Tolerance Strategies of Atriplex Atacamensis and A. Halimus in Response to Multiple Abiotic Stresses 2019

Tolerance strategies of Atriplex atacamensis and

A. halimus in response to multiple abiotic

stresses

Fabiola Alejandra Orrego Márquez

2019

Pontificia Universidad Católica de Chile Facultad de Agronomía e Ingeniería Forestal

Tolerance strategies of Atriplex atacamensis and A. halimus in response to multiple abiotic stresses

Fabiola Alejandra Orrego Márquez

Thesis to obtain the degree of

Doctor en Ciencias de la Agricultura

Santiago, Chile, April 2019

Thesis presented as part of the requirements for the degree of Doctor in Ciencias de la Agricultura, approved by the

Thesis Committee

______Dr. Rosanna Ginocchio, Advisor

______Dr. Claudia Ortíz-Calderón

______Dr. José Miguel Fariña

Santiago, April 2019

Financial funding support for Fabiola Orrego and all thesis activities was obtained

from CONICYT Doctoral Grant (21141059/2014), Center of Applied Ecology &

Sustainability (CAPES) by CONICYT PIA/BASAL FB0002 (2014) and Facultad de

Agronomía e Ingeniería Forestal UC.

Acknowledgements

I would like to thank mi thesis advisor, Rosanna Ginocchio for her support and dedication during this process, and for teaching me to take

care of myself as much as I care for what I do.

To mi co-advisor Claudia, for her precise comments on plant

physiology and biochemistry that shed light on my understanding of

plants.

I thank my family, for accompanying me with curiosity and enthusiasm

on each step of my academic path.

To my beloved Diego, for his permanent support during this process

and for being a man I can look up to.

To all my professional and scientific colleagues in the RESUME lab,

especially to Luz María de La Fuente, that taught me how to work

properly with plants.

To every one of my friends in FAIF and FCB, thanks for help me

broaden my perspectives, and being allies in this beautiful path of

knowledge.

Contents

General introduction ...... 11

Hypothesis and objectives ...... 26

Chapter 1

Diversidad de halófitas chilenas: distribución, origen y hábito ...... 29

Introducción ...... 31

Materiales y métodos ...... 33

Resultados ...... 34

Discusión y conclusiones ...... 38

Agradecimientos ...... 44

Referencias ...... 44

Chapter 2

Effect of single and combined Cu, NaCl and water stress on three Atriplex species with phytostabilization potential ...... 55

Abstract ...... 56

Introduction...... 57

Materials and methods ...... 60

Results ...... 63

Discussion ...... 67

Conclusion...... 75

Acknowledgments ...... 76

References ...... 76

Chapter 3

Growth and physiological effects of combined Copper, NaCl and water stresses on

Atriplex atacamensis and A. halimus ...... 87

Abstract ...... 88

Introduction...... 89

Materials and methods ...... 91

Results ...... 96

Discussion ...... 104

Conclusion...... 112

Acknowledgments ...... 113

References ...... 113

General Conclusions ...... 130

Table Index

Chapter 1

TABLA 1. Distribución de la riqueza de especies halófitas de Chile según su familia...... 36

TABLA 2. Riqueza, hábito y origen de las especies halófitas presentes en Chile según su distribución geográfica ...... 40

ANEXO 1. Listado de especies halófitas presentes en Chile según la base de datos eHALOPH...... 48

Chapter 2

Table 1. Shoot length (SL), dry root weight (DRW) and dry shoot weight (DSW) of Atriplex atacamensis (AA), A. halimus (AH) and A. nummularia (AN) seedlings subjected to available Cu, NaCl and a PEG-induced water stress for seven days.86

Chapter 3

Table 1. Root length (cm.) and root and shoot fresh weight (g) of Atriplex atacamensis and A. halimus seedlings exposed for 10 days to single and combined Cu, NaCl and PEG stresses...... 125

Table 2. Total chlorophyll and carotenoid content of Atriplex atacamensis and A. halimus subjected to single and combined Cu, NaCl and PEG stresses...... 126

Table 3. Root and shoot water content (WC in %) of A. atacamensis and A. halimus seedlings exposed for 10 days to single and combined Cu, NaCl and PEG stresses ...... 127

Table 4. Sodium and K concentration (in mg∙g-1) in roots and leaves of A. atacamensis and A. halimus seedlings exposed for 10 days to single and combined Cu, NaCl and PEG stresses...... 128

Table 5. Reduced and oxidized glutathione (nmol g FW -1) in leaves and roots of Atriplex atacamensis and A. halimus exposed for 10 days to single and combined Cu, NaCl and PEG stresses...... 129

Figure Index

Chapter 2

Figure 1. Root length increase of seedlings of A. atacamensis, A. halimus and A. nummularia subjected to increasing available Cu, NaCl and a decrease of solute potential...... 82

-1 Figure 2. Plant water content (g H2O g ) of Atriplex atacamensis, A. halimus and A. nummularia seedlings subjected to increasing available Cu, NaCl and a decrease of solute potential...... 83

Figure 3. Increase in root length (cm) of Atriplex atacamensis and A. halimus subjected to increasing single Cu concentrations and its combination with NaCl and PEG...... 84

Figure 4. Picture of A. halimus and A. atacamensis seedlings under control, Cu and Cu+PEG treatments of combined stress assay...... 85

Chapter 3

Figure 1. Visual symptoms of single and combined stresses on Atriplex seedlings. (a) Loss of turgor and leaf reddening on Atriplex halimus leaves subjected to 7.85 mM PEG...... 120 Figure 2. Leaf osmotic potential (MPa) of A. atacamensis (left) and A. halimus (right) seedlings exposed for 10 days to single and combined Cu, NaCl and PEG stresses...... 121

Figure 3. Leaf proline (mg g-1 FW) of A. atacamensis (left) and A. halimus (right) seedlings exposed for 10 days to single and combined Cu, NaCl and PEG stresses (n=4; mean± EE)...... 122

Figure 4. Copper concentration (mg∙g-1) in roots and leaves of A. atacamensis (upper) (n=5; means± EE) and A. halimus (lower)...... 123

Figure 5. Non-protein thiols (µmol g-1) in roots and leaves of Atriplex atacamensis (left) and A. halimus (right) seedlings exposed for 10 days to single and combined Cu, NaCl and PEG stresses...... 124

General Introduction

In Chile there are nearly 550 inactive and abandoned tailing storage facilities (TSF) in the north and central part of the country (Servicio Nacional de Geología y

Minería, 2018). Most of these TSF are a byproduct of historical copper production, and as such are not subject to current closure legislation (Servicio nacional de

Geología y Minería, 2018). Exposure of abandoned and non-stabilized TSFs to geographic and climatic conditions that predominate in these areas, turns them into pinpoints of copper enrichment into nearby ecosystems, productive land and human settlements (Navarro et al., 2008; Montenegro et al., 2009). Copper is a widespread component in soils, but an increase in its concentration due to anthropogenic activities can cause toxicity symptoms in plants, such as root damage, leaf chlorosis and wilting (Kabata-Pendias, 2010; Brahim and Mohamed,

2011).

A widely accepted approach for the rehabilitation of copper-enriched sites is phytostabilization, or the use of metal-tolerant plants to immobilize metals in the rhizosphere (Mendez and Maier, 2008; Alford et al., 2010). Previous approaches to phytostabilization focused on metals as the key limiting factor for plant establishment, lead to the search for metal-tolerant plants (methallophytes) or metal hyperaccumulators in the native component of the geographical area of interest (Ginocchio and Baker, 2004; Orchard et al., 2009; Gonzalez et al., 2010;

Tapia et al., 2017). Despite being a valid approach, it is known that successive or simultaneous occurrence of other abiotic limitations besides metal enrichment are

11 present in these systems and can further restrict plant survival and establishment.

The convergence of geographical and climatic conditions such as high temperature, scarce precipitation (Holmgren et al., 2006) and primary salinization of soils in northern and central Chile (Oyarzún and Oyarzún, 2011; Casanova et al., 2013) can heavily restrict phytostabilization efforts in these ecosystems

(Mendez and Maier, 2007; Ginocchio et al., 2017). Under these conditions it is key to consider the simultaneous effects of metals, salt and water stress in plant growth and tolerance traits as a tool to redefine parameters for proper species selection in phytostabilization programs (Mendez and Maier, 2007; Lutts and Lefevre, 2015).

Metal, salt and water stresses cause similar restrictions to plant metabolism and growth when studied separately (Saslis-Lagoudakis et al., 2014). An increase in the assimilation of sodium or metal ions within plant cells caused by external salt or metal enrichment can interfere with protein structure and membrane integrity or trigger oxidative damage (Clijsters et al., 1999; Dietz et al., 1999; Reichman, 2002;

Zhang et al., 2014; Flowers et al., 2015). This causes a decrease in photosynthetic efficiency, and an overall interruption of plant growth (Munns and Tester, 2008;

Stepien and Johnson, 2009). Water stress caused by the osmotic effects of salt assimilation, metal-induced root damage or a decrease in external water supply can also lead to oxidative damage and a decrease in photosynthetic efficiency, which ultimately results in growth impairments or wilting (Moran et al., 1994;

Hernández et al., 1995; Chaves et al., 2002). The convergence of the effects of salt, metal and water stresses suggests that plants with a broad response range to these abiotic conditions could represent a good alternative to assess phytostabilization in arid and semiarid regions (Lutts and Lefevre, 2015). Halophyte 12 species have been identified as good candidates for the rehabilitation of these specific metal enriched sites (Manousaki and Kalogerakis, 2011a).

Halophytes are a highly diverse group of species that complete their life cycle in saline habitats with an NaCl concentration of at least 200 mM (Flowers and

Colmer, 2008) or 4 dS mol-1 of soil electroconductivity (Koyro, 1999). Even though they only represent 0.2% of all flowering species (Flowers and Colmer, 2015), halophytes derive from a wide range of lineages, are present in all life forms and present a wide range of tolerance mechanisms that allows them to colonize saline ecosystems from deserts to mangroves (Flowers et al., 2010; Manousaki and

Kalogerakis, 2011b). One of the main assumptions behind the use of halophyte species for the rehabilitation of metal enriched systems is that halophyte tolerance traits to resist or evade toxicity or osmotic symptoms of salinity can also be effective to bear primary or secondary effects of metal stress (Lutts and Lefevre,

2015; Nikalje and Suprasanna, 2018). Antioxidant, chelating agents and compatible osmolyte synthesis are three tolerance mechanisms present in halophytes that could be specifically used against metal stressors (Figure 1).

Figure 1. Effect of metal toxicity in plants and avoidance or tolerance responses used by halophytes. Modified from Lutts & Lefevre (2015).

Antioxidant response in halophytes

Metal-induced oxidative damage operates through the Fenton reaction, where

2+ unpaired metal electrons (Me ) reduce hydrogen peroxide (H2O2) to form hydroxyl radicals (∙OH) (Seckin Dinler et al., 2010). This reactive oxygen species (ROS) is particularly toxic for plants, because it has the potential to oxidize biomolecules such as lipids, nucleic acids and proteins (Jara-Hermosilla et al., 2017).

Halophyte antioxidant response to metals is naturally augmented due to their permanent relationship with different salts in their growing habitats (Nagajyoti et al.,

2010). However, there are important intraspecific differences regarding antioxidant strategies to overcome an oxidative situation caused by the assimilation of metal ions. For example, Duarte et al., (2013) found that Spartina maritima presents a

14 rather close relationship between metal concentration and superoxide dismutase

(SOD), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and non- enzymatic activity when exposed to a contaminated site (Duarte et al., 2013). A related species, S. densiflora, has a temporal organization of the antioxidant enzymes APX, catalase (CAT), peroxidase (POD) and SOD activity, modulated by

H2O2 fluctuations during the oxidative process (Martínez Domínguez et al., 2010).

Interestingly, a different response arises when salt and metal stresses are combined. In Suaeda fruticosa, leaf enzymatic activity of APX, CAT and GPX decreases when NaCl is combined with Cu or Cd (Bankaji et al., 2016a), which suggests metal damage to the enzyme structure that undermines its antioxidant ability (Schützendübel and Polle, 2002). Evidence on how the combination of salt and metal stress increases oxidative damage above the antioxidant potential of the plant and how this affects plant functioning, raises questions about oxidative thresholds in halophytes, the effect of metal stressors on antioxidant structure and functioning of halophytes.

Synthesis of chelating agents

Synthesis of chelating agents is a critical strategy, triggered when plant tissues reach phytotoxic metal concentrations. Chelating agents combine with ionic forms of metals that normally have the most lethal effects to form inactive complexes

(Memon and Schröder, 2009). These complexes are later stored in cell compartments, mainly the cell wall or vacuole. Synthesis of chelating agents is a widespread trait in the plant kingdom, and several molecules are recognized as

15 metal chelators (Rauser, 1999); one of the most studied chelating agents related to metal tolerance are phytochelatins.

Phytochelatins (PC) are cysteine-rich peptides synthetized from the non-enzymatic antioxidant gluthatione (GSH), which chelate and deactivate several metal components (Grill et al., 1989). Evidence of PC chelation under non-toxic concentrations of metals involved in metabolism, such as Fe, was reviewed early in many sensitive plants (Wagner, 1993; Cobbett, 2000). It has been proposed that the main function of PC is to maintain metal homeostasis in plants, with active chelation and detoxification only in metal stress scenarios (Liu et al., 2015).

Phytochelatins are metal specific chelators; hence they are not present during salt stress. However, PC synthesis occurs when halophytes are subjected to metal stress. In Suaeda fructicosa, application of Cd and Cu increases PC concentration with a direct decrease in GSH concentration in roots and leaves (Bankaji et al.,

2015). Since GSH is a required substrate for PC synthesis, there is an indirect relationship between metal chelation and non-enzymatic antioxidant potential

(Yadav, 2010). For example, an increase of phytochelatin synthase in Atriplex halimus in response to Pb and Cd stress is accompanied by an increase in the expression of gluthatione s-transferase (GST), an enzyme that transports GSH to the site where PC will be synthesized (El-Bakatoushi et al., 2015). Under those circumstances, it is also interesting to explore if PC synthesis decreases the antioxidant potential of halophytes and how this translates into plant growth and metal accumulation.

Compatible osmolyte synthesis 16

Metal assimilation can alter plant water status at three levels: water uptake, water transport and/or water loss. First, water uptake is limited if metal assimilation alters the morphological integrity of the root; then metal assimilation by the plant can influence the number and size of xylem elements, further limiting water transport to upper organs; finally, metals can limit water loss through stomatal modifications or leaf senescence (Barceló and Poschenrieder, 1990). All these events lead to a state of physiological drought that can either aggravate plant health or induce physiological adaptations that allow the plant to reestablish intracellular osmotic balance.

One of the most studied osmotic adjustment mechanisms in water stressed plants is the synthesis of compatible osmolytes. Compatible osmolytes are low weight organic molecules that accumulate in the intracellular space and counteract the osmotic effect of inorganic salts in the medium and maintain protein and membrane stability (Ashraf and Foolad, 2007). One of the most studied compatible osmolytes is proline, a common amino acid in halophytes and sensitive plants

(Anjum et al., 2014).

Proline synthesis in halophytes subjected to metal stress occurs in response to direct structural damage or derived water uptake deficiency. For example, synthesis of proline and other organic acids in Spartina alterniflora is implicated in root chelation and accumulation of Cd (Min-Wei Chai, 2012). In Aeluropus litoralis, proline concentration increases with increasing concentrations of Co, Cd, Au and

Pb (Rastgoo and Alemzadeh, 2011). In contrast, proline synthesis of Atriplex halimus subsp. schweinfurthii seems to occur in response to a decrease in root

17 water conductivity and tissue water content, instead of metal assimilation (Nedjimi and Daoud, 2009).

As can be seen, proline is an essential component of plant abiotic tolerance, because it is present in multiple stress situations (Ashraf and Foolad, 2007).

However, its specific behavior during single and multiple stresses remains poorly explored.

Selection of halophyte candidates

Species selection is crucial in phytostabilization strategies. Native or endemic species of metal-enriched sites are a priority group, because they are well adapted to local conditions, (Mendez and Maier, 2008; Heckenroth et al., 2016; Ginocchio et al., 2017), can trigger ecological succession processes on disturbed sites

(Tapia et al., 2017) and can even prevent the introduction of non-native species

(Testiati et al., 2013) into rehabilitation sites. However, knowledge about halophyte species in the native component of Chilean flora is scarce, so it is necessary to explore which local species could represent good alternatives for phytostabilization strategies.

Atriplex is a halophyte genus of endemic, native and introduced shrubs and herbs present in Chile, whose tolerance to specific metals has been previously assessed with promising results. One of these is Atriplex halimus, an introduced species from

Europe whose growth parameters, physiological response and tolerance mechanisms have been tested under Cd (Lefevre et al., 2010; Soualem et al.,

2014), As (Tapia et al., 2013) Pb (Manousaki and Kalogerakis, 2009) and Zn (Lutts et al., 2004a) stresses. A. nummularia is an Australian species introduced in Chile 18 during the 1970s because of its foraging potential (Lailhacar et al., 1995;

Fernández et al., 2016). Although its tolerance to metal stress has not been thoroughly studied (Jordan et al., 2002; Mok et al., 2013), it is a fast growing plant with high growth potential under saline conditions (De Melo et al., 2016).

One native component of the genus Atriplex is A. atacamensis. This shrub is one of the 23 native or endemic Atriplex species described in Chile (Rosas, 1989); it inhabits dry riverbeds and ravines of the Tarapacá and Antofagasta regions

(Rosas, 1989; Fernández et al., 2016). The geographical overlap of this species with areas affected by mining (Lam et al., 2016) has led to a few studies that have shown its ability to tolerate arsenic without serious growth impediments, through its accumulation in roots and osmotic adjustment in leaves (Tapia et al., 2013;

Vromman et al., 2016a).

The three aforementioned Atriplex species are salt-tolerant shrubs of arid and semiarid areas that can also be found near sites affected by mining. Studies have shown that they can tolerate different metals, which indicates some degree of generality in their tolerance strategies to abiotic stressors. However, there are no studies that address species-specific effects of copper enrichment on growth and physiology, or the way in which its combination with water and salt stress may affect their tolerance strategies.

In recognition of this knowledge gap, I propose to study growth parameters, antioxidant response, synthesis of chelating agents and compatible osmolyte response of Atriplex atacamensis, A. halimus and A. nummularia when subjected to single and combined conditions of copper, water and salt stress.

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Hypotheses and objectives

Hypotheses

Hypothesis 1: Atriplex atacamensis, A. halimus and A. nummularia seedlings have different tolerance thresholds to increasing conditions of copper, salt and water stresses.

Hypothesis 2: Single copper, salt and water stresses have variable effects on growth parameters of Atriplex atacamensis and A. halimus seedlings and induce the expression of chelating, osmoregulative and antioxidant mechanisms.

However, the combination of these stresses negatively affects plant growth and expression of these mechanisms.

General objective

To assess tolerance thresholds and tolerance mechanisms in individuals of three halophyte species of the genus Atriplex when exposed to single and combined copper, salt, and water stresses.

Specific Objectives

1. To describe the Chilean halophyte biodiversity and to identify candidate species for phytostabilization of chemically (i.e. metal enriched and salinized) degraded soils.

2. To assess the effect of increasing conditions of salinity, available copper and water stress on growth parameters of A. atacamensis, A. halimus and A. nummularia seedlings.

3. To assess single and combined effects of copper, salt and water stresses on growth, ion distribution and the expression of antioxidant, compatible osmolytes and chelating agents used by of A. atacamensis and A. halimus individuals.

Chapter 1

Diversidad de halófitas chilenas: Distribución, origen y hábito

Fabiola Orrego1,3, Luz María de la Fuente1,3; Miguel Gómez2; Rosanna Ginocchio1,3

1Departamento de Ecosistemas y Medio Ambiente, Facultad de Agronomía e Ing.

Forestal, Pontificia Universidad Católica de Chile. Avenida Vicuña Mackenna 4860,

Santiago, Chile.

2Departamento de Ciencias Vegetales, Facultad de Agronomía e Ing. Forestal,

Pontificia Universidad Católica de Chile. Avenida Vicuña Mackenna 4860,

Santiago, Chile.

3Center of Applied Ecology and Sustainability, Facultad de Ciencias Biológicas, CP

6513677, Universidad Católica de Chile, Santiago, Chile

This chapter was published in Gayana Botanica – Ediciones de la Universidad de Concepción, Chile (75, 2, 555-567)

RESUMEN

Las especies halófitas son reconocidas por su capacidad de sobrevivir en ambientes salinos. Sin embargo, esta condición varía enormemente entre los diferentes taxones de plantas halófitas, dificultando su correcta clasificación.

Actualmente, la base de datos eHALOPH registra las especies halófitas de todo el mundo bajo un único criterio de salinidad. En este trabajo se presenta el primer listado de halófitas en Chile, elaborado a partir de eHALOPH. Los resultados indican una diversidad de 138 especies, distribuidas en 31 familias. Más del 80% de estas especies son herbáceas y cerca del 55% son exóticas. La mayor riqueza se encontró en las regiones de Coquimbo y Valparaíso, y la menor en el territorio insular. Estos resultados son una primera aproximación a la diversidad de especies halófitas en Chile. Al mismo tiempo, plantean un desafío para avanzar con su reconocimiento con el fin de proponer posibles estrategias para su conservación y uso.

PALABRAS CLAVE: Halófita, diversidad vegetal, ecosistemas salinos, eHALOPH.

ABSTRACT

Halophyte species are recognized for their ability to survive in salty environments.

However, this condition greatly varies among plant taxons, difficulting their proper classification. Currently, the eHALOPH database registers halophyte species from

30 around the world, under a single salinity criterion. In the present study, the first list of halophyte species present in Chile made from eHALOPH is presented. Results indicate a diversity of 138 species, distributed in 31 families. Over 80% of species are herbaceous and nearly 55% are exotic. The largest richness was found in

Coquimbo and Valparaíso regions, and the lowest in the insular territory. These results are a first approximation to halophyte diversity in Chile. At the same time, it poses a challenge to advance in their recognition in order to propose strategies for their conservation and use.

Keywords: Halophyte, plant diversity, saline ecosystems, eHALOPH

INTRODUCCIÓN

El término halófita (halos= sal; phyta= planta de) se utiliza hace más de 200 años para definir a aquellas especies de plantas que han hecho de los ecosistemas salinos su hábitat (Flowers et al., 1986). Sin embargo, la gran diversidad de ambientes en los que existen y la gran cantidad de estrategias que usan para tolerar o evadir el exceso de salinidad en el sustrato, ha derivado en la proliferación de variadas definiciones (Breckle 1990, Grigore et al. 2010). Esto crea una fuente de incertidumbre a la hora de clasificar una especie como halófita, tanto a nivel regional como global (Grigore et al., 2014). A pesar de ello, en la literatura actual se ha llegado a un consenso con respecto al uso de la concentración de cloruro de sodio (NaCl) como el parámetro que permite discriminar a las especies halófitas de aquellas sensibles a la salinidad (Flowers &

Colmer 2008). En base a esta definición, se han creado los primeros listados de especies halófitas a nivel global (Aronson 1989, Menzel & Lieth 2003, Santos et al.

2015).

En la actualidad, perturbaciones tales como la degradación y salinización de los suelos devuelven el interés hacia este grupo de especies. Sus principales usos están enfocados en el desarrollo de la agricultura biosalina (Koyro 2003, Toderich et al. 2008), la rehabilitación de suelos degradados y contaminados con residuos mineros (Al-Farrajii & Al-Hilli 1994, Aronson & Le Floc’h 1995, Lutts & Lefevre

2015), la generación de biomasa para forraje (Lailhacar et al., 1995) y como biofiltro de residuos municipales y aguas residuales (Shpigel et al. 2013, Buhmann et al. 2015). A pesar del gran valor de estas especies, su identificación y clasificación sigue siendo un desafío a nivel local y regional.

En Chile, el estudio de este grupo se ha realizado a través de la descripción de las especies presentes en hábitat salinos (San Martín et al. 1992, Teillier 1998,

Teillier & Becerra 2003, Ramírez & Álvarez 2012, entre otros) y la evaluación de los rasgos fisiológicos y morfológicos que favorecen la tolerancia a la salinidad

(Rhodes & Felker 1988, Poblete et al. 1991, Bartels & Dinakar 2013). Dichos resultados indican que nuestro país posee una riqueza importante de especies halófitas; sin embargo, la información sobre la diversidad de estas especies está descrita sólo a nivel local. Por ello, consideramos esencial identificar la diversidad de este grupo de especies a nivel nacional con el fin de generar estrategias de conservación y definir sus potenciales usos.

En este contexto, el objetivo de este trabajo es proponer el primer listado de halófitas vasculares presentes en Chile y señalar sus principales parámetros 32 ecológicos utilizando el listado global de especies eHALOPH.

MATERIALES Y MÉTODOS

LISTADO DE HALÓFITAS PRESENTES EN CHILE

Este trabajo utilizó como fuente principal la base de datos eHALOPH. Esta consiste en un registro global de especies halófitas basado en las publicaciones de Aronson (1989) y Menzel & Lieth (2003), que se mantiene en permanente actualización gracias al aporte de investigadores de todo el mundo (Santos et al.

2015). La definición de halófita que sustenta este listado corresponde a aquellas especies capaces de “tolerar una conductividad eléctrica de al menos 7,8 dSm-1

(equivalente a 80 mM de NaCl) durante tiempos significativos de su ciclo de vida”

(http://www.sussex.ac.uk/affiliates/halophytes 09/11/17). Este listado es de libre acceso y está publicado en la web http://www.sussex.ac.uk/affiliates/halophytes/.

Para elaborar el listado nacional de halófitas, se contrastó el listado eHALOPH (consultado el 13 de diciembre de 2017) con las especies presentes en

Chile según el Catálogo de las Plantas Vasculares del Cono Sur (Zuloaga et al.,

2008), disponible en la página web http://www2.darwin.edu.ar/. No se consideraron subespecies ni variedades en la elaboración de este listado.

Para cada especie halófita presente en Chile, se registró la familia, hábito, origen y distribución regional. Esta información se recopiló a partir del mismo catálogo y el inventario nacional de especies de Chile (MMA 2017).

La distribución regional de las especies se presentó en base a 13 regiones

33 administrativas y tres zonas insulares: Archipiélago de Juan Fernández, Islas

Desventuradas e Isla de Pascua. Esta forma de presentación responde a las categorías de distribución utilizada en el Catálogo de Plantas Vasculares del Cono

Sur. Adicionalmente, varias publicaciones fueron utilizadas para complementar los datos faltantes (Marticorena & Quezada 1985, Matthei et al. 1995, Ramírez & San

Martín 2006, Fuentes et al. 2013).

RESULTADOS

RIQUEZA ESPECÍFICA DE LAS FAMILIAS DE HALÓFITAS EN CHILE

El listado de especies halófitas de Chile está compuesto por un total de 138 especies (Anexo 1), distribuidas en 31 familias (Tabla 1). Las familias con mayor riqueza de halófitas son Poaceae con 29 especies, Amaranthaceae con 20,

Fabaceae con 11, Asteraceae con 9 y Cyperaceae con 8 (Tabla 1). De las 31 familias registradas, 26 poseen menos de un 5% de representación. Sólo dos familias tienen más de 10% de representación: Poaceae y Amaranthaceae con

21,01% y 14,49%, respectivamente (Tabla 1).

Del grupo de las gimnospermas, solo se encontró la familia Ephedraceae, representada por una especie, Ephedra ochreata. Las otras 30 familias pertenecen al grupo de las angiospermas, dentro de las cuales hay 26 familias de dicotiledóneas y 5 familias de monocotiledóneas.

DISTRIBUCIÓN, ORIGEN Y HÁBITO DE LAS ESPECIES HALÓFITAS DE CHILE La mayor riqueza de especies halófitas se concentra en la zona centro del país, con una disminución progresiva hacia el extremo sur y el territorio insular (Tabla 2).

Las regiones de Coquimbo (COQ) y Valparaíso (VAL) tienen la mayor riqueza, con

81 especies de halófitas cada una. Le siguen las regiones del Biobío (BIO) con 74 y la Metropolitana (RME) con 70. Al contrario, las regiones de Aysén (AIS) y de

Magallanes y de la Antártica Chilena (MAG) presentan la menor riqueza de especies del territorio continental, con 25 y 37 registros respectivamente. El territorio insular también presenta una baja riqueza de especies halófitas, siendo las islas Desventuradas la zona de menor número de especies en el territorio nacional (6 especies).

De las 138 especies de halófitas que hay en Chile, 4 son endémicas (3%),

58 nativas (42%) y 76 introducidas (55%) (Tabla 2). Las cuatro especies de halófitas endémicas registradas son Prosopis tamarugo, Atriplex atacamensis,

Atriplex repanda y Hordeum brachyantherum, todas presentes en la zona norte y centro de Chile (Anexo 1). En la zona sur, la única especie endémica registrada corresponde a Hordeum brachyantherum, presente en la región de Magallanes y de la Antártica Chilena. En el territorio insular no se registran especies endémicas.

TABLA 1. Distribución de la riqueza de especies halófitas de Chile según su familia (n=138). Los resultados se presentan en orden alfabético. / Distribution of chilean halophyte species richness by its family (n=138). Results presented in alphabetical order.

Familia Riqueza %

Aizoaceae 6 4,35

Amaranthaceae 20 14,49

Anacardiaceae 1 0,72

Apiaceae 2 1,45

Araliaceae 1 0,72

Asteraceae 9 6,52

Boraginaceae 1 0,72

Brassicaceae 5 3,62

Caryophyllaceae 2 1,45

Convolvulaceae 2 1,45

Cyperaceae 8 5,80

Ephedraceae 1 0,72

Euphorbiaceae 2 1,45

Fabaceae 11 7,97

Frankeniaceae 2 1,45

Goodeniaceae 1 0,72

Juncaceae 5 3,62

Juncaginaceae 2 1,45

Malvaceae 2 1,45

Plantaginaceae 7 5,07

Plumbaginaceae 1 0,72

Poaceae 29 21,01

Polygonaceae 3 2,17

Portulacaceae 1 0,72

Potamogetonaceae 3 2,17

Primulaceae 2 1,45

Ruppiaceae 1 0,72

Solanaceae 4 2,90

Typhaceae 2 1,45

Verbenaceae 1 0,72

Zygophyllaceae 1 0,72

Riqueza total 138

En Chile continental, las regiones del extremo norte (Arica y Parinacota,

Tarapacá, Antofagasta y Atacama) apenas superan el 50% de especies halófitas nativas y endémicas (Tabla 2). Esta tendencia se invierte en las regiones de la zona centro y sur del país, donde el porcentaje de halófitas introducidas supera el

50%. En los sistemas insulares este número es aún mayor, con valores sobre el

80%.

El hábito predominante entre las halófitas chilenas es el herbáceo, con 114 especies que representan un 82,6% de la riqueza total de halófitas. Las hierbas perennes son el grupo mayoritario, con el 53% del total de las especies descritas

(Tabla 2). Las especies halófitas leñosas constituyen un grupo menos abundante, con 5 árboles, 13 arbustos y 5 subarbustos, los que en conjunto representan casi el 17% del total de especies. De las 138 especies registradas sólo existe una especie con hábito de enredadera, Calystegia sepium (Anexo 1).

Con respecto a la distribución regional de los distintos hábitos, las hierbas perennes son el hábito de mayor presencia en todas las regiones del país, con al menos un 40% de representación en cada una de ellas (Tabla 2). En la zona sur, este valor es aún mayor (60%).

Las herbáceas anuales, corresponden al segundo grupo más abundante después de las herbáceas perennes en todas las regiones y presentan una mayor riqueza en regiones de la zona norte y centro, con una disminución progresiva hacia el sur (Tabla 2).

La mayor frecuencia de especies leñosas (arbóreas, arbustivas y subarbustivas) ocurre en las regiones de Atacama y Coquimbo con 13 y 12 especies respectivamente (Tabla 2). En cambio, la frecuencia de este grupo de especies disminuye notoriamente hacia el extremo sur y en el territorio insular, con menos de 3 especies.

DISCUSIÓN Y CONCLUSIONES

RIQUEZA DE FAMILIAS HALÓFITAS

Este es el primer estudio que compila la diversidad de especies halófitas presentes en Chile, a partir de un listado colaborativo mundial. En él se presentan

138 especies de halófitas, distribuidas en 31 familias, lo que equivale a un 2,3% de las especies y un 16,8% de las familias de flora vascular descritas para Chile

(Marticorena 1990). Las familias más diversas son Poaceae (29 especies),

Amaranthaceae (20 especies), Fabaceae (11 especies), Asteraceae (9 especies) y

Cyperaceae (8 especies). Si bien esta representación coincide con algunas de las familias de mayor riqueza en Chile (Asteraceae, Poaceae y Fabaceae) (Comisión

Nacional del Medio Ambiente, 2008), la familia Amaranthaceae no aparece en este

38 grupo, a pesar de ser la segunda familia de mayor riqueza de halófitas a nivel nacional (Tabla 1) y la primera a nivel mundial (Santos et al. 2015).

A nivel global, las 138 especies halófitas descritas en Chile representan un

9,3% de las 1479 especies identificadas por la base de datos eHALOPH

(https://www.sussex.ac.uk/affiliates/halophytes/). Este valor está muy por debajo de las 673 especies nativas y endémicas descritas para Argentina a través de esta misma herramienta (Cantero et al., 2016) o las más de 300 especies descritas a partir de registros monográficos y trabajos de terreno en países de Europa

(Grigore, 2008) y Asia (Khan & Qaiser 2006, Ghazanfar et al. 2014). La baja riqueza de halófitas a nivel nacional puede ser reflejo de la baja diversidad de especies en Chile comparada a la de otros países en Sudamérica (Comisión

Nacional del Medio Ambiente, 2008); o bien, estar influenciada por la alta presencia de especies de origen euroasiático en los registros de la base de datos eHALOPH.

ORIGEN DE LAS ESPECIES

Un 55% de las especies halófitas presentes en Chile son introducidas. Del 45% restante, un 42% corresponde a especies nativas, y sólo un 3% a especies endémicas (Tabla 2). Este patrón difiere significativamente al descrito para la flora vascular en Chile, compuesta por más de un 85% de especies nativas (Fuentes et al. 2013), de las que casi la mitad son endémicas (Moreira-Munoz, 2011). Al respecto, es importante considerar que la base de datos eHALOPH se construyó desde dos publicaciones (Aronson 1989, Menzel & Lieth 2003), cuyo objetivo fue la recopilación de especies tolerantes a la salinidad con potencial uso económico. 39

En ese sentido, no nos debe sorprender que, al contrastar este listado con la flora de Chile, se encuentre una presencia importante de especies introducidas, o que las familias con mayor diversidad de halófitas también posean la mayor riqueza de especies introducidas (Fuentes et al. 2013, Fuentes et al. 2014). Así también, la distribución multirregional de este grupo de especies (O’Leary & Glenn 1994) y el escaso conocimiento acerca de halófitas autóctonas en Chile (Ramírez & Álvarez

2012), puede ser otra causante de los resultados observados.

TABLA 2. Riqueza, hábito y origen de las especies halófitas presentes en Chile según su distribución geográfica. Distribución: TAR= Región de Arica y Parinacota, Región de Tarapacá; ANT= Región de Antofagasta; ATA= Región de Atacama; COQ= Región de Coquimbo; VAL= Región de Valparaíso; RME= Región Metropolitana; LBO= Región del Libertador General Bernardo O’Higgins; MAU= Región del Maule; BIO= Región del Biobío; ARA= Región de la Araucanía; LLA= Región de Los Ríos y Región de Los Lagos; AIS= Región de Aysén del General Carlos Ibáñez del Campo; MAG= Región de Magallanes y Antártica Chilena; IDE= Islas Desventuradas; IPA= Isla de Pascua; JFE= Archipiélago de Juan Fernández. Origen: EN= endémica; NA= nativa; IN= introducida. Forma de vida: HA= herbácea anual; HAB= herbácea anual o bianual; HP= herbácea perenne; Ar= arbustiva; Sa= subarbustiva; Ab= arbórea; E= enredadera perenne / Richness, habit and origin of halophyte species present in Chile by geographical distribution. Distribution: TAR= Arica y Parinacota Region, Tarapacá Region; ANT= Antofagasta Region; ATA= Atacama Region; COQ= Coquimbo Region; VAL= Valparaíso Region; RME= Metropolitan Region; LBO= Libertador Bernardo O’Higgins Region; MAU= Maule Region; BIO= Biobío Region; ARA= Araucanía Region; LLA= Los Ríos Region and Los Lagos Region; AIS= Aysén del General Carlos Ibáñez del Campo Region; MAG= Magallanes y Antártica chilena Region; IDE= Islas Desventuradas; IPA= Easter Island; JFE= Archipiélago de Juan Fernández. Origin:

EN= endemic; NA= native; IN= Introduced. Life form: HA= anual herb; HAB= anual or bianual herb; HP= perennial herb; Ar= shrub; Sa= subshrub; Ab= tree; E= perennial vine.

Región Riqueza Hábito (n° especies) Origen (n° especies) HA HP Hab Ab Ar Sa E EN NA IN

TAR 49 11 28 1 2 4 3 0 2 25 22 ANT 58 13 32 4 2 3 4 0 2 29 27 ATA 61 15 30 3 2 8 3 0 2 30 29

Zona norteZona COQ 81 24 42 3 2 6 4 0 2 36 43 VAL 81 23 46 4 2 3 2 1 1 32 48

RME 70 19 40 4 2 3 1 1 1 29 40 LBO 44 15 18 4 2 3 2 0 0 18 26 MAU 59 18 34 3 1 2 1 0 1 23 35 Zona centroZona BIO 74 24 39 4 1 3 2 1 0 27 47 ARA 57 17 33 3 1 1 1 1 0 20 37

LLA 58 15 36 4 0 1 1 1 0 24 34 AIS 25 5 16 2 1 0 0 1 0 12 13

Zona surZona MAG 37 6 25 3 0 2 1 0 1 17 19 IDE 6 2 3 0 0 1 0 0 0 0 6

JFE 26 10 12 2 1 0 1 0 0 3 23 Islas IPA 13 2 9 0 1 0 0 1 0 2 11 Total 138 36 73 5 5 13 5 1 4 58 76 Chile % 100 26,1 52,9 3,6 3,6 9,4 3,6 0,7 2,9 42 55,1 Total Chile

HÁBITO DE LAS ESPECIES HALÓFITAS PRESENTES EN CHILE

Más del 80% de las especies halófitas identificadas en Chile son herbáceas. Este grupo, además, presenta la mayor riqueza en todas las regiones del país, incluso en el territorio insular (Tabla 2). Esta predominancia es llamativa, pues a nivel global, se ha descrito que la relación de riqueza entre halófitas leñosas y

41 herbáceas es más bien equitativa (Aronson 1989, Khan & Qaiser 2006, Ghazanfar et al. 2014). La alta representación de este grupo en Chile se puede explicar por la presencia de ciertos tipos de hábitat que favorecen la colonización de hierbas tolerantes a la salinidad, tales como ecosistemas costeros, humedales y lagunas salobres (San Martín et al. 1992, Teillier & Becerra 2003, Ramírez & Álvarez

2012). Al mismo tiempo, la alta proporción de especies introducidas en el grupo de hierbas halófitas puede ser un reflejo de su éxito en el uso de hábitat naturales y perturbados, tal como lo ha descrito Ramírez et al. (2018) en marismas de la

Región del Biobío.

Por otra parte, las halófitas leñosas en Chile apenas representan un 16,6% del listado total. Pero, al contrario de las herbáceas, la mayoría de estas (82,6%) son nativas o endémicas de las zonas norte y central (Tabla 2). La riqueza de especies leñosas en la zona norte se explica principalmente por la presencia de géneros nativos con dos o más especies (Atriplex, Prosopis, Lycium, Frankenia y

Suaeda), cuya distribución regional se encuentra más bien restringida al territorio entre las regiones de Arica y Parinacota y Coquimbo (Anexo 1). En el norte, aparecen también tres de los cuatro árboles endémicos o nativos presentes en

Chile: Prosopis tamarugo, P. chilensis y Geoffroea decorticans. Al respecto, se ha descrito que la condición de halofitismo entre especies arbóreas es rara (O’Leary

& Glenn 1994). Aún más, si se considera que gran parte de los árboles halófitos descritos a nivel global crecen en manglares, y ecosistemas de interfaz marino- terrestre, sistemas radicalmente distintos a los que alojan a este grupo de especies en Chile.

Al igual que las especies arbóreas, la diversidad de arbustos halófitos es bastante baja. Sin embargo, con una riqueza de 18 especies, 15 de las cuales son nativas y/o endémicas, vale la pena resaltar el componente autóctono de este grupo. Estas especies están representadas por variantes nativas y endémicas de géneros con amplia distribución mundial, como Lycium, Atriplex, Suaeda y

Senecio. Esto podría indicar que, dentro de la gran diversidad de estos géneros, ocurre cierto nivel de adaptación local a ecosistemas salinos.

PERSPECTIVAS PARA EL ESTUDIO DE HALÓFITAS EN CHILE

Hoy en día, existe amplio conocimiento acerca de la fisiología y las estrategias de adaptación que utilizan las especies halófitas para enfrentarse a distintos sistemas salinos (Munns & Tester 2008). Sin embargo, existe un gran vacío de información con respecto a su diversidad a nivel nacional y a las distintas estrategias que utilizan para adaptarse a este tipo de ambientes en Chile.

Al realizar una búsqueda online de libros y artículos científicos con las palabras “halófita” y “Chile”, se encuentran listados de especies que existen en hábitats salinos, análisis paleobotánicos de ecosistemas antiguos, estudios de acumulación y tolerancia a metaloides y estudios acerca del uso de halófitas para la nutrición humana y animal. La mayoría de estos artículos fueron publicados entre 1980 y 2000 y denotan la falta de información sistematizada sobre la riqueza de especies halófitas presentes en Chile y de sus características ecológicas. En este contexto, el presente listado, realizado a partir de los registros obtenidos en la

43 base de datos eHALOPH, se constituye como una primera aproximación para compilar la diversidad de las especies halófitas presentes en Chile. Sin embargo, el enriquecimiento de este listado, ya sea a través de una exhaustiva revisión de fuentes bibliográficas nacionales, y la generación de nuevos aportes a dicha base de datos, requiere de la activa colaboración de investigadores y académicos. De este modo, identificar nuevas especies halófitas y ampliar nuestro conocimiento ecológico acerca de ellas, nos permitirá también identificar nuevas oportunidades para relevar la importancia de esta parte del patrimonio natural a través de su uso en diferentes áreas.

AGRADECIMIENTOS

Proyecto CONICYT FB-0002-2014 (Center of Applied Ecology and Sustainability, CAPES UC). Beca CONICYT doctorado 21141059

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ANEXO 1. Listado de especies halófitas presentes en Chile según la base de datos eHALOPH. Distribución: TAR= Región de Arica y Parinacota, Región de Tarapacá; ANT= Región de Antofagasta; ATA= Región de Atacama; COQ= Región de Coquimbo; VAL= Región de Valparaíso; RME= Región Metropolitana; LBO= Región del Libertador General Bernardo O’Higgins; MAU= Región del Maule; BIO= Región del Biobío; ARA= Región de la Araucanía; LLA= Región de Los Ríos y Región de Los Lagos; AIS= Región de Aysén del General Carlos Ibáñez del Campo; MAG= Región de Magallanes y Antártica Chilena; IDE= Islas Desventuradas; IPA= Isla de Pascua; JFE= Archipiélago de Juan Fernández. Origen: EN= endémica; NA= nativa; IN= introducida. Forma de vida: HA= herbácea anual; HAB= herbácea anual o bianual; HP= herbácea perenne; Ar= arbustiva; Sa= subarbustiva; Ab= arbórea; E= enredadera perenne / List of halophyte species present in Chile according to the eHALOPH database. Distribution: TAR= Arica y Parinacota Region, Tarapacá Region; ANT= Antofagasta Region; ATA= Atacama Region; COQ= Coquimbo Region; VAL= Valparaíso Region; RME= Metropolitan Region; LBO= Libertador Bernardo O’Higgins Region; MAU= Maule Region; BIO= Biobío Region; ARA= Araucanía Region; LLA= Los Ríos Region and Los Lagos Region; AIS= Aysén del General Carlos Ibáñez del Campo Region; MAG= Magallanes y Antártica chilena Region; IDE= Islas Desventuradas; IPA= Easter Island; JFE= Archipiélago de Juan Fernández. Origin: EN= endemic; NA= native; IN= introduced. Life form: HA= anual herb; HAB= anual or bianual herb; HP= perennial herb; Ar= shrub; Sa= subshrub; Ab= tree; E= perennial vine.

Taxa Distribución Origen Hábito T A A C V L M B A L A M R I J I A N T O A B A I R L I A M D F P R T A Q L O U O A A S G E E E A Aizoaceae Carpobrotus chilensis (Molina) N.E.Br. x x x x x x x x NA HA Carpobrotus edulis (L.) N.E. Br. x x x IN HA

Mesembryanthemum crystallinum L. x x x x x IN HA Mesembryanthemum nodiflorum L. x x IN HA Sesuvium portulacastrum (L.) L. x IN HP Tetragonia tetragonoides (Pall.) Kuntze x x x x x x x x x IN HA

Amaranthaceae Atriplex atacamensis Phil. x x x EN Ar Atriplex deserticola Phil. x x x NA SAr Atriplex hortensis L. x x IN HA Atriplex nummularia Lindl. x x IN Ar Atriplex patula L. x x x IN HA Atriplex prostrata Boucher ex DC. x x x x x IN HA Atriplex repanda Phil. x x x EN Ar Atriplex rosea L. x IN HA Atriplex semibaccata R.Br. x x x x x IN HP Atriplex tatarica L. x IN HA Beta vulgaris L. x x x x IN HP Chenopodium album L. x x x x x x x x x x x x x IN HA Chenopodium quinoa Willd. x x x x NA HA Dysphania ambrosioides (L.) Mosyakin & Clemants x x x x x x x x x x x x x IN HP Maireana brevifolia (R. Br.) Paul G. Wilson x x IN Ar Salsola kali L. x x x x x x x x IN HA Sarcocornia fruticosa (L.) A.J.Scott x x x x x x x x x x x IN SAr Suaeda argentinensis A. Soriano x NA Ar Suaeda foliosa Moq. x x x x NA SAr Suaeda patagonica Speg. x NA HA Anacardiaceae Schinus polygamus (Cav.) Cabrera x x x x x x x x x NA Ar Apiaceae

Ammi visnaga (L.) Lam. x x x x x x x IN HA Apium graveolens L. x x x IN HAB Araliaceae Hydrocotyle bonariensis Lam. x x x x NA HP Asteraceae Achillea millefolium L. x x x x x x x x IN HP Amblyopappus pusillus Hook. & Arn. x x x x NA HA Baccharis spartioides (Hook. & Arn. ex DC.) J. Remy x x NA Ar Cotula coronopifolia L. x x x x x x x x x x x x IN HP Cressa truxillensis Kunth x x x x x NA HP Picrosia longifolia D. Don x NA HP Senecio subulatus D.Don ex Hook. & Arn. x x NA Ar Senecio filaginoides DC. x x NA Ar Symphyotrichum squamatum (Spreng.) G.L.Nesom x x x x x x x x x NA HP Boraginaceae Heliotropium curassavicum L. x x x x x x x NA HP Brassicaceae Brassica nigra (L.) W.D.J. Koch x x x x x x x IN HA Lobularia maritima (L.) Desv. x x x x x x x x x IN HP Lepidium latifolium L. x IN HP Lepidium spicatum Desv. x x x x x x NA HP Raphanus raphanistrum L. x x x x x x x x x x IN HAB Caryophyllaceae Spergularia marina (L.) Griseb. x x x x IN HAB Spergularia media (L.) C.Presl ex Griseb. x x x x x x x x x x x x x IN HP Convolvulaceae Calystegia sepium (L.) R. Br. x x x x x x x IN E Calystegia soldanella (L.) Roem. & Schult. x x x x x x x NA HP

Cyperaceae Bolboschoenus maritimus (L.) Palla x IN HP Cyperus laevigatus L. x x x IN HP Cyperus rotundus L. x x x x x IN HP Eleocharis pachycarpa E. Desv. x x x x x x x x NA HP Ficinia nodosa (Rottb.) Goetgh., Muasya & D.A. Simpson x x x x x x x x IN HP Isolepis cernua (Vahl) Roem. & Schult. x x x x x x x x x x x x NA HA Schoenoplectus americanus (Pers.) Volkart ex Schinz & R. x x x x x x NA HP Keller Schoenoplectus californicus (C.A.Mey.) Soják x x x x x x x x x x x x x x NA HP Ephedraceae Ephedra ochreata Miers x NA Ab Euphorbiaceae Euphorbia serpens Kunth x x x x x x x x NA HP Euphorbia terracina L. x IN HP Fabaceae Acacia dealbata Link x x x x x x x x IN Ab Geoffroea decorticans (Gillies ex Hook. & Arn.) Burkart x x x x NA Ab Lotus tenuis Waldst. & Kit. x x x x x IN HP Melilotus indicus (L.) All. x x x x x x x x x x x x x x IN HA Melilotus officinalis (L.) Lam. x x x x IN HAB Prosopis chilensis (Molina) Stuntz x x x x x NA Ab Prosopis reptans Benth. x NA Ar Prosopis strombulifera (Lam.) Benth. x x x x x NA Ar Prosopis tamarugo Phil. x x EN Ab Trifolium fragiferum L. x IN HP Trifolium tomentosum L. x x x x IN HA Frankeniaceae

Frankenia salina (Molina) I.M.Johnst. x x x x x x NA SAr Frankenia triandra J. Remy x x NA SAr Goodeniaceae Selliera radicans Cav. x x x x x x x x x x NA HP Juncaceae Juncus acutus L. x x x x x x NA HP Juncus balticus Willd. x x x x x x x x x x x x NA HP Juncus bufonius L. x x x x x x x x x x x x x NA HA Juncus capitatus Weigel x x x IN HA Juncus kraussii Hochst. x NA HP Juncaginaceae Triglochin palustris L. x x x x x x x NA HP Triglochin striata Ruiz & Pav. x x x x x x x x x x x NA HP Malvaceae Malvella leprosa (Ortega) Krapov. x x x x x NA HP Modiola caroliniana (L.) G.Don x x x x x x x x x IN HP Plantaginaceae Bacopa monnieri (L.) Pennell x x x x NA HA Limosella australis R. Br. x x x x x x x x x x x NA HA Plantago australis Lam. x x x x x x x x NA HP Plantago coronopus L. x x x IN HA Plantago lanceolata L. x x x x x x x x x x x x x x IN HP Plantago major L. x x x x x x x x x x x x x IN HP Plantago maritima L. x NA HP Plumbaginaceae Armeria maritima (Mill.) Willd. x x x x x x x x x x NA HP Poaceae Agropyron cristatum (L.) Gaertn. x IN HP

Agrostis stolonifera L. x x x x x x x x x x x x IN HP Arundo donax L. x x x x x x x IN HP Chloris gayana Kunth x IN HP Cynodon dactylon (L.) Pers. x x x x x x x x x x IN HP Distichlis scoparia (Kunth) Arechav. x x x x x x x x x NA HP Distichlis spicata (L.) Greene x x x x x x x x x x NA HP Echinochloa colona (L.) Link x x x x x x IN HA Echinochloa crus-galli (L.) P. Beauv. x x x x x x x x x IN HA Eragrostis curvula (Schrad.) Nees x x IN HP Festuca rubra L. x x x x IN HP Hainardia cylindrica (Willd.) Greuter x x IN HA Hordeum brachyantherum Nevski x x x x EN HP Hordeum jubatum L. x IN HP Hordeum murinum Huds. x x x x x x x x x IN HA Leptochloa fusca (L.) Kunth x x x NA HP Leymus arenarius (L.) Hochst. x IN HP Parapholis incurva (L.) C.E.Hubb. x x x IN HA Paspalum distichum L. x x x x x IN HP Paspalum vaginatum Sw. x x x x x x x x NA HP Phragmites australis (Cav.) Trin. ex Steud. x x x x x x x x x IN HP Poa lanuginosa Poir. x x x x NA HP Polypogon elongatus Kunth x x x NA HP Polypogon maritimus Willd. x x IN HA Polypogon monspeliensis (L.) Desf. x x x x x x x x x x x IN HA Puccinellia glaucescens (Phil.) Parodi x x x x x x x NA HP Setaria viridis (L.) P. Beauv. x x x x x IN HA Sporobolus virginicus (L.) Kunth x IN HP Stenotaphrum secundatum (Walter) Kuntze x x x IN HP

Polygonaceae Polygonum aviculare L. x x x x x x x x x x x x x x IN HAB Polygonum maritimum L. x x x x x x IN HP Rumex crispus L. x x x x x x x x x x x x x x IN HP Portulacaceae Portulaca oleracea L. x x x x x x x x x x IN HA Potamogetonaceae Potamogeton pusillus L. x x x x x x x x x IN HAB Stuckenia pectinata (L.) Börner x x x NA HP Zannichellia palustris L. x x x x x x x x NA HP Primulaceae Anagallis arvensis L. x x x x x x x x x x x IN HA Samolus repens (J.R. Forst. & G. Forst.) Pers. x x x x x x NA HP Ruppiaceae Ruppia maritima L. x x x x x NA HP Solanaceae Lycium chilense Miers ex Bertero x x x x x x x NA Ar Lycium tenuispinosum Miers x NA Ar Physalis viscosa L. x x x x x x IN HP Solanum peruvianum L. x NA HP Typhaceae Typha angustifolia L. x x x x IN HP Typha domingensis Pers. x x NA HP Verbenaceae Phyla nodiflora (L.) Greene x x x x x x x x x x x IN HP Zygophyllaceae Tribulus terrestris L. x x x IN HA

Chapter 2

Effect of single and combined Cu, NaCl and water stress on three

Atriplex species with phytostabilization potential

Orrego, F.1,2, Ortíz-Calderón, C.3, Lutts, S.4 & Ginocchio, R. 1,2

1Departamento de Ecosistemas y Medio Ambiente, Facultad de Agronomía e Ing.

Forestal, Pontificia Universidad Católica de Chile. Avenida Vicuña Mackenna 4860,

Santiago, Chile.

2Center of Applied Ecology and Sustainability, Facultad de Ciencias Biológicas, CP

6513677, Universidad Católica de Chile, Santiago, Chile

3Departamento de Biología, Universidad de Santiago de Chile, Avenida Bernardo

O´Higgins 3363, Santiago, Chile

4 Groupe de Recherche en Physiologie Végétale, Earth and Life Institute -

Agronomy, Université Catholique de Louvain, Louvain-la-Neuve, Belgium.

Abstract

Phytostabilization of copper enriched substrates in arid and semiarid areas considers the use of metal-tolerant or metallophyte plants to avoid metal dispersion. However, the co-occurrence of other abiotic restrictions on plant growth, such as drought and salinity requires a new perspective for plant selection.

Components of salt and drought tolerance traits of halophytes are also present in metal stress. Under this scenario, Atriplex species, typical of dry and salty soils, emerge as good candidates for phytostabilization. However, in order to confirm this potential, it is necessary to explore specific responses to increasing copper, salt and water stress.

In this study, we compared the effect of single and combined copper, NaCl and water stresses on growth parameters the Chilean A. atacamensis, European A. halimus and Australian A. nummularia under controlled hydroponic assays. Results showed that increasing copper had severe effects on root development, with subsequent effects on shoot biomass. Salt stress effects were mostly osmotic, with a decrease in shoot fresh weight and water content. Yet, PEG- induced water stress had no clear effect on growth of roots and shoots. Combination of copper stress with NaCl and PEG- induced water stress caused a further decrease of plant growth, but this effect varied among species. This shows that growth of Atriplex species responds differently to each individual stress and that stressor combination causes overall negative effects in measured parameters.

Keywords: Stressor, metal enrichment, phytotoxicity, halophytes, saltbush 56

Introduction

Anthropogenic enrichment of metals due to industrial activities, is a worldwide issue that causes major environmental consequences (Gidhagen et al., 2002; De

Gregori et al., 2003). Among them, the accumulation of potentially toxic metals in groundwater and soils do not only could pose direct effects on organisms and affect the development of ecological communities, but could also potentially threaten human health by the transport of metals through food chains (Martínez-

Domínguez et al., 2008; Montenegro et al., 2009). In particular, northern and central Chile presents a wide gradient of metal enrichment from natural and anthropogenic sources (De Gregori et al., 2003). Among them, copper (Cu) stands out because of its high constitutive presence in several mineral formations, but mainly because of its historical enrichment as a result of unregulated mining operations during the XIX and XX century (Ginocchio, 2000; Lam et al., 2016).

Copper is considered a micronutrient for plants, but the range between nutritious and phytotoxic concentrations is narrow. Copper tissue concentration among 5 and

10 ppm is necessary for physiological processes such as photosynthesis, cell wall metabolism and ethylene sensing (Yruela, 2005), but tissue accumulation above this threshold is considered phytotoxic (Kabata-Pendias, 2010; Verdejo et al.,

2015). Typical Cu phytotoxicity symptoms are growth inhibition, browning of roots and leaf interveinal chlorosis and reddening (Reichman, 2002). Copper phytotoxicity depends on its total concentration and other soil physicochemical parameters that determine its bioavailability, such as soil organic matter, dissolved

57 organic carbon and pH (Ginocchio et al., 2002; Zeng et al., 2011). Therefore, in places where the presence of Cu is consistently high, such as chemically degraded soils and hard-rock mine wastes, a detrimental effect in plant communities and ecosystems may be expected (Ginocchio, 2000; Ginocchio et al., 2002; Ortiz-

Calderón et al., 2008). One alternative for the ecological rehabilitation of these areas is phytostabilization, where metal-tolerant plants are selected to sequester metals at a rhizosphere level in order to restrict wind and groundwater dispersion and therefore, in situ stabilization of metals (Mendez and Maier, 2008; Alford et al.,

2010).

In Chile and several other countries, Cu-enriched areas occur in arid and semiarid areas that, as a result of low precipitation and high temperatures, also present variable levels of salinization (Oyarzún and Oyarzún, 2011; Casanova et al., 2013).

Therefore, plant research in a phytostabilization context, must consider not only the effects of a primary metal stressor, but also the co-occurrence of other abiotic restrictions, such as drought and salinity (Ginocchio et al., 2017). Under this conditions, halophytes or salt tolerant species have been proposed as good candidates for phytostabilization due to the presence of salt and drought tolerance traits that are also present in metal stress response (Mittler, 2006; Nikalje and

Suprasanna, 2018).

Atriplex is a globally distributed genus of halophyte herbs and shrubs present in arid areas with varying levels of salinity (Manousaki and Kalogerakis, 2009; Walker et al., 2014). Its colonization success in deserts, Mediterranean and coastal areas is based on their ability to tolerate variable conditions of salt and water stresses

(Lutts et al., 2004a; Brignone et al., 2016a). The genus Atriplex has been proposed for phytostabilization, because it has been found growing near mining areas and metal enriched sites (Mendez and Maier, 2007). Further research on these species has found that they not only survive in these areas, but they can cope with different concentrations of metals and metalloids (Manousaki and Kalogerakis, 2009;

Nedjimi and Daoud, 2009; Mateos-Naranjo et al., 2013; Vromman et al., 2016b).

In Chile, Atriplex is present with a great diversity of native and introduced species

(Rosas, 1989; Brignone et al., 2016a). Atriplex atacamensis, a shrub endemic to the Atacama Desert, grows near mining areas and it has been described as an arsenic tolerant species (Tapia et al., 2013; Vromman et al., 2016a). Further south, in the semiarid Coquimbo Regio, Atriplex nummularia and Atriplex halimus are two exotic species that were introduced in the seventies due to its livestock potential

(Lailhacar et al., 1995). A. nummularia, a fast growing shrub originary from

Australia, has been proposed for the remediation of salt enriched sites, and also tested for its metal tolerance (Jordan et al., 2002). A. halimus, native to the

Mediterranean basin, has been profusely studied in the last 15 years for its potential to resist the effect of several metals (Walker et al., 2014; El-Bakatoushi et al., 2015). Although there is evidence about the potential of these species to tolerate metal stress, there is scarce work on their tolerance to copper and the presence of other abiotic stressors, such as drought and salinity occurring at the same time. Therefore, the aim of the present study was to compare, at a laboratory level, growth responses of A. atacamensis, A. halimus and A. nummularia when subjected to single and co-ocurring copper, salt and water stresses.

Materials and methods

Plant material

Fruits of A. atacamensis were collected in February 2017 along the Loa River

(UTM 19 K: 510307 E 7516731 N), Antofagasta Region of Chile. Atriplex halimus fruits were obtained commercially from Spain in 2017 (Weberseeds), and A. nummularia fruits were obtained in 2015 from the Corporación Nacional Forestal

(CONAF) at the Coquimbo Region, Chile. Fruits were kept dry and in darkness until used. After removal of the bracts, seeds were rinsed several times and submerged in distilled water for three hours to dissolve adhered salts. Then, seeds were left to germinate in separate trays filled with perlite and distilled water under controlled laboratory conditions. Temperature was set at 22±1°C, and relative humidity was about 40%. Natural light was supplemented by Phillips lamps in order to maintain an irradiance of 60 µmol m-2s-2 under a 12 h photoperiod.

After five weeks, eight seedlings at a two-leaf stage were transplanted into polystyrene plates floating on 1 L polypropylene boxes with an aerated Hoagland solution containing 0.2 mM of Mg(SO4)2∙7H2O; 0.5 mM Ca(NO3)2∙4H2O; 0.5 mM

KNO3; 0.1 mM of K2HPO4; 0.2 µM Cu(SO4)∙5H2O; 0.2 µM of Zn(SO4)∙7H2O; 2µM of MnCl2∙4H2O; 10 µM of H3BO3; 0.1 µM of MoO3; and 10.7 µM EDTA chelated Fe

(Harper et al., 1998). Before transplant, all roots were cut to 3 cm to promote homogeneity among naturally heterogeneous individuals. Plants were allowed to acclimate for seven days under these conditions, and then individual tolerance essays for Cu, drought and salinity were performed.

Single-stressor assays

In total, nine experiments were carried out; each one lasted for seven days under the laboratory conditions described above. For Cu treatments, Cu(SO4)∙5H2O was added to the nutrient solution in order to achieve nominal concentrations of 0, 10 or

20 µmol L-1. For drought treatments, polyethylene glycol (PEG), a polymer that reduces water availability for the plant without entering to its tissues, was used. In each treatment, PEG 6000 flakes were diluted into the nutrient solution in order to reach 0, -0.1 and -0.25 MPa according to Michel and Kaufmann (1973). For salinity treatments, NaCl was added in order to reach 0, 1% and 2% concentrations.

Reagents used for nutrient solution and treatments were analytical grade obtained from Merck (Germany). Each experiment had four replicas, represented on polystyrene boxes. The nutrient solution was replaced mid-experiment and the pH was adjusted every two days to 5.3±0.05 with KOH or HCl to favor Cu and nutrient availability in the solution.

At the end of each experiment, five plants per treatment were randomly harvested.

Roots were rinsed with deionized water and gently blotted dry. Roots and shoots were then separated for total length and fresh weight measurements. Root increment was calculated as the difference between root length average of four plants measured at the beginning and the end of the experiments. Dry weight was determined after two days of incubation in an oven at 50°C. Plant water content was calculated using fresh and dry weight measurements according to the following formula (Vromman et al., 2011):

Combined stressors assay

After single stress experiments, a combined assay was performed on A. atacamensis and A. halimus seedlings in order to assess combined stress responses. Six seedlings of each species were transplanted into an aireated modified Hoagland solution that consisted of 1.43 mM of NH4NO3; 323 mM of

NaH2PO4∙2H2O; 512 mM of K2SO4; 750 mM of CaCl2∙2H2O; 1.64 mM of

MgSO4∙7H2O; 11.4 mM of MnSO4∙H2O; 14 mM of Na2MoO4∙2H2O; 57.8 mM of

H3BO3; 0.96 mM of ZnSO4∙7H2O; 0.4 mM of CuSO4∙5H2O and 42.7 mM of Fe-

EDTA (Vromman et al., 2016a). After 1 week of acclimation under these conditions, single Cu (10 µM) conditions, and its combination with NaCl (0.5%) and

PEG (-0.1 MPa) were applied for ten days. Room temperature and humidity were the same as the previous experiment and nutrient solution was replaced once mid- treatment. At the end of the experiment, root length and shoot fresh weight was measured on five seedlings per species.

Statistical analysis

Two independent single stress experiments were performed earlier, with similar trends. Experiments were carried out under a completely randomized design, and significances per species were tested by a one-way ANOVA test with significance levels determined at P<0.05. If results were significant, a Tukey’s test was used to identify differences among groups. In case of non-normality or homogeneity of 62 variances, data sets were compared with a Kruskal–Wallis non-parametric test. On combined stress assays, one-way ANOVA was performed in order to compare treatments on each species, and differences among groups were also identified through Tukey tests. All parametric and non-parametric analysis were performed with the statistical package INFOSTAT (Di Rienzo et al., 2008).

Results

Effect of Cu on plant growth

The effect of available Cu on growth parameters varied among species and treatments. Although mortality was only observed in 11% of A. halimus seedlings exposed to 20 µM Cu (data not shown), a general detrimental effect was observed in all three species at this Cu concentration. Also, a reddish color was observed on the abaxial side of leaves of the three species, especially when subjected to 20 µM

Cu.

In control conditions, root growth of A. nummularia was greater than A. atacamensis and A. halimus; yet, this increase was significantly affected by available Cu. Atriplex atacamensis and A. nummularia were the most affected species, with a 49% and 45% decrease in response to 10 uM Cu, respectively.

This magnitude was lower in A. halimus (18%), but still significant. In all cases, no further variation was observed in response to 20 µM Cu (Figure 1).

Figure 1

Root dry weight of A. halimus and A. nummularia was not significantly affected by

Cu. Yet, a significant decrease (27%) was found in A. atacamensis when subjected to 10 µM Cu (Table 1). Interestingly, shoot dry weight of A. atacamensis and A. nummularia had a non-significant decrease under Cu treatments, whilst A. halimus had a significant decrease (P<0.05, H=4.7) in response to 20 µM Cu (Table 1).

Plant water content was also affected by copper treatments (Figure 2), but only A. halimus and A. atacamensis had a significant reduction at 10 µM Cu which continued under 20 µM Cu treatments (Figure 2). At the end of the experiment, total reduction of plant water content of A. halimus and A. atacamensis subjected to 20 µM Cu was 58% and 36%, respectively. In contrast, water content of A. nummularia significantly decreased under 10 µM Cu treatments, with no further significant decrease in response to 20 µM Cu (Figure 2).

Impact of salinity on plant growth

Seven percent of A. nummularia seedlings treated with 2% NaCl died during the experiment. The rest of the plants remained alive, but with different levels of growth disruption. Although an increase in root length was observed on seddlings of the three species of Atriplex treated with 1% NaCl, only root length of A. atacamensis had no significant decrease when compared to control conditions (Figure 1). Two percent NaCl treatments did cause a significant decrease (31%) of A. atacamensis

64 root growth compared with the 42% and 43% decrease of A. halimus and A. nummularia under the same conditions (Figure 1).

Root dry weight of the species was not affected by 1% NaCl, but under 2% NaCl a significant decrease of this parameter was found on A. halimus (17%) and A. nummularia (30%) seedlings. Shoot dry weight of the species did not decrease in response to NaCl, but A. atacamensis and A. nummularia had a non-significant

15% and 21% increase of shoot dry weight in response to 1% NaCl. Plant water content of all three Atriplex species decreased in response to 2% NaCl, but only A. atacamensis was also significantly affected by 1% NaCl (Figure 2). Wilting and a decrease in leaf growth was visible, especially on A. halimus, that showed a 38% decrease of water content at 2% NaCl treatments.

Effect of solute potential reduction on plant growth

A reduction of solute potential by the addition of PEG did not cause evident growth impairments in Atriplex selected species. Still, 5% of Atriplex atacamensis seedlings subjected to -0.25 MPa died during the experiment (data not shown).

Concurrently, the decrease of solute potential did not cause a significant effect on root length in any of the Atriplex species (Figure 1); on the contrary, growth followed a similar pattern to control conditions. Dry weight of Atriplex species also remained unaffected whatever the treatment (Table 1), but the reduction of solute potential did cause a significant decrease of plant water content in A. nummularia

65 and A. halimus, that reached 20% under -0.25 MPa for both species. Atriplex atacamensis was not affected by applied treatments (Figure 2).

Effect of combined stresses on A. halimus and A. atacamensis

As it was observed on single-stress assays (Figure 1), roots of A. atacamensis were bigger than A. halimus in control conditions. When exposed to 10 µM Cu, root growth of A. atacamensis had a significant decrease of 53%, but A. halimus was not affected (Figure 3). Combination of Cu with NaCl or PEG caused a further decrease in root length of A. atacamensis, and whilst the difference was not significant, plants subjected to the combination of Cu and NaCl had the lowest growth.

Similar to root length, shoot fresh weight of A. atacamensis significantly decreased in response to single Cu and its combination with NaCl and PEG. This decrease was more marked under the Cu+PEG treatment, where it reached 55% of control conditions (Figure 3). The response of A. halimus was slightly different; unlike single stress assays, shoot fresh weight did not significantly vary when subjected to 10 µM Cu. Interestingly, when Cu was combined with NaCl, shoot fresh weight had a 40% non-significant increase. Finally, the combination between Cu and PEG had a negative effect on A. halimus, with a significant 65% decrease (Figure 3).

Table 1

Figure 2

Figure 3

Discussion

Growth parameters of Atriplex species vary under control conditions

In this work, nine experiments were performed on three Atriplex species, either native to Chile or from the European Mediterranean and Australia. As expected, plant growth parameters of these species greatly varied under control conditions; whereas A. nummularia showed high shoot biomass production, A. atacamensis had high root elongation. In contrast, A. halimus seedlings had the lowest root and shoot growth. These results differ from other studies, that reported a similar biomass production and height between A. halimus and A. atacamensis cuttings

(Tapia et al., 2013), or a higher biomass production of two A. halimus clones over

A. nummularia seedlings (Silveira et al., 2009). Growth differences among species are to be expected, especially in wild varieties. High variation and differentiation could be a reflection of each species growth potential and its underlying strategies when subjected to different conditions. Therefore, it is important to consider growth under control conditions in order to better understand their response range when subjected to single and combined stress conditions.

Effect of intermediate Cu and NaCl concentrations on growth

As halophytes, Atriplex species would be expected to show a positive response to salinity prior to the appearance of detrimental effects caused either by osmotic or toxicity conditions (Flowers et al., 2015). Yet, on this study, NaCl beneficial effects were not observed. Our data shows that 1% NaCl had no effect on A. halimus shoot dry and fresh biomass, whilst a non-significant increase was found on A. nummularia and A. atacamensis. Other studies show that beneficial effect of NaCl varies among Atriplex species and experimental conditions. For example, Nemat

Alla et al. (2011) found an increase in shoot dry and fresh weight of A. halimus seedlings grown in pots in response to 50 mM (0.3%) NaCl, similar to the results found by Bendaly et al. (2016) on A. halimus cuttings grown in hydroponics.

Vromman et al. (2016a) found no significant variation on growth parameters of A. atacamensis seedlings subjected to 100 (1.7%) mM NaCl and Silveira et al. (2009) found that NaCl concentration between 100 (0.58%) and 300 (1.75%) mM promoted growth on A. nummularia seedlings. Most of these studies show that the transition between beneficial effects and the expression of stress signals seems to occur around 100 mM (0.6%) NaCl. Above this point, there is an onset of detrimental effects, related to an early decrease of substrate water potential and a late response of Na toxicity and nutrient imbalance (Munns, 2002; Verslues et al.,

2006).

Salt concentrations used on this study were equivalent to 170-340 mM, higher than the suggested threshold; also, direct exposition to NaCl provided by the use of an hydroponic design could have increase contact between roots and this stressor,

68 which could explain the absence of a positive effect on growth. In this scenario, growth on halophyte species could be sustained by the expression of tolerance strategies that aim to avoid toxic effects of NaCl ions, and osmotic imbalance. One of these strategies refers to Na accumulation in leaf vacuoles, a response that has been described both as a detoxifying mechanism to avoid Na toxicity in the cytosol and as inexpensive osmolyte for water regulation. On this subject, Belkheiri &

Mulas (2013) found that Na accumulation in A. halimus shoots was two times higher than A. nummularia. In fact, Na levels of A. nummularia under salinity treatments were not significantly different from control conditions. On the other side, Vromman et al. (2016a) found an increase in Na concentration in roots and leaves of A. atacamensis under NaCl treatments. Selective strategies among

Atriplex species, and its possible co-occurrence of other morphological and physiological mechanisms are still a research subject on this group. Therefore, further observations are needed to understand the role of Na and other molecules on the osmotic and toxicity component of NaCl stress on these species.

Similar to salinity, no significant negative effects on plant growth were found for

PEG-induced water stress treatments. Although root length and biomass production were not significantly affected, a decrease tendency was found on shoot fresh biomass on A. atacamensis and A. halimus that was only significant for

A. nummularia. Even though no clear negative effect of PEG was found on

Atriplex, other studies have found that this component can cause metabolic irruptions and has a double effect on water acquisition by the plant: it decreases water availability to roots by changing solute potential of nutrient solution and

69 creating a physical layer that decreases root access to water (Shi et al., 2015).

Therefore, it is possible that the magnitude of the effect of PEG on plant growth was hidden under the slight biomass variation in roots, and small seedling size, which did not allow to observe the full magnitude of the effect.

Detrimental effect of Cu and NaCl on growth parameters of Atriplex species

Our findings indicate that Cu treatments had a negative effect on growth parameters of all three species. However, when species-specific response was compared, we found that A. halimus and A. nummularia were less affected by Cu tan A. atacamensis. One of the first responses of plants to Cu addition into the media is to avoid Cu transport to leaves in order to avoid its oxidative effects on leaf biomolecules (Mateos-Naranjo et al., 2013). Most plants achieve this by root sequestration (Lange et al., 2017), where early Cu toxicity effects, such as root growth inhibition and suberization, can manifest (Marques et al., 2018). Decrease in functional root surface would restrict water and nutrient acquisition and transport to shoots, as observed on A. atacamensis. These species registered severe root damage under Cu treatments, and a significant decrease of shoot biomass and plant water content in response to 10 µM Cu. Since A. atacamensis seeds were collected in a heavily Cu enriched site (62.8 µM available Cu), it is pertinent to ask whether those populations were able to tolerate Cu due to a decrease of soil Cu bioavailability, the expression of other strategies (i.e., avoidance or exclusion) or its association with soil microorganisms, as it has been described for these species and A. nummularia in arid regions of Chile (Aguilera et al., 1998).

In order to avoid the mismatch between field and laboratory conditions, assessment of toxicity symptoms in plants should be closely connected to understandable and replicable metal concentrations. Regretfully, the array of concentrations used on Cu toxicity assays is large (Han et al., 2012; Marques et al., 2018), making proper interpretation and comparison difficult, even among species of the same genus. It is known that Cu bioavailability is mostly governed by substrate pH and organic matter content (Cataldo and Wil, 1978), therefore, we suggest to consider these parameters on the selection of experimental conditions and ecological interpretation.

Root growth of A. nummularia and A. halimus was significantly affected by Cu treatments, but unlike A. atacamensis, positive growth occured even under the highest Cu concentration. Since root elongation measurements were made against day one, we believe that these two species underwent residual growth before Cu toxicity symptoms appeared. The fact that these two species were able to sustain growth under those circumstances, and that overall growth parameters were less affected than A. atacamensis, could indicate a broader tolerance range to Cu, or the presence of evasion strategies in order to limit Cu transport and assimilation. In fact, Mateos-Naranjo et al. (2013) showed that A. halimus is able to tolerate intermediate Cu concentrations, without accumulating Cu in roots. Once a threshold is passed, Cu causes a decrease in photosynthetic rate and growth.

More studies on the effect of Cu on growth, ion accumulation and physiological indicators of Atriplex species could shed some light into different strategies among

71 species to evade or tolerate high concentration of metals in experimental settings and natural conditions.

High NaCl concentrations also caused a significant decrease in Atriplex growth parameters. We propose that they were mostly related to the osmotic component of NaCl stress, because shoot fresh weight and water content decreased in response to 2% NaCl, but root and shoot dry weight did not. This could mean that physiological mechanisms that allow biomass production were not affected.

Studies performed by Nemat Alla et al. (2011) and Boughalleb & Denden (2011) revealed that A. halimus had optimal growth from 50 up to 300 mM NaCl. Silveira et al. (2009) found similar values for A. nummularia cuttings, and Vromman et al.

(2016a) found that A. atacamensis was able to grow under at least 100 mM NaCl.

In this study, we used NaCl concentration ranging from 171 to 342 mM, and the most negative results were found only on the higher treatment. Unlike A. halimus and A. nummularia, whose salt tolerance has been explored under a soil remediation context, there are no studies that explore A. atacamensis tolerance to salinity. A greater understanding of A. atacamensis halophytism could give clues to understand how it deals with the osmotic and ionic component of salt stress, and how that can help to understand its response to metal stress.

Effect of combined stresses on Atriplex

Numerous studies have investigated metal, drought and salt stress independently, but few have examined the consequences of its interaction. Gathered evidence on

72 combined stress research show that the combination of two or more stresses creates a new state, different than the effect of each individual stressor (Mittler,

2006), and that on most cases, this combination has a greater negative impact than single stress conditions (Choudhury et al., 2017). In our experiments, we found that combined stresses caused different responses on root elongation and shoot fresh weight of A. atacamensis and A. halimus. As it was observed on the first assay, root elongation of A. atacamensis was severely affected by single Cu treatments. Here, we found that the combination of Cu and PEG caused a further significant decrease on A. atacamensis but had no effect on A. halimus. Visual observations confirmed that root morphology of A. halimus was less affected than

A. atacamensis under single Cu conditions (Figure 4). However, on a combined stress scenario, root browning and thickening was observed on both species. More research is needed to confirm whether this response was linked to single PEG or its combination with Cu, but a prior study performed by Chazen, Hartung &

Neumann (1995) found that, beside the osmotic effect of PEG on nutrient solution, there was also an additional flow inhibition on roots that was not observed on isosmotic treatments NaCl and KCl on Zea mays seedlings. If this is the case, it could explain why Cu and PEG combined treatments caused root impairments and a significant decrease in shoot fresh weight of both species. Therefore, it would be interesting to explore the effect of single and combined PEG effect on root anatomy and function, and how it may impact the on whole plant water relations.

Figure 4

Single salinity assays established that intermediate concentrations of NaCl could have beneficial effect on Atriplex growth, namely because halophyte species display strategies to tolerate its ionic and osmotic components (Flowers and

Colmer, 2008). Therefore, it could be expected that NaCl treatments would either improve tolerance conditions of Atriplex subjected to Cu, or cause an additive negative effect. Results showed that, once again A. atacamensis and A. halimus had different responses under combined treatments. Shoot fresh weight of A. atacamensis significantly decreased in response to the combination of Cu and

NaCl, but A. halimus was not affected, and even had an increase tendency. The fact that the effect of Cu and NaCl combination on A. atacamensis was not significantly different than single Cu, suggests that the toxicity component of NaCl stress was not operating on A. atacamensis seedlings. The fact that an increase tendency was found on fresh weight of A. halimus shoots could imply that NaCl was either not being perceived as a stress, or it was actively being used to maintain biomass production.

On this context, studies that address the effect of NaCl on metal stress show contrasting results. Ghnaya et al. (2007) found that 400 mM NaCl improved growth rate and biomass of Sesuvium portulacastrum cuttings subjected to Cd, but

Sghaier et al. (2015) observed no effects of added NaCl on growth parameters of the shrub Tamarix gallica subjected to As. The combination of As and NaCl was also studied on A. atacamensis with similar results. But here, it was also found that the lack of a negative effect of As could be related to a decrease of As

74 accumulation in roots when NaCl is present. Bankaji et al. (2014) compared the response of A. halimus and Suaeda fruticosa to the combination of Cu and Cd and the effect of its combination with NaCl, and found that addition of 200 mM NaCl with either 400 µM Cu or 400 µM Cd had no beneficial effect on biomass or chlorophyll content of S. fruticosa, but it did alleviated the detrimental effects of Cu on A. halimus. Overall, it seems that NaCl effects on metal tolerance of halophytes are not a generalized trait, and it depends on their specific metal tolerances. On

Atriplex species, metal tolerance has been explored mostly on A. halimus (Lutts et al., 2004a; Manousaki and Kalogerakis, 2009; Bankaji et al., 2016b) and only recently on A. atacamensis (Tapia et al., 2013). While this does not respond if halophyte species are more tolerant to metals than glycophytes, it does offer a window to further study the performance of multi-tolerant species on challenging environmental scenarios.

Conclusion

It is clear that more studies are needed to explore specific responses or physiological patterns of the effect of metal enrichment among halophytes and the contribution of other stresses to their response. This is particularly relevant on species such as Atriplex that have already been proposed as potential species for the rehabilitation of saline, degraded and metal enriched soils. Information about the specific effect of salt, water and metals stress in Atriplex species, as well as the effect of their combination on their physiology and growth are tools that will allow

75 better understanding the role of these species in their ecosystems and putting toxicity and stress studies into context.

Acknowledgments

This study was funded by the Comisión Nacional de Investigación Científica y

Tecnológica (CONICYT) and Center of Applied Ecology and Sustainability

(CONICYT PIA/BASAL FB0002). F. Orrego was funded by CONICYT-

PFCHA/Doctorado Nacional/2014-21141059. We thank Corporación Nacional

Forestal (CONAF) for providing A. nummularia seeds for the experiments. F.

Orrego would also like to acknowledge Luz María de La Fuente, Rebeca Garay,

Natalia Rodríguez, Rocío Rivera, Romina Jara and Karina Rubio, for their dedication and help during the essays.

References

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7.0

5.3 b 3.5 b 1.8 b a a a a 0.0 a a

0 10 20 Increaselengthrootin (cm) 7.0 Added copper7.0 (umol -1) 7.0 a Atriplex atacamensis Atriplex halimus a a 5.3 Atriplex nummularia5.3 5.3 a b a a b a 3.5 3.5 3.5 a b a b b LR-A.n delta c ab 1.8 1.8 DeltaLR-num 1.8 b a a a a a a a a a a

Increaselengthrootin (cm) 0.0 0.0 0.0 0 10 20 0 1 2 0 -0.1 -0.25 Added copper (umol -1) NaCl concentration Solute potential (MPa)

Figure 1. Root length increase of A. atacamensis, A. halimus and A. nummularia seedlings subjected to increasing Cu, NaCl and a decrease of solute potential.

Bars represent the mean of four replicates ± EE and letters denote significant differences among treatments on each species, according to a one-way ANOVA or

Kruskal Wallis and Tukey test (P≤0.05)

7.0

5.3 b 3.5 b 1.8 b a a a a 0.0 a a 0 10 20 Increaselengthrootin (cm) Added copper (umol -1)

14.0 Atriplex atacamensis14.0 14.0 Atriplex halimus c b Atriplex nummularia b b b b c b ab b 10.5 b 10.5 b 10.5 ab b a a a a b a a a a a a 7.0 a 7.0 a 7.0

3.5 3.5 3.5

Plant water content (g g-1) H2O 0.0 0.0 0.0 0 10 20 0 1 2 0 -0.1 -0.25 Added Copper (µmol-1) NaCl concentration (%) Solute Potential (Mpa)

-1 Figure 2. Plant water content (g H2O g ) of Atriplex atacamensis, A. halimus and A. nummularia seedlings subjected to increasing available Cu, NaCl and a decrease of solute potential. Bars represent the mean of four replicates ± EE and letters denote significant differences among groups of each species when compared to control, according to a one-way ANOVA or Kruskal Wallis and Tukey test (P≤0.05).

24 B 1.6 c

18 1.2 bc C b 12 a 0.8 B a AB a A 6 A a a A A 0.4

0 Shoot fresh weight (gr) 0.0 Increase in root length (cm) Cu10 Cu10+PEG Cu100 Cu10+PEG Cu0 Cu10+NaCl Cu0 Cu10+NaCl Treatment Treatment

Figure 3. Increase in root length (cm) of Atriplex atacamensis and A. halimus subjected to 10 uM Cu and its combination with 0.5% NaCl and 7.85 mM PEG.

Bars represent the mean of five replicates ± EE. Letters denote differences among groups of Atriplex atacamensis (uppercase) and Atriplex halimus (lowercase) when compared to control, according to a one-way ANOVA or Kruskal Wallis and Tukey test (P≤0.05)

A.halimus A. atacamensis

Control

Cu+PEG

Figure 4. Picture of A. halimus and A. atacamensis seedlings under control, Cu and Cu+PEG treatments of combined stress assay. The bar represent 10 cm in length.

Table 1. Shoot length (SL), dry root weight (DRW) and dry shoot weight (DSW) of Atriplex atacamensis (AA), A. halimus

(AH) and A. nummularia (AN) seedlings subjected to available Cu, NaCl and a PEG-induced water stress for seven days.

Data are the means of four replicates ± EE. Considering the same lines, different letters denote significant difference between treatment groups according to a one-way ANOVA or Kruskal Wallis and Tukey test (P≤0.05).

Treatments Copper addition (µmol L-1) Salinity (% NaCl) Solute potential (-MPa) Growth Species 0 10 20 0 1 2 0 0.1 0.25 parameters SL (cm) AA 5.7±0.15a 5.6±0.2a 5.5±0.25a 6.3±0.15b 5.8±0.1b 5.1±0.1a 2.4±0.1a 2.2±0.01a 2.9±0.2b AH 3.9±0.2b 2.9±0.1a 2.9±0.1a 3.9±0.1b 3.7±0.15ab 3.4±0.1a 2.9±0.01a 3±0.1a 2.8±0.1a AN 3.9±0.2b 3.1±0.1a 3.3±0.1a 3.6±0.2a 3.4±0.05a 3.1±0.05a 3±0.1a 3.1±0.1a 2.9±0.1a RFW (mg) AA 15.8±1.64a 11.9±3.4a 7.3±0.2a 15.5±3.9b 10.6±0.5ab 5.6±0.3a 3.1±0.3a 3±0.1a 4.5±0.8a AH 5.8±0.8b 2.2±0.1a 1.9±0.3a 3.7±0.3b 3.2±0.2b 2±0.2a 0.7±0.2a 1.4±0.1a 1.2±0.4a AN 10.4±1.2b 6.4±0.6a 7.1±0.7a 10.1±0.7b 9.3±0.4b 5.5±0.3a 4.6±1.3a 5.9±0.95a 4.1±0.95a SFW (mg) AA 81.7±6.7b 57.8±4.5a 46.2±1.6a 91±9.5ab 101.9±4.4b 70.5±5.0a 18.5±0.4a 15.8±1.5a 15.2±1.5a AH 33.8±5.0b 15.7±0.3a 11.4±0.7a 34.7±2.8b 32.5±3.8ab 22.6±1a 23.9±2.5a 22.7±1.7a 19.2±0.6a AN 64.6±3b 36.3±2.1a 37.4±3.1a 51.9±4.4ab 63.1±4.8b 37.2±6.2a 50.1±3.5b 45.5±5.5ab 29.4±3.5a RDW (mg) AA 1.1±0.1b 0.8±0.1a 0.8±0.04ab 1±0.05a 1±0.05a 0.8±0.1a 0.5±0.1a 0.5±0.1a 0.6±0.04a AH 0.3±0.02a 0.4±0.1a 0.4±0.2a 0.3±0.03b 0.2±0.02ab 0.2±0.02a 0.1±0.04a 0.2±0.05a 0.2±0.03a AN 1.0±0.1a 0.8±0.1a 0.7±0.1a 0.9±0.1b 0.8±0.01ab 0.6±0.07a 0.7±0.1a 0.7±0.1a 0.5±0.1a SDW (mg) AA 7.4±0.6a 5.8±0.5a 5.9±0.2a 8.0±0.5a 9.2±0.3a 8.1±0.6a 2.1±0.1a 1.8±0.2a 1.8±0.2a AH 2.8±0.4b 1.9±0.1ab 1.8±0.1a 3.3±0.3a 3.2±0.4a 3.2±0.2a 1.1±0.1a 1.1±0.1a 1.2±0.01a AN 5.6±0.3a 4.6±0.05a 5±0.5a 4.4±0.3a 5.2±0.4a 4.4±0.5a 3.9±0.2a 3.7±0.3a 2.8±0.3a

Chapter 3

Growth and physiological effects of combined Copper, NaCl and water

stresses on Atriplex atacamensis and A. halimus

Orrego, F.1,2, Ortíz-Calderón, C.3, Lutts, S.4 & Ginocchio, R. 1,2

1Departamento de Ecosistemas y Medio Ambiente, Facultad de Agronomía e Ing.

Forestal, Pontificia Universidad Católica de Chile. Avenida Vicuña Mackenna 4860,

Santiago, Chile.

2Center of Applied Ecology and Sustainability, Facultad de Ciencias Biológicas, CP

6513677, Universidad Católica de Chile, Santiago, Chile

3Departamento de Biología, Universidad de Santiago de Chile, Avenida Bernardo

O´Higgins 3363, Santiago, Chile

4 Groupe de Recherche en Physiologie Végétale, Earth and Life Institute -

Agronomy, Université Catholique de Louvain, Louvain-la-Neuve, Belgium.

Abstract

Halophyte species have been proposed as good candidates for the phytostabilization of metal enriched sites in arid and semiarid ecosystems, but co- occurring conditions, such as salinity and water stress, can affect plant growth and colonization. In this work, we determined the effect of single and combined copper, salt and water stress on growth and tolerance strategies used by two xerohalophyte species of the genus Atriplex: Chilean A. atacamensis and

European A. halimus. Seedlings of both species were subjected to 5 and 10 uM

Cu, 0.5% NaCl and 7.85 mM PEG in hydroponic culture to create single and combined stress treatments. Single Cu decreased growth parameters of both species, with a higher effect on A. atacamensis. Copper accumulation was higher in roots, which can be related to an increase of root non-protein thiols. Single NaCl had no negative effects on growth, but increased leaf Na. Single PEG decreased shoot growth, especially in A. halimus, but had no effect on GSH expression.

Combination of Cu, NaCl and PEG further decreased growth parameters, but did not decrease glutathione, proline or non-protein thiol expression. Atriplex atacamensis and A. halimus are differently affected by combined stresses, but both species show an active tolerance response to the presented conditions.

Keywords

Tolerance mechanisms, phytostabilization, halophytes, combined stresses

Introduction

Copper (Cu) is an element widely spread in soils and a biotic constituent of living organisms (Richardson, 1997; Nagajyoti et al., 2010). At plant level, Cu is an essential micronutrient involved in photosynthetic processes, cellular respiration, cell wall metabolism and lignin formation (Reichman, 2002; Nagajyoti et al., 2010).

However, exposure to high bioavailable Cu concentrations can cause severe effects on plant growth and development, such as photosynthesis inhibition, disruption of cell membrane integrity, root browning, interveinal chlorosis and finally wilting (Reichman, 2002; Ye et al., 2014).

Anthropic soil Cu enrichment, derived from long term mining operations among other sources, has caused serious consequences to productive lands, natural ecosystems, and human health (Tordoff et al., 2000; Wang and Liu, 2003; Lam et al., 2016). In this context, phytostabilization or the use of metal tolerant plants for in situ inmobilization of potential pollutants at soil level has been proposed as a cost-effective alternative to control metal dispersion and reduce environmental risks (Ginocchio and Baker, 2004; Heckenroth et al., 2016). However, depending on site-specific conditions (i.e climate), metal enriched soils could coexist with other co-ocurring stresses, such as drought and salinity, which can further restrict phytostabilization efforts (Mendez and Maier, 2007; Ginocchio et al., 2017). In these conditions, combination of two or more abiotic stressors may result in new conditions for plant development, different from the effect of each stressor by itself

(Mittler, 2006; Zandalinas et al., 2018). Therefore, plant selection for

89 phytostabilization of metal enriched soils must consider not only specific metal stressors, but the presence of multiple co-occurring stresses and their effect on plant growth and development.

It has been proposed that halophyte species could be good alternatives for phytostabilization of metal enriched soils in arid and semiarid environments

(Mendez et al., 2007; Parraga-Aguado et al., 2014). Physiological strategies developed by halophytes to cope with osmotic and ionic effects of salt stress are shared with tolerance mechanisms developed for both metal (Lutts and Lefevre,

2015) and water stresses (Manousaki and Kalogerakis, 2011b; Hamed et al.,

2013). These shared physiological responses correspond to augmented antioxidant response (Ozgur et al., 2013), compatible osmolyte synthesis, ion chelation and sequestration in intracellular compartments (Bankaji et al., 2015).

The genus Atriplex comprises a group of halophyte species that grow in arid and semi-arid regions of the world (Brignone et al., 2016b). The ability of these species to colonize xerophytic areas with different levels of soil salinity has outlined them as candidates for the stabilization of metal enriched soils. In particular, Atriplex halimus, a shrub native to the European Mediterranean and A. atacamensis, endemic to the Atacama Desert, north of Chile, have shown variable levels of tolerance to metals (i.e Cd, Zn, Pb) and metalloids (i.e., As) (Lutts et al., 2004b;

Lefevre et al., 2010; Vromman et al., 2011; Tapia et al., 2013; Walker et al., 2014).

Further studies on these species have shown that combination of metal and salt stresses have a different effect on plant growth and physiological status than single stress conditions (Manousaki and Kalogerakis, 2009; Bankaji et al., 2016b;

Vromman et al., 2016a, 2017). Although work has been done to understand the physiological response of these species when exposed to combined stresses, the effect of copper excess and its combination with water and salt stresses have not been evaluated yet. Therefore, in the present study, we assessed and compared growth parameters and physiological response mechanisms of A. atacamensis and

A. halimus when subjected to single and combined conditions of Cu, water and salt stress under controlled laboratory conditions.

Materials and methods

Plant material and growth conditions

Fruits of Atriplex atacamensis were collected in February 2017 along the Loa River near Calama city (UTM 19 K: 510307 E 7516731 N) at the Antofagasta Region, located in northern Chile. A composed sample from those soils showed that total copper concentration was 15.7 mg Kg-1 with a pH of 8.1 and 28.7 meq L-1 soluble

Na. Fruits of Atriplex halimus collected in Spain were obtained commercially on

August 2017 from Weberseeds.

Seeds from debracted fruits of both species were germinated in 4 L polypropylene boxes with commercial compost. After 4 weeks, a total of 384 seedlings with three pairs of fully developed leaves were transferred into a hydroponic culture on 1.5 L polypropylene boxes with a modified Hoagland solution. The experimental nutrient solutions were changed once during the experimental assays. Nutrient solution consisted of 1.43 mM of NH4NO3; 323 mM of NaH2PO4∙2H2O; 512 mM of K2SO4;

750 mM of CaCl2∙2H2O; 1.64 mM of MgSO4∙7H2O; 11.4 mM of MnSO4∙H2O; 14 mM of Na2MoO4∙2H2O; 57.8 mM of H3BO3; 0.96 mM of ZnSO4∙7H2O; 0.4 mM of

CuSO4∙5H2O and 42.7 mM of Fe-EDTA (Vromman et al., 2016a).

After one week of acclimation, 12 single and combined treatments of water, Cu and salt stresses were applied to nutrient solutions for each species under a factorial design. For water stress treatments, Polyethylene glycol 6000 (PEG) was applied in order to obtain 0 (Control) and -0.1 MPa of solute pressure according to Michel and Kaufmann (1973). For metal stress, plants were exposed to 0 (control), 5 and

10 µM CuSO4∙5H2O. Finally, for salt stress, NaCl was applied in order to obtain 0

(control) and 0.5% of NaCl in the nutrient solution. Reagents used for nutrient solution and treatments were analytical grade obtained from Merck and Sigma-

Aldrich (Germany). Salt and Cu treatments were selected according to Cu measurements in natural and enriched areas of north and central Chile (Badilla-

Ohlbaum et al., 2001; Ortiz-Calderón et al., 2008; Santibáñez et al., 2008; Díaz et al., 2011) and single stress assays performed previously on both Atriplex species

(Orrego et. al in prep). Each treatment was applied to 32 plants (8 plants per box).

Experimental boxes were kept in a phytotron with controlled conditions. Room temperature was maintained at 24 ±1 °C, with a photoperiod of 12 hours and a relative humidity of 55%. Since Cu availability strongly depends on pH, all treatments were adjusted to a pH of 5,3 ± 0.5 with HCl 0.5M and KOH 0.5M.

Plant growth measurements

Five plants per treatment combination were randomly harvested after 10 days. At each harvest, plants were washed with distilled water and blotted dry carefully.

Then, root length was measured and fresh weight of roots and shoots were determined. Afterwards, plants were dried for 48 h at 45°C for dry weight measurement of root and shoot biomass.

Extraction and quantification of chlorophyll and carotenoids

For total chlorophyll (Chl a and b) and total carotenoid quantification, 100 mg leaves were ground with 8 mL of 80% acetone, and then centrifuged at 3000 rpm for 10 min at 4 °C. The absorbance of the samples supernatant was determined

(D) at 663.2 nm, 646.8 nm for chlorophyll, and 470 nm for carotenoids. The pigment concentrations were calculated according to the following equations

(Lichtenthaler, 1987):

Water balance

For plant water content (WC) measurement (%), dry (DW) and fresh weight (FW) of four individuals per treatment were used to calculate root and shoot WC according to the formula:

For osmotic potential (Ψ) determination, the method of (Bajji et al., 1998)was used.

Four leaves of the middle section of three plants per treatment were quickly collected, placed in three Eppendorf tubes with three bottom holes and stored in liquid nitrogen. Frozen leaves were then subjected to three cycles of thawing and refreezing. Then, each Eppendorf tube was encased individually on another tube and centrifuged at 8000 RPM for 15 minutes at 4°C. The collected sap was used to measure osmolarity with a vapor pressure osmometer (Wescor 5500, USA) and converted from mosmoles kg-1 to MPa using the following formula according to the

Van’t Hoff equation (Zhu et al., 2001).

For proline quantification, 200 mg of fresh grinded leaves were extracted in a hot bath with 10 mL of 3% salicylic acid. Then, free proline was quantified according to

Bates et al. (1973) using a spectrophotometer (Beckman DU-640, USA).

Ion quantification

For Cu, Na and K quantification, 50 to 100 mg of leaves and 20 to 30 mg of roots dry weight (DW) were digested in 35% HNO3 (v/v) and evaporated to dryness on a sand bath at 80° C. Minerals were then incubated with a mix of HCl 37% + HNO3

68% (3:1 v/v) until evaporation (Vromman et al., 2016b). The residue was then dissolved with distilled water and filtered. Ion measurements were made in an atomic absorption spectrometer (Thermo scientific, USA) in duplicate.

Non-protein thiols quantification

For non-protein thiols concentration, 50-100 mg of frozen fresh tissue was grinded in 5% (w/v) sulfosalicylic acid and 6.3 mM diethylenetriaminepentaacetic acid at 0

°C. The homogenate was then centrifuged at 10000g for 30 minutes at 4°C. Three hundred microliters of supernatant were mixed with 630 μL of 0.5M K2HPO4 (pH

7.0). After two minutes, the absorbance at 412 nm was recorded. Then, 25 μL of 10 mM 5,5-dithiobis 2- nitrobenzoic acid (pH=7.0) was added to the solution, and once again the absorbance at 412 nm was recorded after 2 minutes according to (De

Vos et al., 1992). Thiol concentration was calculated as the difference between the two measurements, using an extinction coefficient of 13,600 M cm−1.

Reduced and total glutathione quantification

For reduced (GSH) and total (GSHt) glutathione quantification in roots and leaves, 200 mg of frozen tissue was extracted and derivatized by orthophthalaldehyde (OPA) according to Cereser et al. (2001). GSHt was quantified after a reduction step of oxidized glutathione (GSSG) by dithiotreitol

(DTT). Extracts were filtered through 0.45 μm microfilters (Chromafil PES-45/15,

Macherey-Nagel) prior to injection and OPA derivatives were separated on a reversed-phase HPLC column with an acetonitrile-sodium acetate gradient system and detected fluorimetrically. Five μL of sample were injected into a

Shimadzu HPLC system (Shimadzu, ‘s-Hertogenbosch, The Netherlands) equipped with a Nucleodur C18 Pyramid column (125 × 4.6 mm internal diameter;

5 μm particle size) (Macherey-Nagel, Düren, Germany). Derivatives were eluted in acetonitrile gradient in a 50 mM sodium acetate buffer pH 6.2 at 30°C at a flow

95 rate of 0.7 mL min−1. The mobile phase consisted of distilled water (eluent A), acetonitrile (eluent B) and 50 mM sodium acetate buffer pH 6.2 (eluent C). The gradient program was as follows: 0 min, 100 % C; 5 min, 100 % C; 12 min, 30 %

A, 70 %, B; 17 min, 30 % A, 70 % B; 23–33 min, 100 % C, re-equilibration time.

Fluorimetric detection was performed with a Shimadzu RF-20A fluorescence detector (The Netherlands) at 420 nm after excitation at 340 nm. GSH (retention time of 3.02 min) was quantified using nine-point calibration curves with custom- made external standard solutions ranging from 0.0625 to 50 μM, and every ten injections a check standard solution was used to confirm the calibration of the system. The recovery was determined using GSH as an internal standard.

Statistical treatment of data

Growth parameters, water balance, and ion quantification were analyzed using a three-way analysis of variance (copper, salinity and solute potential as factors).

The experimental design was fully factorial with fixed main effects. All data were analyzed by InfoStat statistical package (Di Rienzo et al 2016). Homogeneity of variance was calculated by Levene’s test and non-normal variables were transformed by root or logarithm. When the ANOVA was significant at P=0.05,

Tukey's HSD tests were performed to determine if differences were significant.

Results

Plant Growth Parameters

All plants survived until the end of the experiment, although chlorosis and serious loss of turgor was observed in combined treatments of PEG and Cu on A. halimus since day four. Other visual symptoms included browning of the roots and reddening of the abaxial side of A. atacamensis and A. halimus leaves.

Figure 1

Root length of A. halimus significantly decreased in response to Cu (F=4.1,

P<0.05) and PEG (F=6.0, P<0.05) single effects. Salinity tended to increase root length and no significant effects were observed for combined treatments (Table 1).

In contrast, root length of A. atacamensis significantly varied in response to single and combined effects of Cu, NaCl and PEG (F=7.3, P<0.05). In the absence of

PEG, Cu caused a significant decrease in root length (F=76.3, P<0.001), and when

PEG was spiked, a 50% decrease in root length was observed in all treatments, either single or combined with Cu and NaCl (Table 1).

Root fresh weight of A. atacamensis greatly varied in response to Cu, NaCl, PEG single and combined effects (Table 1). Single 5 µM Cu treatment induced a 20% increase in root fresh weight of A. atacamensis, but 10 µM Cu reversed this effect, and caused a 65% average decrease of root fresh weight, even when combined with PEG or NaCl. In A. halimus, Cu (F=10.7, P<0.05) and PEG (F=21, P<0.001) single effects caused a significant decrease of root fresh weight. Unlike A. atacamensis, Cu and NaCl combination caused a significant increase of root fresh

97 weight of A. halimus, but only in treatments where PEG was absent (Table 1).

Also, single NaCl caused a 33% increase of root fresh weight of A. halimus, much higher than the 3% increase noted for A. atacamensis roots (Table 1).

Shoot fresh weight of A. atacamensis significantly decreased in response to the interaction between Cu and PEG (F=32.3, P<0.001). A significant increase of shoot fresh weight was observed when 5 µM Cu was applied; however, this effect significantly reversed in response to 10 µM Cu. In A. halimus, shoot fresh weight significantly varied in response to NaCl (F=12.5, P<0.001) and PEG (F=160, P<0.

001) single and combined (F=18.45, P<0.001) effects. Notably, in the presence of

PEG, shoot fresh weight of A. halimus decreased to an average of 63% compared to control plants, effect that did not statistically vary in combination with NaCl or Cu

(Table 1).

Table 1

Total chlorophyll content of A. atacamensis and A. halimus varied in response to the interaction between Cu and NaCl and PEG and NaCl; in A. halimus, it also varied in response to the interaction among Cu and PEG (F=4.21, P<0.05) (Table

2). In A. halimus, a significant 23% decrease of chlorophyll in response to single

Cu was observed; however, when combined with NaCl, this effect reversed (Table

2). In A. atacamensis, chlorophyll decreased in all PEG-combined treatments; yet, the magnitude of this decrease was smaller than in A. halimus.

Carotenoid content showed different patterns among both Atriplex species. Whilst single and combined stressors caused an increase of carotenoids in A. atacamensis, in A. halimus, a decrease was observed (Table 2). Carotenoid content in A. halimus varied in response to the combination of Cu, NaCl and PEG

(F=4.43; P<0.05), but in A. atacamensis, it varied only in response to the combination of Cu and PEG (F=125.3; P<0.001) (Table 2).

Table 2

Water Balance

Root and shoot water content of A. atacamensis varied in response to PEG and Cu single and combined effects (Table 3). PEG caused a significant decrease in root

(F=4.35, P<0.05) and shoot (F=5.5, P<0.05) water content, with no significant variation when combined with Cu or NaCl. In the absence of PEG, shoot and root water content of A. atacamensis decreased in response to 10 µM Cu and increased in response to NaCl. In A. halimus, shoot water content varied in response to PEG and Cu (F=3.74, P<0.05) and PEG and NaCl (F=5.5, P<0.05) combined effects. Similar to A. atacamensis, the presence of PEG caused a general decrease in shoot water content of A. halimus that did not increase when combined with NaCl or Cu (Table 3).

Single effects of PEG (F=11.9, P<0.001) and NaCl (F=16.9, P<0.001) caused a significant decrease of leaf osmotic potential in A. atacamensis (Figure 2). Copper

99 treatments did not have a significant effect on leaf osmotic potential, but when combined with NaCl, a marked decrease was noted (F=3.8, P<0.05). A similar effect was observed in A. halimus, where the effect of the combination between

Cu, NaCl and PEG caused a 230% average decrease in leaf water potential

(Figure 2). While the combination of Cu, NaCl and PEG caused a significant decrease of leaf osmotic potential to an average of -3.2 MPa in A. atacamensis and -8.0 MPa in A. halimus (Figure 3).

Leaf proline of A. atacamensis greatly varied in response to the combination of Cu,

NaCl and PEG (F=10.17, P<0.001). A significant increase in proline occurred in response to PEG and its combination with Cu; however, when NaCl was present, proline decreased to control levels (Figure 2). Single Cu did not have an effect on leaf proline, even when combined with NaCl. On the other side, leaf proline of A. halimus varied significantly in response to PEG and its independent interaction with

NaCl (P<0.05) and Cu (P<0.001). PEG caused an increase in leaf proline of A. halimus four times bigger than A. atacamensis. However, unlike A. atacamensis, when NaCl was applied, proline concentration did not decrease (Figure 2).

Table 2

Figure 2

Figure 3 100

Ion Accumulation

Single and combined effects of Cu, NaCl and PEG caused different patterns of ion accumulation on A. atacamensis and A. halimus (Table 4 and Figure 4). Root K in

A. halimus significantly varied according to the interaction of Cu, NaCl and PEG

(F=10.9, P<0.001). Single addition of NaCl caused a moderate, but significant decrease of K in roots (F=16.75, P<0.001), but not in leaves. On the other side, Cu caused a general decrease in K that was more visible in leaves than roots (Figure

4). When K accumulation for both species was compared, similar K content was found on roots of both species (60 mg g-1 DW). Nevertheless, leaf K of A. halimus was almost two times larger than A. atacamensis (Table 4).

NaCl significantly increased sodium accumulation in roots and leaves of both species (Table 4). However, in A. atacamensis, there was also a significant influence of Cu and PEG that was not observed in A. halimus (Table 4). In the presence of NaCl, Cu caused a significant decrease of leaf Na, even when combined with PEG. In A. halimus, only NaCl explained the variation of Na accumulation in roots (F=197.5, P<0.001); in leaves, it was also explained by the interaction between NaCl and Cu (F=4.41, P<0.05). Overall, NaCl treatments caused a near three-fold increase in root Na and a nearly six-fold increase in leaves of both species compared to control conditions (Table 4).

Copper accumulation in roots and leaves of both species significantly increased in response to Cu, and was five to seven fold higher in roots than leaves (Figure 4).

Copper concentration in roots of A. atacamensis, increased in response to Cu single effects, but when combined with NaCl, it decreased a 20%. In leaves, a

101 different response was noted. Although an increase of Cu occurred in response to

Cu treatments, its combination with NaCl caused a further accumulation of Cu

(Figure 4). Copper accumulation in roots and leaves of A. halimus varied significantly according to single and combined effect of Cu, NaCl and PEG (Figure

3). Copper treatments caused a significant increase of Cu in A. halimus leaves and roots, but unlike A. atacamensis, there were no differences in Cu accumulation among 5 µM Cu and 10 µM Cu treatments (Figure 4). In the absence of NaCl, PEG caused a slight but non-significant increase of Cu, in roots (F=16.9, P<0.001) and leaves (F=17.1, P<0.001).

Table 4

Figure 4

Oxidative Response and Chelation

Leaf GSH of A. atacamensis significantly varied in response to Cu, Na and PEG combined effects (F=8.21, P<0.01) (Table 5). Interestingly, there was a near four- fold increase of GSH in response to single NaCl (Table 5) in both Atriplex species.

Leaf GSSG of both species slightly increased in response to 10 µM Cu, but the most important increase was found when combined with NaCl and PEG; In A.

102 atacamensis, this increase was about 870% compared to control conditions

(F=28.6, P<0.001); in A. halimus, it was 440% (F=3.7, P<0.05) (Table 5).

Table 5

Root GSH of both species also varied in response to Cu, NaCl and PEG combined effects (Table 5). Overall, root and leaf GSH and GSSG of A. atacamensis increased in response to Cu, but GSH content nearly doubled GSSH concentration in control and stress treatments. Combination of 5 µM Cu and NaCl caused an increase of GSH and GSSH, but those levels remained unchanged when Cu concentration increased to 10 µM Cu (Table 5).

In A. halimus, root GSH followed a similar pattern as A. atacamensis. The combination of 5 µM Cu and NaCl caused a slight but significant increase of GSH.

However, the combination of 10 µM Cu and NaCl, with or withouth the presence of

PEG resulted in an increase of root GSH, a response that was not observed for

GSSG content (Table 5).

Non-protein thiol concentration significantly increased in response to Cu in roots and leaves of both species. In roots of A. atacamensis, this increase was explained by the interaction between Cu, NaCl and PEG, with a clear effect of Cu treatments

(Figure 5). In A. halimus roots, non-protein thiol concentration was also explained by the interaction among Cu, NaCl and PEG (Figure 5). However, the magnitude of this response was nearly half of that of A. atacamensis.

103

Figure 5

Leaf accumulation of non-protein thiols was about one third of root accumulation in both species (Figure 5), and in A. atacamensis was explained by the individual interactions among Cu and NaCl (F=15.01, P<0.001) and NaCl and PEG (F=4.9,

P<0.05). In A. halimus, the magnitude of the increase was lower than A. atacamensis, but the effect of the interaction among Cu, NaCl and PEG remained

(F=7.7, P<0.001).

Discussion

Studies about the effect of single and combined stressors on plant species are an useful tool for understanding stress physiology, but also give valuable information for defining plant uses in ecological restoration and remediation programs. In particular, discussion about the response of halophyte species to metal toxicity has multiplied in the last years (Walker et al., 2014; Nikalje and Suprasanna, 2018). In this study, growth and tolerance traits of two Atriplex species were measured in the presence of Cu and its combination with salinity (NaCl) and water stress (PEG).

1. Effect of Cu enrichment on Atriplex species

It has often been proposed that salt tolerance traits of halophytes confer them the ability to tolerate ionic stress caused by metals (Lutts and Lefevre, 2015).

104

Therefore, it could be expected that both Atriplex species tolerate Cu concentrations otherwise considered toxic. Our results show that A. halimus and A. atacamensis were able to withstand 5 µM Cu without significant consequences to its growth. Fresh weight, water content and ion acquisition of both species did not vary significantly under these conditions. Leaf chlorophyll, a known target of Cu ions and indicator of the photosynthetic status of the plant, was also unaffected.

Maintenance of near-control conditions in Atriplex plants subjected to intermediate

Cu concentrations could be related to the deployment of physiological mechanisms that allow the plant to cope with variable levels of Cu enrichment without evident morphological symptoms. For example, an increase of the non-enzymatic antioxidant GSH and its oxidized form (GSSG) was found, suggesting that there is an active process of ROS scavenging in roots and leaves (Jozefczak et al., 2012).

An increase in leaf carotenoids, molecules with photoprotective and stress signaling functions was also found, but only on A. halimus.

On the other hand, 10 µM Cu treatments caused significant impairments to both species. The most conspicuous effects were observed in roots, with a marked decrease in root length, root density and fine root suberization, especially on A. atacamensis. Root damage is a known consequence of Cu toxicity, and can be explained by cell membrane damage and alterations to the cell cycle (Pena et al.,

2015) and hormone levels in root proliferation areas (Lequeux et al., 2010). Also,

Cu- induced root suberization, restricts water and ion permeability (Kováč et al.,

2018), causing further damage to the plant. In fact, the decrease in root and shoot water content in response to 10 µM Cu could be related to Cu-induced root 105 damage, namely because Cu treatments did not have a significant effect on leaf osmotic potential or biomass production of none of the species. The lack of an effect of metal toxicity on water content was also described by Vromman et al.

(2017) on A. atacamensis seedlings exposed to As, a non-nutritional metalloid.

Copper-induced root damage had variable effects on ion accumulation of both species. Potassium, an essential nutrient with numerous roles in plant metabolism and plant water balance (Marschner, 2012), decreased in both species. Although root and leaf Na was not affected by single Cu treatments, a notable increase in root and leaf Cu was found. These results are in line with several authors that describe active metal sequestration on Atriplex roots as a possible avoidance strategy (Kachout et al., 2012; Mateos-Naranjo et al., 2013). However, it is interesting to note that root Cu concentration and root:leaf Cu relationship was higher in A. atacamensis than A. halimus, even when combined with other stressors. This difference was particularly evident at 10 µM Cu, where Cu accumulation in roots of A. atacamensis nearly doubled A. halimus. Differences in

Cu accumulation between organs and species could be explained by species- specific strategies or local adaptations to metal toxicity. For example, seeds of A.

Atacamensis used in this study came from a population with high levels of available Cu (15.7 ppm); therefore, a certain level of tolerance could be expected.

Lower Cu accumulation in A. halimus roots and leaves could be explained by the lack of such adaptations, or a metal exclusion strategy, expressed by low metal accumulation in roots and restricted transport to shoots (Mateos-Naranjo et al.,

2013). The ability to capture high concentrations of Cu in tissues without severe effects to the plant functioning can be achieved by its chelation and sequestration 106 in a non-reactive form (Gratão et al., 2005). On this subject, we found that Cu induced non-protein thiols synthesis in roots and leaves of both species, with a similar pattern of root Cu accumulation, where A. atacamensis nearly doubled A. halimus in 10 µM Cu treated plants.

The described effects correspond with other articles that explore Cu toxicity

(Kabata-Pendias, 2010; Brahim and Mohamed, 2011; Dasgupta-Schubert et al.,

2011); however, the magnitude of the effect vary according to the concentration used. For example, Mateos-Naranjo et al. (2013) found that Cu LC50 of Atriplex halimus occurred between 15 y 30 mM, a concentration three orders of magnitude higher than those used in this study. These large differences can be explained not only for the substrate used in the experiment – whether it is in vitro culture, soil or hydroponics-, but also the pH of the nutrient solution used to cultivate or irrigate the plants. Copper bioavailability decreases with rising pH (Ginocchio et al., 2009); therefore, toxic concentration of Cu can be overestimated, along with plant tolerance (Kopittke et al., 2010).

2. Effect of salinity on Atriplex species

No negative effects were found in growth parameters of Atriplex species treated with 0.5% NaCl (85.5 mM). On the contrary, shoot biomass and water content increased under these conditions. These results are in agreement with other studies that found that moderate NaCl concentrations (50-200 mM) can actually promote growth in halophyte species (Han et al., 2012; Walker et al., 2014), while higher concentrations have inhibitory effects (Nemat Alla et al., 2011). 107

The fact that leaf water potential and shoot dry weight were also unaffected by

NaCl treatments, suggests that Atriplex species were actively performing osmotic adjustment in leaves in order to keep growing (Bromham and Bennett, 2014).

Osmotic adjustment in halophyte species can be achieved by the accumulation of compatible osmolytes and/or inorganic ions (K, Na, Cl) in the vacuole. These strategies allow the plant to maintain a more negative leaf water potential than the substrate, favoring continuum water supply (Adolf et al., 2013). The use of each one of these strategies seems to be related to carbon economy, where ion sequestration is a less demanding mechanism than organic compatible solute synthesis (Eisa et al., 2012). In fact, proline synthesis on NaCl-treated plants was not different than control conditions in none of the species.

Our results show that Na assimilation increased under salinity treatments in both

Atriplex species, but unlike Cu, Na was higher in leaves than in roots. This suggests that there is active transport to upper organs in order to perform osmotic adjustment. Previous studies performed in two Tecticornia species (also

Chenopodiaceae), showed that a group of compatible solutes, among which were

Na and glycinbetaine, increased in response to 500 mM NaCl (English and Colmer,

2013). Moreover, Martìnez et al. (2003) found that A. halimus was able to accumulate Na in leaves after 22 days of withholding water, even in the absence of salinity.

The increase of Na in Atriplex leaves can also be explained by its use in carbon metabolism. Halophytes that also exhibit a C4 photosynthetic pathway, such as

Atriplex, use Na ions for PEP regeneration from pyruvate in mesophyll chloroplasts

(Ohnishi et al., 1990; Subbarao et al., 2003). Therefore, active Na transport into 108 leaves could also be a strategy to favor photosynthesis under salt stress or water scarcity.

3. Dual effect of PEG on Atriplex

PEG-induced water stress had a negative impact on root and shoot growth of both

Atriplex species. Notably, morphological and physiological responses of Atriplex seedlings to PEG were similar to the most stressful treatments of this experiment, and its most visible effects were observed at shoot level.

Whilst PEG is considered the best component to imitate water stress in nutrient solutions, some studies have also suggested that, in addition to a decrease of solute potential, this molecule also has a physical effect around roots that could explain the observed effects on Atriplex shoots (Shi et al., 2015). On this subject,

Slama et al. (2007) reported that growth inhibition caused by PEG was more severe than other isosmotic treatments, such as mannitol or NaCl, probably due to an increase of viscosity that restricts gas and water transport to the plant. In this line, they proposed that growth restrictions in PEG- treated plants could be better explained by a combination between osmotic stress and nutrient deficiency. In fact, our results show that K accumulation in shoot decreased in the presence of PEG, regardless of the accompanying stressor. PEG induced stress was particularly visible as a severe loss of turgor and wilting in A. halimus shoots, and whilst no significant variation in leaf GSH or carotenoids was found, a 10-15 fold proline increase occurred, suggesting that stress sensing and response of this stressor has another timing or a different pathway.

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4. Atriplex species response to combined stresses

The combination of more than one abiotic stressor caused different responses in both Atriplex species, but, as it was proposed by Choudhury et al., (2017) stressor combination caused a synergistic effect, where decrease in plant growth parameters had a greater magnitude when more than one stressor was present.

Of particular relevance is the effect of Cu and its individual combination with NaCl and PEG. Several studies have proposed that salinity conditions alleviate metal toxicity effects on halophyte species (Wali et al., 2015; Cheng et al., 2018). Our results show that salinity conditions do not alleviate or improve growth parameters of A. halimus and A. atacamensis subjected to Cu enrichment, particularly on 10

µM Cu treatments; instead, the only parameters that varied in response to the combination between Cu and NaCl were carotenoids and glutathione.

Total glutathione increased in response to the combination of Cu and NaCl treatments. GSH:GSSH ratio showed that the decrease of GSH in roots and leaves that occurred in response to Cu, did not vary when combined with NaCl. This suggest that reduced glutathione was being oxidized to GSSG at a higher rate than its recycling capacity, or there was a detrimental effect on glutathione reductase synthesis (Anjum et al., 2014). It is also possible that this decrease in GSH was related to phytochelatins synthesis as Bankaji et al., (2015) observed in Suaeda fruticosa seedlings, or for the sequestration of Cu ions in the vacuole. In fact, the observed increase in root NPT in response to Cu remained in conditions of salinity.

This implies that active sequestration of Cu ions could occur in parallel to glutathione ROS scavenging activity.

110

Single NaCl and Cu enrichment are two abiotic disturbances that modify ion availability in the substrate. However, ion transport and assimilation also depends on the ability of the plant to acquire and transport water through the xylem (De

Boer and Volkoc, 2003). Therefore, it could be expected that PEG-induced water stress also restricted ion assimilation, especially when combined with Cu or NaCl treatments. However, our results indicate otherwise: in conditions of salinity, PEG did not have an effect on leaf Na of none of the studied species. Similarly, it was found PEG treatments did not reduce Cu accumulation in roots or leaves of

Atriplex species subjected to Cu; in fact, it caused an increase in Cu accumulation in leaves and roots of A. halimus.

Another component with an interesting response to stress combination was proline.

This molecule has been described as a compatible osmolyte, but also as a stress signaling molecule (Ashraf and Foolad, 2007). Our results show that proline did not varied in response to single Cu stress in none of the species; but it did in response to single PEG treatments. These results show that in Atriplex, proline is synthetized in response to water stress. However, proline response to the combination of NaCl and PEG varied among the two species: whilst A. halimus proline levels remained high, A. atacamensis proline remained similar to control levels. The difference among these responses could reflect that A. atacamensis uses NaCl as a substrate to decrease leaf water potential, and tolerate PEG- induced water stress, as it has been described on A. halimus (Martínez et al., 2005). On the other side, it could be that the detrimental effect of PEG on A. halimus was so severe, that proline is assuming more than one role in plant response to PEG.

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Overall, stressor combination caused a neutral or synergistic effect, where the sum of more than one condition caused a similar or worse effect than single-stressor conditions, especially with PEG. However, further comparison among species, and search for tolerance patterns in response to specific stress combinations were limited by the chosen factorial design. In this line, we believe that the presented results could be used as a base to perform more specific research on combined stress response at a species level, or broaden the picture on Atriplex specific responses.

Stress combination experiments are a valid approach to describe the nature of plant responses to multiple stressors on the field (Suzuki et al., 2014). With this in mind, we observed that stressor combination on Atriplex species resulted in a decreased condition and an augmented synthesis and accumulation of different defense components. Yet, physiological effects and its visual expression greatly varied among A. atacamensis and A. halimus on different stressor combinations.

The ability to interpret stressor magnitude and its effect on Atriplex species will allow us to understand further about the ecophysiology of these two species used in the phytostabilization of metal enriched sites.

Conclusion

Single and combined stressors had different effects between the two Atriplex species. Intermediate Cu treatments did not affect Atriplex species, but high Cu treatments did affect growth and physiological response of A. atacamensis. NaCl did not have detrimental effects on plant growth, but induced physiological

112 responses, such as ion accumulation and GSH synthesis. PEG treatments affected water balance parameters of both species, especially in A. halimus, but did not have an effect on ion transport and accumulation. Finally, growth and physiological responses of A. atacamensis and A. halimus to combined stressors showed to be contrasting. Sodium chloride did not alleviate Cu-induced stress; PEG did not decrease ion acquisition in Cu and NaCl treatments, and caused detrimental effects that depended on the presence of other stressors.

Acknowledgments

This study was funded by the Comisión Nacional de Investigación Científica y

Tecnológica (CONICYT) and Center of Applied Ecology and Sustainability

(CONICYT PIA/BASAL FB0002). F. Orrego was funded by CONICYT-

PFCHA/Doctorado Nacional/2014-21141059. F. Orrego would like to acknowledge

Hélène Dailly and Andrea Ávila for their help with glutathione, Proline and non- protein thiols analysis.

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doi: 10.1016/j.scitotenv.2011.09.085. Vromman, D., I. Lefèvre, Z. Šlejkovec, J.-P.P. Martínez, N. Vanhecke, et al. 2016a. Salinity influences arsenic resistance in the xerohalophyte Atriplex atacamensis Phil. Environ. Exp. Bot. 126: 32–43. doi: 10.1016/j.envexpbot.2016.01.004. Vromman, D., J.P. Martínez, and S. Lutts. 2017. Phosphorus deficiency modifies As translocation in the halophyte plant species Atriplex atacamensis. Ecotoxicol. Environ. Saf. 139(January): 344–351. doi: 10.1016/j.ecoenv.2017.01.049. Vromman, D., B. Paternostre, M. Briceño, C. Teixeira-cardoso, and A. Flores- bavestrello. 2016b. Bioremediation & Biodegradation Arsenic Distribution in Shoots of the Halophyte Plant Species Atriplex atacamensis Growing in an Extreme Arid Mining Area from Northern. J. bioremediation Biodegrad. 7(3). doi: 10.4172/2155-6199.1000349. Wali, M., E. Fourati, N. Hmaeid, R. Ghabriche, C. Poschenrieder, et al. 2015. NaCl alleviates Cd toxicity by changing its chemical forms of accumulation in the halophyte Sesuvium portulacastrum. Environ. Sci. Pollut. Res. doi: 10.1007/s11356-015-4298-9. Walker, D.J., S. Lutts, M. Sánchez-García, and E. Correal. 2014. Atriplex halimus L.: Its biology and uses. J. Arid Environ. 100–101: 111–121. doi: 10.1016/j.jaridenv.2013.09.004. Wang, Z.-L., and C.-Q. Liu. 2003. Distribution and partition behavior of heavy metals between dissolved and acid-soluble fractions along a salinity gradient in the Changjiang Estuary, eastern China. Chem. Geol. 202(3–4): 383–396. doi: 10.1016/j.chemgeo.2002.05.001. Ye, N., H. Li, G. Zhu, Y. Liu, R. Liu, et al. 2014. Copper suppresses abscisic acid catabolism and catalase activity, and inhibits seed germination of rice. Plant Cell Physiol. 55(11): 2008–2016. doi: 10.1093/pcp/pcu136. Zandalinas, S.I., R. Mittler, D. Balfagón, V. Arbona, and A. Gómez-Cadenas. 2018. Plant adaptations to the combination of drought and high temperatures. Physiol. Plant. 162(1): 2–12. doi: 10.1111/ppl.12540.

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a b c

Figure 1. Visual symptoms of single and combined stresses on Atriplex seedlings.

(a) Loss of turgor and leaf reddening on Atriplex halimus leaves subjected to 7.85 mM PEG. (b) Browning of Atriplex atacamensis roots subjected to 10 uM Cu. (c)

Leaf chlorosis in A. atacamensis subjected to combined 10 uM Cu and 0.5% NaCl.

120

Figure 2. Leaf osmotic potential (MPa) of A. atacamensis (left) and A. halimus

(right) seedlings exposed for 10 days to single and combined Cu, NaCl and PEG stresses (n=3; mean± EE). Different letters denote significant differences at p ≤

0.05 among treatments.

121

c c c

20 b b b 10

Leafproline g-1(mgFW) a a a a a a 0

Control

Cu 5 uM 5 Cu

Cu 10 uM 10 Cu 0.5% NaCl

Cu 5uM+ NaCl0.5% 5uM+ Cu

Cu 10 uM+ NaCl 0.5% NaCl uM+ 10 Cu 30 30 Withouth P.E.G With P.E.G c c c

20 20 b b b

10 10 e d d

c bc Leaf proline (mg g-1 FW) g-1 (mg proline Leaf Leaf proline (mg g-1 FW) g-1 (mg proline Leaf ab ab b ab a ab ab a a a a a a

0 0 Control

Control Cu 5uM Cu

Cu 5 uM 5 Cu Cu 10uM Cu

Cu 10 uM 10 Cu NaCl 0.5% NaCl

NaCl 0.5% NaCl Cu 5uM + NaCl 0.5% +NaCl 5uM Cu

Cu 5 uM+ NaCl 0.5% uM+ NaCl 5 Cu Cu 10uM + NaCl 0.5% +NaCl 10uM Cu

Cu 10 uM+ NaCl 0.5% uM+ NaCl 10 Cu

Figure 3. Leaf proline (mg g-1 FW) of A. atacamensis (left) and A. halimus (right) seedlings exposed for 10 days to single and combined Cu, NaCl and PEG stresses

(n=4; mean± EE). Different letters denote significant differences at p ≤ 0.05 among treatments.

122

Figure 4. Copper concentration (mg∙g-1) in roots and leaves of A. atacamensis

(upper) (n=5; means± EE) and A. halimus (lower) (n=5 means± EE) seedlings exposed for 10 days to single and combined Cu, NaCl and PEG stresses. Different letters denote significant differences at p ≤ 0.05 among treatments.

123

Figure 5. Non-protein thiols (µmol g-1) in roots and leaves of Atriplex atacamensis

(left) and A. halimus (right) seedlings exposed for 10 days to single and combined

Cu, NaCl and PEG stresses (n=5; means± EE). Different letters denote significant differences at p ≤ 0.05 among treatments.

124

Table 1. Root length (cm.) and root and shoot fresh weight (g) of Atriplex atacamensis and A. halimus seedlings exposed for 10 days to single and combined Cu, NaCl and PEG stresses (n=5; mean± EE). At the end of the table, a summary of analyses of variance of all sources of variation are given.

Atriplex atacamensis Atriplex halimus

Treatments Root Root fresh Shoot fresh Shoot fresh Root length Root length fresh weight weight weight weight

Control 35.8 ± 1.4 0.3 ± 0.02 0.92 ± 0.07 19.6±2.3 0.33±0.1 0.87±0.2

Cu 5 µM 24.9 ± 1.7 0.36 ± 0.03 1.08 ± 0.2 22±1 0.24±0.03 0.99±0.1

Cu 10 µM 18.9 ± 0.9 0.11 ± 0.002 0.52 ± 0.03 17.9±1.2 0.18±0.03 0.79±0.1

NaCl 0.5% 34.2 ± 1.2 0.31 ± 0.01 1.11 ± 0.02 25.3±2.6 0.44±0.03 1.39±0.1

Cu 5 µM + NaCl 0.5% 17.7 ± 0.5 0.17 ± 0.01 1.05 ± 0.03 21.6±2.1 0.32±0.03 1.38±0.1

Cu 10 µM + NaCl 0.5% 18 ± 0.5 0.09 ± 0.002 0.63 ± 0.03 16.5±2.6 0.23±0.03 1.32±0.1

PEG 7.85% 18.8 ± 0.7 0.08 ± 0.004 0.27 ± 0.02 18.7±1.9 0.23±0.1 0.43±0.1

Cu 5 µM + PEG 7.85% 13.7 ± 0.6 0.06 ± 0.003 0.28 ± 0.04 17.3±1.9 0.15±0.03 0.28±0.1

Cu 10 µM + PEG 7.85% 19.8 ± 0.8 0.1 ± 0.01 0.42 ± 0.03 16.7±2 0.19±0.04 0.33±0.1

PEG 7.85% + NaCl 0.5% 18.2 ± 1.3 0.09 ± 0.01 0.36 ± 0.04 17.7±3.9 0.22±0.1 0.29±0.04

Cu 5 µM + NaCl 0.5% + PEG 7.85% 16.6 ± 0.8 0.06 ± 0.01 0.36 ± 0.02 18.3±2.7 0.14±0.03 0.31±0.1

Cu 10 µM+ NaCl 0.5% + PEG 7.85% 19.2 ± 0.6 0.09 ± 0.004 0.56 ± 0.01 17.3±1.7 0.16±0.01 0.31±0.04

Source of variation F, P F, P F,P F, P F, P F, P

Cu 87.8; <0.001 29.9; <0.001 0.5; NS 4.1; <0.05 10.7; <0.05 0.7; NS

NaCl 5.3; <0.05 5.7; <0.05 11.7; <0.05 2.9; NS 1.4; NS 12.5; <0.05

PEG 153.9; <0.001 305.6; <0.001 206.9; <0.001 6.0; <0.05 21; <0.001 160; <0.001

Cu*NaCl 0.5; NS 5.7; <0.05 0.33; NS 2.3; NS 0.3; NS 0.03; NS

Cu*PEG 76.3; <0.001 68.1; <0.001 32.3; <0.001 1.4; NS 2.5; NS 0.55; NS

NaCl*PEG 10.87; <0.05 11.2; <0.05 1.6; NS 0.01; NS 4.7; <0.05 18.45;<0.001

Cu*NaCl*PEG 7.3; <0.05 5.8; <0.05 0.2; NS 1.4; NS 0.04; NS 0.57; NS

125

Table 2. Total chlorophyll and carotenoid content of Atriplex atacamensis and A. halimus subjected to single and combined Cu, NaCl and PEG stresses (n=5; mean± EE). At the end of the table, a summary of analyses of variance of all sources of variation are given.

Atriplex atacamensis Atriplex halimus Treatments Chl a+b Car Chl a+b Car Control 2.0±0.02 0.45±0.01 1.8±0.06 0.31±0.01 Cu 5 µM 1.9±0.03 0.57±0.01 1.7±0.03 0.23±0.01 Cu 10 µM 1.8±0.03 0.66±0.01 1.4±0.02 0.21±0.01 NaCl 0.5% 2.0±0.03 0.47±0.02 1.8±0.04 0.37±0.01 Cu 5 µM + NaCl 0.5% 1.8±0.03 0.69±0.01 1.8±0.03 0.26±0.01 Cu 10 µM + NaCl 0.5% 1.8±0.01 0.76±0.01 1.7±0.03 0.18±0.01 PEG 7.85% 1.9±0.02 0.46±0.01 1.9±0.03 0.36±0.00 Cu 5 µM + PEG 7.85% 1.8±0.02 0.56±0.02 1.6±0.03 0.29±0.01 Cu 10 µM + PEG 7.85% 1.7±0.02 0.64±0.01 1.4±0.03 0.23±0.01 PEG 7.85% + NaCl 0.5% 1.8±0.02 0.49±0.01 1.7±0.03 0.34±0.01 Cu 5 µM + NaCl 0.5% + PEG 7.85% 1.6±0.03 0.71±0.01 1.5±0.03 0.27±0.01 Cu 10 µM+ NaCl 0.5% + PEG 7.85% 1.6±0.03 0.74±0.01 1.4±0.04 0.16±0.01 Source of variation F;P F;P F;P F;P Cu 45.27; <0.001 277; <0.001 100.43; <0.001 96.5; <0.001 NaCl 32.19; <0.001 0.06; NS 0.02; NS 11.7; <0.05 PEG 90.08; <0.001 47.63; <0.001 37.44; <.0.001 59.5; <0.001 Cu*NaCl 5.44; <0.05 0.43; NS 8.87; <0.05 3.6; <0.05 Cu*PEG 0.56; NS 125.3; <0.001 4.21; <0.05 136.8; <0.001 NaCl*PEG 5.57; <0.05 0.33; NS 42.1; <0.001 14.7; <0.001 Cu*NaCl*PEG 1.84; NS 2.07; NS 1.06; NS 4.43; <0.05

126

Table 3. Root and shoot water content (WC in %) of Atriplex atacamensis and A. halimus seedlings exposed for 10 days to single and combined Cu, NaCl and PEG stresses (n=4; mean± EE). At the end of the table, a summary of analyses of variance of all sources of variation are given.

Atriplex atacamensis Atriplex halimus Treatments Root Shoot Root Shoot Control 92.9 ± 0.2 83.5 ± 1.8 94.5 ± 0.2 88.5 ± 1.6 Cu 5 µM 93.1 ± 0.96 84.6 ± 0.9 93.4 ± 0.5 86.7 ± 0.9 Cu 10 µM 85.7 ± 2.8 73.8 ± 2.5 92.5 ± 0.1 84.7 ± 0.3 NaCl 0.5% 94.3 ± 0.59 88.2 ± 0.2 94.5 ± 0.3 89.5 ± 1.7 Cu 5 µM + NaCl 0.5% 92.3 ± 1.76 87.9 ± 0.6 93.6 ± 0.4 88.9 ± 0.4 Cu 10 µM + NaCl 0.5% 88.7 ± 0.56 82.6 ± 0.5 91.6 ± 0.7 89.6 ± 2.1 PEG 7.85% 90.5 ± 0.73 73.6 ± 0.7 92.7 ± 0.8 68.1 ± 8.5 Cu 5 µM + PEG 7.85% 88.8 ± 0.81 73.2 ± 0.7 91.9 ± 1.3 73.1 ± 2.2 Cu 10 µM + PEG 7.85% 86.7 ± 1.09 72.6 ± 1 92.3 ± 0.6 63.1 ± 4.4 PEG 7.85% + NaCl 0.5% 89.8 ± 0.64 74 ± 0.6 94.2 ± 0.3 61.6 ± 7.8 Cu 5 µM + NaCl 0.5% + PEG 7.85% 87.6 ± 0.75 75.1 ± 0.7 93.1 ± 0.5 80 ± 1.5 Cu 10 µM+ NaCl 0.5% + PEG 7.85% 89.7 ± 0.46 79.3 ± 0.4 93 ± 0.4 74.2 ± 4.6 Source of variation F;P F; P F;P F; P Cu 6.2; <0.05 3.72; <0.05 24; <0.001 2.7; NS NaCl 1.1; NS 10.5; <0.05 1.27; NS 11.1; <0.05 PEG 14; <0.001 37.3; <0.001 0.88; NS 126.8; <0.001 Cu*NaCl 0.16; NS 0.03; NS 0.81; NS 0.16; NS Cu*PEG 4.35; <0.05 5.5; <0.05 3.97; <0.05 3.74; <0.05 NaCl*PEG 0.8; NS 3.32; NS 1.4; NS 5.5; <0.05 Cu*NaCl*PEG 0.49; NS 0.3; NS 0.64; NS 0.44; NS

127

Table 4. Sodium and K concentration (in mg∙g-1) in roots and leaves of Atriplex atacamensis and A. halimus seedlings exposed for 10 days to single and combined Cu, NaCl and PEG stresses (n=4; mean± EE). At the end of the table, a summary of analyses of variance of all sources of variation are given.

Atriplex atacamensis Atriplex halimus Treatments Root Na+ Leaf Na+ Root K+ Leaf K+ Root Na+ Leaf Na+ Root K+ Leaf K+ Control 4.5 ± 0.3 5.4 ± 0.4 53.7 ± 6 69.4 ± 10.3 6.4 ± 1.4 6 ± 0.4 59.2 ± 8.1 144.6 ± 9.7 Cu 5 µM 5.9 ± 1.4 7.8 ± 0.9 42.9 ± 1.1 63.6 ± 2.8 6.1 ± 2.6 7.4 ± 1 72.7 ± 8.4 77.5 ± 2.1 Cu 10 µM 4.3 ± 0.1 28.6 ± 4.4 42.7 ± 5.9 43.7 ± 5.9 7.6 ± 0.2 11.7 ± 2.2 42.3 ± 2.9 95.6 ± 9.8 NaCl 0.5% 22.7 ± 3.4 4.9 ± 0.2 30.1 ± 3.7 39.3 ± 1.5 29.3 ± 5.2 70.5 ± 9.1 40.6 ± 5.9 102.3 ± 20.3 Cu 5 µM + NaCl 0.5% 12.6 ± 0.7 4.5 ± 0.1 19.3 ± 1.5 37.5 ± 2.3 22.1 ± 2.3 66.4 ± 7.9 43.3 ± 2.9 104.5 ± 3.1 Cu 10 µM + NaCl 0.5% 18.9 ± 0.8 9.2 ± 2.5 27.2 ± 1.5 30.9 ± 3.9 21.7 ± 0.7 45.3 ± 1 57.5 ± 1.3 83.4 ± 7.8 PEG 7.85% 4.9 ± 0.2 9.6 ± 1.2 30.8 ± 4.5 41.5 ± 2.9 11.6 ± 2.1 10.2 ± 1.6 40 ± 3.3 109.1 ± 14.6 Cu 5 µM + PEG 7.85% 4.5 ± 0.1 31.7 ± 2.2 26 ± 2.1 30.7 ± 5.4 9.6 ± 0.01 6.4 ± 1 47.4 ± 5.5 88.3 ± 11.3 Cu 10 µM + PEG 7.85% 9.2 ± 2.5 11.4 ± 2.1 29.9 ± 4.8 39.8 ± 5.9 6 ± 1.1 9.8 ± 0.6 31.2 ± 1.7 76.7 ± 11.5 PEG 7.85% + NaCl 0.5% 20.7 ± 1.1 63.9 ± 8.3 27.8 ± 1.3 30.8 ± 3.5 24.7 ± 0.1 54 ± 6.9 51.4 ± 5.5 85.8 ± 10.7 Cu 5 µM + NaCl 0.5% + PEG 7.85% 16.4 ± 0.6 45.7 ± 1.7 24.9 ± 3.1 34.4 ± 1.5 23.2 ± 1.3 52.4 ± 3.5 21.6 ± 0.3 70.8 ± 12 Cu 10 µM+ NaCl 0.5% + PEG 7.85% 16.9 ± 1.6 48.1 ± 2.6 20.9 ± 2.7 34.7 ± 0.8 23.4 ± 1.9 51.8 ± 1.3 22.4 ± 1.9 87.2 ± 8.4 Source of variation F; P F; P F; P F; P F; P F; P F; P F; P

Cu 7.4; <0.05 5.2; <0.05 5.1; NS 1.3; NS 3.2; NS 1.7; NS 6.4; <0.05 6.7; <0.05 NaCl 337; <0.001 525.3; <0.001 30.5; <0.001 13.7; <0.001 197.5; <0.001 382.8; <0.001 16.75; <0.001 2.3; NS PEG 0.01; NS 4.31; <0.05 9.2; <0.05 10.9; <0.05 0.6; NS 2.4; NS 52.5; <0.001 5.5; <0.05 Cu*NaCl 9.6; <0.001 78.5; <0.001 0.7; NS 1.1; NS 0.7; NS 4.41; <0.05 14.2; <0.001 3.3; <0.05 Cu*PEG 0.9; NS 61.7; <0.001 1.1; NS 3.7; <0.05 0.4; NS 1.6; NS 8.4; <0.05 0.8; NS NaCl*PEG 0.005; NS 12.4; <0.05 6.2; <0.05 5; <0.05 1.6; NS 2.95; NS 0.01; NS 0.01; NS Cu*NaCl*PEG 3.2; NS 68.7; <0.001 2.6; NS 1.24; NS 2.7; NS 3.1; NS 10.9; <0.001 2.9; NS

128

Table 5. Reduced (GSH) and oxidized (GSSG) glutathione (nmol g FW -1) in leaves and roots of Atriplex atacamensis and

A. halimus exposed for 10 days to single and combined Cu, NaCl and PEG stresses. At the end of the table, a summary of analyses of variance of all sources of variation are given.

Atriplex atacamensis Atriplex halimus Treatments Leaf GSH Leaf GSSG Root GSH Root GSSG Leaf GSH Leaf GSSG Root GSH Root GSSG Control 47±4.8 16.4±1.2 123.4±5.4 17.2±1.3 52.6±2.18 21.4±1.0 151.6±4.6 24.2±4.6

Cu 5 µM 141.6±7.7 15.6±1.8 541.2±33.2 129.4±11.7 151.2±18.3 39.6±5.2 346.4±24.3 245±21.7

Cu 10 µM 245.4±15.7 40.8±4.3 627.6±39.2 257.6±18.6 255.4±16.9 54.0±7.4 420.4±12.7 311.2±27.3

NaCl 0.5% 165.0±6.5 17.8±2.1 156.6±17.5 45.0±6.1 142.6±20.9 40.2±7.0 162.2±15.1 28.6±5.1

Cu 5 µM + NaCl 0.5% 251.0±12.8 30.8±1.3 409.4±37.4 255.8±0.7 251.8±15.3 58.0±8.6 419.8±37.3 270.6±0.7

Cu 10 µM + NaCl 0.5% 282.8±8.4 33.4±7.1 338.8±43.1 339.6±0.7 287.0±9.7 49.6±4.4 512.2±34.2 299.6±0.7

PEG 7.85% 90.6±8.7 14.2±3.2 93.6±5.7 16.2±1.7 70.6±4.3 30.8±2.0 111.4±8.1 32.4±4.0

Cu 5 µM + PEG 7.85% 215.2±16.8 24.4±3.2 421.6±27.6 114.4±8.6 148±16.5 42.4±3.9 284.6±32.5 226.6±27.9

Cu 10 µM + PEG 7.85% 285.8±16.7 26.8±3.2 590.0±14.9 267.6±18.9 341.0±11.8 48.2±9.6 460.6±10.1 558±20.5

PEG 7.85% + NaCl 0.5% 178.8±8.8 25.2±2.1 99.6±6.9 87.8±9.1 149.2±10.3 53.8±6.2 267.0±66.8 53.4±9.19

Cu 5 µM + NaCl 0.5% + PEG 7.85% 337.8±24.1 141.0±2.9 444.2±16.6 224.6±10.6 323.6±26 111.8±10.9 465.6±28.8 429.2±30.6

Cu 10 µM+ NaCl 0.5% + PEG 7.85% 431.6±11.9 159.2±12.1 470±22.7 187.8±23.7 421.8±15.1 116.0±15.7 493.2±29.0 225.0±8.0

Source of variation F; P F; P F;P F; P F; P F; P F; P F; P Cu 270.7; <0.001 102.1; <0.001 263.5; <0.001 200.6; <0.001 209.5; <0.001 20.7; <0.001 178.2; <0.001 292.0; <0.001

NaCl 234.4; <0.001 218.8; <0.001 28.4; <0.001 38.1; <0.001 109.2; <0.001 50.1; <0.001 47.2; <0.001 1.8; NS

PEG 85.1; <0.001 140.4; <0.001 0.8; NS 7.1; <0.05 34.8; <0.001 20.2; <0.001 0.8; NS 25.9; <0.001

Cu*NaCl 8.0; <0.001 31.7; <0.001 19.3; <0.001 13.9; <0.001 7.3; <0.05 0.7; NS 2.1; NS 54.9; <0.001

Cu*PEG 1.93; NS 29.9; <0.001 4.0; <0.05 8.4; <0.001 11.1; <0.001 0.06; NS 0.8; NS 3.5; NS

NaCl*PEG 0.06; NS 166.6; <0.001 10.8; <0.05 6.0; <0.05 4.4; <0.05 10.8; <0.05 5.9; NS 3.5; NS

Cu*NaCl*PEG 8.21; <0.001 28.6; <0.001 4.4; <0.05 11.2; <0.001 2.06; NS 3.7; <0.05 5.6; NS 42.1; <0.001

129

General Conclusion

We identified 138 halophyte species in Chile, 45% of which are native or endemic.

Most of these species are perennial herbs and have a homogeneous distribution along the continental territory. Chilean richness and endemism of halophyte species is low when compared with other countries. We propose that the genus

Atriplex, represented among other species by the endemic shrub Atriplex atacamensis, and the exotic species A. nummularia and A. halimus, has good candidates to assess the effect of single and combined copper, salt and water stresses on plant growth and tolerance mechanisms (antioxidant, chelating and osmotic).

At early plant stages (seedling), responses of selected Atriplex species (A. atacamensis, A. halimus and A. nummularia) to single stresses, under hydroponic and laboratory-controlled conditions were the following: Single Cu decreased root elongation and induced root suberization of all species. A concentration of 10 µM

Cu caused generalized toxicity symptoms in experimental seedlings, but growth of

A. halimus and A. nummularia was less affected than in A. atacamensis seedlings.

Single NaCl caused a decrease in both shoot fresh biomass and water content.

Two percent NaCl had severe effect on growth of all species, but 1% NaCl only affected A. halimus seedlings. A different pattern was found under PEG-induced water stress, where the greatest impact was found on shoot fresh weight and water content. Here, the Australian A. nummularia had the biggest decrease, while the other species were mainly unaffected. 130

Combined stress conditions caused a more negative response than single stress in the two studied species, A. atacamensis and A. halimus. Root and shoot growth decreased, GSH synthesis increased and osmotic and chelating mechanisms were disrupted. Contrary to what had been found in other halophytes, NaCl did not alleviate stressful conditions caused by Cu. Ion accumulation occurred on both species when subjected to Cu and NaCl. Whilst Cu mainly accumulated in roots,

Na was transported to leaves. Combination of these treatments with PEG-induced water stress was expected to reduce ion transport and accumulation, but this effect was not observed in none of the species. Synthesis of proline also varied among species; Atriplex atacamensis synthetized proline only in response to PEG-induced water stress, but A. halimus also did it when combined with NaCl.

All things considered, available Cu and PEG-induced water stress were the most relevant limiting factors for survival and growth of studied Atriplex species at seedling stage, affecting specifically root development and shoot growth, respectively.

Stress thresholds and tolerance strategies of both Atriplex species followed no clear pattern. Atriplex atacamensis was able to tolerate intermediate salinity conditions but was sensitive to bioavailable copper and PEG-induced water stress.

On the other side, A. halimus was able to grow under high salinity, and tolerate intermediate conditions of Cu; however, it was highly affected by PEG-induced water stress and its combination with other stresses.

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We can conclude that Atriplex atacamensis can be used for substrate rehabilitation under proper management strategies that control metal bioavailability and water acquisition on early stages. At the same time, A. halimus can be used for metal phytostabilization in sites with higher bioavailable Cu. However, since this is an exotic species, it is important control plant propagation and offer an appropriate irrigation program to favor its growth.

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