Differentiation between two strains of microalga Parachlorella kessleri using modern spectroscopic method

The differentiation between wild type of Parachlorella kessleri and its mutant strains PC Mut2, PC Mut4 by using the Multi-functional Plant Efficiency Analyzer (М-РЕА-2) was studied. Mutant algal cells of P. kessleri have been obtained by UV-C during 3 and 10 min respectively. Light-induced kinetics of prompt fluorescence (OJIP transients), delayed fluorescence and modulated reflection at 820 nm (redox transitions of P700 in PSI) showed disturbance of electron transport flow in photosystem II (PSII) and an increase fraction of non-reducing centers of secondary quinone acceptors of electron (QB). In addition, the amplitudes of the fast and slow peak in the kinetics of the delayed light emission and non-photochemical fluorescence quenching ( NPQ) were significantly reduced in mutant cells, indicating low level of the membrane energization of photosynthetic membranes. Changes of photosynthetic reactions of mutants may lead to an increase of the carotenoids content, which protect cells against the light stress. It is suggested to use parameters of induction curves of prompt and delayed fluorescence to characterize mutant algal cells in biotechnological studies.


Background
The solar radiation sustains life on Earth. However significant increase of UV-C radiation may lead to inhibition of many biological processes. The major cellular targets of UV-C are various biomolecules, which directly absorb this radiation or are indirectly affected by various UV-C induced photochemical reactions. The biological and, ultimately, ecological consequences are numerous (Charles 2008). It is known that UV-C induces some physiological effects such as declining photosynthetic rates, which can be related not only to damaged biomolecules, but also to ultrastructural changes in organelles or membranes (Holzinger and Lütz 2006). Typical alterations include swollen mitochondrial cristae, disrupted thylakoids or detached phycobilisomes in chloroplasts, bent-shaped dictyosomes, and damaged plasmalemma. An intact ultrastructure of the algal cell is a prerequisite for the optimum functioning of all physiological processes (Charles 2008). Parachlorella kessleri was described as a new genus, derived from the true spherical Chlorella. The wild strain SAG 211-11 h was formerly referred to as Chlorella vulgaris but later reassigned as Chlorella kessleri (Fott and Nováková 1969). Today this species is referred to as P. kessleri (Krienitz et al. 2004).
Fluorescence methods are used in biotechnological experiments for monitoring photosynthesis processes, which give detailed information of primary defects of cell metabolism, mainly at the membrane level (Schreiber 2004;Antal et al. 2009;Matorin et al. 2013;Schansker et al. 2014). The basis of fluorescence methods lies in the ability of chlorophyll located in photosynthesis membranes to serve as a natural detector for the algae cells, emitting quanta fluorescence. Importantly, such methods allow to gain information of alga state in real time. Measurement of the ratio between the fluorescence intensity under the photosynthesis saturating illumination (F M ) and under low light intensity, which induces no changes in the state of the photosynthetic apparatus (F O ) makes it possible to determine the maximum efficiency of the PSII processes, which is equal to F V /F M= (F M -F O )/F M . The F V /F M ratio presents dimensionless characteristics of the efficiency of photosynthesis, which is independent of the species of organisms. Measurements of fluorescence induction curves are carried out during several seconds by using PAM or PEA instrument. Measurements of fluorescence induction curves with high resolution (starting from 20 μs) have been recently used in studies of photosynthetic reactions in higher plants and algae cultures (Schreiber 2004;Strasser et al. 2005;Matorin and Rubin 2012). The M-PEA-2 instrument allows to measure separate reactions in PSI and PSII simultaneously (Strasser et al. 2010;Oukarroum et al. 2013;Bulychev et al. 2013). Moreover, it monitors induction changes of delayed fluorescence, which indicate the level of the membrane energization (Goltsev et al. 2009).
In the present paper we investigated processes in PSI and PSII of wild type of P. kessleri and two mutant strains by using a newly designed Multi-functional Plant Efficiency Analyzer (M-PEA-2, Hansatech). This instrument gives a comprehensive picture of primary photosynthetic events by simultaneous recording the prompt and delayed fluorescence kinetics and reflectance at 820 nm at high time resolution. Here we introduce results of the first attempt of assessment of photosynthetic characteristics of the mutant strains of algal cells by using M-PEA-2.

Strains and growth conditions
The strain of Parachlorella kessleri microalga was obtained from Al-Farabi Kazakh National University, Biotechnology Department culture collection. P. kessleri wild type strain was grown in tris-acetate-phosphate (TAP) medium (pH 6-7) (Gorman 1965), in 250 ml Erlenmeyer flask at 28°C under continuous illumination of photosynthetic photon flux density (PPFD) 120 μmol photons m −2 s −1 and constant shaking.

UV irradiation and mutagenesis
According to Harris (1989), 5 ml of the liquid culture with a density of 1×10 6 /ml algal cells were placed in 9 cm Petri dish, where the culture formed a thin layer, covering the bottom. The dish was placed on shaker at 20 rpm and exposed to UV-C lamp (254 nm and 40 erg/mm 2 ) at distance 15 cm during 3 and 10 min respectively. After UV irradiation, the cells were inoculated in solid TAP medium and incubated in darkness for 24 h to prevent photoreactivation. After the 24 h, some dishes were incubated photoautotrophically (photon flux density 120 μmol photons m −2 s −1 ), others were incubated heterotrophically (in dark) during 15 days.

Selection of the mutant strains
After the incubation period, two mutant strains were selected based on their phenotypic characteristics, using colonies whose color and size differed from the wild type colonies. These mutant strains, PC Mut2 and PC Mut4, were obtained from P. kessleri after irradiation time of 3 and 10 min respectively. These mutants were transferred from the solid to liquid medium and kept under phototrophic growth conditions (photon flux density 120 μmol photons m −2 s −1 ).

Analysis of chlorophyll a and b content
Spectrophotometry method was used according to Merchant et al. (2007). The calculation of the concentration of the pigments was determined by the optical density of pigment solutions at appropriate wavelength.

Chlorophyll fluorescence and absorbance measurements
Light-induced kinetics of prompt Chl fluorescence (PF), delayed Chl fluorescence (DF) and modulated reflectance at 820 nm (MR) were recorded by using Multi-functional Plant Efficiency Analyzer (M-PEA-2, Hansatech Instrument Ltd., King's Lynn, Norfolk, UK). Prior to the measurements, the cells were concentrated on a membrane filter. Then a thin layer of cells was fixed by a special clip on the surface of the measuring chamber and incubated in the darkness for 10 min. It has previously been verified in algal suspension by using Aqua-Pen fluorometer (Photon System Instruments, Czech Republic) that concentration algal cells on the filter surface doesn't affect the physiological state of cells .
The design of the measuring chamber, timing protocol of a single measurement, and data acquisition procedure for M-PEA-2 can be found in Strasser et al. (2010). PPFD and duration of actinic illumination were set to 1300 μmol photons m −2 s −1 and 3 s, respectively. In the M-PEA-2, the DF induction curves are constructed from the DF intensities at certain delay time-point in the kinetics of DF decay recorded during the dark interruptions of the actinic light, which induces OJIP (intermediate levels of the light-induced fluorescence curve) rise. In our experiments, the delay time point in the DF decay curve was set to 50 μs. It is assumed that submillisecond DF components are generated from PSII reaction centers in the state Z + P 680 Q A − (Goltsev et al. 2009), where Z is a tyrosine Z residue, P 680 is a primary electron donor, and Q A is a primary quinone acceptor in PSII.
Fluorescence was measured by using Water-PAM fluorometer (Heiz Walz, Effeltrich, Germany). Ratio F V /F M is the maximum quantum yield of PSII photochemistry was measured in dark-adapted sample, where F V = F M -F O . Measurements in the light were performed with progressive increase of light intensity from 0 to 800 μmol photons m −2 s −1 . At the end of each illumination session, F M ' and fluorescence yield in light F(t) were measured using saturating flash (0.8 s, 3000 μmol photons m −2 s −1 ) (Schreiber 2004;Herlory et al. 2007). These parameters were used to calculate non-photochemical quenching of chlorophyll fluorescence NPQ = (F M -F M ')/F M ', the quantum yield of photochemical conversion of light energy in PSII, as Y = (F M '-F(t))/F M ', and the relative yield of noncyclic electron transport at given light intensity (Lippemeier et al. 1999). Parameters are designated according to generally accepted nomenclature (Schreiber 2004).
Light absorption spectrum of samples was measured by using HITACHI-557 spectrophotometer. Fluorescence spectra, excitation spectra, and fluorescence decay times in algae were measured by using Fluorolog-3 (Horiba Jobin Yvon) spectrofluorometer with the TCSPC (time correlated single photon counting) option (O'Connor and Philips 1984). Fluorescence was measured at 685 nm under excitation with pulse LED at 390 nm. Fluorescence decay kinetics was approximated with the sum of two exponential curves.
All measurements were repeated no less than five times. The figures present data of three replicate experiments at least.

Results and discussion
In this study mutations were induced by UV-C radiation. UV-C mutagenesis offers many advantages such as less pollution, simple operation, and sterile cultivation conditions. After UV-C irradiation, some colonies appeared with phenotypic characteristics that differed in color and size from the control colonies. The colonies were selected based on their phenotypic appearance, transferred into liquid medium and then subcloned several times to insure that the mutation was stable and no changes in their phenotypic characteristics occurred. The major cellular targets of UV-C are different biomolecules, which directly absorb this radiation, or which are indirectly affected by various UV-C induced photochemical reactions (Charles 2008). In the present paper two mutant strains of P. kessleri after irradiation during 3 and 10 min respectively were selected and designated as PC Mut2 and PC Mut4. It was shown microscopically that mutant cells are smaller, than wild type cells. All strains were grown in the liquid TAP medium under phototrophic conditions. The algal cultures of the wild and mutant strains were different in color.
Absorption spectra of wild type and mutant strains of cells are in Figure 1. The spectra have been normalized to the maximum chlorophyll absorption in red region at 675 nm. Relative increase of carotenoid absorption at the Soret band was noted in mutant strains of P. kessleri. The highest increase was observed in PC Mut4. This was confirmed by measurements of fluorescence excitation spectra ( Figure 2В) The se spectra showed an increase in the region of carotenoid absorption. Fluorescence spectra did not differ significantly ( Figure 2А), which may be related to the fact that carotenoids in algae do not fluorescence, but efficiently transfer excitation energy of chlorophyll a, which is fluorescence emitter.
It is known that the constant fluorescence level F O highly correlates with the total content of pigments in the photosynthetic apparatus of algal cells, that harvest light energy, and F O level correlates with cell number (Matorin et al. 2004). The ratio F V /F M reflects the maximum quantum yield in PSII. This value is related to the processes of water splitting and oxygen evolution in PSII. Changes of fluorescence parameters (F O , F V /F M ) are shown in Figure 3. Figure 3 shows fast increase of algal cells of wild type (curve 1) after the lag-phase (1 st day), as measured by F O . PC Mut2 (curve 2) and PC Mut4 (curve 3) mutants demonstrate a lower growth rate during investigation period. Photosynthetic activity (F V /F M ratio) of wild type cells slightly increased during growth, reaching the maximum value at the 3 rd day. After prolonged culturing (6 th day) photosynthetic activity, judged by F V /F M ratio, was decreased, possibly due to exhaustion of mineral nutrients in the growth medium. Mutants PC Mut2 and PC Mut4 had a lower F V /F M values. For more detailed investigation of photosynthetic activity of wild type and two mutant strains P. kessleri cells, fluorescence parameters were measured by using M-PEA-2 ( Figure 4). M-PEA-2 provides extended possibilities to evaluate the functional state of PSII and PSI by measuring light-induced kinetics of the PF (OJIP transients), DF, and MR with a high resolution (Strasser et al. 2010;Oukarroum et al. 2013). Although there is no full consensus on the origin of the complex OJIP rise kinetics, it is commonly accepted that the underlying mechanisms may involve successive decrease in photochemical and non-photochemical fluorescence quenching due to the light-induced PSII closure and PQs reduction (Antal et al. 2009;Strasser et al. 2010). PSII closure refers to reduction of the primary quinone acceptor, Q A , in PSII. Figure 4 presents kinetic curves of fluorescence induction after switching the light on, normalized against the O level. In the control cells, the shape of the fluorescence curve corresponded to that described in the literature (Lazár 2009;Strasser et al. 2010). Usually, several components are observed in the kinetics of fluorescence induction in response to intense illumination, namely, the O-J-I-P transitions. The initial level O corresponds to chlorophyll fluorescence intensity with open RCs of PSII (F O ) when all Q A are oxidized. The time period required to reach this level is up to 20 μs. The O-J phase is caused by light induced reduction of Q A , while the subsequent phases reflect mainly its further accumulation in reduced state, caused by a decrease in its reoxidation rate as a result of reduction of Q B acceptors and the pool of quinone. The shape of the O-J-I-P curve was changed in the mutants (Figure 4). The mutant PC Mut4 showed marked decrease in the contribution of the photochemical phase J-I-P, which indicates interruption of the electron flow from PSII to plastoquinone pool. To quantitatively analyze the characteristics of the primary processes of photosynthesis on the basis of O-J-I-P kinetic curve parameters, the so called JIP test (Strasser et al. 2010) was used. The JIP test uses the following parameters of the fluorescence induction kinetic curve: (a) fluorescence intensity at 20 μs (F O ), 300 μs (F300 μs), 2 ms (F J ), 30 ms (F I ), 6 s (F 6s ), and F P (F M , the maximum fluorescence yield); (b) time to reach the maximum fluorescence (t F M ), and (с) area under the kinetic curve below the F M level. These characteristics were used to calculate the following parameters presented in (Table 1)  From the V I and S M parameters, it is possible to derive information on further accumulation of reduced which occurs due to reduction of Q B and the quinone pool.
It is assumed that the O-J phase of fluorescence induction reflects the accumulation of both reducing and non-reducing PSII (Antal et al. 2009;Strasser et al. 2010). The hypothesis is supported by the fact that DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea), which inhibits electron transport between Q A and Q B , leads to fast growth of fluorescence to the maximum level within 2 ms, corresponding to the appearance of the J peak in the control. Analysis demonstrated that, in cells of the strains obtained, the number of non-Q B -reducing centers of PSII, which are incapable of the quinone pool reduction, was increased. This was supported by a number of other parameters (Table 1). Accordingly, values of the parameter М O , which reflects the initial slope of the induction curve, increased. Total flow of photons, absorbed by the pigments of the PSII antenna and normalized against the RC number (ABS/RC), increased in the cultures of mutant strains P. kessleri, as compared to the control values. At the same time, the relative amplitude of the J-I phase (V I ) was practically unchanged.
Prompt fluorescence induction curves also demonstrated the suppression of fluorescence decay after reaching the maximum due to ΔpH dependent nonphotochemical quenching (q E = (F M -F 6s )/F V )). The value of this parameter decreased in mutant strains, which evidences a decrease in the membrane energization. The ability of the quinone pool to quench fluorescence q PQ = (F M -F I )/F V in algae of mutant strains was also reduced. Figure 5 shows the kinetics of fluorescence decay in the wild type and mutant strains of P. kessleri cells. Biexponential fitting of decay kinetics was applied to distinguish two components, one of them is related to energy trapping (fast component) and the other to charge stabilization and recombination in PSII reaction centers (slow сcomponent) (Volgusheva et al. 2007.).
In our experiments we found a fast component with half-time about 1 ns and a slower component with halftime more than 2 ns (Table 2). In the wild type and PC Mut2, the ratio of these components was 40 and 60%, respectively. In PC Mut4, the half-time of the slow component increased from 2.2 to 2.5 ns, and its relative magnitude also increased to 72%.
Slower fluorescence decay and an increase in the contribution of the slow component, generally, reflect a higher reduction of the PSII Q A acceptor. It is clearly seen in the presence of DCMU, an inhibitor of electron transport. The fact of slower fluorescence decay in PC Mut4 agrees well with the increase in the concentration of non-Q Breducing centers of PSII in cells of this mutant strain.
Reflectance changes at 820 nm (MR) are assigned to the redox transitions of P 700 , a primary electron donor in PSI, and plastocyanin, an electron donor for P 700 + . For the simplicity, further in the text we refer MR signal to P 700 . In the dark adapted state, P 700 is in the neutral state and the reflectance at 820 nm is high. After the onset of illumination MR value rapidly declines, reaching a dip at about 10 ms, followed by the slow recovery to the initial level. These changes reflect the rapid oxidation of P 700 by the acceptor side of PSI, and the subsequent reduction of P 700 + by electrons arriving from PSII ( Figure 4B) shows MR transients normalized to the initial value at 20 μs (MR/MR 0 ) recorded simultaneously with the OJIP curves described above.
In PC Mut2 mutant strain, the initial oxidation, as well as reduction of P 700 was similar to those in the wild type. In PC Mut4 mutant strain, P 700 oxidation was reduced, indicating an inhibition of electron transport via PSI.
Both DF and PF emission originates from radiative deactivation of the singlet excited state of antennae Chl in PSII (Goltsev et al. 2009). In the case of PF, excited states are created mainly through the absorption of photons by photosynthetic pigments, whereas DF emission is coupled to excited states formed as a result of the backward reactions of electron transport. In order to separate a low yield DF emission from high yield PF signal, DF is recorded in the dark after the excitation pulse offset. DF intensity is proportional to the rates of the corresponding recombination reactions which are strongly affected by the energization of a thylakoid membrane (Wraight and Crofts 1971). Thus, changes of DF yield during the induction period depend on the formation of electrical and proton differences across the membrane. Fast and slow phases of the DF induction kinetics recorded with M-PEA in plant leaves have recently been analyzed (Goltsev et al. 2009;Oukarroum et al. 2013). It is assumed that the fast component (I 1 ) (~10-100 ms) reflects transient formation of the electrical gradient across the membrane, likely, due to generation of charge pairs in PSI (P 700 + [Fe-S] − ). The presence of the second DF peak, I 2 , in the seconds range, is associated with the light induced formation of the proton transmembrane gradient (ΔpH) that increases the constant of rate of emission transitions in the RC of PSII.
Peaks on DF curve at 20-50 ms, and 1 s were reduced in mutant strains, indicating a decrease in the formation of electrical and proton potential differences across the thylakoid membrane. However, the fast component of DF was reduced to a greater extent in PC Mut4 strain. The slow phase of the DF induction kinetics (ΔpH) was reduced more significantly in PC Mut2, compared to the wild type. The last fact agrees with the reduced non-photochemical quenching q E = (F M -F 6s )/F V in PC Mut2 (Table 1).
These data were confirmed by measurement of fluorescence parameters in algae with a Water-PAM pulse fluorometer. The relative rate of the noncyclic electron transport (ETR), calculated as rETR = Y × E i × 0.5, and nonphotochemical fluorescence quenching NPQ = (F M -F M ')/ F M ' by electrochemical gradient of protons were reduced in mutants (Table 3).
Thus, simultaneous registration of the kinetics of prompt, delayed fluorescence and redox state of P 700 made it possible to monitor the individual reactions of accumulation of reduced carriers between the photosystems and kinetics of electrochemical proton gradient on a thylakoid membrane of mutant strains P. kessleri.
It is known that the constant fluorescence level F O highly correlates with the total content of pigments in the photosynthetic apparatus of algal cells that harvest light energy (Matorin et al. 2004). The chlorophyll a and b content in the wild type were 53.3 ± 5.7, 30.2 ± 2.4 mg/l respectively while in the PCMut2 were 48.3 ± 2.3, 25.2 ± 2.3 mg/l respectively and in the PCMut4 were 33.3 ± 3.2, 10.2 ± 3.6 mg/l respectively. Fluorescence parameters are used long ago in hydrobiology and oceanology for an assessment of the concentration of microalgae in water and their photosynthetic activity. There is a large number various the submersible fluorometers of different firms for these purposes. Researchers have therefore been trying to find a method of determining the chlorophyll a concentration from in situ fluorescence measurements. These would cover not only fluorescence induced naturally by sunlight but also that induced by artificial light sources. Measurements of the latter are either contact measurements carried out in situ with submersible fluorometers or remote methods using lidars (Pogosyan and Matorin 2005).

Conclusion
Microalgae, which use sun energy for synthesis of organic matter, are the main energy source in aquatic systems and serve as the food for other organisms. The linear chain of photosynthetic electron transport comprises two sequentially operating photosystems (PSII and PSI) interconnected through intermediary quinone pool of electron carriers. According to the Z-scheme of electrontransport chain (ETC.), activities of each photosystem affect the redox state of the other system. Such a relation between PSII and PSI is evident from chlorophyll fluorescence, whose level depends on the redox state of the quinone acceptor Q A , as well as from the redox state of chlorophyll P 700 , the primary electron donor of PSI. The photoreaction of PSII reduces Q A , thus raising chlorophyll fluorescence and the extent of P 700 reduction, while the PSI reaction converts P 700 into its oxidized form (P 700 + ), which promotes Q A oxidation and fluorescence lowering. Simultaneous chlorophyll fluorescence and 820 nm transmission measurements by using М-РЕА-2 allow evaluating the functional state of PSII and PSI. Analysis of fluorescence induction curves demonstrated inhibition of electron transport in PSII and an increase fraction of non-Q B -reducing centers. This manifested itself also in an increase of the slow component in decay curve of fluorescence with half-time 2 ns in PC Mut4. In addition, analysis of induction curves of the The kinetic parameters of fluorescence induction were recorded with M-PEA-2 instrument under actinic illumination of 1300 μmol photons m −2 s -1 .

Figure 5
Kinetics of fluorescence decay. In wildtype (curve 1) and mutant strains of Parachlorella kessleri cells (PCMut2-curve 2) and (PCMut4 -curve 3). The measurements were performed with a Fluorolog-3 spectrofluorometer at 685 nm, using TCSPC method with sample activation with light at 1 Mhz frequency with a LED 390 nm by pulses with half-widths of 0.9 ns. delayed fluorescence and a decrease of NPQ revealed a decrease in the energization of photosynthetic membranes in mutant cells. Our study showed that the most sensitive parameters are induction curves of prompt and delayed fluorescence in mutant algal cells. Selected parameters are indeed viable tools for diagnostic alga state in biotechnological studies during obtaining mutant forms.
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