Effect of light stress on maximum photochemical efficiency of photosystem II and chloroplast structure in cryptogams Cladonia mitis and Pleurozium schreberi

doi:10.21203/rs.3.rs-2746070/v1

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Effect of light stress on maximum photochemical efficiency of photosystem II and chloroplast structure in cryptogams Cladonia mitis and Pleurozium schreberi

https://doi.org/10.21203/rs.3.rs-2746070/v1

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Earlier beliefs suggested that lichens thrived in open and warm environments, whereas mosses prefer shaded places with high humidity. However, excessive light can be a stress factor for both lichens and bryophytes, as it can damage their photosynthetic apparatus, particularly the chlorophyll pigments located in the chloroplasts. The measurement of photosynthetic activity and the use of fluorescence microscopy allows the assessment of the influence of light as a stress factor on the state of preservation of the thallus. The main goal of our research was to determine the changes that occur in the photosynthetic activity and structure of chloroplasts of selected species lichen Cladonia mitis (Sandst.) and moss Pleurozium schreberi (Willd. Ex Brid.) under the influence of different intensity of light in laboratory conditions: FL − 100% light, RL − 30% maximum light, and NL - natural sunlight. The results of the two-factor analysis of variance (ANOVA) tests showed a variation in the activities of the selected species over time depending on the amount of light energy supplied. The results showed that Cladonia mitis showed the lowest level of chloroplast fluorescence in the strongest and reduced light, which coincides with its low level of photosynthetic activity. The moss showed high and intense fluorescent activity of the chloroplasts, indicating their much higher resistance to light radiation.

Photosynthetic activity

light stress

fluorescence

chloroplast

lichens

bryophyte

Lichens are very common in microhabitats where they can be exposed to adverse environmental conditions, including extreme temperatures, desiccation, or solar radiation (Beckett et al. 2021; Dziurowicz et al. 2022; Nguyen et al. 2013). These tiny poikilohydric organisms have evolved defence mechanisms to protect themselves against changing environmental conditions, including reducing metabolism when water availability decreases, tolerating the loss of virtually all free water without dying, and resuming growth after rehydration. In turn, photoprotection focusses on light scattering, radiation shielding, heat dissipation, activation of antioxidant and macromolecular defences, and membrane repair (Beckett et al. 2021; Dziurowicz et al. 2022; Nguyen et al. 2013). Bryophyte, on the other hand, unlike lichens, prefer moist habitats, which allows them to be fully hydrated and metabolically active for extended periods of time (Dziurowicz et al. 2022; Green et al. 2011). However, due to their distinctive morphology and physiology, bryophyte rely heavily on their external environment and are vulnerable to the impacts of climate change, which can alter the availability of water and nutrients, as well as affect the temperature and humidity conditions in their habitat (He et al. 2016). Therefore, the bryophyte faces great challenges associated with the drying and rehydration cycle and often high temperatures, which causes them to be metabolically inactive (Liu et al. 2003). One way to reduce water loss is for mosses to form dense clusters that reduce water evaporation (Charron and Quatrano 2009). Assessing the physiological state of plants and their activity, in addition to monitoring their physical existence and the intensity of environmental stress, can provide important information about ecosystems. Various experimental and analytical methods have been developed to monitor the physiological state of plants (Chen et al. 2019). One of the methods uses the phenomenon of chlorophyll fluorescence. It is a simple tool widely used to analyse the state and function of the photosystem II (PSII). The maximum photochemical efficiency of photosystem II (Fv/Fm), otherwise called photosynthetic activity, is easy to measure as a representation of the photosynthetic performance of a studied organism. Changes in the functioning of photosynthetic systems can be detected without any damage to the studied samples by measuring chlorophyll fluorescence. This is because a fraction of the light is absorbed by chlorophyll molecules in PSII and used for energy conversion in photosynthesis, while excess energy can be either dissipated as heat or remitted as light (Maxwell and Johnson 2000). Chlorophyll fluorescence variations reflect the response of photosystem II (PS II) to changes in photosynthetically active radiation (PAR) intensity (Demmig-Adams and Adams 1992; Dziurowicz et al. 2022; Wu et al. 2017). Studies conducted over the years using such measurements have identified several relationships, including the level of hydration and photosynthetic activity. Among the results, we find information that dehydration damages the photosynthetic apparatus and therefore causes inactivation of the photosynthetic process (Liu et al. 2014). Other studies indicate that in the case of desiccated mosses and lichens, wetting the thallus leads to its reactivation (Orekhova et al. 2022; Schlensog and Schroeter 2001). Those so-called desiccation-tolerant organisms (DT) can be subdivided into two groups. Homoiochlorophyllous (HDT) are those that maintain their chlorophyll content during periods of desiccation, while poikilochlorophyllous (PDT) lose most or all their chlorophyll during these periods (Bewley 1979; Hambler 1961; Gaff 1977; 1989; Tuba et al. 1994; Tuba et al. 1996). In opposition to complete desiccation is the equally negative effect of too much water content, one that prevents optimal CO2 exchange and contributes to stopping the process (Dziurowicz et al. 2022; Lakatos 2011; Lange et al. 1993). Apart from water availability, the efficiency of photosynthesis can be significantly impacted by light conditions. Although light is its integral element, necessary for it to be carried out, the relationship between the amount of available light and the intensity of photosynthesis is not linear since the rate of photosynthesis reaches a saturating point at a consuming certain level. Research over many years has shown that too much light becomes a stressor and can be harmful (Beckett et al. 2021; Dziurowicz et al. 2022; Rochaix 2011). Damage occurs when photon absorption of photons exceeds the capacity of the downstream reactions to consume the products of the light phase, and reaction centers become saturated (Allorent et al. 2013; Barber and Andersson 1992; Wobbe et al. 2016).

To date, most work using photosynthetic activity to assess the state of plant viability has focused on interpreting photosynthetic activity alone. However, the evaluation of photosynthetic activity combined with chloroplast fluorescence imaging by fluorescence microscopy can more accurately show the changes that occur in photosynthetically active organisms under stress. It is very easy to observe chloroplasts by chlorophyll fluorescence using fluorescence microscopy (Wada 2013), and it offers a way to test the viability status of lichen and moss thalli (Kauppi 1980).

Our research carried out consisted of collecting selected lichens and bryophyte in the natural environment and then growing them in strictly defined changing light conditions while maintaining the same other conditions (temperature and humidity). For this study, we selected Cladonia mitis (Sandst.) and Pleurozium schreberi (Willd. Ex Brid.) Mitt. as representatives of lichens and bryophyte, respectively, which occur in this same type of pine forest habitat. The main goal of our research was to determine the changes that occur in photosynthetic activity and presence and location of chloroplasts of selected species under the influence of different amounts of light in laboratory conditions. We made the following hypotheses: (1) photosynthetic activity was variable over time, depending on the amount of light energy supplied (2) the photosynthetic activity of the lichen is higher than that of the moss in all light types tested, (3) soaked thalli of lichen Cladonia mitis and moss Pleurozium schreberi show higher photosynthetic activity compared to non-soaked thalli, and (4) the structure of chloroplasts in both study species degrades under the influence of the strongest light (full light).

Establishment and maintenance of laboratory cultures of lichen and bryophytes

Species of lichen C. mitis and bryophyte P. schreberi were randomly collected in Bory Tucholskie National Park within the lichen Scots pine forest community (Cladonio-Pinetum Ass.) in February 2022 (Węgrzyn et al, 2020; 2021a; 2021b). The collected samples were cleaned of plant debris and soil particles and divided into 3 study samples, which were later placed in 3 experimental glass containers (aquariums) with different light treatments. Within each container, we placed 6 subsamples of C. mitis (C) and 6 subsamples of P. schreberi (P) as pseudoreplications (6 chambers per container eq. study sample) (Fig. 1, Table 1). As treatment within containers, we used two types of artificial light conditions: 1) full light (FL) – 100% of artificial light; 2) reduction light (RL) – 30% of artificial light. The reduction was achieved by covering the glass of the aquarium with a dark film. Both light conditions were created by application of artificial lighting which was generated by lamps with the following specifications: type GROWY LED 354, light PAR − 224.3 µmol/m2s and wavelength range 50% max. 450 nm and 50% max. 650 nm. For the control container identified as natural light (NL), we used natural sunlight (Table 1). The substrate for the study species was sterile sand. We kept the boxes in a climatic chamber at 21 ° C.

Table 1

Segregation of six study samples (experimental containers) of *Cladonia mitis* (C) and *Pleurozium schreberi* (P) in fieldwork into types of light: FL – 100% light; RL – 30% maximum light; NL – natural sunlight (control sample), with the values: Light energy (LE) and photosynthetically active radiation (PAR).
Species	Types of Light	LE [W/m²]	PAR [µmol/m²]
Cladonia mitis	FL	122.7	506.9
Pleurozium schreberi	FL	125.2	495.1
Cladonia mitis	RL	40.6	151.4
Pleurozium schreberi	RL	42.0	165.3
Cladonia mitis	NL	11.5	178.6
Pleurozium schreberi	NL	11.5	178.5

When FL samples with RL samples, the first ones mentioned were characterized by a more than three times higher light energy. In the case of the RL samples, the energy was about four times higher than in the NL samples (Table 1). As the artificial lighting was active 18 hours a day. The lighting was automatically turned off for 6 hours from 21 pm to 3 am. Lichens and bryophyte were systematically watered so that their thallus and tissues were constantly moist. The experimental cultivation was carried out for 150 days.

Measurements of photosynthetic activity

Photosynthetic activity measurements were performed six times, at different time intervals, to assess the viability of the examined samples. The first measurement was made on the day the culture was established; the next measurements took place after 30, 60, 90, 120 and 150 days from the beginning of the experiment. During measurement, 36 samples were taken from each compartment, three each from all six subspecies, both to measure soaked and non-soaked samples: 1) soaked samples: Cladonia mitis (CM-S) and Pleurozium schreberi (PS-S) were placed in Eppendorf filled with distilled water for a period of 2 h, and after this, the material was transferred to the HPEA/LC clips for a 15-minute dark adaptation aimed at extinguishing the activity of the photosystem II; 2) non-soaked samples: Cladonia mitis (CM-NS) and Pleurozium schreberi (PS-NS) were measured directly after being transferred to the HPEA/LC clips without water treatment. That was repeated for both species. Measurements were made using a Handy PEA + fluorometer (Handy PEA, Hansatech Instrument Ltd, King's Lynn, Norfolk, England). The fluorometer light probe emitted the dose of excitation light, which allowed recording the maximum photochemical efficiency of photosystem II (Fv/Fm) (Chowaniec and Rola 2020; Dziurowicz et al. 2022; Węgrzyn et al. 2021b).

Microscopic fluorescence imaging of chloroplasts

At the designated dates of photosynthetic activity measurements, small samples of lichen thalli and bryophyte gametophyte were also collected directly from the culture to make microscopic preparations showing the preservation status of the chloroplasts. In the case of lichens, the top fragments of the thallus were collected, where there are numerous cells of Asterochloris sp. algae of the lichen photobiont belonging to the Trebouxiaceae family within the Chlorophyta cluster. The thallus fragments were cut into thin flat sections, which were placed in a water solution of the microscopic preparation. In the case of bryophyte, gametophyte leaflets were collected, from which a water microscope preparation was made directly. In visible and fluorescent light with filter V-2A, EX 380–420, DM 430, BA450, the Nikon Eclipse 7000 microscope was used to observe the state of preservation of chloroplasts in the organisms tested.

Statistical analysis

A two-way analysis of variance (ANOVA) followed by Tukey’s HSD test (Honestly Significant Difference) for an equal sample size (p < 0.05) was performed to reveal significant differences in Fv/Fm between the duration of the experiment (1, 30, 60, 90 and 150 days) and light types (FL, RL, NL) separately for C. mitis (N = 648), and P. schreberi (N = 648) as well as soaked or non-soaked. A graphical interpretation of the interaction between the duration of the experiment, the species, and the type of sample (soaked and non-soaked) for different types of light was presented. Before analysis, the normality of the distribution was verified using the Kolmogorov-Smirnov test (p > 0.05) and the Levene test (p > 0.05) to assess the equality of variances. Box plots were presented to illustrate the differences in individual comparisons.

Photosynthetic activity

The results of the statistical analysis indicated visible differences in the photosynthetic activity of Cladonia mitis and Pleurozium schreberi depending on the type of light used and the duration of the experiment (Table 2, Fig. 2).

Our results shown in Fig. 1 include four graphs that illustrate the photosynthetic activity of CM-NS (Fig. 2A), CM-S (Fig. 2B), PS-NS (Fig. 2C) and PS-S (Fig. 2D) depending on light intensity (FL, RL, NL) and the duration of the experiment (1, 30, 90, 120, 150 days). CM-NS indicates the lowest values of photosynthetic activity under FL conditions, higher values in the RL sample, and the highest values in the control sample. The photosynthetic activity values range from 0.1 to 0.8 Fv/Fm for the FL sample (Fig. 2A). CM-S indicated the lowest values of photosynthetic activity under FL conditions, the highest in RL, and the highest in NL. As with the CM-NS sample, CM- S was characterised by the highest decrease in photosynthetic activity for FL light, higher values for the RL sample, and the lowest decrease for the NL sample. The values of photosynthetic activity range from 0.38 to 0.8 (Fig. 2B). For the PS-NS sample, photosynthetic activity values range from 0.55 to 0.8. The lowest level of photosynthetic activity was observed for the FL conditions, which are close to the values presented by the RL conditions. The highest values were observed in the NL sample (Fig. 2C). For PS-S, the average values of photosynthetic activity reach the range of 0.55 to 0.78 Fv/ Fm. PS-S reaches the lowest activity level in FL conditions, then higher in the RL sample, and the highest in the NL conditions. Bryophyte (PS-NS, PS-S) and lichens (CM-NS, CM-S) showed the highest level of photosynthetic activity in NL and the lowest levels in FL.

Table 2

Results of multivariate ANOVA for the effects of the following variables on the Fv/Fm of the samples: A) CM-NS, B) CM-S, C) PS-NS, and D) PS-S on the duration of the experiment (1, 30, 90, 120 and 150 days), types of lighting (FL, RL, NL), and interaction between time and types of light. Significant effects (p < 0.05) are shown in bold.
		F	p
A	Measurement Day	92.903	p < 0.001
	Type of Light	88.071	p < 0.001
	Measurement Day x Type of Light	8.185	p < 0.001
B	Measurement Day	58.420	p < 0.001
	Type of Light	57.105	p < 0.001
	Measurement Day x Type of Light	5.224	p < 0.001
C	Measurement Day	9.32	p < 0.001
	Type of Light	6.23	p = 0.002
	Measurement Day x Type of Light	5.13	p = 0.003
D	Measurement Day	13.39	p < 0.001
	Type of Light	21.22	p < 0.001
	Measurement Day x Type of Light	2.82	p = 0.002

The graphical interpretation of the interaction for three different types of light (respectively: Fig. 3 - Fl, Fig. 4 - RL, Fig. 5 - NL) and the selected species CM-S and CM-NS and PS-S and PS-NS were shown in Figs. 3–5.

On each measurement day, CM-NS showed the lowest photosynthetic activity values among all selected species, and PS-NS species showed the highest values for FL conditions (Fig. 3). CM-NS noted decreasing values from the beginning of the experiment, with the lowest value occurring on day 60, and then increased until the end of the experiment. In turn, for PS-NS, the lowest activity values were observed on day 30 and then increased to day 150. Species PS-S showed activity slightly lower than that of its non-hydrated form while maintaining an increase and decrease trend analogous to that of the PS-NS species. In turn, CM-S showed higher values of photosynthetic activity than its non-hydrated CM-NS species, with a progressive decrease in photosynthetic activity with each day of measurement (Fig. 3).

The highest values of the photosynthetic activity are recorded by the PS-NS species and slightly lower or similar PS-S species for the RL sample (Fig. 4). Species P. schreberi, both in soaked and non-soaked samples, showed photosynthetic activity in the range of 0.6–0.7. The lowest values of the photosynthetic activity are shown by CM-NS, which reaches a critical level on day 90 and then increases to day 120. The values Fv/Fm of the CM-S thallus are higher than those of CM-NS, but still lower than those of both samples of P. schreberi (soaked and non-soaked (Fig. 4).

The activity level of all samples was very high and oscillated between 0.7–8.0 Fv/Fm for the control sample (Fig. 5). The highest activity values were recorded for PS-NS and PS-S because they did not differ much from each other. In turn, the CM-NS species has the lowest values on each measurement day, with it was lower limit on day 60. The CM-S species showed photosynthetic activity higher than that of the CM-NS and are closely comparable to that of the PS-NS and PS-S species (Fig. 5).

Microscopic fluorescence imaging of chloroplasts

The fluorescence of the chloroplasts of Cladonia mitis varied depending on the type of light and the length of the experiment. Initially, in all types of light, the fluorescence of chloroplasts was at a level similar to that of fluorescence. For the FL sample, the lowest fluorescence was on day 60 and day 90, and during the following days, the chloroplast fluorescence increased significantly. For the RL sample, a decrease in fluorescence was observed, with the lowest fluorescence occurring on day 90. On subsequent days, an increase in fluorescence could be observed. The control sample was characterised by similar chloroplast fluorescence for all days. The FL sample had the lowest chloroplast activity, while the control sample had the highest chloroplast activity relative to the rest of the tested groups (Fig. 6).

The fluorescence of chloroplasts of the moss Pleurozium schreberi varied over time depending on the type of light. Referring to the FL sample, a decrease in the fluorescence of the chloroplasts was evident from day 30 to day 90. On subsequent days, an increase in the fluorescence of chloroplasts was observed. For the RL sample, slight decreases in fluorescence could be observed on day 30 and day 90, however, there was a resurgence later. For the control sample, there are no major differences in the change in chloroplast fluorescence (Fig. 7).

Photosynthetic activity

Bryophyte and lichens are two distinct groups of organisms, often co-occurring in the same habitats that share certain ecological and morphological characteristics. It is well-known that both hydration and access to light are crucial for the photosynthesis and growth of the organisms mentioned. Both groups tolerate full desiccation without suffering photooxidative damage under strong light. The results of two-factor analysis of variance (ANOVA) tests showed a variation in photosynthetic activity in C. mitis and P. schreberi over time depending on the amount of light energy supplied. The results obtained in the present work refer to earlier research (Dziurowicz et al. 2022). Current research is another step in understanding the mechanisms which confirmed that changes in photosynthetic activity occur in mosses and lichens depending on the amount of PAR energy supplied (Beckett et al. 2021; Dziurowicz et al. 2022; Wu et al. 2017). Evaluation of the methods and findings resulted in updated methodology and data collection, this time using both non-soaked and soaked samples, as well as fluorescence imaging.

Under non-stressful conditions, Fv/Fm levels usually remain at the same level, but this balance can be disturbed by the influence of stress factors, which include water, high temperature, and excessive radiation (PAR) (Lan et al. 2012). Absorption of more light that can be used for photosynthesis can lead to oxidative reactions that cause damage. This is especially dangerous for poikilohydric photoautotrophs that retain their chlorophyll during periods of dehydration (Heber and Lüttge 2011). In the present study, the photosynthetic activity of P. schreberi and C. mitis was lowest at the highest light intensity, which consequently coincides with the adaptation of lichens and mosses to perform photosynthesis under reduced sunlight and even shade (Tobias and Niinemets 2010; Wegrzyn et al. 2021). Species that are adapted to photosynthesise in the dark show maximum photochemical potential and the highest solar energy conversion efficiency in PS II when the reaction centres are completely open (Krause 1988; Franklin et al. 1992). On the other hand, the presence of excess light affects the partial illumination of the PS II reaction center, thereby determining its partial closure, resulting in an inevitable reduction in Fv/Fm levels (Lan et al. 2012). In extreme cases, excess energy can activate oxygen, thus creating reactive oxygen species (ROS), which in turn can damage the photosynthetic apparatus and other cellular components leading to photoinhibition and photooxidative stress (Mkhize et al. 2022). The first line of defence against oxidative stress in lichens involves the inactivation of reactive oxygen species (ROS) by enzymatic systems and the accumulation of lipophilic and hydrophilic compounds within cells (Beckett et al. 2021). However, under moderate light conditions, ROS is also produced, and a small amount of it is a signaling element responsible for acclimation and programmed cell death (Pospíšil, 2016). Results from the early 2000s (de la Torre et al. 2002; 2004; de Vera et al. 2002; 2004a; 2004b; de la Torre Noetzel et al. 2007) have shown that the ability to withstand high UV radiation can be credited to the fungus (mycobiont) that protects the more-sensitive photosynthetic active algae (photobiont) of the lichen with a dense cortex it develops (Vera et al. 2010). Furthermore, the concentration of specific secondary metabolites, such as depsidones, and dibenzofurans, often increases in lichens under stress conditions (Holger 2014). Responsible for the radiation shielding effects is the production of metabolites that absorb radiation, such as parietin (Solhaug et al. 2003; Solhaug and Gauslaa 2004; Wynn-Williams et al. 2002) and usnic acid (e.g. Galloway 1993; Quilhot et al. 1994; 1998; Rikkinen 1995; Solhaug and Gauslaa 1996).

Lichens are specialised to live in environments with limited access to water and can quickly recover from dehydration when water becomes available again. On the contrary, bryophytes are better suited for occupying moist habitats, often those that are only available seasonally, but have slower recovery times after experiencing dehydration (Green and Proctor 2016). The higher the PAR energy, the faster the drying of the thallus (Tobias and Niinemets 2010; Veres et al. 2022b), which in turn leads to photoinhibition and confirms the first research hypothesis that photosynthetic activity varies over time depending on the amount of PAR energy supplied. The response to high PAR is observed in both C. mitis and P. schreberi, and is more pronounced in lichens (Kappen et al. 1996). Lichens under conditions of water scarcity become dehydrated very quickly and go into a state of anabiosis, limiting or completely stopping photosynthesis (Kappen et al. 1996; Lange et al. 1993). An example is the CM-NS sample for which very low and sometimes critical Fv/Fm values were observed in full and reduced light. The completely opposite situation was observed for the PS-NS trial in which very high levels of photosynthetic activity were recorded throughout the experiment. The indicated differences are due to the different physiology of the studied species, and thus the degree of hydration of their thallus. Single bryophyte shoots have low resistance to water loss, while packed shoots forming turf have an increased capacity to store and transport water, significantly prolongs their physiological activity (Tobias and Niinemets 2010).

As part of our investigation, we not only measured the photosynthetic activity of the lichens in their non-soaked state, but also irrigated them for a period of two hours, since the importance of hydrating the samples under investigation before measuring their physiological parameters has already been shown in the previous study (Węgrzyn et al. 2021b). Exposure to the sunlight of the dry lichen thallus can cause the accumulation of severe damage in PS II, and water is the endogenous factor for the regeneration of the damaged apparatus (Veres et al. 2022a). In the study, the hydrated form of C. mitis showed significantly higher levels of photosynthetic activity than the non-soaked thallus, which is a natural process that occurs in lichens (Grimm et al. 2021; Wegrzyn et al. 2021). The process of desiccation is responsible for reducing the amount of light that can pass through the upper cortex of a lichen thallus (Büdel and Lange 1994; Ertl 1951). Additionally, it is believed to create a functional disconnection of components of the photochemical apparatus (Sigfridsson 1980; Bilger et al. 1989). On the other hand, soaked P. schreberi thallus mosses showed lower Fv/Fm values in each light, which may be the result of the supersaturation phenomenon (Green et al. 2011) and limited CO2 diffusion (Green et al. 2011; Möller et al. 2022). The results presented here did not confirm the second research hypothesis that lichens in any type of light show higher values of photosynthetic activity than mosses, however, they partially confirmed the third hypothesis, as we found that the lichens that were soaked in water exhibited a significant increase in photosynthetic activity when compared to the non-soaked specimens. It can be explained by the fact that both lichens and bryophytes are classified as homoiochlorophyllous, which means that they can rapidly resume photosynthesis after rewetting. However, a disadvantage of this trait is that chlorophyll continues to absorb photons even after desiccation, and the energy from the excess light cannot be used for photochemical work (Gasulla et al. 2021; Heber et al. 2006; Kranner et al. 2003). To prevent photooxidative damage in the desiccated state, homoiochlorophyllous poikilohydric cryptogams have evolved a highly efficient mechanism of photoprotection intended to prevent photodamage through controlled dissipation of thermal energy that is assessed by measuring non-photochemical quenching (NPQ) (Hájek et al. 2009; Heber and Lüttge 2011; Mkhize et al. 2022). NPQ is induced rapidly on a time scale of seconds to a few minutes, making it ideal for dealing with sudden fluctuations in light intensity (Goss and Lepetit 2015). In the case of lichens, NPQ is induced only after algae have been acclimated to prolonged light, so it would be a mistake to calculate NPQ values while measuring photosynthetic activity (Goss and Lepetit 2015; Maxwell and Johnson 2000).

Chloroplast structure

Fluorescence microscopy is a technique that allows the study of changes in the viability of single and intact cells with the accompanying autofluorescence of chloroplasts being verifiable with the naked eye (Boluda and Hawksworth 2014; Kauppi 1980; Takahashi 2019). Assessing chloroplast cell viability based on fluorescence microscope images alone is not precise enough (Bolhàr-Nordenkampf and Öquist 1993). However, in combination with the measurement of photosynthetic activity, it provides a reliable picture of the changes that occur in the studied species caused by excessive light exposure (Dutta et al. 2015). Chloroplasts can move depending on the amount of light energy provided to them, and their movement is directly proportional to the amount of PAR energy demand (Davis et al. 2011; Wada 2013). Under conditions of low illumination, chloroplasts carry out an accumulation process, in which case they expose themselves to light, but with excess light, there is an avoidance process in which chloroplasts hide behind each other (Dutta et al. 2015; Suetsugu and Wada 2012). The combination of accumulation and avoidance responses presumably allows plant cells to strike a balance between maximising light uptake and minimizing photodamage (Davis and Hangarter 2012). Photos of the changes under the microscope are an illustration of the trends of photosynthetic changes that occur in C. mitis and P.schreberi along with their chloroplast structure. At low fluorescence of chloroplasts, the preparation showed low intensity, the dominant colour of the cell walls (blue), while in the situation of full fluorescence the red colour dominated. C.mitis showed the lowest level of chloroplast fluorescence in the strongest and reduced light, which coincides with its low level of photosynthetic activity. This observation may indicate that chloroplast degradation or its avoidance reaction is occurring. As noted by other authors (Beckett et al. 2021; Derks et al. 2015; Veres et al. 2022b), high light intensity leads to progressive changes in the cell, which in most cases are irreversible. However, lichens in the sample of natural light show very high photosynthetic activity and thus are accompanied by intense fluorescence, which informs about the normal state of chloroplast behaviour. In P.schreberi, the identification of chloroplasts is much simpler, as they are arranged in a single layer, which was a problem in lichens in which chloroplasts were grouped in algal cells. The moss species suffer the greatest loss of chloroplast fluorescence in the trial of intense light. However, in bryophyte, the movement of chloroplasts is more likely to take place than their complete degradation. As photosynthetic activity decreased, fluorescence decreased, but at the final stages of the experiment, activity increased significantly, translating into intense fluorescence of chloroplasts. In a situation where the structure of chloroplasts was destroyed, they would not show fluorescence activity, as the chlorophyll in them is degraded (Otegui 2018). The activity of the repair apparatus depends on the regeneration time and the rate of progressive photodamage. This condition is known as photoinhibition of photosynthesis and occurs when the rate of photodamage exceeds the repair capacity (Derks et al. 2015; Melis 1999). Under reduced and natural light conditions, the mosses showed both high photosynthetic activity and intense fluorescence of the chloroplasts, indicating their much higher resistance to light radiation. Thus, the results presented partially confirm the fourth hypothesis, which assumes that chloroplasts are degraded under the influence of the strongest light. In lichens, this phenomenon is already noticeable in the first days of measurement, and the damage caused by PAR energy leads to the degradation of chloroplast cells. However, the degradation is small, or at least imperceptible, at a given stage of analysis. More research is needed to assess the viability of chloroplasts along with their photosynthetic activity.

Measurements of photosynthetic activity, combined with fluorescence microscopy imaging, allow observation of the conservation and viability of chloroplasts. In lichens and mosses, there is a phenomenon of displacement and relocation of chloroplasts depending on the given PAR energy demand. The intensification of light causes degradation of the photosynthetic apparatus in lichens, leading to photoinhibition of photosynthesis under oxidative stress. Mosses under conditions of excessive hydration exhibit superstorage properties, which slows down photosynthesis. The adequate hydration of the thallus and the availability of water are essential elements that affects the repair processes that take place in both lichens and mosses.

Author Contributions: PD, PF and MHW conceptualization, methodology, supervised the findings of this work. PD performed statistical analysis of the data. PD, PF, MHW and KW field material collection, writing original draft preparation. PD, PF, MHW, KW and PW-P writing review and editing. MHW. project management, performed microscopic fluorescence imaging of chloroplasts. All authors have read and agreed to the published version of the manuscript.

Funding: This research was funded by the Forest Fund of the State Forests, grant number EZ.0290.1.12.2021

Acknowledgments: We are grateful to Janusz Kochanowski Director of “Bory Tucholskie” National Park for agreeing to conduct research in the national park; to Agnieszka Turowska from “Bory Tucholskie” National Park for all assistance during the implementation of the project and assistance during collected material; and to anonymous reviewers for their very valuable suggestions and remarks on manuscript.

Data availability: All data generated or analysed during this study are included in this published article. Raw data are available from the corresponding author on reasonable request.

Conflict of interest: The authors have no relevant financial or non-financial interests to disclose.

Allorent G, Tokutsu R, Roach T, Peers G, Cardol P, Girard-Bascou J, Seigneurin-Berny D, Petroutsos D, Kuntz M, Breyton C, Franck F, Wollman F-A, Niyogi K, Krieger-Liszkay A, Minagawa J, Finazzi G (2013) A dual strategy to cope with high light in Chlamydomonas reinhardtii. TPJ 25(2):545–557. https://doi.org/10.1105/tpc.112.108274
Barber J, Andersson B (1992) Too much of a good thing: light can be bad for photosynthesis. TIBS 17(2): 61–66. https://doi.org/10.1016/0968-0004(92)90503-2
Beckett R, Minibayeva F, Solhaug K, Roach T (2021) Photoprotection in lichens: Adaptations of photobions to high light. Lichenologist 53(1):21–33. https://doi.org/10.1017/S0024282920000535
Bewley JD (1979) Physiological aspects of desiccation tolerance. Annu Rev Plant Physiol 30(1) 195–238. https://doi.org/10.1146/annurev.pp.30.060179.001211
Bilger W, Rimke S, Schreiber U, Lange OL (1989) Inhibition of energy-transfer to photosystem II in lichens by dehydration: different properties of reversibility with green and blue-green phycobionts. J Plant Physiol 134(3):261–268. https://doi.org/10.1016/S0176-1617(89)80240-8
Bolhàr-Nordenkampf HR, Öquist G (1993) Chlorophyll fluorescence as a tool in photosynthesis research. In: Hall DO, Scurlock JMO, Bolhàr-Nordenkampf HR, Leegood RC, Long SP (eds.) Photosynthesis and Production in a Changing Environment. Springer, Dordrecht. https://doi.org/10.1007/978-94-011-1566-7_12
Boluda CG, Rico V, Hawkswort DL (2014) Fluorescence microscopy as a tool for the visualization of lichen substances within Bryoria thalli. Lichenologist 46(5):723–726. https://doi.org/10.1017/S0024282914000292
Büdel B, Lange OL (1994) The role of cortical and epinecral layers in the lichen genus Peltula. Cryptogamic Botany 4:262–269.
Charron AJ, Quatrano RS (2009) Between a rock and a dry place: the water-stressed moss. Mol Plant 2(3):478–486. https://doi.org/10.1093/mp/ssp018
Chen YE, Wu N, Zhang ZW, Yuan M, Yuan S (2019) Perspective of monitoring heavy metals by moss visible chlorophyll fluorescence parameters. Front Plant Sci 10(35). https://doi.org/10.3389/fpls.2019.00035
Chowaniec K, Rola K (2022) Evaluation of the importance of ionic and osmotic components of salt stress on the photosynthetic efficiency of epiphytic lichens. Physiol Mol Biol Plants:1–15. https://doi.org/10.1007/s12298-022-01134-2
Davis PA, Caylor S, Whippo CW, Hangarter RP (2011) Changes in leaf optical properties associated with light-dependent chloroplast movements. Plant Cell Environ 34:2047–2059. https://doi.org/10.1111/j.1365-3040.2011.02402.x
Davis PA, Hangarter RP (2012) Chloroplast movement provides photoprotection to plants by redistributing PSII damage within leaves. Photosynth Res 112:153–161. https://doi.org/10.1007/s11120-012-9755-4
de la Torre Noetzel R, Sancho LG, Pintado A, Rettberg P, Rabbow E, Panitz C, Deutschmann U, Reina M, Horneck G (2007) BIOPAN experiment LICHENS on the Foton M2 mission: pre-flight verification tests of the Rhizocarpon geographicum-granite ecosystem. ASR 40(11):1665–1671. https://doi.org/10.1016/j.asr.2007.02.022
de la Torre R, Horneck G, Sancho LG, Pintado A, Scherer K, Facius R, Deutschmann U, Reina M, Baglioni P, Demets R (2004) Studies of lichens from high mountain regions in outer space: the BIOPAN experiment. In: Harris RA, Ouwehand L (eds.) ESA Publications Division, Noordwijk, The Netherlands:193–194.
de la Torre R, Horneck G, Sancho LG, Scherer K, Facius R, Urlings T, Rettberg P, Reina M, Pintado A (2002) Photoecological characterization of an epilithic ecosystem at high mountain locality (central Spain). In: Lacoste H (eds.) ESA Publications Division Noordwijk the Netherlands:443–444.
De Vera JP, Horneck G, Rettberg P, Ott S (2002) The potential of the lichen symbiosis to cope with extreme conditions of outer space – I. Influence of UV radiation and space vacuum on the vitality of lichen symbiosis and germination capacity. IJA 1(4):285–293. https://doi.org/10.1017/S1473550403001216
De Vera JP, Horneck G, Rettberg P, Ott S (2004a) The potential of lichen symbiosis to cope with the extreme conditions of outer space. II: Germination capacity of lichen ascospores in response to simulated space conditions. Adv Space Res 33:1236–1243. https://doi.org/10.1016/j.asr.2003.10.035
De Vera JP, Horneck G, Rettberg P, Ott S (2004b) In the context of panspermia: may lichens serve as shuttles for their bionts in Space?. In: Harris RA, Ouwehand L (eds.) ESA Publications Division Noordwijk The Netherlands:197–198.
Demmig-Adams B, Adams WW (1992) Photoprotection and Other Responses of Plants to High Light Stress. Annu Rev Plant Physiol Plant Mol Biol 43:599–626. https://doi.org/10.1146/annurev.pp.43.060192.003123
Derks A, Schaven K, Bruce D (2015) Diverse mechanisms for photoprotection in photosynthesis. Dynamic regulation of photosystem II excitation in response to rapid environmental change. BBA-Bioenergetics 1847(4-5):468–485. https://doi.org/10.1016/j.bbabio.2015.02.008
Dutta S, Cruz JA, Jiao Y, Chen J, Kramer DM, Osteryoung KW (2015) Non‐invasive, whole‐plant imaging of chloroplast movement and chlorophyll fluorescence reveals photosynthetic phenotypes independent of chloroplast photorelocation defects in chloroplast division mutants. TPJ 84(2):428–442. https://doi.org/10.1111/tpj.13009
Dziurowicz P, Fałowska P, Waszkiewicz K, Wietrzyk-Pełka P, Węgrzyn MH (2022) Changes in photosynthetic activity of the lichen Cladonia mitis and the Pleurozium schreberi under artificial high-energy lighting in laboratory. ABCbot 64/2 accepted article. https://doi.org/10.24425/abcsb.2022.143381
Ertl L (1951) Über die Lichtverhältnisse in Laubflechten. Planta 39:245–270. https://doi.org/10.1007/BF01909397
Franklin LA, Levavasseur G, Osmond CB, Henley WJ, Ramus J (1992) Two components of onset and recovery during photoinhibition of Ulva rotundata. Planta 187:399–408. https://doi.org/10.1007/BF00195321
Gaff DF (1977) Desiccation tolerant vascular plants of Southern Africa. Oecologia 31(1):95–109. https://doi.org/10.1007/BF00348713
Gaff DF (1989) Responses of desiccation tolerant ‘resurrection’ plants to water stress. In: Kreeb KH, Richter H, Hinckley TM (eds) Structural and Functional Responses to Environmental Stresses SPB Academic Publishing:264–311.
Galloway DJ (1993) Global environmental change: lichens and chemistry. Bibl Lichenol 53:87–95.
Gasulla F, del Campo EM, Casano LM, Guéra A (2021) Advances in Understanding of Desiccation Tolerance of Lichens and Lichen-Forming Algae. Plants 10(4):807. https://doi.org/10.3390/plants10040807
Goss R, Lepetit B (2015) Biodiversity of NPQ. J Plant Physiol 172:13–32. https://doi.org/10.1016/j.jplph.2014.03.004
Green TA, Proctor MC (2016) Physiology of photosynthetic organisms within biological soil crusts: their adaptation, flexibility, and plasticity. Biocrusts: an organizing principle in drylands:347–381. https://doi.org/10.1007/978-3-319-30214-0_18
Green TGA, Sancho LG, Pintado A (2011) Ecophysiology of Desiccation/Rehydration Cycles in Mosses and Lichens. In: Lüttge U, Beck E, Bartels D (eds.) Plant Desiccation Tolerance. Ecol stud 215. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-19106-0_6
Grimm M, Grube M, Schiefelbein U, Zühlke D, Bernhardt J, Riedel K (2021) The lichens’ microbiota, still a mystery?. Front Microbiol 12:714. https://doi.org/10.3389/fmicb.2021.623839
Hájek T, Tuittila ES, Ilomets M, Laiho R (2009) Light responses of mire mosses–a key to survival after water‐level drawdown?. Oikos 118(2):240–250. https://doi.org/10.1111/j.1600-0706.2008.16528.x
Hambler DJ (1961) A poikilohydrous, poikilochlorophyllous angiosperm from Africa. Nature 191(4796):1415–1416.
He X, He KS, Hyvönen J (2016) Will bryophytes survive in a warming world?. PPEES 19:49–60. https://doi.org/10.1016/j.ppees.2016.02.005
Heber U, Lange OL, Shuvalov VA (2006) Conservation and dissipation of light energy as complementary processes: homoiohydric and poikilohydric autotrophs. J Exp Bot 57(6):1211–1223. https://doi.org/10.1093/jxb/erj104
Heber U, Lüttge U (2011) Lichens and Bryophytes: Light Stress and Photoinhibition in Desiccation/Rehydration Cycles – Mechanisms of Photoprotection. In: Lüttge U, Beck E, Bartels D (eds.) Plant Desiccation Tolerance ECOLSTUD 215. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-19106-0_7
Holger T, Marie-Laurence A, David D, Joël B (2014) Oxidative stress regulation in lichens and its relevance for survival in coastal habitats. Advances in Botanical Research 71:467–503. https://doi.org/10.1016/B978-0-12-408062-1.00016-0
Kappen L, Schroeter B, Scheidegger C, Sommerkorn M, Hestmark G (1996) Cold resistance and metabolic activity of lichens below 0 C. ASR 18(12):119–128. https://doi.org/10.1016/0273-1177(96)00007-5
Kauppi M (1980) Fluorescence microscopy and microfluorometry for the examination of pollution damage in lichens. Ann Bot Fenn:163–173.
Kranner I, Zorn M, Turk B, Wornik S, Beckett RP, Batič F (2003) Biochemical traits of lichens differing in relative desiccation tolerance. New Phytol 160:167–176. https://doi.org/10.1046/j.1469-8137.2003.00852.x
Krause GH (1988) Photoinhibition of photosynthesis. An evaluation of damaging and protective mechanisms. Physiol Plant 74:566–574. https://doi.org/10.1111/j.1399-3054.1988.tb02020.x
Lakatos M (2011) Lichens and Bryophytes: habitats and species. In: Lüttge U, Beck E, Bartels D (eds.) Plant Desiccation Tolerance. Ecol stud 215:65–87. https://doi.org/ 10.1007/978-3-642-19106-0_5
Lan S, Wu L, Zhang D, Hu C (2012) Composition of photosynthetic organisms and diurnal changes of photosynthetic efficiency in algae and moss crusts. Plant Soil 351(1):325–336. https://doi.org/10.1007/s11104-011-0966-9
Lange OL, Büdel B, Heber U, Meyer A, Zellner H, Green TGA (1993) Temperate rainforest lichens in New Zealand: high thallus water content can severely limit photosynthetic CO2 exchange. Oecologia 95(3):303–313. https://doi.org/10.1007/BF00320981
Liu Y, Cao T, Glime J (2003) The Changes of Membrane Permeability of Mosses under High Temperature Stress. Bryologist 106(1):53–60. https://doi.org/10.1639/0007-2745(2003)106[0053:TCOMPO]2.0.CO;2
Maxwell K, Johnson GN (2000) Chlorophyll fluorescence—a practical guide. J Exp Bot 51(345):659–668. https://doi.org/10.1016/B978-0-12-408062-1.00016-0
Melis A (1999) Photosystem-II damage and repair cycle in chloroplasts: what modulates the rate of photodamage in vivo?. Trends Plant Sci 4(4):130–135. https://doi.org/10.1016/S1360-1385(99)01387-4
Mkhize KGW, Minibayeva F, Beckett RP (2022) Lichen photobionts can be hardened to photoinhibition by pretreatment with light. Acta Physiol Plant 44(11):1–10. https://doi.org/10.1007/s11738-022-03458-8
Möller T, Porada P, Petersen I (2022) The effect of water potential on the water balance of lichens and mosses – distribution patterns of internal and external water. PREPRINT Version 1 Res Sq https://doi.org/10.21203/rs.3.rs-1915047/v1
Nguyen KH, Chollet-Krugler M, Gouault N, Tomasi S (2013) UV-protectant metabolites from lichens and their symbiotic partners. Nat Prod Rep 30(12):1490–1508. https://doi.org/10.1039/C3NP70064J
Orekhova A, Barták M, Hájek J, Morkusová J (2022) Species-specific responses of spectral reflectance and the photosynthetic characteristics in two selected Antarctic mosses to thallus desiccation. Acta Physiol Plant 44(1):1–19. https://doi.org/10.1007/s11738-021-03339-6
Otegui MS (2018) Vacuolar degradation of chloroplast components: autophagy and beyond. J Exp Bot 69(4):741–750. https://doi.org/10.1093/jxb/erx234
Pospíšil P (2016) Production of reactive oxygen species by photosystem II as a response to light and temperature stress. Front Plant Sci 7:19–50. https://doi.org/10.3389/fpls.2016.01950
Quilhot W, Fernandez E, Hidalgo ME (1994) Photoprotection mechanisms against UV radiation. Brit Lichen Soc Bull 75:1–5.
Rikkinen J (1995) Whats behind the pretty colours. A study on the photobiology of lichens. Bryologist 4:1–239. https://doi.org/10.2307/3244316
Rochaix JD (2011) Regulation of photosynthetic electron transport. BBA-Bioenergetics 1807(3):375–383. https://doi.org/10.1016/j.bbabio.2010.11.010
Schlensog M, Schroeter B (2001) A new method for the accurate in situ monitoring of chlorophyll a fluorescence in lichens and bryophytes. Lichenologist 33(5):443–452. https://doi.org/10.1006/lich.2001.0340
Sigfridsson B (1980) Some effects of humidity on the light reaction of photosynthesis in the lichens Cladonia impexa and Collema flaccidum. Physiol Plant 49:320–6. https://doi.org/10.1111/j.1399-3054.1980.tb02671.x
Solhaug KA, Gauslaa Y (1996) Parietin, a photoprotective secondary product of the lichen Xanthoria parietina. Oecologia 108:412–418. https://doi.org/10.1007/BF00333715
Solhaug KA, Gauslaa Y (2004) Photosynthates stimulate the UV-B induced fungal anthraquinone synthesis in the foliose lichen Xanthoria parietina. Plant Cell Environ 27:167–176. https://doi.org/10.1111/j.1365-3040.2003.01129.x
Solhaug KA, Gauslaa Y, Nybakken L, Bilger W (2003) UV induction of sun-screening pigments in lichens. New Phytol 158:91–100. https://doi.org/10.1046/j.1469-8137.2003.00708.x
Suetsugu N, Wada M (2012) Chloroplast photorelocation movement: a sophisticated strategy for chloroplasts to perform efficient photosynthesis. In Advances in Photosynthesis — Fundamental Aspects. InTech:215–234. ISBN 978-953-307-928-8.
Takahashi T (2019) Routine management of microalgae using autofluorescence from chlorophyll. Molecules 24(24):44–41. https://doi.org/10.3390/molecules24244441
Tobias M, Niinemets Ü (2010) Acclimation of photosynthetic characteristics of the moss Pleurozium schreberi to among‐habitat and within‐canopy light gradients. Plant Biol 12(5):743–754. https://doi.org/10.1111/j.1438-8677.2009.00285.x
Tuba Z, Csintalan Z, Proctor MC (1996) Photosynthetic responses of a moss, Tortula ruralis, ssp. ruralis, and the lichens Cladonia convoluta and C. furcata to water deficit and short periods of desiccation, and their ecophysiological significance: a baseline study at present‐day CO2 concentration. NEPHAV 133(2):353–361. https://doi.org/10.1111/j.1469-8137.1996.tb01902.x
Tuba Z, Lichtenthaler HK, Csintalan Z, Nagy Z, Szente K (1994) Reconstitution of chlorophylls and photosynthetic CO2 assimilation upon rehydration of the desiccated poikilochlorophyllous plant Xerophyta scabrida (Pax) Th. Dur. et Schinz. Planta 192:414–420. https://doi.org/10.1007/BF00198578
Vera JPD, Möhlmann D, Butina F, Lorek A, Wernecke R, Ott S (2010) Survival potential and photosynthetic activity of lichens under Mars-like conditions: a laboratory study. Astrobiology 10(2):215–227. https://doi.org/10.1089/ast.2009.0362
Veres K, Csintalan Z, Laufer Z, Engel R, Szabó K, Farkas E (2022a) Photoprotection and high-light acclimation in semi-arid grassland lichens–a cooperation between algal and fungal partners. Symbiosis 86(1):33–48. https://doi.org/10.1007/s13199-021-00823-y
Veres K, Sinigla M, Szabó K, Varga N, Farkas E (2022b) The long-term effect of removing the UV-protectant usnic acid from the thalli of the lichen Cladonia foliacea. Mycol Prog 21(9):1–13. https://doi.org/10.1007/s11557-022-01831-y
Wada M (2013) Chloroplast movement. Plant Science 210:177–182. https://doi.org/10.1016/j.plantsci.2013.05.016
Węgrzyn MH, Fałowska P, Alzayany K, Waszkiewicz K, Dziurowicz P, Wietrzyk-Pełka P (2021b) Seasonal changes in the photosynthetic activity of terrestrial lichens and mosses in the lichen scots pine forest habitat. Diversity 13. https://doi.org/10.3390/d13120642
Węgrzyn MH, Fałowska P, Kołodziejczyk J, Alzayany K, Wężyk P, Zięba-Kulawik K, Hawryło P, Turowska A, Grzesiak B, Lipnicki L, Wietrzyk-Pełka P (2021a) Tree height as the main factor causing disappearance of the terricolous lichens in the lichen Scots pine forests. Sci Total Environ 771. https://doi.org/10.1016/j.scitotenv.2020.144834
Węgrzyn MH, Kołodziejczyk J, Fałowska P, Wężyk P, Zięba-Kulawik K, Szostak M, Turowska A, Grzesiak B, Wietrzyk-Pełka P (2020) Influence of the environmental factors on the species composition of lichen Scots pine forests as a guide to maintain the community (Bory Tucholskie National Park, Poland). GECCO 22. https://doi.org/10.1016/j.gecco.2020.e01017
Wobbe L, Bassi R, Kruse O (2016) Multi-level light capture control in plants and green algae. Trends Plant Sci 21(1):55–68. https://doi.org/10.1016/j.tplants.2015.10.004
Wu L, Lei Y, Lan S, Hu C (2017) Photosynthetic recovery and acclimation to excess light intensity in the rehydrated lichen soil crusts. PLoS One 12(3). https://doi.org/10.1371/journal.pone.0172537
Wynn-Williams DD, Edwards HGM, Newton EM, Holder JM (2002) Pigmentation as a survival strategy for ancient and modern photosynthetic microbes under high ultraviolet stress on planetary surfaces. Int J Astrobiology 1:39–49. https://doi.org/10.1017/S1473550402001039

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Effect of light stress on maximum photochemical efficiency of photosystem II and chloroplast structure in cryptogams Cladonia mitis and Pleurozium schreberi

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Introduction

Materials And Method

Establishment and maintenance of laboratory cultures of lichen and bryophytes

Measurements of photosynthetic activity

Microscopic fluorescence imaging of chloroplasts

Statistical analysis

Results

Photosynthetic activity

Microscopic fluorescence imaging of chloroplasts

Discussion

Photosynthetic activity

Chloroplast structure

Conclusion

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References

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