As a source of energy, light is one of the essential environmental factors for plant life in additition to water, mineral nutrients and carbon dioxide. Light intensity, spectral quality, duration and direction of illumination are highly variable, which remarkably influences the dynamics and efficiency of the photosynthesis process and consequently plant growth. To alleviate the effect of rapid light fluctuation on the photosynthetic apparatus and to adjust light availability with the metabolic demands of an organism, regulatory mechanisms of photosynthesis harmonize light capture and its utilization (Roach and Krieger-Liszkay 2019, Vojta et al. 2019).

Photosynthetic pigments – chlorophyll a (Chl a), chlorophyll b (Chl b) and carotenoids (Cars) have a crucial role in light energy capturing. They are assembled into light harvesting antenna complexes (LHCs) which deliver excitation energy into the reaction center of photosystems I and II (PSI and PSII) (Morishige and Dreyfuss 1998). Special Chl molecules in the reaction center of PSI and PSII pass excited electrons into electron transport chain in thylakoid membranes of chloroplasts. Carotenoids have a dual role in photosynthesis. They are accessory light-harvesting pigments that transfer light energy to Chls, and so expand the wavelength range of absorbed light. They also have a protective role and prevent harmful effects of exposure to high light intensity (Hashimoto et al. 2016). By altering the content of Chls and Cars, plants adjust photosynthesis to variable light conditions. In many studies, Chls and Cars contents and their ratios have been used as an indicator of plant acclimation to light availability (Lichtenthaler and Burkart 1999, Gonçalves et al. 2001, García-Plazaola et al. 2002, Kitajima and Hogan 2003, Walters 2005, Silvestrini et al. 2007).

During absorption of light energy by Chls, and its transfer through LHCs to the reaction centers of PSI and PSII, a small portion of energy is dissipated as heat and re-emitted as light. Energy released as light is called chlorophyll fluorescence (Maxwell and Johnson 2000). Laboratory methods based on induction and analysis of Chl a fluorescence, such as the saturation pulse method as well as Chl a transient induction and JIP test, have been applied for the evaluation of photosynthetic efficiency in higher plants. These methods have been demonstrated to be very powerful tools for in vivo analysis of the plants’ photosynthetic performance, since they are inexpensive, nondestructive, potentially repetitive and highly informative concerning the gained data (Maxwell and Johnson 2000, Strasser et al. 2000, Mkandawire et al. 2014).

Duckweeds (family Lemnaceae) are freshwater macrophytes that are broadly distributed in the natural environment and widely used in ecotoxicological and environmental studies. Plant species belonging to this family have a simple structure, small size, rapid growth rate and vegetative reproduction. Easiness of handling in laboratory conditions, high sensitivity and possibility of evaluation of a wide range of endpoints make them suitable organisms for estimation of the effect of various abiotic environmental factors, including light intensity. Among five genera of duckweed family (Spirodela, Landoltia, Lemna, Wolffiella, and Wolffia) the species Lemna minor L. and Lemna gibba L. have been the most frequently used for environmental and laboratory studies (Wang 1990, Mkandawire et al. 2014).

The objective of the present study was to compare growth characteristics, photosynthetic pigment content and PSII performance in duckweed plants grown at two different irradiation regimes: low light (LL) 50 µmol_PHOTONS m^-2 s^-1 and high light (HL) – 500 µmol_PHOTONS m^-2 s^-1. Based on the reports on Lemna minor plants growing at different irradiation levels (Artetxe et al. 2002, García-Plazaola et al. 2002) as well as on a broad knowledge of photosynthesis in sun and shade grown plants (Mathur et al. 2018) we hypothesized that: 1) duckweed plants exposed to LL would reveal retarded growth and decrease in biomass gain due to limited photosynthetic potential; and 2) HL grown plants would adjust their photosynthetic performance by decreasing the amount of photosynthetic pigments and by modulating the PSII functioning in such a way as to obtain the most efficient photosynthetic performance associated with effective protection from photodamage. Our aim was to analyze the energy fluxes through the PSII with the intention of detecting which part of electron transport is predominantly adjustable to different irradiation levels.

In this work, plant growth, photosynthetic pigments content and chlorophyll fluorescence-based parameters were measured to determine the effect of two light intensities (LL and HL) on photosynthetic efficiency in duckweed (L. minor).

Photosynthesis is the basic metabolic process that influences plant growth and biomass production. It is dynamic process, highly dependent on environmental conditions including light as a source of energy (Kaiser et al. 2019). Therefore, the evaluation of plant growth can indicate photosynthesis efficiency under different light conditions. Growing under changing irradiation levels, plants have to adjust their photosynthetic machinery in the way to equilibrate the light capturing with its efficient utilization in photosynthesis. This is especially important in conditions of high irradiation when photoinhibition of photosynthesis might occur, which could have a broad range of detrimental effects on plants, from decreased growth to plant death (Roach and Krieger-Liszkay 2019). During the first seven days of our experiment, LL and HL generally had similar effects on multiplication of plants (expressed as RG_N). An exception was the 4^th day, when plants exposed to LL showed more prominent growth (Fig. 1). Since stock cultures of plants were grown at 70 µmol_PHOTONS m^-2 s^-1, a possible explanation for such observation at this time-point could be stress in plants exposed to HL due to an increase of light intensity to 500 µmol_PHOTONS m^-2 s^-1. In a further experimental period (from the 7^th till the 10^th day), plants exposed to HL showed enhanced multiplication which could be a result of acclimation to HL and in this time period plants used benefits from higher energy irradiation.

Consideration of the effect of LL and HL on growth parameters of duckweed, RG_m showed a greater difference among two groups of plants (Fig. 1). Higher sensitivity of fresh biomass parameter in comparison to frond number has already been observed by Smith and Kwan (1989) and Naumann et al. (2007). They revealed that duckweeds exposed to metal salts continued to multiply but produced smaller fronds, which resulted in similar frond number but significantly lower biomass. Furthermore, our results are in accordance to data published by Artetxe et al. (2002). In their study L. minor acclimated to 500 µmol_PHOTONS m^-2 s^-1 showed remarkably higher biomass increase than plants exposed to 50 µmol_PHOTONS m^-2 s^-1. Starch could have a significant contribution in total plant biomass. The proportion of starch in duckweed can vary from 3% to 75% of dry weight (Yin et al. 2015), depending on growth conditions, including light intensity.

One of the first characteristics of high light grown or sun-grown plants islower chlorophyll and carotenoid content than in low light grown or shade plants, which is considered to be a long-term regulation mechanism that controls light absorption capacity (Ruban 2009). Such dynamics of the photosynthetic pigments under high irradiation levels, typically present in land plants, is appreciated as a very efficient adaptive plant mechanism to ever-changing natural environmental conditions (Mlinarić et al. 2016) and presently it is of potential interest for genetic improvement of photosynthesis and plant productivity (Nowicka et al. 2018).

Decreased content of Chl (a+b) and Cars in plants exposed to HL (Fig. 2) is in accordance with reports on the photosynthetic pigments in L. minor plants grown at low and high irradiation levels. Artetxe et al. (2002) measured higher Chl content in plants acclimatized to LL (50 µmol_PHOTONS m^-2 s^-1) than in those grown at HL (500 µmol_PHOTONS m^-2 s^-1). For the same light regimes García-Plazaola et al. (2002) determined decreased chlorophyll and total carotenoid content and a higher Cars/Chl (a+b) ratio in HL grown plants, while the Chl a/Chl b ratio varied very little between the light regimes. Paolacci et al. (2018) studied the effect of a range of different light intensities on two species of the genus Lemna (L. minor and L. minuta) and found that chlorophyll content decreased with increasing light intensities. However, the Chl a/Chl b ratio did not change significantly. Biochemical and molecular mechanisms that control photosynthetic pigment accumulation under different irradiation levels are shown to be closely associated with the accumulation of light-harvesting proteins of PSII (LHCII) (Mathis and Burkey 1989, Sato et al. 2015). It is noticed especially in Chl b accumulation (Tanaka and Tanaka 2000). It indicates direct correlation of pigments accumulation with the regulation of the PSII efficiency and PSII driven electron-transport rate. Our results on the Cars content (Fig.2) are in accordance with the investigation done by García-Plazaola et al. (2002) who revealed increased level of total Cars in L. minor plants grown under low irradiation. In their research, the most of the total Cars in LL plants consisted of lutein and β-carotene.

The revealed decrease of F_v/F_m was less (5.7%) than the decrease in PI_ABS (28%) in plants exposed to HL (Fig. 3) indicated that the high irradiation level caused only minor effects to the primary photochemistry of PSII. The primary photochemistry of the PSII is influenced by the absorption and trapping processes. So, we compared the values of the PSII energy fluxes per active reaction center (RC) in plants exposed to LL and HL (Tab. 1). The plants grown in HL revealed increased ABS/RC, TR₀/RC and DI₀/RC, while there was no difference between HL and LL plants for the ET₀/RC value. The ABS/RC parameter is considered as measure for the average PSII antennae size and its increase in plants exposed to HL likely indicates overloading of PSII RC with electrons. This increased the values of dissipation of excess excitation energy per active reaction centers (DI₀/RC) in HL-grown plants and, in turn, decreased the density of their active reaction centers (RC/CS) for 57% in comparison to plants exposed to LL (Tab. 1).

The performance index (PI_ABS) is a multi-parametric expression that takes into account the properties of several PSII energy fluxes, such as absorption of light energy, trapping of excitation energy, conversion of excitation energy to the photosynthetic electron transport and the dissipation of excess excitation energy as heat. Therefore, it is considered a very good indicator of overall plant vitality (Tsimilli-Michael et al. 2000, Van Heerden et al. 2007). All parameters that are included in PI_ABS calculation (RC/ABS, TR₀/DI₀ and ET₀/(TR₀ –ET₀) revealed increased values in LL-grown plants in comparison to HL-grown plants (Tab. 1). This clearly showed that plants under LL assembled their PSII in such a way as to handle the absorbed and trapped light energy better than HL-grown plants in the given growth conditions.

Further, we investigated several chlorophyll a fluorescence derived parameters (relETR, qP and NPQ) with the aim of revealing whether some in situ short-term functional differences in photochemical and non-photochemical quenching of chlorophyll a fluorescence under high irradiance levels (800 µmol m^-2 s^-1) were developed by different growth conditions (LL and HL). The values of effective quantum yields of PSII (ΔF/F_m’) and PSII driven electron-transport rate (relETR) in HL- and LL-grown plants showed no difference (Tab. 2).

The PSII driven relETR is calculated on the tripartite basis of the ΔF/F_m’, photosynthetically active irradiation and the factor that accounts for the energy distribution between PSII and PSI (Schreiber et al 1995). Although relETR under a certain physiological condition corresponds very well (almost linearly) to the CO₂ fixation rate (Krall and Edwards 1992), it cannot be taken unambiguously as a reliable measure for the overall photosynthetic efficiency, as under some other physiological conditions different processes that compete for CO₂ fixation might take place (Maxwell and Johnson 2000). Also, changes in the antennae size and distribution are reported to occur under high irradiance conditions (Ruban 2009) and this might be highly misleading if relETR were taken as an exclusive parameter in photosynthetic capability assessment of plant samples. In order to make more accurate assessment of the absorbed light fraction that is used in photosynthesis, relETR is usually combined with photochemical quenching coefficient (qP), a parameter also obtained by employing the saturation pulse method (Schreiber et al. 1995). When exposed to actinic light of 800 µmol m^-2 s^-1 plants grown in HL had increased qP values in comparison to plants grown in LL (Tab. 2). In contrast to the relETR that relies on the absorbed light energy used in PSII photochemistry, the qP indicates the fraction of the open PSII reaction centers (RCs that are capable for photochemical performance, i.e. Q_A reduction) in light adapted plant samples (Baker and Rosenqvist 2004). Accordingly, the qP value is a direct consequence of the photosynthesis saturation by incident irradiance. When plants were challenged by high irradiance level (800 µmol m^-2 s^-1) PSII appeared to be equally downregulated in both LL- and HL-grown plants, which was also reflected as similar values of relETR (Tab. 2). However, increased qP values in HL-grown plants indicated that they were capable of directing a greater fraction of absorbed light into the photosynthesis than LL-grown plants, when exposed to 800 µmol m^-2 s^-1. This can also be related to the almost two-fold increase of fresh weight of HL-grown plants in comparison to the LL-grown plants (Fig. 1). Better photosynthetic performance under high irradiance conditions enable plants to increase their primary metabolism, particularly starch content, which is of great recent interest in biofuel production (Cui and Cheng 2015).

As described by Schreiber at al. (1995) only a fraction of light absorbed by PSII is directed into the photosynthesis due to electron-transport chain (photochemical energy conversion). Another two fractions are harmlessly dissipated as heat due to non-photochemical processes, and the red auto-fluorescence of chlorophyll molecules. Since these three processes are in competition, which portion of the absorbed light will be used to drive photosynthesis depends on several regulatory mechanisms. The major one is non-photochemical quenching of chlorophyll fluorescence (NPQ), which is a measure of plant efficiency in converting excess energy to heat, with the aim of avoiding photo-damage of the PSII RC’s (Maxwell and Johnson 2000). Decreased NPQ values in HL plants nicely corroborates their decreased Cars values in comparison to LL plants. It is also in accordance with other fluorescence parameters obtained by the saturation pulse method (Tab. 2) and the data on the heat dissipation per irradiated leaf cross-section (DI₀/CS) obtained by the method of the chlorophyll a induction transient and JIP test (Tab. 1). Although investigations of NPQ have been performed for a long time (Bilger and Björkman 1990), its exact molecular mechanism is under constant debate (Niyogi 2000, Nayak et al. 2001, Ruban 2016). What is well known by now is that, basically, NPQ involves conversion of violaxanthin to zeaxanthin through the xanthophyll cycle, which is closely related to structural rearrangements of LHCII complexes that enable xanthophyll conversion and effective excess excitation energy dissipation as heat. However, this is just a piece of the puzzle, since other protective mechanisms such as redistribution of excitation energy between PSII and PSI (Allen 2003) as well as protective role of β-carotene in the PSII RC (Choudhury and Behera 2001) are known to operate in parallel with NPQ.

Based on the presented data it can be concluded that duckweed plants revealed great capability to build their PSII in such a way as to accommodate absorption and trapping processes most effectively at low irradiance growth conditions as well as photochemical energy conversion and NPQ under high irradiance. The described differences in the photosynthetic apparatus assembly and functioning only partially supported our first hypothesis. That is, increased levels of chlorophylls and carotenoids as well as decreased overall photosynthetic performance in LL plants was greatly reflected in biomass production but only slightly in their multiplication in comparison to the HL plants. Our second hypothesis was entirely confirmed, since HL plants adjusted their photosynthetic performance by short-term up-regulation of the part of absorbed light that was directed into the photosynthesis. This was accompanied by photoprotective modification of the fraction of a certain number of RC, from active to dissipative. Furthermore, long-term accommodation of HL plants was evident due to decreased accumulation of photosynthetic pigments. The observed adjustments might have an important role in the photoprotection of duckweed plants growing under different irradiation conditions when challenged by various environmental pollutants. Therefore, the obtained results can serve as a platform for further research into the effects of environmental pollutants on duckweed plants under different irradiation levels.