Introduction

Plants growing under natural conditions are constantly exposed to ultraviolet radiation. Considering the components of this radiation, UV-A (315-400 nm) has the lowest biological activity, but compared with UV-B (280-315 nm) and UV-C (100-280 nm) it penetrates into deeper tissues due to longer wavelengths (McKenzie et al. 2004). Regardless of the condition of the ozone layer, in the spectrum of solar radiation reaching the Earth’s surface UV radiation constitutes up to 6%, in which almost 95% is UV-A radiation and only about 5% is UV-B radiation (Moan 2001; Hollósy 2002).

The influence of UV-B radiation on plants is widely discussed in the literature, however, there are no similar studies regarding the response of plants to UV-A (Verdaguer et al. 2017). The UV radiation effect on plants leads to a number of changes in physiological processes, affecting especially the process of photosynthesis. One of the effects is photoinhibition of the photosystem II (PSII), comprehended as an imbalance between PSII photodamage caused by high irradiance, fluctuating light as well as short wavelength, and its subsequent repair (Li et al. 2018). It is believed that: the main site of direct damage is the catalytic cluster Mn4O5Ca; the resulting reactive oxygen species (ROS) additionally degrade the protein subunits D1 and D2 from the PSII reaction centre and damage the binding sites of QA and QB (Kale et al. 2017). On the other hand, there are reports about a positive effect of UV-A radiation on the photosynthetic apparatus, especially when UV-A radiation was used as a plant acclimation factor for UV-B under specific conditions (Štroch et al. 2015).

Although the UV-B:UV-A ratio undergoes quite significant changes both during the day and the year (due to unstable UV-B and constant UV-A transmission to the Earth’s surface), there is a lot of information about the influence of these UV components on plants (Moan 2001; Verdaguer et al. 2017). Direct influence of ionizing radiation on plant cells is associated with: membrane lipid peroxidation (Urban et al. 2016), protein polymerization and enzyme deactivation (Urban et al. 2016); destruction of double bonds C=C leading to changes in the structure of DNA and proteins (Urban et al. 2016 and literature therein), disorder of ion transport, depolarization of membranes and increase in their permeability (Wuytack et al. 2003). Moreover, UV radiation influences the photosynthesis process which is associated with (1) disturbances in the synthesis of chlorophyll pigments (degradation of alpha amino levulinic dehydrogenase ALAD) (Urban et al. 2016); (2) disorders in the synthesis of carotenoid pigments (MEP pathway) (Giuliano 2014) as well as (3) degradation of pigments related to radiation-induced senescence and/or radiation-induced programmed cell death (Kataria et al. 2014; Urban et al. 2016; Verdaguer et al. 2017). However, the influence of UV-A radiation in relation to the UV-A:PAR ratio is poorly understood (Barnes et al. 2013; Verdaguer et al. 2017).

Grass pea (Lathyrus sativus L.) is a legume species resistant to harsh growing conditions, such as drought, salinity or periodic flooding (Piwowarczyk et al. 2014, 2016, 2017). Due to the advantageous composition of seeds, high level (approx. 30% of eed dry weight) of protein rich in exogenous amino acid–lysine (Grela et al. 2010), it is used for consumption in Central and South Asia or East Africa (Vaz Patto et al. 2006). In these regions (near the equator) the intensity of UV radiation is the highest (Moan 2001).

We hypothesized that red light, improving the light and/or dark reactions of photosynthesis, would reduce the risk of ROS action on PSII RC under UV-A radiation. Therefore the aim of the work was to examine whether UV-A:red light radiation would enable quick acclimation of the photosynthetic apparatus of grass pea to the UV-A negative effect.

Materials and methods

Plant material and experimental conditions

The plant material comprised of the Polish grass pea cultivar ‘Krab’. Seeds were sown in pots (12 cm in diameter) containing horticultural soil (sandy-loam) (Biovita, Tenczynek, Poland). The pots were placed in a grow room at a temperature of 23 ± 2 °C under white LED lighting (intensity 120 μmol [quanta] m−2·s−1, spectral composition Fig. 1a) and a 16-h photoperiod. The plants were watered every 2–3 days. 14-day-old seedlings were placed under UV-A:red light (ratio 10:90, intensity 110 μmol [quanta] m−2·s−1, spectral composition Fig. 1b) for 48 h. Control plants were kept under initial conditions. Twenty seedlings (five in each of four pots) were grown in each treatment.

Fig. 1
figure 1

Spectral composition of a white LED light and b UV-A: red light, RU relative units

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Measurements and assays

The youngest, fully developed leaves (the 3rd node counting from the top of the plant) were used. Measurements were performed using 10 biological replicates (one replicate = an individual leaf from an individual plant).

Photosynthetic pigment concentration was assayed spectrophotometrically (Double Beam spectrophotometer U-2900, Hitachi High-Technologies Corporation) using the method developed by Lichtenthaler (1987) and the equations as described by Wellburn (1994) [Chl a = 12.21*A663 −2.81*A646; Chl b = 20.13*A646 −5.03*A663; Car = (100*A470 −  3.27*Chl a − 104*Chl b)/198]. The lipid peroxidation level, based on the malondialdehyde (MDA) content, was assessed according to Dhindsa et al. (1981). Chlorophyll a fluorescence measurement was performed using a Handy-PEA (Hansatech, UK) fluorometer according to standard procedures. The leaves were dark-adapted for 25 min. The fluorescence was induced by red light: λmax = 650 nm, 2000 μmol [quanta] m−2·s−1. Selected functional and structural photosynthetic parameters were extracted from the recorded curves using the fluorometer producer’s software (PEA-Plus) and/or calculated according to Jiang et al. (2008) (Table 1–according to Piwowarczyk et al. 2018). Gas exchange measurements were carried out with a portable open gas-exchange system (LCpro-SD; ADC BioScientific Ltd UK) with a 6.24 cm2 cuvette with a mixed Red/Blue LED Light Source Head. Before recording the leaves were adapted for 2 min in the cuvette to allow photosynthesis to reach the steady state. Measurements were done under CO2 saturated conditions (650 µmol·mol−1); 300 µmol·s−1 of air flow, 50–55% relative humidity within the cuvette, leaf temperature of 25 °C and under the 130 µmol [quanta] m−2·s−1 red light intensity. Photosynthetic light response curves were done on the same plants used for net photosynthesis for a stepwise reduction of PAR ranging from 1500 to 0 µmol (quanta) m−2·s−1 (in 100, 50, 20, 0, 100, 300, 500, 1000, 1500, 300 and 100 µmol (quanta) m−2·s−1 steps). The leaves were adapted to each of the light intensities for 5, 3, 2, 5, 5, 5, 5, 5, 5, 5 and 5 min, respectively before data point recording. Air flow, relative humidity and CO2 concentration inside the cuvette were the same as described in the case of gas exchange measurement.

Table 1 Abbreviations and descriptions of extracted and calculated photosynthetic parameters (Jiang et al. 2008; Kalaji et al. 2011; Piwowarczyk et al. 2018)
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Statistical analyses

The results were subjected to statistical analyses using Statistica 12.0 (StatSoft Inc., Tulsa, OK, USA). The significant differences between the means of each treatment were determined using Duncan’s test at p < 0.05.

Results and discussion

The effect of UV-A radiation on the photosynthetic apparatus in a short period of time consisted in a rapid decrease in the chlorophyll pigment content. Salama et al. (2011) observed in the leaves of plants treated with UV-A + PAR radiation a decrease in the level of chlorophyll pigments ranging from 34% (Rumex vesicarius) to even 99% (Plantago major). Also the level of carotenoid pigments, whose function in the photosynthetic apparatus is connected on the one hand with supplying additional excitation energy to the reaction centre of the photosystem II (RC PSII), and on the other with the protection of PSII against photoinhibition and photooxidation by dissipating the excess absorbed light energy in the xanthophyll cycle, undergoes significant changes as a result of UV-A radiation. In the studies of Salama et al. (2011), UV-A radiation caused a statistically significant decrease in the carotenoid pigment content in most of the studied species. Our results revealed that UV-A radiation had no effect on chlorophyll a, chlorophyll b and total chlorophyll as well as carotenoid contents in grass pea shoots (Fig. 2a). It seems that in grass pea plants chlorophyll and carotenoids synthesis pathways were not disturbed by UV-A radiation.

Fig. 2
figure 2

a Photosynthetic pigment content, b lipid peroxidation level expressed as the malondialdehyde (MDA) content, c extracted and calculated chlorophyll a fluorescence parameters, d CO2 assimilation efficiency of photosynthesis of grass pea seedlings after 48 h of UV-A:red radiation treatment, a–b error bars–standard deviations, d numbers are given as a percentage of the control (= 100%), abbreviations–see Table 1, *significant difference between treatments according to Duncan’s test at p < 0.05

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Alterations in the pigment content could also be associated with chloroplast damage. Besides direct destruction of plasmatic membranes by high energy UV radiation, plastid membranes might be damaged as a result of reactive oxygen species (ROS) activity (Urban et al. 2016). The source of ROS can include PSII malfunction as well as impairment of antioxidant enzymes (Urban et al. 2016). ROS cause changes in membrane lipid composition (increased level of unsaturated vs. saturated fatty acids) as well as potassium leakage and lipid peroxidation (Singh et al. 2010; Tokarz et al. 2018). In the case of grass pea, UV radiation had no significant effect on the MDA content (Fig. 2b), indicating lack of changes in the level of lipid peroxidation. Lack of changes in lipid peroxidation in leaves under stress conditions results in sufficient antioxidant system efficiency or efficient linear electron transport between PSII and PSI, or the conjunction of these two mechanisms (Piwowarczyk et al. 2018; Tokarz et al. 2018).

Moreover, the direct impact of UV on the photosynthetic apparatus is related to the damage to the oxygen-evolving complex (OEC), degradation of D1 and D2 proteins of PSII, degradation of the plastoquinone pool (PQ) as well as cytochrome b6f (Kataria et al. 2014; Urban et al. 2016; Verdaguer et al. 2017), which leads to disturbances in electron transport between PSII and PSI (Tyystjarvi and Vass 2004). In isolated spinach chloroplasts, UV-A radiation caused damage to PSII arising from disturbed electron transport on both the donor (OEC) and acceptor (PQ) sides (Tyystjarvi and Vass 2004). Damage to OEC, which is sensitive to high PAR intensities and UV-A radiation, leads to degradation of RC PSII from the electron ‘donor side’ (Zsiros et al. 2006). This process is caused by direct oxidation of P680 + or indirect inactivation of PSII RC by ROS formed on the donor side as a result of OEC degradation by UV-A radiation (Tyystjärvi 2008; Turcsányi and Vass 2000). Disturbances on the acceptor side of PSII are associated with degradation of PQ by UV radiation (Vass et al. 2005), which results in the permanently reduced state of the QA electron acceptor of PSII leading to photoinhibition of PSII RC (Tyystjärvi 2008). Measurement of chlorophyll a fluorescence of grass pea leaves subjected to 48 h of UV-A:red light revealed that the donor side of PSII- OEC remained unchanged (Fv/Fo) (Fig. 2c). Furthermore, the electron flux beyond PSII RC (φEo and ψEo) and the electron transport flux (ET0) per reaction center and per cross section of thylakoid increased significantly in leaves of stressed plants (Fig. 2c). Moreover, in grass pea plants exposed to UV-A:red radiation higher efficiency of RC was associated with a significant increase in total electron carriers per RC (Sm) (Fig. 2c). On the other hand, the dissipated energy flux (DI0) per RC decreased (Fig. 2c). Similar changes in the photosynthetic apparatus efficiency were found in grass pea genotypes under drought stress conditions (Silvestre et al. 2014). We hypothesize that more efficient linear electron transport associated with the stable state of the donor side of PSII RC makes it possible to avoid the direct and/or indirect impact of UV-A radiation.

Most of the reports on the harmful effects of UV radiation on the photosynthetic apparatus concerned isolated chloroplasts or thylakoids. The research conducted on intact leaves indicated significantly higher resistance of the photosynthetic apparatus. Studies conducted on lettuce and Arabidopsis that were pre-illuminated with red light followed by UV-A revealed that pre-illumination with red light had a protective effect on the photosynthetic apparatus. Nevertheless, a significant decrease in net photosynthesis was observed (Kreslavski et al. 2013, 2016). Moreover, other authors observed a slight increase in the photosynthesis rate as a result of enhanced stomatal conductance and/or a reduction in the functional size of PSII (Joshi et al. 2007; Štroch et al. 2015; Verdaguer et al. 2017). Furthermore, plants exposed to shortwave radiation, but in the PAR range (Purple and Blue), were characterized by higher Rubisco activity and higher transcriptional levels of 10 genes of the key Calvin-Benson Cycle enzymes, which in turn did not result in higher rates of photosynthesis due to inactivation of PSII caused by that radiation (Wang et al. 2009).

In our studies, plants exposed to 48 h of UV-A:red light showed an increased rate of photosynthesis without changes in stomatal conductance and rate of transpiration (Table 2). Furthermore, the increasing rate of photosynthesis was pronounced at higher light intensities (Fig. 2d).

Table 2 Net photosynthesis [PN (µmol CO2 m−2·s−1)], stomatal conductance [Gs (mmol H2O m−2·s−1)] and rate of transpiration [E (mmol H2O m−2·s −1)] in 14-day-old grass pea seedlings after 48 h of UV-A:red radiation treatment
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In summary, acclimatization of the photosynthetic apparatus of grass pea to UV-A stress conditions results from efficient linear electron transport due to maintaining the effective OEC complex (Fv/Fo value) and increased total electron carriers and/or effective CO2 carboxylation.

Author contribution statement

Krzysztof Tokarz designed and performed experiments, discussed the results and wrote the paper; Barbara Piwowarczyk analyzed data and wrote the paper; Anna Wysocka and Wojciech Makowski gave technical support and performed experiments; Tomasz Wójtowicz discussed the results and wrote the paper; Elżbieta Golemiec gave scientific and technical support; All authors proofread manuscript.