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27 December 2024 UNVEILING NATURE'S SUNSCREEN: THE POTENTIAL ROLE OF LIVERWORT SCALES IN UV PROTECTION
Riya Jain, Atra Shahryari, Ixchel S. González-Ramírez
Author Affiliations +
Abstract

UV radiation is important for plant life as it induces essential processes such as growth, germination, and secondary metabolite pathways. However, prolonged exposure to UV-A and UV-B radiation can have negative effects on organisms. For example, UV radiation can directly or indirectly damage DNA molecules and cause enzyme denaturation. Sessile organisms have multiple mechanisms, such as the evolution of various UV-absorbent pigments, that protect them from the negative effects of constant UV radiation exposure. While liverworts are a group of plants commonly associated with mesic and shaded environments, there are multiple species within this group that inhabit dry and sunlight-exposed environments. For example, the complex thalloid liverwort Calasterella californica (Hampe ex Austin) D.G.Long & T.X.Zheng lives in exposed outcrops across California, where it is exposed to a great amount of solar radiation. During the dry season, C. californica thalli dehydrate and become surrounded by their large purple ventral scales. The plants remain in this “dormant” state until the subsequent wet season, when the thalli rehydrate. Since the purple-red pigmentation in plants is often associated with light-damage protection, we hypothesize that the purple scales of xerophytic liverworts—like C. californica—protect the particularly vulnerable desiccated thalli from potential damage associated with UV radiation. As a first step to test this idea, we document the sunlight absorbance spectrum of the purple scales of C. californica and compare it to the more-common, translucent liverwort scales and to commercial sunscreen. We found that purple scales absorb UV radiation in a pattern that matches that of sunscreen, particularly for UV-A and UV-B radiation. Both sunscreen and purple liverwort scales absorb two to three times more UV-A radiation than translucent scales. These results are consistent with the idea that purple scales play a role in protecting dormant xeric liverworts against UV damage. Although we focused on the scales of C. californica, there are other liverwort species (like Reboulia hemisphaerica (L.) Raddi and Targionia hypophylla L.) that have similar purple scales and rolling mechanism; this points to a relatively widespread adaptive mechanism used by distantly related liverworts that could allow them to thrive in dry regions.

While sunlight is essential to plant life, the intense exposure to UV rays can be harmful (Hollósy 2002) as it can cause DNA damage, polymerization of proteins, and enzyme denaturation (Vanhaelewyn et al. 2020). Based on wavelength, UV rays are categorized into three groups: ultraviolet A (UV-A, 315–400 nm), ultraviolet B (UV-B, 280–315 nm), and ultraviolet C (UV-C, 200–280 nm). While UV-C rays are largely filtered out by Earth's atmosphere, UV-A and UV-B rays reach the surface of our planet and thus interact with life (Tedetti and Sempéré 2006). The excessive exposure to UV-A and UV-B radiation is more likely to have a negative effect on plants (Hollósy 2002).

Like other organisms, plants can perceive UV radiation through specific photoreceptors (Vanhaelewyn et al. 2020). These UV-photoreceptors have important roles in the induction of key processes such as growth (through phototropism), germination, and production and allocation of secondary metabolites (Vanhaelewyn et al. 2020). Nevertheless, high exposure to UV radiation can damage DNA through direct and indirect mechanisms that alter DNA structure. Direct damage occurs via the direct absorption of UV radiation, primarily high-energy UV-B wavelengths, by the DNA molecules. On the other hand, UV-A wavelengths are not preferentially absorbed by the DNA molecules, but they can generate highly reactive molecules that interact with DNA. These indirect DNA reactions are often associated with UV-A radiation because it is filtered the least by the atmosphere and accounts for most of the exposure experienced by organisms on the Earth's surface (Maverakis et al. 2010). UV-B rays can also interfere with photosynthesis, depriving plants of organic carbon (Teramura and Sullivan 1994), and can cause a reduction in the size, quality, and health of plants (Shi and Liu 2021).

While mobile organisms often avoid excessive exposure to potentially-damaging UV rays, sessile organisms like plants have evolved various mechanisms to withstand constant exposure to this radiation, from UV-reflectance of cuticle wax and cellular DNA repair mechanisms (Vanhaelewyn et al. 2020) to the presence of a variety of UV-protective pigments such as phenylpropanoids, flavonoids, and carotenoids that absorb UV radiation (Frohnmeyer and Staiger 2003; Jordan 2004; Valenta et al. 2020). While liverworts are commonly associated with wet and shady environments, there are many species of liverworts, like Calasterella californica (Hampe ex Austin) D.G.Long & T.X.Zheng, that occur in warmer and drier conditions. C. californica is a species of complex thalloid liverwort that is widespread in California. It is prevalent in rock and soil outcrops in areas that are exposed to continuous direct sunlight. To survive in these water-deprived environments, these liverworts—like other bryophytes—are capable of losing their internal water and pausing metabolic activity until they are rehydrated in a mechanism referred to as desiccation tolerance (Gaff 1977; Proctor et al. 2007; Stark 2017). During this metabolically-diminished, desiccated state, liverworts like C. californica are exposed to intense sunlight, including UV radiation. C. californica, like other dryland-specialized complex thalloid liverworts, displays a “rolling-up” strategy during the dry season.

The “roll-up” strategy of some complex thalloid liverworts has been described in taxonomic treatments, but it has not received much attention in terms of its ecological implications. Complex thalloid liverworts have two to four rows of ventral scales, which are flattened structures (varying in shape, size, and color) attached to the underside of the thallus, often in contact with the substrate. In mesic species, like Marchantia polymorpha L. or Lunularia cruciata (L.) Dumort. ex Lindb., ventral scales are inconspicuous and translucent, and they are thought to enhance plant attachment to the substrate. However, in species with the “roll-up” strategy, like C. californica and Targionia hypophylla L., the ventral scales are purple-red (Fig. 1c) and comparatively larger. When the thallus of these species is desiccated and shrinks, the ventral scales fold over the thallus (i.e., “roll up”), covering it (Fig. 1a and 1b). So, when we observe a desiccated plant of C. californica or T. hypophylla from a dorsal perspective, we are observing the ventral scales surrounding the dormant thallus. Although there is no formal study on the potential effects of this rolling strategy, it seems plausible that the scales of liverworts that roll up help protect the desiccated thallus from physical damage. Furthermore, to our knowledge, all the complex thalloid liverworts that display the roll-up strategy also have highly colored scales, from red to dark purple. Across different groups of plants, red pigments have been associated with protection from stressors like drought, herbivory, and light damage (Green et al. 2005; Hooijmaijers and Gould 2007; Martínez-Abaigar and Núñez-Olivera 2022). For example, liverworts such as Isotachis lyallii Mitt. are polymorphic for leaf color and present a rose-red color when growing in unshaded areas (Allison and Child 1975; Schuster and Engel 1997). Here, we hypothesize that the purple scales of xerophytic liverworts with the roll-up strategy protect them against UV radiation damage while they are desiccated. As a first step to test this idea, we test the potential UV protection effect of purple scales by analyzing its UV absorbance spectrum and comparing it to translucent liverwort scales and commercial sunscreen. If purple scales have the potential to protect against excessive UV radiation, we expect that purple scales will have high absorbance of wavelengths corresponding to the UV-A and UV-B spectra.

Fig. 1.

a. Thalli of C. californica with arrows pointing to the dark purple scales present on the ventral surface. b. Desiccated thalli of C. californica—the underside (scales) is curled up and inwards. c. Isolated ventral scale of C. californica under the microscope, which naturally appears red/maroon. d. Thalli of L. cruciata, which is glossy green-colored and possesses characteristic crescent-shaped cups (Tully 2020). e. Dried thalli of L. cruciata showing browning from the edges (Freyman 2021). f. Ventral view of L. cruciata showing translucent scales arranged in parallel rows (Jensen 2024).

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Methods

Collection of Biological Materials

We used two liverworts with contrasting scales for the experiment: Calasterella californica and L. cruciata. C. californica has purple ventral scales and displays the rolling-up strategy when the thallus is dry, while L. cruciata has white/translucent scales that always remain on the underside of the thalli and do not perform the rolling strategy when dry. We used fresh samples of C. californica (collected at Briones State Park under the permit 21-1083 of the East Bay Regional Park District) and L. cruciata (collected in the UC Berkeley Botanical Garden greenhouses). The liverwort material was collected in hard plastic containers, transported to the UC Berkeley campus, and maintained at room temperature until we performed the experiments. We manually cleaned the liverwort thalli using ionized water and tweezers to remove the soil particles and rhizoids and manually removed the scales under a dissecting microscope.

Scales and Sunscreen Spectra

Ventral scales samples. Due to the negligible weight of liverwort scales, we standardized the quantity of scales by area. To do this, we superposed scales of the two species of liverworts over graph paper until each covered 50 mm2, i.e., two 5 × 5 mm squares. To avoid the potentially confounding effect of scale shape or density in the solvent, we pulverized the scales in two grinding cycles (first, 5000 rpm × 31 × 20 sec and next, 6000 rpm × 3 × 30 sec) using a tissue pulverizer (PRECELLYS® Evolution Touch). We placed 400 microliters of distilled water into each tube using a micropipette and vortexed the samples to suspend the scale tissue. We replicated this procedure to create a replication of both L. cruciata and C. californica.

Sunscreen samples. We decided to compare scales absorbance to the absorbance of sunscreen. For this, we used Neutrogena Sport Face oil-free sunscreen SPF 70+. We weighed 0.50 grams of the sunscreen using a weighing boat and a scale. Then, we added 0.50 grams of distilled water on the weighing boat with the sunscreen and mixed it using a stirring rod to create a 50:50 solution of sunscreen: water. Using the same method, we created two more dilutions of sunscreen. We used 0.50 g of sunscreen and 1.03 g of distilled water to create 32.68% solution of sunscreen and we used 0.11 g of sunscreen and 0.93 g of distilled water to make 10.58% solution of sunscreen. Finally, we prepared a control sample with only distilled water so we could account for how water affected the absorbance of our samples.

Spectroscopy. We vortexed each sample immediately before placing it into the well of our spectrophotometer to ensure a homogenous mixture and accurate results from the spectrophotometer. We placed 300 microliters of each sample in a well in the SpectraMax M3 spectrophotometer and Softmax Pro 7. The spectrophotometer was set up to measure the absorbance of wavelength spanning from 200 to 800 nm, every 5 nm. These wavelengths span the UV–visible spectrum of light.

Data processing. The data and figures were made in R version 4.1.1 (R Core Team, R Foundation for Statistical Computing, Vienna, Austria). We used the packages ggplot2 and gridExtra. We created two absorbance curves, the first using the raw absorbance data and the second by subtracting the absorbance of water to isolate the absorbance of the scales. We have made available the code for producing the figures (Appendix S2). Figure 1 was composed in Canva.

Imaging of Ventral Scales

To visually contrast the absorbance of UV light by intact scales, we photographed the ventral side of scales and rolled-up thalli of C. californica. The imaging was done using a Zeiss LSM710 microscope in the Biological Imaging Facility at UC Berkeley under UV lighting.

Results and Discussion

In Figure 2, we show the absorbance spectra for translucent scales, purple scales, and three different concentrations of sunscreen. The absorption spectrum of sunscreen did not vary with its concentration. When focusing on the UV radiation spectrum (highlighted in orange colors in Fig. 2), we found that translucent (L1 and L2) and purple (C1 and C2) scales have an overlapping low absorption pattern in the UV-C wavelength range (100–280 nm). However, starting at ∼280 nm, colored scales absorb more UV-B radiation than translucent scales. Furthermore, for UV-A wavelengths, which make up 95% of all radiation reaching Earth's surface, the absorbance spectrum of the purple scales of C. californica is identical to the absorption spectrum of sunscreen. In contrast, the absorption of the translucent scales of L. cruciata decreases considerably to almost half of the absorbance of purple scales and sunscreen in the UV-A range.

Fig. 2.

UV-vis absorbance spectrum of the scales of C. californica, L. cruciata, and sunscreen in water solutions. The left panel uses the raw data, while the right panel shows the absorbance of the solution minus the water absorbance. Curves are colored by the type of material (C1 and C2: C. californica, L1 and L2: L. cruciata, S1, S2 and S3: sunscreen at 50%, 32.68% and 10.58% solutions, respectively). The dashed and solid lines convey repetition. In panel (a), the green line corresponds to the absorption spectrum of water, the control. The graphs have three colored regions representing the UV-A (315–400 nm), UV-B (280–315 nm), and UV-C (100–80 nm) wavelengths.

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Additionally, we conducted a set of observations on intact scales. First, we observe that the ventral scales are darker under UV light than the rest of the tissue, indicating a higher absorbance of UV wavelengths by scales (Fig. 3b). Interestingly, this observation is also true for desiccated plants with rolled thalli (Fig. 3c and d) where we observe that the apical sections of the thalli that are enclosed by ventral scales (arrows) appear very dark under UV illumination, consistent with ventral scales absorbing UV radiation when the thalli are rolled-up. Furthermore, the reflectance of intact scales (Fig. S1 in Appendix S1) and the lack of UV excitement response when photosynthetic tissue is covered by ventral scales (Fig. S2 in Appendix S1) are results consistent with the ventral scales of C. californica containing pigments that absorb UV radiation.

Fig. 3.

Intact scales of C. californica observed under the microscope. a. Ventral view of one thallus of C. californica showing the two rows of purple ventral scales. b. Ventral view of the thallus of C. californica under UV light illumination, darker areas correspond to high UV absorbance. Image within the circle is a close-up. c. Rolled-up thalli (dorsal view) showing the ventral scales (arrows) under normal light. d. Rolled-up thalli under UV light, darker areas correspond to high UV absorbance. Bar = 1 mm.

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The purple scales of C. californica have a UV-A and UV-B absorption that is comparable to the absorption of sunscreen (Fig. 2), supporting our hypothesis that the purple scales of C. californica play a role in UV protection during the rolled-up stage of this liverwort. Although we focused on the scales of C. californica, there are other liverwort species, like Reboulia hemisphaerica (L.) Raddi and Targionia hypophylla, that have similar purple scales and rolling mechanism (Whittemore 1982). This points to a relatively widespread and potentially adaptive mechanism used by distantly-related complex thalloid liverworts (documented in at least three different families: Aytoniaceae, Ricciaceae, Targionaceae) that allows them to thrive in dry regions (Scott 1982; Longton 1988).

Our findings align with a study that identified a newly described anthocyanidin pigment, riccionidin A, in the cell walls of the Antarctic liverwort Cephaloziella varians (Gottsche) Steph. (Waterman et al. 2018). This purple pigment, potentially analogous to the one underpinning the coloration of C. californica scales, was demonstrated to have a role in UV protection. The study suggests that cell wall pigments like riccionidin A may be more prevalent and essential for UV tolerance in bryophytes with purple cell wall pigmentation than previously thought. In fact, anthocyanin-like pigments have been documented to play an important role in UV absorbance across bryophytes (Martínez-Abaigar and Núñez-Olivera 2022). But unlike the case of C. varians, where riccionidin was found in photosynthetic leaves of the leafy liverwort, the purple pigment in liverworts with the rolling-up strategy seems to be particularly concentrated in the ventral scales. Exploring the potential fitness associated with the purple scales in the context of the roll-up mechanism of liverworts could provide further insights into their adaptive value. Furthermore, we speculate that a compound similar to riccionidin is responsible for the purple coloration of the scales found in C. californica and other rolling-up complex thalloid liverworts; however, additional studies are needed to identify the pigments in these scales. To our knowledge, there are no comprehensive studies that document the rolling-up strategy across species of complex thalloid liverworts. Such studies would allow us to investigate the protective role that ventral scales might have had in the evolutionary history of complex thalloid liverworts.

Acknowledgments

We would like to thank José Vásquez-Medina for allowing us to use his spectrophotometer. Thank you to Emily Lam and Kaitlin Allen for training and facilitation on using this equipment. Thank you to Sonia Nosratinia for technical support in obtaining the lab materials necessary for this experiment. Thank you to Susan Malisch for facilitating the collection of L. cruciata in the UC Berkeley Botanical Garden. We thank the RCNR Biological Imaging Facility at UC Berkeley, in particular manager Denise Schichnes, for helping with the UV absorbance pictures. Thank you to Ben Karin for helping with the reflectance measurements. Thank you to Jesús Martínez-Gómez for his feedback. Thank you to three anonymous reviewers for their constructive feedback. We thank Will Freyman and Tully for their open access liverwort observations through iNaturalist.

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Riya Jain, Atra Shahryari, and Ixchel S. González-Ramírez "UNVEILING NATURE'S SUNSCREEN: THE POTENTIAL ROLE OF LIVERWORT SCALES IN UV PROTECTION," Madroño 71(4), 145-150, (27 December 2024). https://doi.org/10.3120/0024-9637-71.4.145
Published: 27 December 2024
KEYWORDS
absorbance
Calasterella californica
liverworts
Lunularia cruciata
Marchantiophyta
UV protection
ventral scales
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