and PSI, and abs is the total light absorption of the leaf taken as 0.84.

Φ NPQ
The quantum yield of regulated non-photochemical energy loss in PSII, that is the quantum yield for dissipation by down regulation in PSII The quantum yield of non-regulated energy loss in PSII, a loss process due to PSII inactivity Calculated as F s /F m 1 − q P Excitation pressure of PSII, or the fraction of closed PSII reaction centers Calculated as 1 − q P

Measurements of Leaf Absorbance Changes at 820 nm
A Hansatech P 700 + measuring system was employed to monitor light-induced changes in leaf absorbance at around 820 nm according to Havaux et al. [57], as described before [58]. The fraction of closed PSI reaction centers (B 1 ) was calculated as: B 1 = ∆S/(∆S)max = (Rfr − R')/(Rfr − R).

Statistical Analysis
Data are presented as the mean ± SD. Statistical analysis was performed using the Student's t-test. Differences were considered statistically significant at p < 0.05.

Allocation of the Absorbed Light Energy in PSII under Normal Growth and Al 3+ Exposure
Under control growth conditions at pH 6.5, the durum wheat 'Appulo E' presented higher effective quantum yield of photochemical energy conversion in PSII (Φ PSII ) ( Figure 2a (Figure 2), but higher Φ NO (that did not differ from control conditions) (Figure 3a), than the durum wheat 'Appulo E'. However, 'Appulo E' due to the efficient photoprotective mechanism, that is the quantum yield for dissipation by down regulation in PSII, possessed lower Φ NO even though from control conditions ( Figure 3a).

Non-Photochemical Quenching under Normal Growth and Al 3+ Exposure
The triticale 'Dada' had higher non-photochemical fluorescence quenching (NPQ) under control growth conditions (pH 6.5) than the durum wheat 'Appulo E' but under 148 μM Al at pH 4.5 it was the reverse (Figure 3b).

Electron Transport Rate and the Redox State of PSII under Normal Growth and Al 3+ Exposure
Under control growth conditions the durum wheat 'Appulo E' presented higher electron transport rate (ETR) ( Figure 4a) and a more oxidized redox state of PSII (qP) (Figure 4b), than the triticale 'Dada'. Under Al exposure the triticale 'Dada' had higher ETR than the durum wheat

Non-Photochemical Quenching under Normal Growth and Al 3+ Exposure
The triticale 'Dada' had higher non-photochemical fluorescence quenching (NPQ) under control growth conditions (pH 6.5) than the durum wheat 'Appulo E' but under 148 μM Al at pH 4.5 it was the reverse (Figure 3b).

Electron Transport Rate and the Redox State of PSII under Normal Growth and Al 3+ Exposure
Under control growth conditions the durum wheat 'Appulo E' presented higher electron transport rate (ETR) (Figure 4a) and a more oxidized redox state of PSII (qP) (Figure 4b), than the triticale 'Dada'. Under Al exposure the triticale 'Dada' had higher ETR than the durum wheat

Non-Photochemical Quenching under Normal Growth and Al 3+ Exposure
The triticale 'Dada' had higher non-photochemical fluorescence quenching (NPQ) under control growth conditions (pH 6.5) than the durum wheat 'Appulo E' but under 148 µM Al at pH 4.5 it was the reverse (Figure 3b).

Electron Transport Rate and the Redox State of PSII under Normal Growth and Al 3+ Exposure
Under control growth conditions the durum wheat 'Appulo E' presented higher electron transport rate (ETR) (Figure 4a) and a more oxidized redox state of PSII (q P ) (Figure 4b), than the triticale 'Dada'. Under Al exposure the triticale 'Dada' had higher ETR than the durum wheat 'Appulo E' (Figure 4a), but the same redox state of PSII (q P ) (Figure 4b) with durum wheat 'Appulo E'.

The Maximum PSII Quantum Efficiency (Fv/Fm) and PSII Maximum Efficiency in Light (Fv'/Fm') under Normal Growth and Al 3+ Exposure
The maximum quantum efficiency of PSII (Fv/Fm) under normal growth conditions was higher in the durum wheat 'Appulo E', but under 148 μM Al it was higher in the triticale 'Dada' (Figure 5a

Oxidative Damage under Normal Growth and Al 3+ Exposure
Under Al exposure the level of lipid peroxidation measured as malondialdehyde (MDA) content and expressed as nmol (MDA) g −1 fresh weight increased compared with control growth conditions, but it was the same in both the triticale 'Dada' and the durum wheat 'Appulo E', while under normal growth conditions it was higher in 'Dada' (Figure 6).

The Maximum PSII Quantum Efficiency (F v /F m ) and PSII Maximum Efficiency in Light (F v '/F m ') under Normal Growth and Al 3+ Exposure
The maximum quantum efficiency of PSII (F v /F m ) under normal growth conditions was higher in the durum wheat 'Appulo E', but under 148 µM Al it was higher in the triticale 'Dada' (Figure 5a). The maximum efficiency of PSII in the light (F v '/F m ') was similar under control growth conditions (Figure 5b), but under 148 µM Al it was higher in the triticale 'Dada' suggesting a higher quantum yield of the open, functional reaction centers, than in the durum wheat 'Appulo E' (Figure 5b). 'Appulo E' (Figure 4a), but the same redox state of PSII (qP) (Figure 4b) with durum wheat 'Appulo E'.

The Maximum PSII Quantum Efficiency (Fv/Fm) and PSII Maximum Efficiency in Light (Fv'/Fm') under Normal Growth and Al 3+ Exposure
The maximum quantum efficiency of PSII (Fv/Fm) under normal growth conditions was higher in the durum wheat 'Appulo E', but under 148 μM Al it was higher in the triticale 'Dada' (Figure 5a

Oxidative Damage under Normal Growth and Al 3+ Exposure
Under Al exposure the level of lipid peroxidation measured as malondialdehyde (MDA) content and expressed as nmol (MDA) g −1 fresh weight increased compared with control growth conditions, but it was the same in both the triticale 'Dada' and the durum wheat 'Appulo E', while under normal growth conditions it was higher in 'Dada' (Figure 6).

Oxidative Damage under Normal Growth and Al 3+ Exposure
Under Al exposure the level of lipid peroxidation measured as malondialdehyde (MDA) content and expressed as nmol (MDA) g −1 fresh weight increased compared with control growth conditions, but it was the same in both the triticale 'Dada' and the durum wheat 'Appulo E', while under normal growth conditions it was higher in 'Dada' (Figure 6). Materials 2018, 11, x FOR PEER REVIEW 7 of 12

Excitation Pressure in PSI and PSII under Normal Growth and Al 3+ Exposure
The fraction of closed PSI reaction centers (B1) or PSI excitation pressure under both control growth conditions and Al exposure was higher in 'Appulo E' (Table 2), while the fraction of closed PSII reaction centers (PSII excitation pressure) under control growth conditions was higher in the triticale 'Dada', but under Al exposure was higher in 'Appulo E' ( Table 2). Under 148 μM Al, PSII excitation pressure in both triticale 'Dada' and durum wheat 'Appulo E' was higher than PSI excitation pressure (Table 2).

Discussion
In a hydroponic solution as summarized by Famoso et al. [2], Al may be found either (a) as free Al 3+ , that actively inhibits root growth; (b) precipitated with other elements and essentially non-toxic to plant growth; (c) different hydroxyl Al monomers also non-toxic to roots [62]; or (d) complexed with other elements in an equilibrium between its active and inactive states. Thus, the degree of Al toxicity to plants is primarily related to the activity of free Al 3+ in solution [63]. In our experiment, according to the GEOCHEM-EZ speciation program [59], the free Al 3+ activities in the nutrient solutions were calculated to be 16.8 μM.
The significant lower quantum efficiency of PSII photochemistry in 'Dada' (ΦPSII) under control growth conditions (Figure 2a) was compensated by a significant higher regulated heat dissipation, a loss process serving for protection (ΦNPQ) (Figure 2b), that was sufficient enough to retain the same quantum yield of non-regulated energy dissipated in PSII (ΦNO) in both 'Dada' and 'Appulo E' (Figure  3a). Under Al exposure we observed a reverse situation, with the significant higher photoprotective heat dissipation (ΦNPQ) in 'Appulo E' (Figure 2b) not only to compensate the significant lower quantum efficiency of PSII photochemistry (ΦPSII) (Figure 2a), but even more, to lower the quantum yield of non-regulated energy dissipated in PSII (ΦNO) compared to 'Dada' (Figure 3a).
The most vulnerable component of the photosynthetic machinery to abiotic stresses is considered to be PSII [64]. However, despite the fact that PSI was shown to be more resistant to mild

Excitation Pressure in PSI and PSII under Normal Growth and Al 3+ Exposure
The fraction of closed PSI reaction centers (B 1 ) or PSI excitation pressure under both control growth conditions and Al exposure was higher in 'Appulo E' (Table 2), while the fraction of closed PSII reaction centers (PSII excitation pressure) under control growth conditions was higher in the triticale 'Dada', but under Al exposure was higher in 'Appulo E' ( Table 2). Under 148 µM Al, PSII excitation pressure in both triticale 'Dada' and durum wheat 'Appulo E' was higher than PSI excitation pressure ( Table 2).

Discussion
In a hydroponic solution as summarized by Famoso et al. [2], Al may be found either (a) as free Al 3+ , that actively inhibits root growth; (b) precipitated with other elements and essentially non-toxic to plant growth; (c) different hydroxyl Al monomers also non-toxic to roots [62]; or (d) complexed with other elements in an equilibrium between its active and inactive states. Thus, the degree of Al toxicity to plants is primarily related to the activity of free Al 3+ in solution [63]. In our experiment, according to the GEOCHEM-EZ speciation program [59], the free Al 3+ activities in the nutrient solutions were calculated to be 16.8 µM.
The significant lower quantum efficiency of PSII photochemistry in 'Dada' (Φ PSII ) under control growth conditions (Figure 2a) was compensated by a significant higher regulated heat dissipation, a loss process serving for protection (Φ NPQ ) (Figure 2b), that was sufficient enough to retain the same quantum yield of non-regulated energy dissipated in PSII (Φ NO ) in both 'Dada' and 'Appulo E' (Figure 3a). Under Al exposure we observed a reverse situation, with the significant higher photoprotective heat dissipation (Φ NPQ ) in 'Appulo E' (Figure 2b) not only to compensate the significant lower quantum efficiency of PSII photochemistry (Φ PSII ) (Figure 2a), but even more, to lower the quantum yield of non-regulated energy dissipated in PSII (Φ NO ) compared to 'Dada' (Figure 3a).
The most vulnerable component of the photosynthetic machinery to abiotic stresses is considered to be PSII [64]. However, despite the fact that PSI was shown to be more resistant to mild water deficit than PSII, it was heavily damaged by prolonged water deficit [64]. PSI is impaired when electron flow from PSII to PSI exceeds the capability of PSI electron carriers to manage the electrons [65,66]. Proton gradient (∆pH)-dependent slow-down of electron transfer from PSII to PSI protects PSI from excess electrons [66]. As occurred in our experiment, PSI in Appulo E was more inhibited under control growth conditions than in Dada. During control growth conditions the high activity of PSII in Appulo E led to a rather higher electron flow to PSI, causing probably the formation of ROS within PSI complex [67,68], which induced a higher PSI excitation pressure in Appulo E than in Dada ( Table 2) that presented a lower PSII photochemistry (Figure 2a) and lower PSI excitation pressure ( Table 2). This higher PSI photoinhibition in Appulo E than in Dada under control growth conditions was alleviated by the absence of PSII photoinhibition in Appulo E as indicated by the F v /F m value ( Figure 5). The absence of the photoprotective mechanism of NPQ in Appulo E under control growth conditions (Figure 3b), that slows-down the electron transfer from PSII to PSI, could not protect PSI from excess electrons. However, this absence of the photoprotective mechanism of NPQ in Appulo E (Figure 3b) did not cause any problem on the fraction of open PSII reaction centers (Figure 4b). Thus, under control growth conditions Appulo E shows lower PSII excitation pressure and a better PSII function, despite a higher PSI photoinhibition. Hence, regardless of the ROS formation within PSI complex in Appulo E, the level of lipid peroxidation, measured by MDA accumulation that reflects ROS formation and corresponds to oxidative damage, was shown to be less than in Dada, under control growth conditions ( Figure 6). Current evidence suggests that ROS production can serve as the signal that triggers the expression of genes that may serve to alleviate electron pressure on the reducing side of PSI [69].
After Al exposure, the electron flow from PSII to PSI in 'Appulo E' was suppressed, but excitation pressure was increased in both photosystems, although more in PSII. This slow-down of electron transfer from PSII to PSI in 'Appulo E' protects PSI from excess electrons. A proper regulation of ETR is crucial in the protection of PSI against photoinhibition [70]. However, PSI photoinhibition may represent a kind of protective mechanism against over-reduction of PSI acceptor side, diminishing creation of huge amount of ROS and avoiding extensive cell injury [71,72]. The controlled photoinhibition of PSII in 'Appulo E', under Al exposure, as indicated by the F v /F m value (Figure 5a), was also able to protect PSI from permanent photodamage [66].
The high excitation pressure in PSII (1 − qp) under Al exposure, observed in 'Appulo E' (Table 2), indicates an imbalance between energy supply and demand [73]. However, the significant increase of NPQ processes in PSII (Figure 3b), that reflects the dissipation of excess excitation energy in the form of harmless heat [47,51,52,74], seems that protected 'Appulo E' plants under Al exposure from the destructive effects of ROS. It appears that under Al exposure, NPQ increase in PSII was sufficient enough to protect 'Appulo E' plants from ROS production since the quantum yield of non-regulated non-photochemical energy loss (Φ NO ) decreased significantly (Figure 3a), thus exhibited lower singlet oxygen ( 1 O 2 ) production. The quantum yield of non-regulated non-photochemical energy loss (Φ NO ) consists of chlorophyll fluorescence internal conversions and intersystem crossing, which leads to the formation of 1 O 2 via the triplet state of chlorophyll ( 3 chl*) [75][76][77]. The increased NPQ in 'Appulo E' under Al exposure (Figure 3b) was also capable to keep the same fraction of open reaction centers as in Dada (Figure 4b) and also the same level of lipid peroxidation (Figure 6), thus the same degree of oxidative damage. The photoprotective mechanism of NPQ can divert absorbed light to other processes, such as thermal dissipation, preventing the photosynthetic apparatus from oxidative damage [47,48,[78][79][80][81].

Conclusions
In conclusion, we confirmed that the triticale cv. 'Dada' was more tolerant to Al phytotoxicity than durum wheat 'Appulo E', as reflected by the better PSII functionality under Al acidic conditions. However, under normal growth conditions (−Al, pH 6.5), durum wheat 'Appulo E' displayed a better PSII functionality. Yet, under Al exposure, PSII was more affected than PSI from Al 3+ phytotoxicity in both cultivars.