Hattoria 10: 53–60. 2019

Desiccation rate affects chlorophyll and carotenoid content and the recovery of the aquatic antipyretica ()

Ricardo CRUZ DE CARVALHO1, 2, Cristina BRANQUINHO2 & Jorge MARQUES DA SILVA3

1 MARE, Marine and Environmental Sciences Centre, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, Edifício C2, Piso 5, 1749–016 Lisboa, Portugal 2 cE3c, Centre for Ecology, Evolution and Environmental Changes, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, Edifício C2, Piso 5, 1749–016 Lisboa, Portugal 3 BioISI, Biosystems and Integrative Sciences Institute and Departamento de Biologia Vegetal, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, Edifício C2, Piso 4, 1749–016 Lisboa, Portugal Author for correspondence: Ricardo CRUZ DE CARVALHO, [email protected]

Abstract In the Mediterranean climate, during the hot and dry summer, small streams dry out exposing the aquatic bryophyte L. ex Hedw. to desiccation, losing all cellular water content. Previous works showed that fast dehydration (less than two hours) is extremely severe, unabling recovery upon rehydration. On the other hand, slow dehydration allows the recovery of the bryophyte through induction of desiccation tolerance mechanisms. To explore how the photosynthetic apparatus responds to contrasting dehydration rates, we measured the chlorophyll a fluorescence parameter Fv/Fm (maximum potential quantum efficiency of Photosystem II) as a proxy to photosynthetic fitness and analysed the content of the pigments (chlorophylls and carotenoids). In slowly dehydrated Fontinalis antipyretica, the content of the pigments remained constant down to RWC of 40–50%, while the loss was striking in fast dehydrated samples as early as RWC of 70–80% RWC, showing different threshold values for different desiccation rates.

Introduction Bryophytes were among the first to colonize the Earth surface. Throughout the evolutionary process, these organisms developed mechanisms that allow them to tolerate the harsh conditions outside of a water environment from which life originated (Alpert 2006). Upon a drying environment that leads to the loss of water content, they evolved into forms with desiccation tolerance (DT) mechanisms, which allowed them to survive in the desiccated state (Oliver et al. 2005). Photosynthesis is one of the most important processes to protect from desiccation, during

53 which it decreases to a full stop below a certain water content threshold, a fact already studied in many bryophytes (Dilks & Proctor 1979; Proctor 1982; Nakatsubo et al. 1989; Tuba et al. 1996; Zotz et al. 1997; Schipperges & Rydin 1998; Cruz de Carvalho et al. 2011). Therefore, chlorophyll is one of the main targets to protect under desiccation stress in bryophytes (Martin & Warner 1984). Although carotenoids are important mechanisms for energy dissipation and photoprotective mechanisms in plants diverting energy away from the reaction centres (Demmig-Adams 1990; Adams & Demmig-Adams 1995; Ruban & Horton 1995), no relation has been observed in relation to the DT level in bryophytes (Seel et al. 1992). Recent works in Fontinalis antipyretica showed that even an aquatic bryophyte can be induced into desiccation tolerance and recover upon rehydration if slowly dried (Cruz de Carvalho et al. 2011, 2012, 2014, 2015, 2017), and, therefore, we addressed how this recovery translates in terms of the variation of the photosynthetic pigments, hypothesizing that slowly dried shoots maintain its higher pigment content as compared to fast dried ones.

Materials and Methods material and culture conditions The aquatic bryophyte Fontinalis antipyretica L. ex Hedw. was collected in a small stream at the Natural Park of Serra de S. Mamede, central Portugal, with very low human interference, and transported to the laboratory under cooling conditions (about 5ºC) where they were rinsed in distilled water. Afterwards, the bryophyte was transferred to the culture medium (Traubenberg & Ah-Peng 2004) and grown under controlled conditions (17ºC day/13ºC night, 20–30 µmol m－2 s－1 PAR and photoperiod of 16 hours). Ten bryophyte shoot tips with 1 cm each (five replicates) were collected for each treatment. Relative water content (RWC) was calculated according to Cruz de Carvalho et al. (2011).

Desiccation induction and recovery Bundles of ten shoots with 1 cm length were desiccated by placing samples in small containers over saturated salt solutions of Ca(NO3)2.4H2O (50% relative humidity [RH]; [－100 MPa]) and K2SO4 (95% RH; [－6 MPa]), referred for here forward as fast and slow drying, respectively. The containers were kept under controlled temperature (21ºC) for several periods of time (30 minutes up to 2 hours in 50% RH and 30 minutes up to 7 days in 95% RH) at low photosynthetic active radiation (PAR) (2–5 µmol m－2 s－1). Recovery was made through immersion in individual containers with culture medium under similar growth conditions for three days.

Chlorophyll a fluorescence analysis Chlorophyll a fluorescence was measured with a PAM 101 Chlorophyll Fluorometer (Heinz Walz GmbH, Effeltrich, Germany) connected to a PAM Data Acquisition System PDA 100 (Heinz Walz GmbH, Effeltrich, Germany) and controlled by the software WinControl v2.08 (2003) (Heinz Walz GmbH, Effeltrich, Germany). Right before the end of the dark period (10 minutes), a saturating light pulse (approximately 4000 µmol m－2 s－1) (KL 2500 LCD, Schott AG, Mainz, Germany) was applied over the measuring light to determine the

54 maximum quantum efficiency of photosystem II (Fv/Fm). Measurements were made before dehydration, and 30 minutes and three days after rehydration (recovery time).

Chlorophylls and carotenoids quantification In an independent assay, samples were desiccated and recovered as previously described. Pigments were extracted from bryophyte shoot tips in 10 ml methanol (100%) for 72 hours. The extracts were read against a control (methanol 100%) at wavelengths 470, 652 and 665 nm in a spectrophotometer (UV500 UV-Visible Spectro, Unicam, Waltham, USA).

Equations from Lichtenthaler (1987) were used for quantification of chlorophylls (Ca, Cb, Ca＋b) and carotenoids (Cx＋c). The ratios chlorophyll a and b (Chl a/b) and chlorophylls and carotenoids (Chls/Carot) were also calculated. Measurements were made in one set of samples before dehydration, another set of samples at the end of dehydration time and another set of samples after three days of recovery.

Statistical analysis Linear regression was applied to the data sample and significant differences were determined using the statistical software GraphPad Prism 6.07 for Windows (2015) (GraphPad Software, San Diego California USA).

Results Chlorophyll a fluorescence

The recovery of the parameter Fv/Fm manifested differently in samples submitted to fast (Fig. 1A) and slow dehydration (Fig. 1B) both after 30 minutes and 3 days, in the same range of RWC achieved at the end of the stress (down to 40% RWC). Slowly dehydrated samples practically remained constant in this parameter, presenting only a slight decrease (ca. 0.700) (Fig. 1B) when compared with fully hydrated control values (0.738±0.009; dotted box).

Figure 1. Maximum efficiency of photosystem II (Fv/Fm) variation with final relative water content (RWC) after (A) fast and (B) slow dehydrated samples of Fontinalis antipyretica with RWC higher than 40%, under non-stress conditions (full circle), after 30 minutes rehydration (open squares) and 3 days rehydration (full triangles). Shaded box indicates the variation of non-stressed samples.

55 On the other hand, Fv/Fm in fast dehydrated samples, present a high decrease at the samples that reached 40% RWC after 30 minutes recovery (ca. 0.350) but presenting a similar recovery as slowly dehydrated samples after 3 days (ca. 0.700) (Fig. 1A).

Chlorophylls and carotenoids content Regarding chlorophylls and carotenoids (Fig. 2), contents remained at control levels during dehydration (10.1±0.8 mg g－1 DW and 2.2±0.1 mg g－1 DW, respectively), both in fast and slow dried samples. However, upon rehydration, and reaching similar RWC, fast dehydration led to a higher loss in chlorophyll a and b, and thus in total chlorophyll, and carotenoids content (Fig. 2A, C, E, G) than in slowly dehydrated samples (Fig. 2B, D, F, H). Nevertheless, the Chl a/b and Chls/Carot ratios remained constant throughout the dehydration process (Fig. 3). In fast dehydration, Chl a/b increases after recovery (Fig. 3A), while the Chls/Carot decreases particularly after RWC ca. 80% (Fig. 3C). In slow dehydration, both ratios slightly decrease being more evident at RWC ca. 50% (Fig. 3B, D).

Discussion Dehydration rate is a very important factor which allows the aquatic bryophyte Fontinalis antipyretica time to prepare and induce desiccation tolerance mechanisms, as previous works showed (Cruz de Carvalho et al. 2011, 2012, 2014, 2015, 2017). Membranes are affected during dehydration, but fast dehydration prevents a correct folding of the membranes and induction of synthesis of proteins to help in this process. Therefore, upon rehydration, membranes are highly disrupted if samples are fast dried when compared with slow dried ones, leading to solute leakage (Cruz de Carvalho et al. 2017), including chlorophylls and carotenoids. However, in bryophytes with daily cycles of dehydration/rehydration, such as Syntrichia ruralis, chlorophyll a and b do not undergo any changes, probably due to the presence of neoxanthin, a carotenoid which may bind the LHCP (light-harvesting chlorophyll protein) to the Photosystem II chlorophyll core, stabilizing it (Tuba 1985), being an evolutionary advantage to survive in such harsh environments. In terms of recovery, it was already shown that the threshold was different for both fast (around 70% RWC) and slow (around 40% RWC) dried samples, both through chlorophyll a fluorescence (Cruz de Carvalho et al. 2011) and solute leakage (Cruz de Carvalho et al. 2017). In the present work, we demonstrate that the same threshold applies chlorophyll and carotenoid content loss. Even when fast samples recover pre-dehydration control values in terms of chlorophyll fluorescence, the net photosynthesis never does (Cruz de Carvalho et al.

2011). This observation is due to the fact that some cells survive, thus presenting Fv/Fm recovery, but most die due to high reactive oxygen species production that reacts with almost every molecule in the cell (Mayaba et al. 2002; Cruz de Carvalho et al. 2012). This work indicates that photosynthetic pigments are one of the groups of molecules that are targeted, both due to ROS and membrane disruption. The fact that carotenoids are also lost during fast but not slow dehydration also suggests protection against such hazards, a process that can be similar to the observed by other authors in the protection of chlorophyll against light during desiccation by zeaxanthin, another carotenoid, which binds to the chlorophyll-containing

56 Figure 2. Chlorophyll a (A, B), chlorophyll b (C, D), total chlorophyll (E, F) and carotenoids (G, H) content variation with final relative water content (RWC) after fast (left column) and slow (right column) dehydrated samples of Fontinalis antipyretica with RWC higher than 40%, under non-stress conditions (full circle), before rehydration (open squares) and after 3 days recovery (full squares). Shaded box indicates the variation of non-stressed samples.

57 Figure 3. Chlorophyll a/b ratio (A, B) and Chlorophylls/Carotenoids ratio (C, D) variation with final relative water content (RWC) after fast (left column) and slow (right column) dehydrated samples of Fontinalis antipyretica with RWC higher than 40%, under non-stress conditions (full circle), before rehydration (open squares) and after 3 days recovery (full squares). Shaded box indicates the variation of non-stressed samples.

thylakoid protein (Deltoro et al. 1998; Heber et al. 2001). Fontinalis antipyretica has a very high chlorophyll content when compared with most bryophytes (Marschall & Proctor 2004), probably to its aquatic environment and usually forest shaded margins, decreasing the amount of light that reaches chloroplasts. Therefore, apart from the molecular protections that are induced during slow drying (Wang et al. 2009; Cruz de Carvalho et al. 2014), a correct folding of the membranes in which the photosynthetic pigments are integrated and thus maintain them intact also allows for a fast and full recovery under these conditions, as seen previously (Cruz de Carvalho et al. 2011). Slow dehydration allows time for desiccation tolerance induction, establishing protective mechanisms for photosynthetic pigments, that will ensure a fast recovery upon rehydration, following rehydration. This slow dehydration process is probably achieved in nature through the complexity of the colony structure that slows water loss, preparing individuals for the upcoming desiccation stress. A similar principle can be assumed for other bryophytes that are assumed to be fully desiccation tolerant.

58 Acknowledgements This work has been supported by Fundação para a Ciência e Tecnologia (FCT) grants SFRH/BD/31424/2006 and PTDC/ATP-ARP/5826/2014, Lisbon, Portugal.

References Adams W. W. III & Demmig-Adams B. 1995. The xanthophyll cycle and sustained thermal energy dissipation activity in Vinca minor and Euonymus kiautschovicus in winter. Plant, Cell and Environment 18: 117–127. Alpert P. 2006. Constraints of tolerance: why are desiccation-tolerant organisms so small or rare? Journal of Experimental Biology 209: 1575–1584. Cruz de Carvalho R., Branquinho C. & Marques da Silva J. 2011. Physiological consequences of desiccation in the aquatic bryophyte Fontinalis antipyretica. Planta 234: 195–205. Cruz de Carvalho R., Catalá M., Marques da Silva J., Branquinho C. & Barreno E. 2012. The impact of dehydration rate on the production and cellular location of reactive oxygen species in an aquatic moss. Annals of Botany 110: 1007–1016. Cruz de Carvalho R., Silva A. B., Soares R., Almeida A., Coelho A. V., Marques da Silva J. & Branquinho C. 2014. Differential proteomics of dehydration and rehydration in bryophytes: evidence towards a common desiccation tolerance mechanism. Plant, Cell & Environment 37: 1499–1515. Cruz de Carvalho R., Silva A. B., Branquinho C. & Marques da Silva J. 2015. Influence of dehydration rate on cell sucrose and water relations parameters in an inducible desiccation tolerant aquatic bryophyte. Environmental and Experimental Botany 120: 18–22. Cruz de Carvalho R., Catalá M., Branquinho C., Marques da Silva J. & Barreno E. 2017. Dehydration rate determines the degree of membrane damage and desiccation tolerance in bryophytes. Physiologia Plantarum 159: 277–289. Deltoro V. I., Catalaynd A., Gimeno C., Abadía A. & Barreno E. 1998. Changes in chlorophyll a

fluorescence, photosynthetic CO2 assimilation and xanthophyll cycle interconversions during dehydration in desiccation-tolerant and intolerant liverworts. Planta 207: 224–228. Dilks T. J. K. & Proctor M. C. F. 1979. Photosynthesis, respiration and water content in bryophytes. New Phytologist 82: 97–114. Demmig-Adams B. 1990. Carotenoids and photoprotection in plants. A role for the xanthophyll zeaxanthin. Biochimica et Biophysica Acta 1020: 1–24. Heber U., Bukhov N. G., Shuvalov V. A., Kobayashi Y. & Lange O. L. 2001. Protection of the photosynthetic apparatus against damage by excessive illumination in homoiohydric leaves and poikilohydric and lichens. Journal of Experimental Botany 52: 1999–2006. Lichtenthaler H. K. 1987. Chlorophylls and carotenoids: Pigments of photosynthetic biomembranes. Methods in Enzymology 148: 350–382. Marschall M. & Proctor M. C. F. 2004. Are bryophytes shade plants? Photosynthetic light responses and proportions of chlorophyll a, chlorophyll b and total carotenoids. Annals of Botany 94: 593–603. Martin C. E. & Warner D. A. 1984. The effects of desiccation on concentrations and a/b ratios of chlorophyll in Leucobryum glaucum and Thuidium delicatulum. New Phytologist 96: 545–550. Mayaba N., Minibayeva F. & Beckett R. P. 2002. An oxidative burst of hydrogen peroxide during

59 rehydration following desiccation in the moss Atrichum androgynum. New Phytologist 155: 275–283. Nakatsubo T., Takamine Y. & Ino Y. 1989. Response patterns of net photosynthesis to moisture of mosses in xeric habitats. Botanical Magazine, Tokyo 102: 63–73. Oliver M. J., Velten J. & Mishler B. D. 2005. Desiccation tolerance in bryophytes: A reflection of the primitive strategy for plant survival in dehydrating habitats? Integrative and Comparative Biology 45: 788–799. Proctor M. C. F. 1982. Physiological ecology: water relations, light and temperature responses, carbon balance. Pages 333–381. In: Smith A. J. E. (ed.), Bryophyte Ecology. Chapman & Hall, London/ NewYork. Ruban A. V. & Horton P. 1995. An investigation of the sustained component of nonphotochemical quenching of chlorophyll fluorescence in isolated chloroplasts and leaves of spinach. Plant Physiology 108: 721–726. Schipperges B. & Rydin H. 1998. Response of photosynthesis of Sphagnum species from contrasting microhabitats to tissue water content and repeated desiccation. New Phytologist 140: 677–684. Seel W. E., Baker N. R. & Lee J. A. 1992. Analysis of the decrease in photosynthesis on desiccation of mosses from xeric and hydric environments. Physiolia Plantarum 86: 451–458. Traubenberg R. C. & Ah-Peng C. 2004. A procedure to purify and culture a clonal strain of the aquatic moss Fontinalis antipyretica for use as a bioindicator of heavy metals. Archives of Environmental Contamination and Toxicology 46: 289–295. Tuba Z. 1985. Photosynthetic pigment responses in Tortula ruralis during daily desiccation. Abstracta Botanica 9: 231–239. Tuba Z., Csintalan Z. & Proctor M. C. F. 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. New Phytologist 133: 353–361. Wang X. Q., Yang P. F., Liu Z., Liu W. Z., Hu Y., Chen H., Kuang T. Y., Pei Z. M., Shen S. H. & He Y. K. 2009. Exploring the mechanism of Physcomitrella patens desiccation tolerance through a proteomic strategy. Plant Physiology 149: 1739–1750.

Zotz G., Büdel B., Meyer A., Zellner H. & Lange O. L. 1997. Water relations and CO2 exchange of tropical bryophytes in a lower montane rain forest in Panama. Botanical Acta 110: 9–17. manuscript received June 24, 2019; accepted July 18, 2019

60