Desiccation stress in intertidal seaweeds: Effects on morphology, antioxidant responses and photosynthetic performance

Accepted Manuscript Title: Desiccation stress in intertidal seaweeds: effects on morphology, antioxidant responses and photosynthetic performance Author: Mar´ıa R. Flores-Molina Daniela Thomas Carlos Lovazzano Alejandra N´un˜ ez Javier Zapata Manoj Kumar Juan A. Correa Loretto Contreras-Porcia PII: DOI: Reference:

S0304-3770(13)00167-8 http://dx.doi.org/doi:10.1016/j.aquabot.2013.11.004 AQBOT 2625

To appear in:

Aquatic Botany

Received date: Revised date: Accepted date:

6-6-2013 5-11-2013 17-11-2013

Please cite this article as: Flores-Molina, M.R., Thomas, D., Lovazzano, C., N´un˜ ez, A., Zapata, J., Kumar, M., Correa, J.A., Contreras-Porcia, L.,Desiccation stress in intertidal seaweeds: effects on morphology, antioxidant responses and photosynthetic performance, Aquatic Botany (2013), http://dx.doi.org/10.1016/j.aquabot.2013.11.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Desiccation stress in intertidal seaweeds: effects on morphology,

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antioxidant responses and photosynthetic performance

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Running title: Air exposure in seaweeds

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María R. Flores-Molinaa,b, Daniela Thomasc, Carlos Lovazzanoc, Alejandra Núñezc, Javier

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Zapatac, Manoj Kumard, Juan A. Correaa, Loretto Contreras-Porciac, *

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(CASEB), Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Postal

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code 6513677, Santiago, Chile

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Departamento de Ecología, Center for Advanced Studies in Ecology and Biodiversity

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Chile, Casilla 567, Valdivia, Chile

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Universidad Andres Bello, República 470, Santiago, Chile

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PO Box 6, Bet Dagan 50250, Israel

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*Corresponding author: Loretto Contreras-Porcia

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Universidad Andres Bello, Chile, Santiago, República 470.

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Tel: +56 227703371.

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E-mail: [email protected]

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Instituto de Ciencias Marinas y Limnológicas, Facultad de Ciencias, Universidad Austral de

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Departamento de Ecología y Biodiversidad, Facultad de Ecología y Recursos Naturales,

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Institute of Plant Sciences, Agricultural Research Organization (ARO), The Volcani Center,

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Highlights

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•Air exposure induces a desiccation stress condition in intertidal seaweed species• High

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activities of antioxidant enzymes and low ROS production were the responses in higher

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intertidal species• Photosynthesis capacity was recovered during rehydration only in higher

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intertidal species• Lower intertidal distribution of certain algal species is explained by their

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insufficient mechanisms of stress tolerance

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Abstract

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Seaweeds are differentially distributed between the upper and lower limits of the intertidal

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zone of rocky coasts around the world. Daily changes in tide height cause water loss,

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triggering desiccation stress as a consequence. How this stress affected some of the

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morphological characteristics and physiological responses in representative intertidal

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seaweeds with contrasting vertical distributions was explored in the present work. The

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selected species were Mazzaella laminarioides (upper-middle distribution), Scytosiphon

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lomentaria and Ulva compressa (middle distribution), and Lessonia spicata and Gelidium rex

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(lower distribution). To assess tolerance response to desiccation, cellular and morphological

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alterations, ROS production, enzymatic activity of catalase (CAT) and ascorbate peroxidase

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(AP) and photosynthesis performance were measured after a simulated emersion stress

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experiment. Results show different tolerance responses to desiccation, with seaweeds having

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higher intertidal distributions displaying greater antioxidant enzymatic activity, suggesting a

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higher capacity to buffer reactive oxygen species (ROS) excess induced during desiccation.

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Contrarily, this capacity seems to be absent or deficient in low intertidal species (i.e. Lessonia

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spicata and Gelidium rex), where AP and CAT activities were below detection limits, ROS

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were higher than normal and caused an over-oxidation of bio-molecules and photosynthetic

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disarray, explaining from a functional stand point their low distribution in the intertidal zone.

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Keywords: Desiccation stress, Zonation pattern, Seaweeds

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Abbreviations: AP, ascorbate peroxidase; CAT, catalase; DHAR, dehydroascorbate

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reductase; GR, glutathione reductase; PRX, peroxiredoxin; ROS, reactive oxygen species;

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RWC, relative water content

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1. Introduction

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Marine macroalgae are pre-eminent producers occupying a basal position in aquatic food

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webs (Lobban and Harrison, 1994). Rocky intertidal zones are dynamic and stressful habitats

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for seaweeds as a result of the rapid changes in physical conditions associated with tides, in

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addition to the changes brought by seasonal variations (Kumar and Reddy, 2012). Seaweeds

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are distributed in bands parallel along the rocky intertidal zone, where distribution and

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relative abundance along the lower limits is mainly controlled by biotic factors such as

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predation and intra and inter-specific competition, while the upper limit distribution is mostly

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determined by abiotic factors such as UV radiation, light, salinity, temperature, nutrient

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availability and air exposure (Zaneveld, 1969). Davison and Pearson (1996) and Ji and

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Tanaka (2002), while studying the photosynthetic and respiratory performance of various

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seaweeds collected from different intertidal regimes following 2 h of desiccation, suggested

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that the ability to withstand desiccation stress (fast recovery during rehydration) and not that

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to avoid desiccation (water retaining ability), is the key factor determining their vertical

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distribution.

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Exposure of seaweeds during low tidal emersions demands the alga to prepare not

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only for desiccation but also for subsequent rehydration and eventual cellular damage (Burritt

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et al., 2002). Extended desiccation may cause a decline in photosynthesis rate while

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interrupting the electron flow between photosystem (PS) I and II (Heber et al., 2010; Gao et

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al., 2011). Additionally, fluctuating and dynamic environmental conditions in the intertidal

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zone trigger the accumulation of reactive oxygen species (ROS) (Collén and Davison, 1999a,

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1999b; Contreras et al., 2005, 2009; Kumar et al., 2010, 2011) that, if not buffered, result in

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an oxidative stress condition. Further, cellular dehydration resulting from desiccation

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increases electrolyte concentration in the cell and brings changes to membrane structures,

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including thylakoids (Kim and Garbary, 2007). Acclimation to an adverse environment 4 Page 4 of 38

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involves complex enzymatic and non-enzymatic antioxidant mechanisms functioning in an

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orchestrated manner to mitigate changes in cellular osmolarity, ion disequilibrium and ROS

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excess (Sampath-Wiley et al., 2008; Contreras et al., 2009; Kumar et al., 2011). The effects of desiccation on the vertical distribution of macroalgae are poorly

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understood. Only few reports highlight the induction of ROS and activation of the antioxidant

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system in seaweeds in response to desiccation (Collén and Davison, 1999a, 1999b; Burritt et

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al., 2002; Ross and Alstyne, 2007; Contreras-Porcia et al., 2011; Kumar et al., 2011;

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Contreras-Porcia et al., 2013; López-Cristoffanini et al., 2013, unpubl. results). Independent

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studies investigating algal responses to fluctuating environmental conditions show alterations

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in photosynthetic performance (Fv/Fm), cellular morphology and ontogenetic development

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(e.g. Abe et al., 2001; Varela et al., 2006; Kumar et al., 2011; Contreras-Porcia et al., 2012;

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Gao and Wang, 2012). However, integrative studies addressing cellular responses to

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desiccation are lacking.

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Along the Chilean coast, the upper-most part of the intertidal zone is characterized by

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a seasonal dominance of Porphyra and Pyropia species such as Pyropia columbina

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(Montagne) WA Nelson (formerly Porphyra columbina Montagne) (Bangiales, Rhodophyta).

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The upper-middle zone is dominated, among others, by Mazzaella laminarioides

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(Gigartinales, Rhodophyta) and the middle zone by Ulva compressa (Ulvales, Chlorophyta),

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Scytosiphon lomentaria (Ectocarpales, Heterokontophyta), Ceramium spp. and Polysiphonia

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spp. (Ceramiales, Rhodophyta). The lower intertidal zone is dominated by Codium spp.

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(Bryopsidales, Chlorophyta), Lessonia spp. (Laminariales, Heterokontophyta), Gelidium spp.

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(Gelidiales, Rhodophyta) and also several species of crustose calcareous red algae (Hoffmann

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and Santelices, 1997). Experimental studies on the littoral zone have been important in

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unraveling the factors regulating the vertical distribution of these species (Alveal, 1970;

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Moreno and Jaramillo, 1983; Buschmann, 1990). Recently, Contreras-Porcia et al. (2011)

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demonstrated that P. columbina exposed to natural desiccation during low tide loose 90 to

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95% water and displays an excess of ROS, elevated activities of antioxidant enzymes and

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high concentration of photosynthetic pigments. Furthermore, desiccation results in over

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expression of tolerance genes, as those coding for ABC (ATP binding cassette) transporter

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proteins, antioxidant enzymes, heat shock proteins, cytochrome P450, cell wall proteins and

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specific transcriptional factors, among others (López, 2012; Contreras-Porcia et al., 2013). It

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is highly likely that these mechanisms, present in P. columbina, represent part of the

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functional tools available to the plants to tolerate desiccation and, at the end, may help to

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explain its ecological dominance in the higher part of the intertidal zone. In this context, we

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assessed the general validity of the responses in P. columbina by testing the hypothesis that

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the presence and adequacy of functional responses to desiccation stress in a selected group of

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common intertidal seaweeds defines their altitudinal position in the intertidal zone.

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2. Materials and Methods

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The presence and adequacy of functional responses to desiccation in Mazzaella laminarioides,

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Scytosiphon lomentaria, Ulva compressa, Lessonia spicata and Gelidium rex were determined

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by monitoring and recording i) cellular alteration, ii) attenuation of ROS over-production, iii)

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oxidation of biomolecules, iv) antioxidant enzymatic activity, and v) photosynthetic response

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after desiccation. These responses were recorded in plants affected by desiccation in vitro, and

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compared with those in plants rehydrated in fresh seawater.

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2.1. Sampling

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Hydrated vegetative individuals of each species were collected along 250 to 300 m of

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coastline in Maitencillo beach (32º 39.5’ S, 71º 26.6’ W) during low tide. Plants were kept in

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plastic bags with seawater and transported to the laboratory in a cooler with ice packs at 5 to

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7°C. Once in the laboratory, these hydrated individuals were exhaustively rinsed with 0.45

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µm-filtered seawater, cleaned using an ultrasonic bath (575T, Crest, NJ, USA) and acclimated

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to laboratory conditions in a growth chamber for 24 h at 14 ± 2°C, 12:12 light: dark

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photoperiod and 30 to 40 µmol photon m-2 s-1 irradiance.

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2.2. In vitro experiment of desiccation and recovery

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In vitro experiments were conducted according to previous work in Pyropia columbina

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(Contreras-Porcia et al., 2011), where responses to 4 h desiccation were similar in magnitude

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to those recorded after 4 h of natural desiccation. In vitro desiccation experiments included an

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initial blot drying of the hydrated plants followed by air exposure in a growth chamber at

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16ºC and 70-80 µm photon m-2 s-1 irradiance for 4 h. In addition, a subset of dehydrated

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fronds was immediately re-hydrated in 0.45 µm-filtered seawater, during 4 h, to characterize

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the recovery from oxidative stress caused by desiccation. This timing was selected because

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plant tissues of all species under study have reached 95-100% relative water content (RWC).

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Control plants were obtained after acclimation, and frozen immediately in liquid nitrogen.

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2.3. Level of desiccation

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The level of desiccation experienced by different algal species during the in vitro trials was

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expressed as relative water content (RWC%) following the formula: RWC%=[(Wd-

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Wdo)x(Wf-Wdo)-1]x100, where Wf is the wet weight of fully hydrated fronds, Wd is the 7 Page 7 of 38

dehydrated weight after desiccation, and Wdo is the dry weight determined after drying for 48

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h at 80°C. In this context, the RWC is the complement of desiccation status and thus, a fully

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hydrated thallus has RWC of 100% and a fully dehydrated thallus has RWC near to 0 %;

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decreasing RWC means increasing desiccation.

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2.4. Morphological effects of desiccation

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Hand-made cross sections of naturally hydrated, dehydrated and rehydrated fronds (five of

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each category) were used to characterize morphological changes at light microscopy level.

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The hydrated sections were mounted in seawater and the dehydrated section in a synthetic,

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water-free resin (Permount™, Electron Microscopy Sciences, PA, USA) to prevent

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rehydration during analysis. Cell size in each category of fronds was defined as the mean

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value from 100 cell measurements. Images were captured with a Nikon Microscopy Unit

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(Nikon Corp., Tokyo, Japan) coupled to a digital recording system (CoolSNAP-Procf, Media

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Cybernetics, MD, USA) and analyzed using the Image Pro Plus Version 4.5 software (Media

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Cybernetics, MD, USA).

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Changes in ultrastructure as a result of desiccation stress were analyzed by

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transmission electron microscopy (TEM) using triplicate samples of hydrated, dehydrated and

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rehydrated fronds. Tissue samples were fixed in 0.22 µm-filtered seawater containing 3%

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(v/v) glutaraldehyde and 1% (v/v) p-formaldehyde for 3 days at 5ºC (Correa and McLachlan,

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1991). Post-fixation for 2 h at 5ºC in 0.05 M sodium cacodylate buffer (pH 7.8) with the

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addition of 2% (w/v) OsO4 and 1% (w/v) potassium hexacyanoferrate, was followed by

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dehydration in ethanol series (10-100%, v/v) and embedding in Spurr’s resin (Spurr, 1969) for

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1 week. Sections for TEM were stained with uranyl acetate-lead citrate and analyzed in an

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electron microscope (Tecnai 12, Philips, Holland), operated at 60 kV. 8 Page 8 of 38

2.5. Quantification of hydrogen peroxide (H2O2)

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H2O2 was determined according to Contreras et al. (2009) in hydrated, dehydrated and

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rehydrated fronds. Fresh tissue (1-2 g) were incubated in 100 mL of 5 µM 2´,7´

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dichlorohydrofluorescein diacetate (DCHF-DA, Calbiochem, CA, USA) for 1 h at room

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temperature. After incubation, the tissue was ground in liquid nitrogen, suspended in 5 mL of

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40 mM Tris-HCl buffer pH 7.0 and centrifuged at 20,600 g for 15 min. Fluorescence was

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measured in a Perkin-Elmer LS-5 spectrofluorometer at an excitation wavelength of 488 nm

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and at an emission wavelength of 525 nm. Final fluorescence values were obtained using a

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standard curve prepared with 0-500 nM of DCF (Sigma, St. Louis, USA). The in situ

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production of H2O2 was detected in hydrated and desiccated plants immediately after

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incubation in DCHF-DA. After rinsing the plants in filtered seawater, hand-made cross-

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sections of the tissues were observed using a Nikon Optiphot II fluorescence microscope with

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an emission filter of 459-490 nm, coupled to a digital recording system (CoolSNAP-Procf,

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Media Cybernetics, MD, USA) and analyzed using the Image Pro Plus Version 4.5 software

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(Media Cybernetics, MD, USA).

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2.6. Lipid peroxidation and protein oxidation

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Lipid peroxidation levels were determined as the amount of thiobarbituric acid (T-BARS)

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reactive species (i.e. lipoperoxides) according to Ratkevicius et al. (2003). Protein oxidation

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levels were determined according to Contreras-Porcia et al. (2011) based on the reaction of

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carbonyls resulting from free radical modification of proteins and 2,4-dinitrophenyl hydrazine

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(DNPH). For that, 4 to 5 g fresh algal tissue were frozen in liquid nitrogen and homogenized

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in a pre-chilled mortar with a pestle. A total of 5 mL of 50 mM sodium buffer pH 7.0,

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containing 0.1 mM EDTA and 1% (w/v) polyvinyl-polypyrrolidone were added during the

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homogenization. The homogenate was centrifuged at 7,400g for 15 min at 4ºC and the 9 Page 9 of 38

supernatant stored at -20ºC. Two aliquots, each containing 1 mg of proteins, were mixed with

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an equal volume of 20% (v/v) trichloroacetic acid (TCA) and centrifuged at 7,400g for 15 min

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at 4ºC. Pellets were suspended with 300 µL of 2 N HCl with or without (blank) 10 mM

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DNPH and left for 1 h at room temperature. Samples were then precipitated with 500 µL of

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20% (v/v) TCA for 10 min at -20ºC, centrifuged at 7,400g for 15 min at 4ºC, and the

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supernatant discarded. After rinsing with 500 µL ethanol: ethyl acetate (1:1, v/v), pellets were

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dissolved in 3 mL 20 mM sodium phosphate buffer (pH 6.8) containing 6M guanidinium

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hydrochloride and centrifuged at 7,400g for 15 min at 4ºC. Finally, the carbonyl concentration

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was calculated from the difference in absorbance recorded at 380 nm for DNPH-treated and

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HCl-treated (blank) samples (ε= 22 mM-1 cm-1) and expressed in µmol of DNPH incorporated

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per mg of protein.

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2.7. Antioxidant enzyme activities

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Protein extracts and the activities of antioxidant enzymes were determined according to

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Contreras et al. (2005, 2009). Briefly, algal tissue (20 g fresh weight) was frozen in liquid

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nitrogen and homogenized in a pre-chilled mortar using a pestle. A total of 60 mL of 0.1 M

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phosphate buffer (pH 7.0), containing 5 mM β-mercaptoethanol was added during the

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homogenization. The homogenate was filtered through Miracloth paper (Calbiochem, San

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Diego, CA, USA) and centrifuged at 7,400g for 15 min at 4ºC. Proteins were precipitated by

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addition of 0.5 g of ammonium sulfate per mL of extract during 2.5 h at 4ºC. The protein

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pellet was dissolved in 2 mL of 0.1 M phosphate buffer (pH 7.0), containing 2 mM β-

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mercaptoethanol and 20% (v/v) glycerol, and the protein concentration was determined using

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the bicinchoninic acid assay according to the protocols by Smith et al. (1985). Final protein

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extracts (2-4 mg mL-1) were stored at -80ºC and the activities were determined using 50-200

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µg of proteins. The specific activity of the antioxidant enzymes catalase (CAT) was measured

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in a reaction mixture containing 0.1 M phosphate buffer, pH 7.0, and 18 mM H2O2. After the

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addition of H2O2, its consumption was determined at 240 nm for 1 min and the activity was

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calculated using the extinction coefficient of H2O2 (ε = 39.4 mM-1cm-1). For AP activity, the

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reaction mixture contained 0.1 M phosphate buffer pH 7.0, 400 µM ascorbate (ASC) and 20

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mM H2O2. After the addition of ASC, its consumption was determined at 290 nm for 1 min

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and the activity was calculated using the extinction coefficient of ASC (ε= 2.8 mM-1cm-1).

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2.8. Photosynthetic efficiency activity

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Maximum photosynthetic efficiency (Fv/Fm) was assessed in 5 to 10 plants, according to

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Contreras-Porcia et al. (2011). Before recording fluorescence, and to allow complete re-

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oxidation to photosystem (PSII) reaction centers, fronds were kept in plastic bags and dark-

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acclimated with leaf-clips for 30 min for M. laminarioides and U. compressa, 25 min for G.

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rex, and 20 min for S. lomentaria and L. spicata. The fluorescence emission rates were

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measured using a portable fluorometer (Plant Efficiency Analyser PEA, Hansatech

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Instruments Ltd., UK) with a maximum light intensity of 2,000 µmol m-2 s-1 for M.

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laminarioides and U. compressa, 2,500 µmol m-2 s-1 for S. lomentaria and L. spicata, and

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3,000 µmol m-2 s-1 for G. rex. The different saturating intensities were chosen based on

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saturation kinetics of Fv/Fm.

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2.9. Statistical analyses

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Significance of the differences were determined by two-way analysis of variance (ANOVA)

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followed by Tukey’s multiple comparisons tests (T), considering species and treatment as

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fixed effects factors with three levels in the latter (control or natural hydration, desiccation

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and rehydration). Prior to the statistical analyses, data were checked for variance homogeneity

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using Levene and Anderson-Darling tests and for normal distribution using Kolmogorov-

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Smirnov and Bartlett tests (Zar, 2010). Data were transformed using the Box-Cox method

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when necessary. Subsequently, the effect sizes in each dependent variable were determined

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estimating Cohen’s d (Cohen, 1988), where an effect size of 0.2 to 0.4 is considered a small

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effect, 0.5 to 0.7 is considered a medium effect, and ≥ 0.8 is considered a large effect.

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Differences between mean values were considered to be significant at a probability of 5%

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(P≤0.05).

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3. Results

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3.1. Morphological changes

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Plants exposed to desiccation displayed low relative water content (RWC %), ranging

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between 4 and 22%. For example, M. laminarioides, U. compressa and S. lomentaria lose

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more water (i.e. ca. 94%, 98% and 97%, respectively) than plants growing at the lower

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intertidal zone (i.e. L. spicata ca. 83% and G. rex ca. 78%). Moreover, cross-sections of

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dehydrated fronds showed a decrease in size of cortical cells and medullar zone in relation to

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hydrated fronds (Table 1). For example, the most evident alterations were observed in U.

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compressa. In their hydrated fronds, cells appeared polygonal, 19.1 ± 2 µm in length and 9 ±

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2 µm in width, whereas in dehydrated individuals the size decreased by 53 to 56% (i.e. 9 ± 2

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µm in length and 4±2 µm in width). In M. laminarioides and L. spicata the medullar zone

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decreased by ca 41% and in S. lomentaria and G. rex the cortex in a 43 to 25%, respectively.

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Major negative effects induced by desiccation were recorded in L. spicata and seemed

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to have an impact on the fine structure of the cells. In naturally hydrated fronds, cellular 12 Page 12 of 38

structure of the cortical cells was normal (Fig. 1a), with the parietal chloroplast (Fig. 1a) and

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characteristic distribution of thylakoids (Fig. 1b). In contrast, during desiccation there was a

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considerable retraction of the protoplast (Fig. 1c) and thylakoid disorganization (Fig. 1d). It is

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important to mention that the fine structure of L. spicata did not recover during rehydration

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(Fig. 1e and 1f).

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3.2. Over-production of hydrogen peroxide (H2O2)

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Desiccation had a different effect on H2O2 production according to the species (Table 2).

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Desiccation increased H2O2 in cortical cells (Fig. 2a), from 9.4 to 25 fold above levels

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recorded in hydrated plants. The smallest increase in H2O2 during desiccation was recorded in

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M. laminarioides (T= 8.71, P<0.001, Cohen´s d= 5.78) and S. lomentaria (T= 10.18, P<0.001,

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Cohen´s d= 7.5); 9.4 and 14 times the level of hydrated plants, respectively. Moreover, this

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increase was more significant in middle and low intertidal species (U. compressa, L. spicata

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and G. rex) than in upper-middle intertidal species, with L. spicata displaying the highest

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increase (25 times) (T= 12.27, P<0.001, Cohen´s d= 9.42). For example, in sections of L.

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spicata and G. rex, ROS-fluorescence was patchy and appeared in both meristoderm and

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cortex (Fig. 2b). During rehydration, the concentration of H2O2 declined in M. laminarioides

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(T= 2.39, P=0.540, Cohen´s d= 1.93) and S. lomentaria (T= 3.16, P=0.156, Cohen´s d= 1.73)

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to the basal levels. However, in lower intertidal species the levels of H2O2 persisted 7 to 11

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times higher than those registered in hydrated plants (in all cases P<0.001, Cohen´s d >3.5)

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(Fig. 2a).

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3.3. Molecular oxidation

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Desiccation had a significant effect on the oxidation of biomolecules (Table 2). Levels of

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lipoperoxides increased significantly (P<0.050, Cohen´s d >2) during desiccation in tissues of

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all species, except S. lomentaria (T= 1.63, P=0.300, Cohen´s d= 1.81), in comparison with

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hydrated plants (Fig. 3a). This increase varied from 2.4 to 8 fold higher than the values

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recorded in hydrated plants, and the effect was stronger in low intertidal species (Fig. 3a).

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During rehydration, lipoperoxide content in all species reached levels even higher than under

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desiccation, except for M. laminarioides (T= 0.69, P=1.0, Cohen´s d= 1.38) (Fig. 3a).

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Protein oxidation (Fig. 3b) under desiccation stress increased 1.5 to 4.5 times in all

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species (P<0.050), except in M. laminarioides (T= 1.11, P=0.997, Cohen´s d= 1.01) and S.

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lomentaria (T= 2.12, P=0.712, Cohen´s d= 1.18). Differences in tolerance to desiccation,

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however, were clear during rehydration. The results showed that carbonyl levels did not

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increased in M. laminarioides and S. lomentaria undergoing rehydration. Contrarily, in U.

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compressa it was higher than in hydrated plants (T= 4.80, P=0.003, Cohen´s d= 5.69), and in

322

L. spicata (T= 7.93, P=0.0001, Cohen´s d= 6.54) and G. rex (T= 6.47, P=0.0001, Cohen´s d=

323

6.32), carbonyl level was higher even than under desiccation (Fig. 3b). L. spicata presented

324

the highest levels of oxidized proteins.

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3.4. Antioxidant responses

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Desiccation and rehydration had significant effects on the antioxidant responses and it varied

328

according to the species (Table 2). With the exception of the low intertidal L. spicata, CAT

329

activity increased significantly (in all cases P<0.01 and Cohen´s d >2) during desiccation

330

compared to hydrated plants (Fig. 4a), ranging from 2.6 to 9.4 times the basal activity, with

331

the highest values recorded in M. laminarioides and S. lomentaria (Fig. 4a). During

332

rehydration, CAT activity returned to the basal levels except in U. compressa, where 14 Page 14 of 38

333

remained elevated (T= 5.64, P=0.0004, Cohen´s d= 5.98). Whereas in L. spicata CAT activity

334

was not detected during rehydration, in G. rex it displayed levels lower than basal activity (ca.

335

23%) (T= 5.15, P=0.0012, Cohen´s d= 6.03) (Fig. 4a). During desiccation, AP activity increased significantly in M. laminarioides (T= 3.64,

337

P=0.05, Cohen´s d= 2.5) and in S. lomentaria (T= 3.71, P=0.041, Cohen´s d= 2.74) (Fig. 4b).

338

On the contrary, in U. compressa this activity decreased 87% in comparison with the basal

339

activity (T= 4.67, P=0.004, Cohen´s d= 4.76). In L. spicata (T= 0.67, P=1.0, Cohen´s d= 0.8)

340

and G. rex (T= 3.44, P=0.086, Cohen´s d= 1.97) AP activity was the same as in hydrated

341

plants (Fig. 4b). During rehydration, AP activity in M. laminarioides (T= 1.68, P=0.924,

342

Cohen´s d= 4.39) and S. lomentaria (T= 1.50, P=0.967, Cohen´s d= 1.74) returned to basal

343

levels, but in U. compressa it dropped below basal levels by 88% (T= 5.73, P<0.001, Cohen´s

344

d= 5.613). Finally, whereas in L. spicata AP activity was not recorded, in G. rex it was the

345

same as in hydrated plants (T= 0.39, P=1.0, Cohen´s d= 0.961) (Fig. 4b).

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3.5. Effects of desiccation on photosynthetic efficiency

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Maximum basal photosynthetic efficiency (Fv/Fm) in hydrated plants varied from 0.470 to

349

0.740 (Fig. 5). Even though during desiccation Fv/Fm decreased 92-98%, after rehydrated M.

350

laminarioides and S. lomentaria Fv/Fm was restored to the maximum levels (Fig. 5). In U.

351

compressa, however, Fv/Fm increased only by 30% with respect to the basal level (T= 16.39,

352

P<0.001, Cohen´s d= 5.04). Finally, the low intertidal L. spicata and G. rex displayed an

353

Fv/Fm below the maximum levels by 2% (T= 13.08, P<0.001, Cohen´s d= 3.56) and 3% (T=

354

11.59, P<0.001, Cohen´s d= 6.69), respectively (Fig. 5), demonstrating an irreversible

355

inactivation of the photosystem.

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4. Discussion

358

Our results support the hypothesis that the presence and adequacy of functional responses to

359

desiccation in a selected group of common intertidal seaweeds defines their bathymetric

360

position in the intertidal zone. In summary, our results show that representative intertidal

361

seaweeds responded differently to desiccation, and those living higher in the intertidal regime

362

display a better tolerance to this stress. Desiccation -triggered by in vitro air exposure- led to

363

significant increase in ROS, inhibition of antioxidant enzymes, cellular alterations, and

364

photosynthetic inactivation, mainly in the lower intertidal Lessonia spicata and Gelidium rex.

365

Detrimental effects observed during rehydration were also stronger in the lower intertidal

366

species. Hence, these results help to explain the bathymetric segregation of seaweed species

367

within the intertidal zone.

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4.1. Antioxidant mechanisms and desiccation tolerance

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It was clear that different seaweed species displayed different desiccation tolerance and such a

371

difference was reflected in their antioxidant metabolism. Control of ROS during desiccation

372

by the antioxidant system has been demonstrated in intertidal seaweeds during tide-driven

373

desiccation (Collén and Davison, 1999b; Contreras-Porcia et al., 2011). For example, the

374

medium to high intertidal Mastocarpus stellatus (Gigartinales, Rhodophyta) (Collén and

375

Davison, 1999b) and Pyropia columbina (Contreras-Porcia et al., 2011) regulate ROS

376

production during desiccation activating their antioxidant metabolism more effectively than

377

lower intertidal species. Some land plants have similar responses to desiccation. In the

378

primitive Selaginella bryopteris, desiccation leads to ROS concentrations three times higher

379

than basal levels; however this excess is efficiently buffered during rehydration by its

380

antioxidant metabolism (Pandey et al., 2010). Antioxidant control of ROS has been also

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16 Page 16 of 38

reported in resurrection plants- a heterogeneous group, mostly angiosperms- that can revive

382

after extended periods of dehydration (Alpert, 2006), a feature used to explain their high

383

tolerance to desiccation. Among ROS, hydrogen peroxide has been described as an important

384

cellular signalling molecule involved in diverse physiological responses (Rhee, 2006).

385

However, under conditions of high concentration and low buffering capacity of the stressed

386

organisms, ROS can be extremely harmful. This is coincident with our current results, where

387

middle to lower intertidal species (Ulva compressa, Lessonia spicata and Gelidium rex)

388

displayed high levels of hydrogen peroxide accumulation even during rehydration and,

389

consequently, they are less tolerant to desiccation, which is demonstrated by cellular

390

alteration and antioxidant enzymatic inactivation. The high intertidal species P. columbina, on

391

the other hand, rapidly buffered ROS excess after a brief rehydration period (Contreras-Porcia

392

et al., 2011) and, as expected, this is a species highly tolerant to desiccation. Based on the

393

above, it is suggested that low intertidal species, as those used in this study, do not have the

394

appropriate capacity to attenuate ROS excess during desiccation. This physiological

395

limitation, instead, could make them poorly suited to inhabit the higher intertidal. Contrarily,

396

species well adapted to flourish in the higher intertidal, like P. columbina, keep ROS under

397

control by activating their antioxidant system, minimizing or eliminating oxidative damage of

398

the cells.

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4.2. ROS excess and cell damage

401

The accumulation of lipoperoxides and carbonyls are a clear indication that ROS buffering

402

mechanisms are not effective (Achary et al., 2008; Jackie et al., 2011). Our results

403

demonstrated an over-production of these oxidative markers in low intertidal species

404

concurrent to high ROS production (i.e. H2O2). Exceptions to this pattern were evident in M. 17 Page 17 of 38

laminarioides and S. lomentaria where lipoperoxides remained high during rehydration even

406

though ROS levels were drastically reduced. A similar situation was reported in S. lomentaria

407

(Contreras et al., 2009), where exposure to copper stress induced arachidonic acid-dependent

408

lipoxygenase activity (LOX), a defense enzyme involved in the production of derivatives of

409

fatty acid oxylipins (Howe and Schilmiller, 2002). Thus, S. lomentaria, and probably M.

410

laminarioides, might require the induction of additional tolerance pathways (e.g. oxylipins

411

production) to buffer oxidative stress caused by desiccation. In contrast, in desiccation

412

tolerant plants of P. columbina there was no evidence of excess lipoperoxides during the

413

desiccation/rehydration cycle (Contreras-Porcia et al., 2011). This can be explained by the

414

activation of peroxiredoxins (PRXs) (Contreras-Porcia et al., 2011; 2013) that use fatty acid

415

hydroperoxides as substrate, inhibiting the accumulation of oxidised molecules under a stress

416

condition (Lovazzano et al., 2013). In resurrection plant under desiccation stress the

417

activation of PRX is also an important part of desiccation stress mechanisms (Leprince and

418

Buitink, 2010). Thus, our results indicate that lower intertidal species are more exposed to

419

oxidative damage caused by desiccation due to their limited antioxidant capabilities.

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4.3. Ecological consequences of limited antioxidant capacity

422

In spite the scarcity of information, the occurrence and effectiveness of antioxidant

423

mechanisms seems to be a requirement in plants inhabiting environments which cause

424

desiccation. This is true in groups of plants phylogenetically quite distant, such as the

425

resurrection plants and the high intertidal seaweeds (Collén and Davison, 1999b; Farrant,

426

2000; Hoekstra et al., 2001; Burritt et al., 2002; Alpert, 2006; López-Cristoffanini et al. 2013,

427

unpubl. results). For example, antioxidant activity in resurrection plants increases during

428

desiccation (Le and McQueen-Mason, 2006; Moore et al., 2009), and this has been used as

429

argument for explaining the high tolerance of these plants to their dry habitats. In seaweeds, 18 Page 18 of 38

two studies have reported the relationship between the abatement of ROS excess by enhanced

431

antioxidant activity and tolerance to desiccation stress (Stictosiphonia arbuscula (Ceramiales,

432

Rhodophyta), Burritt et al., 2002 and Pyropia columbina, Contreras-Porcia et al., 2011).

433

Consistent with the previous argument, desiccation intolerant species seem to have less

434

effective or inefficient antioxidant systems. A possible role of the antioxidant enzymes,

435

including AP, on desiccation tolerance become apparent from our findings where, in middle

436

and lower intertidal species like Ulva compressa, Lessonia spicata and Gelidium rex, they

437

were inactivated or had an activity below detection limits. We further believe that high levels

438

of protein oxidation during desiccation and rehydration in these species may be an indication

439

that enzymes, including AP, are inactivated. Sampath-Wiley et al. (2008) studied the seasonal

440

effects of sun exposure and rehydration during tidal emersion in Porphyra umbilicalis

441

(Bangiales, Rhodophyta) suggested that the elevated level of antioxidant enzymes and

442

carotenoids during emersion contributes to its colonization in upper intertidal region.

443

Similarly, López-Cristoffanini et al., (2013) suggested that consequences of limited

444

antioxidant capacity in seaweeds may also affect the geographic distribution in species such

445

as L. spicata, wherein desiccation was accompanied with inactivation of antioxidant enzymes

446

(AP and CAT). Thus, the inability of middle-low intertidal species to grow in the high

447

intertidal zone could be explained by an inefficient antioxidant system.

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4.4. Photosynthetic performance and desiccation tolerance

450

The functional status of the photosynthetic system of various seaweeds and plant models has

451

been widely used as an indicator of stress caused by desiccation (Bell, 1993; Skene, 2004;

452

Varela et al., 2006; Gylle et al., 2009; Pandey et al., 2010, Schagerl and Möstl, 2011). For

453

example, in the resurrection plant Selaginella bryopteris, the rapid and complete recovery of

454

Fv/Fm after rehydration clearly indicates the absence of marked photoinhibitory or thermal

19 Page 19 of 38

injury to PSII during desiccation. In intertidal and sublittoral ecotypes of Fucus vesiculosus

456

(Fucales, Heterokontophyta) responded to desiccation with a different efficient photosynthetic

457

intensity, and the intertidal ecotype appeared more resistant (Gylle et al., 2009). Similar

458

responses were reported in individuals of Mazzaella laminarioides, where high intertidal

459

individuals had ratios significantly higher than those plants from the lower intertidal when

460

exposed to desiccation stress (Varela et al., 2006)[0]. In both resurrection plants and in

461

Pyropia columbina (Farrant et al., 2003; Contreras-Porcia et al., 2011), photoinhibition was

462

suggested as the tolerance mechanism to cope with desiccation. This is the result of a

463

reversible reduction of the photosynthetic quantum yield due to down-regulation of PSII,

464

where energy is thermally dissipated. Even though carbon fixation could be inhibited during

465

desiccation, electron flow might continue and thus form ROS (Dinakar et al., 2012). Given

466

this, photoinhibition could prevent ROS formation and help to overcome desiccation (e.g.

467

Figueroa et al., 1997; Korbee et al., 2005; Contreras-Porcia et al., 2011). However, the current

468

results suggest that in species of lower intertidal distribution (U. compressa, L. spicata, and

469

G.rex) the poorer photosynthetic performance under desiccation/rehydration cycle is

470

generated by a disarray of the photosynthetic machinery under this stressful condition and not

471

by means of a photoinhibition mechanism. Thus, diverse tolerance mechanisms need to be

472

activated to cope with this environmental condition, which are deficient and/or absent in

473

lower intertidal species.

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5. Conclusion

476

The results of this study supported the hypothesis that the presence and adequacy of

477

functional responses to desiccation in common seaweeds defines their altitudinal position in

478

the intertidal zone. These differences seem to be independent of the algal division or the

479

functional-form groups to which the plants belong. High intertidal species are tolerant to 20 Page 20 of 38

desiccation and that tolerance seems to be based on the various mechanisms that efficiently

481

attenuate ROS excesses caused by desiccation (high antioxidant activity, biomolecule

482

oxidation attenuation and photoinhibition tolerance). By the contrary, lower intertidal species

483

are less tolerant to desiccation and this is likely due to their inefficiency in attenuating the

484

ROS excess produced during desiccation and early recovery.

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6. Acknowledgements

487

This work was supported by FONDECYT 11085019, FONDECYT 1120117 and DI-59-12/R

488

to LC. Additional funding comes from FONDAP 1501-0001 funded by CONICYT to the

489

Center for Advanced Studies in Ecology and Biodiversity (CASEB) Program 7 and ICA to

490

JC. We are especially grateful to Aníbal Contreras for field assistance, and we appreciate the

491

constructive comments from the Editor and from two anonymous reviewers that helped

492

improved the manuscript.

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pigments and photosynthetic efficiency in the red algae Porphyra umbilicalis Kützing

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(Rhodophyta, Bangiales). J. Exp. Mar. Biol. Ecol. 361, 83–91.

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Schagerl, M., Möstl, M., 2011. Drought stress, rain and recovery of the intertidal seaweed

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Fucus spiralis. Mar. Biol. 158: 2471–2479.

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Skene, K.R., 2004. Key differences in photosynthetic characteristics of nine species of

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intertidal macroalgae are related to their position on the shore. Can. J. Bot. 82, 177–184.

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Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Garthner, F.H.; Provenzano, M.D.,

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Fujimoto, E.K., Goeke, N.M., Olson, B.J., Klenk, D.C., 1985. Measurement of protein using

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bicinchoninic acid. Anal Biochem. 150, 76-85.

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Spurr, A.R., 1969. A low-viscosity epoxy resin embedding medium for electron microscopy.

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J. Ultrastruct. Res. 26, 31–43.

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Varela, D.A., Santelices, B., Correa, J.A., Arroyo, M.K., 2006. Spatial and temporal variation

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of photosynthesis in intertidal Mazzaella laminarioides (Bory) Fredericq (Rhodophyta,

626

Gigartinales). J. Appl. Phycol. 18, 827–838.

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Zaneveld, J.S., 1969. Factors controlling the delimitation of littoral benthic marine algal

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zonation. Am. Zool. 9, 367–391.

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Zar, J.H., 2010. Biostatistical analysis, 5th ed. Pearson Prentice-Hall, Upper Saddle River, NJ,

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USA.

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Table 1. Cellular dimensions (μm) of hand-cut cross sections of vegetative hydrated and

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dehydrated fronds of Mazzaella laminarioides, Scytosiphon lomentaria, Ulva compressa,

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Lessonia spicata and Gelidium rex under hydration and desiccation stress. Images show the

634

major cellular changes observed during desiccation in U. compressa, L. spicata and G. rex.

635

Scale bar = 10 ! m. me= meristoderm, c= cortex, m=medulla. Each value is an average of 100

636

measurements ± SD. * indicates significant (P<0.05) differences among hydrated and

637

dehydrated values. Zone tissue

Treatment

(! m)

Hydration

Cortical cell (length)

7.3 ± 2

Medulla (width)

133 ± 13

Scytosiphon lomentaria

an

Mazzaella laminarioides

Cortical cell (length)

6.3 ± 1

3.6 ± 1*

19.1 ± 2 x 9 ± 2

9 ± 2* x 4 ± 2*

4.4 ± 2

2.0 ± 1*

79.1 ± 8

46.4 ± 3*

6.0 ± 1

4.5 ± 1*

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Cortical cell (length)

Lessonia spicata

81 ± 19*

(length x width)

d

Ulva compressa

cell

5.2 ± 2*

M

Poligonal

Desiccation

us

Species

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Medulla (width) Cortical cell (length)

Gelidium rex

638 639 640 641

29 Page 29 of 38

Table 2. Summary of 2-way-ANOVA analyses to compare hydrogen peroxide levels, lipid

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peroxidation, protein oxidation production, ascorbate peroxidase and catalase activities, and

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photosynthetic efficiency (Fv/Fm), in the species M. laminarioides, S. lomentaria, L. spicata

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and G. rex under hydration, desiccation and rehydration treatments. df (degrees of freedom)

646

treatment= 2; df species= 4, df treatment x species= 8.

647 F-ratio

P-value

Hydrogen peroxide Treatment (T) Species (S) TxS

256.51 19.76 3.69

<0.001 <0.001 0.004

Lipid oxidation Treatment (T) Species (S) TxS

192.77 16.87 5.18

<0.001 <0.001 <0.001

66.35 23.01 4.73

<0.001 <0.001 0.001

Ascorbate peroxidase Treatment (T) Species (S) TxS

58.41 96.28 10.40

<0.001 <0.001 <0.001

Catalase Treatment (T) Species (S) TxS

144.17 59.96 17.00

<0.001 <0.001 <0.001

Fv/Fm Treatment (T) Species (S) TxS

653.01 12.84 14.16

<0.001 <0.001 <0.001

648 649 650

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Protein oxidation Treatment (T) Species (S) TxS

cr

Sources of variation

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651 652 653 654 655

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Figure captions

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Fig. 1. Ultrastructure of Lessonia spicata during hydratation (a-b), desiccation (c-d) and

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rehydration (e-f). (a) General view of cortical cells with parietal chloroplast (ch), central

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nucleus (n) and cell wall (cw) in contact with plasmatic membrane. (b) Chloroplast detail with

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the typical arrangement of thylakoids (t). Nuclear pore is also observed. (c) Cortical cell

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detail, with clear protoplast retraction (arrows) and separation between plasmatic membrane

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and cell wall. (d) Abnormal arrangement of thylakoids and diffuse cytoplasm. (e) Cortical cell

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detail of rehydrated plants, showing the same ultrastructural pattern than cells under

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desiccation. (f) Chloroplast and nucleus detail of rehydrated plants, where limited cellular

665

structure is observed. Scale bar = 1 µm.

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Fig. 2. Hydrogen peroxide (H2O2, nmol DCF (dichlorofluorescein) g-1 FT (fresh tissue))

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levels (a) in Mazzaella laminarioides, Scytosiphon lomentaria, Ulva compressa, Lessonia

668

spicata and Gelidium rex during hydration (white bars), desiccation (black bars) and

669

rehydration (grey bars). Values represent the mean of 3-6 independent replicates ± SD. Letters

670

indicate significant differences (P<0.05) between treatments for each species. (b) Localization

671

of hydrogen peroxide, visualized by fluorescence microscopy, in species with high H2O2

672

levels registered during desiccation stress (i.e. U. compressa surface view, and cross sections

673

of L. spicata and G. rex).

674

Fig. 3. Lipid peroxidation (a) and protein oxidation (b) in Mazzaella laminarioides,

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Scytosiphon lomentaria, Ulva compressa, Lessonia spicata and Gelidium rex during hydration

676

(white bars), desiccation (black bars) and rehydration (grey bars). Values represent the mean

677

of 3-6 independent replicates ± SD. Letters indicate significant differences (P<0.05) between

678

treatments for each species. DT, dry tissue.

679

Fig. 4. Activity of the antioxidant enzymes CAT (a) and AP (b) in Mazzaella laminarioides,

680

Scytosiphon lomentaria, Ulva compressa, Lessonia spicata and Gelidium rex during hydration

681

(white bars), desiccation (black bars) and rehydration (grey bars). Values represent the mean

682

of 3-6 independent replicates ± SD. Letters indicate significant differences (P<0.05) between

683

treatments for each species.

684

Fig. 5. Photosynthetic efficiency (Fv/Fm) in Mazzaella laminarioides, Scytosiphon

685

lomentaria, Ulva compressa, Lessonia spicata and Gelidium rex during hydration (white

686

bars), desiccation (black bars) and rehydration (grey bars). Values represent the mean of 5-10

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31 Page 31 of 38

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independent replicates ± SD. Letters indicate significant differences (P<0.05) between

688

treatments for each species.

689

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690

cr

691

us

692 693

an

694

M

695

699 700 701 702

te

698

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703 704

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Highlights

705

•Air exposure induces a desiccation stress condition in intertidal seaweed species•High

706

activities of antioxidant enzymes and low ROS production were the responses in higher

707

intertidal species• Photosynthesis capacity was recovered during rehydration only in higher

708

intertidal species• Lower intertidal distribution of certain algal species is explained by their

709

insufficient mechanisms of stress tolerance

cr

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Figure 3

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Figure 4

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Figure 5

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