Photosynthetic Pigments

Photosynthetic activity and chlorophyll pigment concentration in Medicago x varia T. Martyn leaves treated with the Tytanit growth regulator

The purpose of the research was to determine the effect of the foliar use of a growth regulator with the trade name of Tytanit, containing titanium ascorbate, on photosynthetic activity and chlorophyll content in Medicago × varia T. Martyn leaves. There were two kinds of plots: C – control series; Ti – plants treated with Tytanit, containing 8.5 g of titanium in 1 dm 3 . The following parameters were determined: maximum photosystem II efficiency (F v /F m ) in a dark-adapted state, actual photosystem II efficiency (ΔF/F m’ ) in a light-adapted state, photochemical quenching factor (QP), non-photochemical quenching factor (QN), and chlorophyll a and b content. The Fisher-Snedecor test was used to determine whether the impact of experimental factors was significant, and the HSD 0.05 value was calculated using Tukey’s test. Compared to control, the photosynthetic apparatus performance of alfalfa was positively affected by the regulator compared to control. Tytanit applied to plant leaves increased their photosynthetic activity as a result of an increase in the content of chlorophyll pigments. It was also found that periods of rainfall deficiency did not affect the beneficial effects of the regulator.

The aim of the experiment was to determine the effect of the foliar use of titanium ascorbate growth regulator (Tytanit) on photosynthetic activity and chlorophyll content in Medicago × varia T. Martyn leaves. The studies were intended to demonstrate its effect on photosynthetic activity determined by measuring the induction of chlorophyll fluorescence and chlorophyll a and b content, treating plants twice during each growth cycle over three years.

Titanium is found in soil mainly in the form of minerals such as ilmenite, consisting of titanium-iron oxide and titanium oxide (IV), but in this form it is not bioavailable. Titanium has a significant biological effect on plants, being beneficial in low concentrations and toxic in higher concentrations ( Kuželet al., 2003 ). Optimal doses and time of titanium application to many crops have not yet been determined. In the present experiment titanium was applied to plant leaves in the form of a growth regulator called Tytanit. Manufactured by Intermag Ltd., this product contains 8.5 g Ti per dm 3 (0.8% m/m), in the form of Ti-ascorbate ( Wadas and Kalinowski, 2017a ). There are many studies in the available literature on the impact of the Tytanit growth regulator on the yield of agricultural plants, vegetables and fruits ( Du et al., 2010 , Grajkowski and Ochmian, 2007 , Radkowski and Radkowska, 2010 , Wadas and Kalinowski, 2017b ). However, there is a lack of studies directly relating to the effect of Tytanit on individual parameters determining the photosynthetic activity of plant leaves. Therefore, the studies presented in this paper are innovative, as they complement the lack of knowledge in this area. Used in the experiment the method of evaluating photosynthesis based on chlorophyll fluorescence (CF) in large part replaces conventional measurements of its intensity. CF is a highly sensitive attempt at assessing photosynthetic capacity, being completely non-invasive and allowing studying photosynthesis in vivo. It is particularly useful in situations of the impact of various environmental factors on plants. In the production of plant biomass, the size of photosynthetically active radiation (PAR), with its effect on dry matter production, is crucial. Energy absorbed in the photosynthesis process can be used for carbon assimilation, or can be lost when radiated as fluorescent radiation. Thus, there is a close relationship between the amount of fluorescence and the intensity of photosynthesis. Therefore, chlorophyll fluorescence is a measure of the state and condition of the photosynthetic apparatus ( Zhanga et al., 2000 ).

The results of the research were processes statistically using ANOVA for repeated (three years), multi-factor, and recurrent measurements (three harvest in a growing season). The Fisher-Snedecor test was used to determine whether the effect of Tytanit was significant, while the value of the HSD 0.05 was calculated with Tukey's test. The Statistica program version 12.0 (Dell Inc., Tulsa, Oklahoma, USA) was applied for all other calculations. Means in the tables marked with the same letters in lines/columns do not differ significantly.

The K values are presented in . According to Radzka et al. (2015) , extreme conditions occur when the value of the K-factor is below 0.7 and above 2.5. Thus, according to optimal temperature and humidity conditions were only in April 2017 and September 2015. Throughout the experiment, the best conditions were at the beginning of each growing season. The most difficult situation for plants was in 2015 and 2017, when apart from May and the end of the growing season, the weather ranged from moderately dry to very dry. There was a lack of periods with extreme droughts in the growing season of 2016, when wet and quite wet spells prevailed.

Sielianinov’s hydrothermal coefficient was calculated in order to determine temporal variation of meteorological conditions and their effects on plant growth and development. The hydrothermal coefficient (K) was calculated on the basis of monthly precipitation (P) and monthly sums of daily air temperatures (t), using the following formula ( Radzka et al., 2015 ):

Chlorophyll a and b content was determined according to Khaleghi et al. (2012) . The dates of material collection for the determination of pigment content in each growth cycle are presented in . The optical density of supernatants was determined with the Bio-RADSmartSpect TM Plus Spectrophotometer (equipment and instruments used in experimental work should be mentioned by their common name and between parentheses model, brand, city, state, and country of the manufacturer) at 440, 465, and 663 nm. Next, the results were calculated according to the following formulas:

All measurements were made during the growing season with six replicates, using well-developed leaves. The 2030-B clip, a light emitting diode at 650 nm and a standard intensity of 0.15 μmol m-2 s-1 PAR were used. During the dark-adapted stage leaves were kept in darkness for 15 min.

Measurement of photosynthetic activity of plant leaves was carried out in each growth cycle. For this purpose, on the seventh day after the second spray of alfalfa with the regulator ( ), leaves were collected from 10 randomly selected plants on each plot. Photosynthetic activity was determined by measuring the induction of chlorophyll fluorescence by means of the fluorometer (PAM 2000, Heinz Walz GmbH, Effeltrich, Germany). The following parameters were determined ( Bolhàr-Nordenkampf and Öquist, 1993 ):

The spraying liquid was made by dissolving 0.4 dm 3 ha -1 of Tytanit in 200 dm 3 ∙ha −1 of water, with the control plants treated with the same amount of water. The dose of the regulator was measured according to the manufacturer's recommendations.

The regulator was used in the form of two sprays during each of the three growth cycles (altogether six sprays in the growing season). The application time was based on the growth and development stages of Medicago × varia T. Martyn, adopted according to the European BBCH scale, with following characteristics of the BBCH growth stages:

The research was carried out as a field experiment conducted at the experimental facility of University of Natural Sciences and Humanities in Siedlce (52°10′03″N; 22°17′24″E) between 2015 and 2017. In the autumn of 2014, experimental plots of 6 m 2 (2 m × 3 m) were established. Between plots paths of 1.5 m were kept as herbicide fallow. The soil on which the experiment was founded had a granulometric composition of loamy sand. Organic carbon content (C org ) in the soil was 13.5 gkg -1 DM, with total nitrogen of 1.30 gkg -1 DM and the C:N ratio of 10.4:1. Soil pH of 6.8 was close to neutral. In addition, soil contained high amounts of forms of available phosphorus and magnesium, but forms of available potassium were within the limits of moderate content. Due to relatively high content of soil nutrients, neither pre-sowing nor post-sowing mineral fertilizer treatment was applied to Medicago × varia T. Martyn. Testing of the Tytanit growth regulator, the experiment was conducted in a split-plot design with three replicates.

The smallest concentration of chlorophyll a and b in plants was in 2015 and 2017, when, according to the distribution of Selianinov’s coefficient ( ), there was a drought in the summer and autumn seasons. Then, there were extremely dry periods in October 2017 and August 2015, with very dry July and September in 2017. Drought stress during those months caused a significant decrease in the content of chlorophyll pigments in leaves. The smallest chlorophyll a content (211 mg 100 g -1 FM) was recorded in 2017, 14.6% lower than in 2016. In turn, the concentration of chlorophyll b was reduced most by the drought in 2015, when it was 13.8% lower than in 2016. However, studying the interaction between growing seasons and regulator effects, it turned out that the content of chlorophyll pigments across harvests was significantly higher in years with extremely dry periods.

It is therefore worth noting that the periodic absence of precipitation, with warm sunny weather, did not result in a decrease in the efficiency of the primary photosynthesis reactions, that is to say, non-cyclic transport of electrons proceeded in these conditions smoothly.

The findings indicated that none of the regulator doses significantly affected the photochemical quenching coefficient (QP), the values of which ranged from 0.537 to 0.556 ( ). However, the values of certain parameters of photosynthetic activity were dependent on weather conditions. The photochemical quenching coefficient (QP) and the non-photochemical quenching coefficient (QN) across the doses and growing seasons assumed the highest values in plants of the summer harvest with QN of 0.135 and QP of 0.592 ( , ). As the meteorological data indicate ( ), dry periods alternated with humid ones in each summer season. A very dry summer in 2015 was exceptional, with very large water deficits in both July and August.

It can therefore be assumed that the treatment of Medicago × varia T. Martyn plants with the Tytanit regulator may have caused better nutrition of plant cells with nitrogen, as evidenced by the increases in photosynthetic parameters such as the maximum (F v /F m ) and actual (ΔF/F m' ) photosystem efficiency of alfalfa leaves ( , ).

The smallest value of the maximum photosystem II efficiency with the F v /F m ratio of 0.555 was recorded for control plants in 2015 during the summer growth cycle and in 2016 during the spring growth cycle. In turn, the highest value of the Fv/Fm (0.669) was in plants treated with the regulator in the autumn of 2015. Furthermore, the statistical analysis of the data showed a significant increase in the maximum photosystem II efficiency in plants treated with Tytanit; its average increase relative to control was 12.2%. A similar trend was noted in the case of the actual photosystem II efficiency ( ). For plants treated with the regulator, there was a 19.5% increase in the actual photosystem II efficiency (ΔF/F m’ ). In plants treated with Tytanit its higher values were recorded during the spring growth cycle. In turn, for all growing seasons, the highest value of the ΔF/F m’ was recorded in 2016.

The results of the studies indicate ( , , , , , ) a multidirectional effect of the growth regulator Tytanit, compared to the control series, on the photosynthetic activity of Medicago × varia T. Martyn leaves, as evidenced by the values of chlorophyll content and fluorescence induction parameters.

4. Discussion

The increase in the F v /F m parameter increased the plant demand for photosynthetic products and it lowered plant stress in the growth and development process. Furthermore, as Demmig-Adams and Adams (1992) indicate, increasing maximum photosystem II efficiency means activating the photosystem in a dark-adapted state resulting from the absence of photo inhibition in nitrogen-deficient plant cells. Thus, according to Khaleghi et al., 2012, Laisk et al., 2014, the energy consumed for the transport of electrons is not reduced. At the same time, according to Nishiyama et al.(2006), an increase in the activity of reaction centres of PSII cells in a dark-adapted state is the effect of supplying them with the right amount of nitrogen, which translates into high activity of the photosynthetic apparatus and into increased efficiency of light energy conversion.

According to Michałek and Sawicka(2002), genetic conditions have a very strong impact on the formation of fluorescence parameters, which may explain a lack of variation in the value of the photochemical quenching coefficient (QP) as a response to Tytanit regulator application. Studying the effects of Tytanit on the photochemical indicators of Dactylis glomerata leaves Sosnowski et al. (2020) recorded similar results. According to the authors, none of the Tytanit doses significantly affected this parameter.

In addition to the present experiment, effects of drought stress on chlorophyll fluorescence rates were studied by other researchers. Kianiet al. (2008) observed that increasing water stress did not result in a long-term decrease in the photosynthesis indicators of Helianthus annuus L. plants, but it reduced the actual transport efficiency of PSII electrons. In addition, the authors' analysis of QTL (the Quantitative Trait Loci) showed that several genomic areas were involved in the complete variability of chlorophyll fluorescence parameters during drought stress. Most QTL were specific to a given stress condition. This shows that photosynthetic control of gene expression varied under changing water conditions.

In the present experiment the Tytanit growth regulator positively affected chlorophyll a and b content in Medicago × varia T. Martyn. As reported by Yokoya et al., 2007, Zhao et al., 2016, these photosynthetic pigments are responsible for collecting and transmitting absorbed light to photosynthetic reaction centres, and their concentration is linked to the effectiveness of photosynthesis. In addition, according to Zhao et al. (2016), increased content of these pigments may be one of the factors increasing photosynthetic activity. Thus, as evidenced in the present experiment, the Tytanit regulator increased the concentration of chlorophyll pigments in Medicago × varia T. Martyn leaves, which in turn increased their photosynthetic activity ( , ). Similar results were reported by Wadas and Kalinowski (2017b), who examined the effect of Tytanit on the leaf assimilation surface and on chlorophyll content in very early varieties of potato. They found that foliar application of the Tytanit growth regulator resulted in a stimulating effect of titanium ions on the leaf assimilation surface and chlorophyll content in potato plants. As a response to Tytanit application, the plants formed a larger leaf assimilation surface area, also under stressful conditions. In addition, according to studies conducted in China (Tan and Wang, 2011), after triple use of foliar fertilizer containing titanium, leaves were dark green, shiny and dense, which was also confirmed after application of Tytanit to Medicago × varia T. Martyn crops.

Other authors (Kováčik et al., 2014, Radkowski, 2013) found that Tytanit stimulated chlorophyll content in Phleum pratense L., winter wheat and winter rape leaves. In addition, the authors reported that varied doses and times of Tytanit application only slightly affected chlorophyll content in the leaves of crop species. Kováčiket al. (2014) recorded a positive effect of Mg-Tytanit double application on chlorophyll content in winter wheat and winter rape leaves. Chlorophyll content was higher in plants treated with a Mg-Tytanit dose of 0.2 dm3 ha−1 than in plants treated with a dose of 0.4 dm3 ha−1. The third spray of both Mg-Tytanit doses tended to reduce the content of chlorophyll in leaves. In turn, the present studies indicated that the content of chlorophyll pigments in Medicago × varia T. Martyn leaves was also dependent on weather conditions.

The literature confirmed the effect of drought stress on chlorophyll a and b content in crops. Kiani et al. (2008) observed that chlorophyll a and b content in sunflower leaves decreased as the water deficit increased. Reductions in chlorophyll content in cotton leaves during drought conditions were also noted by Massacci et al. (2008). Similar results were recorded by Arji and Arzani (2008) in their research on the effects of drought stress on selected Olea europaea physiological parameters.

Substantial near-infrared radiation-driven photosynthesis of chlorophyll f-containing cyanobacteria in a natural habitat

Hyperspectral reflectance imaging on vertical cross-sections of beachrock submerged in seawater (23°C and salinity = 35) revealed the presence of a dense ~1 mm thick surface biofilm with high amounts of Chl a, while a more patchy zone containing Chl f, and less Chl a was found below the surface biofilm of the beachrock (Figure 1A,B), exhibiting localized hot spots of Chl f concentration (Figure 1C). Representative reflectance spectra from these regions carrying spectral signatures of maximal Chl a and Chl f absorption at 670–680 nm and 715–725 nm, respectively, are presented in the Supplementary Materials (Figure 1—figure supplement 1) along with additional examples of hyperspectral imaging of beachrock cross-sections (Figure 1—figure supplement 2). The presence of Chl f (and absence of microalgal Chl b and c) in our samples was confirmed by HPLC analysis of beachrock pigment extracts (see Materials and methods), both in a ~ 0–2 mm thick black beachrock sample, and in a more distinct green endolithic layer (~2–5 mm below the beachrock surface) (Figure 1—figure supplement 3). The amount of Chl f relative to Chl a in these samples ranged from 3.5% in the mixed layer to 6% in the green layer, while Trampe and Kühl (2016) reported Chl f amounts ranging from 0.5% to 5% of Chl a in beachrock.

Figure 1 with 8 supplements with 8 supplements see all Download asset Open asset Spatial distribution of photopigments and near-infrared radiation-driven oxygenic photosynthesis in beachrock as mapped with hyperspectral reflectance imaging and chemical imaging of O 2 . (A) RGB image composite, constructed from the hyperspectral image stack (R = 650 nm, G = 550 nm, B = 450 nm), showing ‘true’ colors of beachrock material and the biofilm community in a cross-section of the top layer. (B) False color coded image of the same hyperspectral image stack as in panel A mapping pixels with Chl a absorption (670–680 nm) in green, and Chl f absorption (718–722 nm) in red. Representative reflectance spectra of the two regions are given in Figure 1—figure supplement 1. (C) Overlay of beachrock structure obtained in panel A and the Chl a signature from panel B with map of the relative abundance of Chl f obtained from the amplitude of Chl f absorption (color coded between 0 and 1), as acquired from hyperspectral image analysis. (D) Distribution of O 2 concentration (color coded in units of % air saturation) in the beachrock under illumination of 740 nm light (half-bandwidth = 25 nm; photon irradiance = 28 µmol photons m−2 s−1) when immersed in anoxic seawater, as imaged with the beachrock section covered with a thin paint of agarose containing O 2 –sensitive nanoparticles. The O 2 concentration image was superimposed onto the structural image of the beachrock cross section. The insert is a digital zoom corresponding to the insert in panel C. Additional data on two other beachrock sections are available in the Suppl. Materials (Figure 1—figure supplements 2 and 6).

To further describe the microscale distribution of cells with different photo-pigmentation, we employed hyperspectral fluorescence imaging with confocal laser scanning microscopy (CLSM; 488 nm excitation) on beachrock cross-sections (Figure 1—figure supplements 4 and 5). The CLSM data confirmed the occurrence of patches of filamentous and unicellular Chl f-containing cyanobacteria with a characteristic fluorescence peak around 740–750 nm (cf. Majumder et al., 2017) in deeper endolithic zones (Figure 1—figure supplements 4C and 5A–D). Besides filamentous morphotypes, brightfield microscopy of Chl f hot spots revealed the presence of larger round cell aggregates (Figure 1—figure supplement 5E,F) typical of pleurocapsalean cyanobacteria (Waterbury and Stanier, 1978).

In order to confirm the apparent dominance of cyanobacteria over microalgal oxygenic phototrophs, we employed 16S rRNA gene amplicon sequencing on black beachrock samples taken from the same area as the samples used for hyperspectral and O 2 imaging. Among ~39,000 cyanobacterial-like sequences obtained from the black beachrock, none were classified as chloroplasts. In contrast, among ~17,000 cyanobacterial-like sequences obtained from two samples of seawater, 18% were classified as chloroplasts (Supplementary file 2). Microalgae are thus likely to be completely absent from the black beachrock, where the oxygenic phototrophic community consists exclusively of cyanobacteria.

Analysis of a distinct green layer below the black beachrock surface biofilm showed that most cyanobacterial OTU’s were clustering with Halomicronema (harboring the Chl f-containing species Halomicronema hongdechloris; Chen et al., 2012) as well as coccoid Pseudocapsa and Chroococcidiopsis (Supplementary file 2). The surface layer of the black beachrock harbored a higher cyanobacterial diversity with most OTU’s clustering with Rivularia, Calothrix, and Halomicronema, as well as numerous smaller populations clustering with Chroococcidiopsis and other coccoid cyanobacteria. These data confirm earlier findings of i) Chl f (and only minor amounts of Chl d) in beachrock (Trampe and Kühl, 2016), and ii) a predominance of cyanobacteria as the major oxygenic phototrophs in beachrock (Cribb, 1966; Díez et al., 2007). We note that a comprehensive description of the cyanobacterial diversity associated with beachrock was beyond the scope of the present study, and a detailed study of the microbial diversity in beachrock (based on 16S rRNA amplicon and metagenomic sequencing) will be presented elsewhere. Here, we focus on the photosynthetic activity of Chl f-containing cyanobacteria in beachrock.

By coating beachrock cross-sections with a thin (<1 mm) layer of an O 2 -sensitive nanoparticle-agarose paint (see Materials and methods and Figure 1—figure supplement 6) and subsequent immersion in anoxic water, it was possible to map the local O 2 production over the beachrock cross-section when illuminated with weak NIR levels (740 nm, 25 nm half bandwidth; 28 µmol photons m−2 s−1). We observed hot spots of NIR-driven photosynthesis driving local O 2 levels from 0% to >40–50% air saturation within 15–20 min (Figure 1D, Figure 1—figure supplement 7), which overlapped with regions of high Chl f absorption (Figure 1C, Figure 1—figure supplement 3). The build-up of O 2 in the hotspots harboring Chl f occurred rapidly after onset of NIR illumination and dissipated rapidly back to anoxia within a few minutes after darkening (see Video 1). Based on O 2 concentration images recorded at 5 min intervals after experimental light-dark shifts, we calculated images of apparent dark respiration and NIR-driven net and gross photosynthesis that could be mapped onto the beachrock structure (Figure 2A–D) showing that hotspots of activity aligned with the presence of Chl f (see Figure 1B,C). We extracted estimates of maximum O 2 conversion rates in particular regions of interest (ROI) showing high rates of NIR-driven gross photosynthesis of ~5–15 µmol O 2 L−1 min−1 in the beachrock under the given actinic irradiance of 28 µmol photons m−2 s−1; a similar range was found for two other beachrock cross-sections (data not shown). These volume-specific rates fall among the upper range of maximal gross photosynthesis rates in aquatic phototrophs, and are comparable with photosynthetic rates found in benthic microalgae (Krause-Jensen and Sand-Jensen, 1998).

Figure 2 Download asset Open asset Oxygen consumption and NIR-driven oxygenic photosynthesis in beachrock. Cross-sectional images of initial O 2 consumption after onset of darkness (A) and maximum net photosynthetic O 2 production (B) after onset of actinic NIR illumination (740 nm, 28 µmol photons m−2 s−1) of the beachrock cross-section shown in Figure 1. (C) The NIR-driven gross photosynthesis was estimated by summing the absolute rates of net photosynthesis under NIR and O 2 consumption in the dark. (D) Overlay of gross photosynthesis distribution over a structural image of the beachrock cross-section. (E) Data for O 2 consumption, and NIR-driven net and gross photosynthesis were extracted for 11 regions of interest (ROI) in panel C and are presented as means ± standard deviation within the ROI.

Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Animation of NIR-driven O 2 dynamics over a beachrock cross-section (see Figures 1 and 2) coated with a thin (<1 mm) agarose layer with luminescent O 2 sensor nanoparticles. The movie sequence shows the decline in O 2 concentration (recorded at 5 min interval) starting from steady-state conditions under a NIR irradiance (740 nm; 25 nm HBW) of 28 µmol photons m−2 s−1 approaching steady-state dark conditions after 35 min, followed by the rise in O 2 concentration over 25 min after switching the NIR irradiation on again. The colored scale bar relates the colors to O 2 concentrations.

The Chl f-driven oxygenic photosynthesis by endolithic cyanobacteria thus seems very efficient given the low actinic NIR level applied. High photosynthetic efficiency of Chl f-containing cyanobacteria under NIR has been shown in previous ex situ studies on cultivated strains and enrichments. Gan et al. (2014) thus found that Leptolyngbya strain JSC-1 cells showed a 40% higher O 2 production rate with NIR after undergoing FaRLiP relative to cells grown under red light (Gan et al., 2014), and a similar high photosynthetic efficiency was found in a Chroococcidiopsis strain (Nürnberg et al., 2018). Using similar NIR levels as used in the present study, Behrendt et al. (2015) showed rapid saturation of NIR-driven oxygenic photosynthesis already at 25–30 µmol photons m−2 s−1 (740 nm) in a cell enrichment with Chl f-containing, Aphanocapsa-like cyanobacteria from a cavernous biofilm.

Our structural and chemical imaging of beachrock showed that Chl f and NIR-driven O 2 production was confined to a relative narrow zone 1–2 mm below the beachrock surface in the investigated samples (Figure 1, Figure 2; Figure 1—figure supplements 2, 4 and 5). Assuming a NIR-driven oxygenic photosynthesis rate of 10 µmol O 2 L−1 min−1 (=nmol O 2 cm−3 beachrock min−1) in a 1 mm thick layer (Figure 2), and a conservative estimate of beachrock porosity of ~0.4 in the uppermost 1–2 mm (Vousdoukas et al., 2007), we can estimate the areal NIR-driven gross photosynthesis rate to (10 µmol L−1 min−1 × 0.4×0.1 cm x 10−6 x 10000 x 60 =) ~0.24 mmol O 2 m−2 beachrock h−1. Total beachrock primary productivity remains to be quantified in detail, but it is well known that beachrock habitats sustain high grazing rates of epifauna (McLean, 1974; McLean, 2011) and herbivorous reef fish (Stephenson and Searles, 1960).

To our knowledge, beachrock primary production has only been reported by Krumbein, who studied a Red Sea beachrock habitat (Krumbein, 1979). Based on his data on O 2 exchange under water covered conditions (cf. Figure 13 in Krumbein, 1979), we estimated a total gross photosynthesis of ~10 mmol O 2 m−2 beachrock h−1. While based on several crude assumptions and comparing different habitats, these rough calculations indicate that NIR-driven photosynthesis could account for at least 2–3% of total areal photosynthesis in beachrock habitats. Furthermore, we note that we base this estimate on O 2 dynamics over minutes measured in a thin agar layer adjacent to the actual phototrophic cells, which will underestimate the true dynamics (see Materials and methods), and our measurements were conducted at low NIR levels that may not represent saturated photosynthesis rates. Obviously, the realized total daily primary productivity will also be strongly modulated by the actual biomass distribution, porosity and diel light exposure within the beachrock, as well as the pronounced diel cycles of environmental conditions on the beachrock platform (Petrou et al., 2014; Schreiber et al., 2002). Nevertheless, we argue that our experimental data point to a significant role of NIR-driven oxygenic photosynthesis in beachrock and potentially other endolithic habitats.

Based on the origin of isolates shown to employ FaRLiP (Supplementary file 1), it has been speculated that shaded soils, caves, plant canopies and thermal springs may be prime terrestrial habitats for cyanobacteria with Chl f (Gan et al., 2015; Zhang et al., 2019). Beachrock is widespread in intertidal zones on a global scale (Vousdoukas et al., 2007) and our study shows that these habitats may present a major ecological niche for cyanobacteria with FaRLiP and NIR-driven oxygenic photosynthesis in marine intertidal habitats. While we present the hitherto most detailed insight into the distribution and in situ activity of Chl f-containing cyanobacteria in intact samples from a natural habitat, there is now a need for more precise characterization of the microenvironment and metabolic activities of microorganisms in beachrock to assess the quantitative importance of NIR-driven oxygenic photosynthesis for system productivity.

The present study and the few other studies of natural habitats (Supplementary file 1) demonstrate that a key trait of such cyanobacteria is the formation of biofilms in strongly shaded environments below other algae, cyanobacteria or terrestrial plants, or in the twilight zone of caves (Behrendt et al., 2019). It remains to be explored to what extent the presence of cyanobacteria with FaRLiP-capability or constitutive high Chl d levels play a role for overall photosynthetic productivity in such habitats. That is, does the complementary photopigmentation of cyanobacteria with far-red shifted photopigments in the ‘understory’ of other oxygenic phototrophs lead to higher photosynthetic efficiency and productivity at the community/system level, for example in analogy to plant canopies? Such studies are complicated due to the compacted and often stratified structure of the natural communities, but the novel combination of structural and chemical imaging presented here seems a promising toolset for unraveling the ecological importance of FaRLiP and cyanobacteria with far red-shifted photopigments.

Photosynthetic Pigments

Photosynthetic Pigments

Pigments are colorful compounds.

Pigments are chemical compounds which reflect only certain wavelengths of visible light. This makes them appear "colorful". Flowers, corals, and even animal skin contain pigments which give them their colors. More important than their reflection of light is the ability of pigments to absorb certain wavelengths.

Because they interact with light to absorb only certain wavelengths, pigments are useful to plants and other autotrophs --organisms which make their own food using photosynthesis. In plants, algae, and cyanobacteria, pigments are the means by which the energy of sunlight is captured for photosynthesis. However, since each pigment reacts with only a narrow range of the spectrum, there is usually a need to produce several kinds of pigments, each of a different color, to capture more of the sun's energy.

There are three basic classes of pigments.

Chlorophylls are greenish pigments which contain a porphyrin ring. This is a stable ring-shaped molecule around which electrons are free to migrate. Because the electrons move freely, the ring has the potential to gain or lose electrons easily, and thus the potential to provide energized electrons to other molecules. This is the fundamental process by which chlorophyll "captures" the energy of sunlight. There are several kinds of chlorophyll, the most important being chlorophyll "a". This is the molecule which makes photosynthesis possible, by passing its energized electrons on to molecules which will manufacture sugars. All plants, algae, and cyanobacteria which photosynthesize contain chlorophyll "a". A second kind of chlorophyll is chlorophyll "b", which occurs only in "green algae" and in the plants. A third form of chlorophyll which is common is (not surprisingly) called chlorophyll "c", and is found only in the photosynthetic members of the Chromista as well as the dinoflagellates. The differences between the chlorophylls of these major groups was one of the first clues that they were not as closely related as previously thought.

Carotenoids are usually red, orange, or yellow pigments, and include the familiar compound carotene, which gives carrots their color. These compounds are composed of two small six-carbon rings connected by a "chain" of carbon atoms. As a result, they do not dissolve in water, and must be attached to membranes within the cell. Carotenoids cannot transfer sunlight energy directly to the photosynthetic pathway, but must pass their absorbed energy to chlorophyll. For this reason, they are called accessory pigments. One very visible accessory pigment is fucoxanthin the brown pigment which colors kelps and other brown algae as well as the diatoms.