Ecophysiological and genomic analysis of salt tolerance of Cakile maritima

Environmental and Experimental Botany 92 (2013) 64–72

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Ecophysiological and genomic analysis of salt tolerance of Cakile maritima Ahmed Debez a,1 , Kilani Ben Rejeb a,b,1 , Mohamed Ali Ghars a , Mohamed Gandour a , Wided Megdiche a , Karim Ben Hamed a , Nader Ben Amor a , Spencer C. Brown c , Arnould Savouré b , Chedly Abdelly a,∗ a b c

Laboratoire des Plantes Extrêmophiles, Centre de Biotechnologie de Borj-Cedria (CBBC), BP 901, Hammam-Lif 2050, Tunisia Physiologie Cellulaire et Moléculaire des Plantes, UR5, EAC 7180 CNRS, Université Pierre et Marie Curie (UPMC), Case 156, 4 place Jussieu, 75252 Paris cedex 05, France Institut des Sciences du Végétal, CNRS UPR2355 & Imagif, Centre de Recherche de Gif, bâtiment 23, 91198 Gif-sur-Yvette, France

a r t i c l e

i n f o

Article history: Received 7 July 2012 Received in revised form 1 December 2012 Accepted 3 December 2012 Keywords: Brassicaceae Cakile maritima Halophyte Plant model Valorization

a b s t r a c t Arabidopsis thaliana L. (Brassicaceae) and its close relative Thellungiella salsuginea (Pallas) O.E. Schulz have been widely used as genetic models by researchers in their quest of understanding salt tolerance mechanisms in plants. Despite the fact that significant knowledge has been gained, both of these plants present some limitations mainly in relation to their response to salinity. Indeed, Arabidopsis is a glycophyte, whereas Thellungiella is a facultative halophyte. Among the Brassicaceae, Cakile maritima Scop. is an annual succulent obligate halophyte with a small size genome (1C = 719 Mb) and short life cycle. With these attributes, C. maritima presents a potential as a genetic model system to address salt stress adaptations at the molecular level in the quest to identify salt stress tolerance mechanisms. Beside their potential as promising model species, halophytes might also be valued for their potential as cash crops themselves. The present paper aims to highlight the main results gained on C. maritima using multidisciplinary approaches in complement to those obtained on plant model species of the Brassicaceae family. © 2013 Elsevier B.V. All rights reserved.

1. Introduction With the projected increase in populations of 2.4 billion people over the next four decades coupled with increased urbanization in developing countries, world agriculture is faced with an enormous challenge to increase the present level of food production. The progressive salinization of arable lands at the rate of 3 ha/min worldwide is a major concern for agricultural crop production. The Food and Agriculture Organization of the United Nations (FAO) estimates that approximately 20% of agricultural land and 50% cropland in the world including all continents are salt-affected (Flowers and Yeo, 1995). It means that 7% of the land surface and 5% of cultivated lands are affected by salinity (Flowers and Yeo, 1995). Most of this salt affected land has arisen from the accumulation of salts in the soil or groundwater through natural processes over a long period of time (Munns, 2005). Sodium chloride (NaCl) is the most commonly encountered source of salinity (Li et al., 2006). Soils are classified as saline when the electric conductivity (EC) reaches 4 dS m−1 or more, which is equivalent to approximately 40 mM NaCl (Munns and Tester, 2008). Most plants, including the majority of crop species, are glycophytes,

∗ Corresponding author. Tel.: +216 79 325 848; fax: +216 79 325 638. E-mail address: [email protected] (C. Abdelly). 1 Both authors contributed equally. 0098-8472/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.envexpbot.2012.12.002

which means they are very sensitive to salinity. For glycophytes, salinity triggers ionic imbalance, osmotic stress, and secondary stresses such as nutritional disorders and oxidative stress (Zhu, 2001). Halophytes on the other hand are plants that can grow in the presence of high concentrations of salt and present a competitive advantage over non-halophytes in this environment (Debez et al., 2011). The two main mechanisms for salt tolerance utilized by halophytes are minimizing the entry of salt into the plant with the help of highly selective ion transporters and avoiding the build up of salt at toxic concentrations in the cytoplasm. The latter relies on (i) compartmentalizing Na+ and Cl− ions into vacuoles whereas organic solutes accumulate in cytoplasm, thereby reducing osmotic potential and contributing to maintaining water homeostasis (Flowers and Colmer, 2008) and (ii) the selective excretion of salt via special anatomical structures located on a leaf surface (Barhoumi et al., 2007). The problem of salinity can be tackled by either improving farming practices to prevent soil salinization, or by implementing schemes to reclaim and rehabilitate salt-affected soils (Tester and Davenport, 2003). These techniques are usually expensive and generally considered as only temporary solutions. The alternative option is to select and grow salt-tolerant crops. There are about 350 halophyte species, based on the definition of Aronson (1989) and Flowers et al. (2010), which stipulates that ‘halophytes are plants that survive to complete their life cycle in at least 200 mM salt’. The main objective of this paper is to focus on the ecophysiological and

A. Debez et al. / Environmental and Experimental Botany 92 (2013) 64–72

genomic insights into salt responses of the halophyte Cakile maritima, in comparison of the related species Arabidopsis thaliana and Thellungiella salsuginea, two widely used plant models. 2. The need for a model plant to investigate biological functions Much effort in the last decades has been dedicated to understanding the biology of plant stress adaptation with the ultimate objective of identifying key stress-tolerance criteria that could be transferred via traditional breeding and/or transgene technology to crop plants. Despite their apparent diversity, plants use mostly similar biological functions encoded by genes orthologous from one species to another. However, the complexity and the large size of many crop plant genomes are major obstacles to genetic and molecular investigations. 2.1. Arabidopsis thaliana is a glycophyte model plant species In the early 1980s, a small community of plant molecular biologists adopted A. thaliana as a model system to tackle genome organization and biological functions in higher plants (Meinke et al., 1998; Somerville and Koornneef, 2002). A. thaliana (2n = 10), a member of the Brassicaceae, has one of the smallest nuclear DNA content of any higher plants (estimated as 160 Mb) that was fully sequenced in 2000 (The Arabidopsis Genome Initiative, 2000) and this provided the identification of over 27,059 protein-coding complementary DNAs distributed on five chromosomes. A. thaliana was also chosen for a number of practical reasons: it is a small plant (∼20–30 cm) with a short life cycle, taking only 10 weeks from germination to mature seed, and produces numerous self progeny (around 8000 seeds per plant). A number of research groups have exploited genetic diversity available in A. thaliana for salt tolerance studies. However, as a typical glycophyte, Arabidopsis is highly sensitive to salinity, making it a useful model for saltexcluding glycophytes rather than for salt tolerance (Bressan et al., 2001). 2.2. Halophyte model plant species Due to their high plasticity in terms of salt tolerance, halophytes are the plants of choice to search for tolerance genes and to study salt tolerance mechanisms (Flowers et al., 2010). The facultative halophyte Mesembryanthemum crystallinum has been frequently used as a model in salt tolerance studies (Bohnert and Cushman, 2000). While for this plant, a large expressed sequence tag database has been gathered (Cushman and Bohnert, 2000) and a mutant collection established (Cushman et al., 2008), the ability to transform M. crystallinum efficiently is still lacking. Indeed, salt tolerance of M. crystallinum is based on its ability to switch from C3 photosynthesis to Crassulacean acid metabolism (CAM) in response to salt and to sequester salt ions into anatomical structures, called trichomes or bladder cells, making it difficult to transfer salt adaptation from M. crystallinum to other species such as crops. Another plant in the Brassicaceae that is well-established as a model system for understanding the molecular response to salt-stress is T. salsuginea (previously misidentified as T. halophila) (Amtmann, 2009), a halophyte recently discovered as a native living on the saline soils of the Shandong province in eastern China, the Yukon territory of Canada and the rocky mountains of the United States (Inan et al., 2004; Amtmann, 2009). T. salsuginea (2n = 14) is closely related to Arabidopsis with 90–95% cDNA sequence identity between the two species, and it can be easily transformed by using the floral dipping method (Zhu, 2001). Thellungiella has a number of other features useful for genetic research, such as small size, short life cycle, high seed

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number (4000–8000 seeds per plant) (Bressan et al., 2001) and small genome, which is approximately twice that of Arabidopsis (Table 1). Using a comparative genomic approach, microarray analysis showed that salt tolerance of T. salsuginea is due to constitutive expression of large gene clusters which are stress-induced in Arabidopsis (Taji et al., 2004; Kant et al., 2006). A draft genome of T. parvula (2n = 14) showing to be only 15% larger than that of A. thaliana has been published by Dassanayake et al. (2011). The difference in genome sizes seems to be due at least in part to duplication of single copy genes. Recently a draft sequence of the T. salsuginea genome was released based on ∼134fold coverage to seven chromosomes showing a coding capacity of at least 28,457 genes (Wu et al., 2012). These genome data provides resources that should contribute to our understanding of the genetic basis underlying plant abiotic stress tolerance. This invaluable genome information provides exciting prospects to investigate its genome structure and complexity with that of the related A. thaliana and to investigate the evolution of environmental stress tolerance. 3. Contrasted responses of Thellungiella and Arabidopsis toward salinity 3.1. Effect of the developmental stage Salt tolerance thresholds in A. thaliana and T. salsuginea have been mostly addressed using molecular approaches whereas ecophysiological responses have been less considered until now. In recent comparative studies, Orsini et al. (2010) and Guo et al. (2012) reported that the most tolerant genus at the vegetative stage, i.e. Thellungiella, was much more sensitive than Arabidopsis at the germination stage. T. salsuginea and T. parvula were unable to germinate when NaCl concentration exceeded 150 mM, while the germination rate of A. thaliana was around 12%. Interestingly, inhibition of seed germination of T. salsuginea was not due to ion toxicity because germination recovery after transferring seeds to distilled water was higher in seeds of T. salsuginea than of A. thaliana. At the seven-day-old seedling stage, T. salsuginea was more salt tolerant than A. thaliana, as evidenced by higher values of growth parameters such as root length, fresh and dry mass. This may partly explain why A. thaliana is excluded from saline locations while T. salsuginea can survive in these biotopes (Guo et al., 2012). At the vegetative stage, the comparative analyses of growth under similar salinities revealed that salt treatment led to a significant restriction of leaf development (M’rah et al., 2006). This inhibition was stronger in Arabidopsis leaves compared to Thellungiella and was associated with strong inhibition of both leaf initiation and leaf expansion. Analysis of growth, estimated by the relative growth rate (RGR) that reflects biomass production per unit of time, was not significantly affected up to 200 mM NaCl in Thellungiella and was approximately two to three-fold higher than that observed in Arabidopsis (Ghars et al., 2008). Hence, it appears that T. salsuginea is not a salt-requiring halophyte since its growth activity is not improved by the presence of salt (Inan et al., 2004; M’rah et al., 2006; Ghars et al., 2008). 3.2. Na+ accumulation, and K+ versus Na+ selectivity Thellungiella accumulates less Na+ in its shoots compared to Arabidopsis (Inan et al., 2004; Volkov et al., 2004; Ghars et al., 2008). The limited capacity of Thellungiella to accumulate Na+ in leaves suggests that this halophyte does not use the inclusive strategy when challenged with salinity. Moreover, the negative relationship between water and Na+ contents (Ghars et al., 2008) suggests that in

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A. Debez et al. / Environmental and Experimental Botany 92 (2013) 64–72

Table 1 Nuclear DNA content of A. thaliana, T. salsuginea and C. maritima. Seeds were kindly provided by (A) R.A. Bressan, Purdue University, USA; (B) T. Colmer, University of Western Australia, Australia; (C) C. Magné, Université de Bretagne Occidentale, France; (D) A. Eshel, Tel Aviv University, Israel; (E) C. Cassaniti, University of Catania, Italy; (F) C. Abdelly, Centre de Biotechnologie de Borj Cédria (CBBC), Tunisia; and (G) T. Flowers, University of Sussex, United Kingdom. For assessment of DNA content by flow cytometry, leaves were collected from seedlings and chopped in a modified Galbraith buffer (Galbraith et al., 1983) with addition of 1% (w/v) polyvinylpyrrolidone 10,000 and 5 mM metabisulfite. Fluorescence analysis of the propidium iodide stained nuclei was conducted on a Partec CyFlow with 532 nm laser. The internal standard was Lycopersicon esculentum cv. Montfavet 63-5 with 2C = 1.99 pg (Marie and Brown, 1993). Generally, values are means of 6–10 measurements on leaves from 2 to 4 individuals. Species

Origin

A. thaliana a T. salsuginea C. maritima

Columbia Shandong (China) Dongarra (W. Australia) Yallingup (W. Australia) Augusta (W. Australia) b

Conquet (France)

Beyt Yanai beach (Israel) Sicily (Italy) Bizerte (Tunisia) Enfidha (Tunisia) Jerba (Tunisia)

Localization

◦





33 41 31.5 S 114◦ 59 34.0 E 33◦ 41 31.5 S 114◦ 59 34.0 E 34◦ 21 21.6 S 115◦ 07 43.7 E 48◦ 21 31.75 N 4◦ 46 47.87 O 32◦ 23 30 N 34◦ 52 00 E 37◦ 15 N 9◦ 54 E 36◦ 07 N 10◦ 28 E 3◦ 50 N 11◦ E

Sussex (United Kingdom) a b

Source

2C genome size (pg)

SD (pg)

1C genome (Mb)

A B

0.33 0.67 1.60

0.01 0.02

161 328 782

B

1.57

0.02

768

B

1.58

0.04

773

C

1.40

0.02

670

D

1.52

0.02

743

E F

1.36 1.39

0.02 0.03

665 680

F

1.43

0.01

699

F

1.39

0.05

680

G

1.54

0.05

753

T. salsuginea collected in Shandong (China) was subject of a report Inan et al. (2004) where it was misidentified as T. halophila (Amtmann, 2009). C. maritima ssp. integrifolia (Hornem.) Hyl. ex Greuter & Burdet [=ssp. maritima].

Thellungiella, sodium is not the major ion used in osmotic adjustment, and that this species may be deprived of efficient systems for Na+ vacuolar compartmentalization leading to an apoplastic accumulation of Na+ in leaves. Comparative studies between Thellungiella and Arabidopsis emphasizing K+ /Na+ selectivity, a major trait of salt tolerance, highlighted a marked difference in K+ /Na+ selectivity between the two species. When challenged with high Na+ concentrations, Thellungiella shows higher leaf K+ concentrations (Ghars et al., 2008) and higher K+ /Na+ ratios (Volkov et al., 2004) than Arabidopsis. This was partly ascribed to specific characteristics of root ion channels (Volkov et al., 2004; Volkov and Amtmann, 2006) and low unidirectional fluxes of Na+ (Wang et al., 2006) in Thellungiella in comparison with Arabidopsis. Other studies showed that high Na+ concentrations inhibited to a lesser extent the induction of the gene encoding the high-affinity K+ transporter HAK5 in Thellungiella than in Arabidopsis leading to higher rates of high-affinity K+ uptake in the former species when the plants are grown under salinity conditions (Alemán et al., 2008). 3.3. Photosynthesis The effects of short-term salt stress on gas exchange and the regulation of photosynthetic electron transport were examined in Arabidopsis and Thellungiella (Stepien and Johnson, 2009). Exposure of Arabidopsis to sublethal salt concentrations resulted in stomatal closure and inhibition of CO2 fixation. This led to an inhibition of electron transport through photosystem II (PSII), an increase in cyclic electron flow involving only PSI, and increased nonphotochemical quenching of chlorophyll fluorescence. In contrast, in Thellungiella, although gas exchange was marginally inhibited by high salt and PSI was unaffected, there was a large increase in electron flow involving PSII. However, under long-term salinity, other studies have reported a decrease in CO2 assimilations in the leaves of Thellungiella even at 100 mM NaCl although this detrimental effect is much less pronounced than that observed in Arabidopsis (M’rah et al., 2006).

4. T. salsuginea presents some limitations as a model halophyte One hurdle that plant biologists face in using T. salsuginea as a model is that this species requires a prolonged period of vernalization treatment (four weeks at 4 ◦ C) to induce flowering. Furthermore, according to Inan et al. (2004), ten to fourteen days of low temperature pretreatment (4 ◦ C) are normally required to break seed dormancy of this species. T. salsuginea is also significantly less productive than most crops. Among other disadvantages of using T. salsuginea as a model halophyte for studying salinity tolerance is that its seed germination is strongly inhibited by low salinity (Inan et al., 2004). At the vegetative stage, an increase in salinity inhibited growth of T. salsuginea (Ghars et al., 2008). The depressive effects of salinity on plant growth appeared to result from the inhibition of both leaf initiation and expansion (M’rah et al., 2007), rather than from a damage to the functional integrity of the photosynthetic apparatus (M’rah et al., 2006). The low rate of Na+ accumulation in leaves is a major mechanism by which T. salsuginea is able to preserve photosystem II integrity. It appears that T. salsuginea is not as salt tolerant as most halophytic plants, and that this plant avoids ion translocation to shoots. Hence, the limitation of Na+ influx (Wang et al., 2006) and maintaining a high K+ /Na+ ratio (Volkov et al., 2004; Ghars et al., 2008) are the main mechanisms of salt tolerance in this species in comparison with A. thaliana. 5. Cakile maritima: a potential halophytic plant model for studying salinity tolerance 5.1. General description and economical potential C. maritima (sea rocket) is an annual diploid species (2n = 18), characterized by petioled leaves and C3 pathway of photosynthesis. Flowers are lavender or white. The fruit (1–1.25 cm) consists of a two-segmented silique, each segment generally containing a single seed (Rodman, 1974). The upper fruit segment, which has

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a conic to cylindrical shape, is deciduous and contributes to the plant dispersal when transported away by the wind and waves, owing to its long-term floating ability (Barbour and Rodman, 1970; Maun and Payne, 1989). This species is found in sandy habitats along the coasts of North Atlantic and Mediterranean Sea, Canary Islands, southwest Asia and Australia, with climates ranging from arid to humid (Clausing et al., 2000). According to phylogenetic studies based on sequences of ribosomal DNA, C. maritima seems to be more closely related to A. thaliana than T. salsuginea (Bailey et al., 2006). C. maritima, with a ∼719 Mb genome size (Table 1), 4.5-fold that of A. thaliana and 2.2-fold that of T. salsuginea, shows botanical traits suitable for a genetic model system. Foliar nuclei display strong endoreplication (data not shown). It has a short life cycle (three months from seed to seed) and produces a large number of seeds (10,000 seeds per plant). Interestingly, DNA contents between Italian (Sicilia 1C = 665 Mb) and Sussex accessions (average 1C = 753 Mb) show a considerable increase of 13% in genome size in the second accession. For a comparable example of intra-specific variation, the smallest Medicago truncatula genome observed is ecotype 108-1 with 1C = 0.49 pg (38.1% GC) and the largest is Jemalong with 1C = 0.57 pg (38.6% GC) (Blondon et al., 1994), which corresponds to a 16% increase. Indeed, these M. truncatula ecotypes are difficult to cross. The average plant size is higher than that of Arabidopsis but still relatively small (100–120 cm), making it easy to grow C. maritima plants under controlled laboratory conditions. In addition, C. maritima plant biomass facilitates biochemical work, which is a particularly useful trait. 5.2. Salt stress response 5.2.1. Germination The majority (75%) of seeds of C. maritima can germinate in a concentration of salts close to 200 mM NaCl without showing adverse effects (Debez et al., 2004), whereas T. salsuginea germination was fully suppressed at salinities above 120 mM NaCl (Guo et al., 2012). High concentrations of NaCl induced secondary dormancy in seeds of both species, and impaired germination process mainly through an osmotic effect, as evidenced by the high final germination rate (87% and 81%, respectively for C. maritima and T. salsuginea) after transferring non-germinated seeds onto distilled water. C. maritima seeds can survive up to four months immersed in sea water and show up to one year floating capacity on sea water (Gandour et al., 2008). Recently, proteomic investigations revealed that salinity inhibited seed germination in C. maritima by delaying the degradation of seed storage proteins, whereas lipid degradation appeared to be less impacted (Debez et al., 2012). Interestingly, a set of constitutively present proteins involved in several metabolic pathways like glycolysis, amino acid metabolism, photosynthesis, and protein folding was identified. These proteins showed significantly increased abundance during germination. This pattern was yet less pronounced under salinity. 5.2.2. Behavior at the vegetative stage The salt tolerance of a plant species is usually characterized by assessing the growth performance under increased soil salinity. At the vegetative stage, C. maritima exhibited a typical behavior of an euhalophyte, requiring the presence of a moderate salt concentration (50–100 mM NaCl) to express its maximal growth potentialities (Debez et al., 2004). The stimulation of plant growth at optimal salinity was ascribed to higher leaf expansion (as evidenced by the increase in leaf number and leaf surface area per plant) and improved water status (Debez et al., 2006, 2008). Comparing the growth activity (expressed as the whole plant relative growth rate, RGR) of C. maritima with other Brassicaceae (A. thaliana, T. salsuginea, and Brassica napus) under increasing salinity

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highlights the better salt tolerance of C. maritima as the latter showed: (i) higher RGR in the absence of salt, (ii) a stimulation of RGR at moderate salinities and (iii) an aptitude to maintain growth potentialities even at 200 mM NaCl (Table 2). Higher salinities caused a significant decrease in the plant growth, but C. maritima was able to maintain growth activity, flowering and seed production up to 500 mM NaCl. 5.2.2.1. Na+ accumulation. In C. maritima, the Na+ concentration of the leaves increased with increasing NaCl concentration in the medium (Debez et al., 2004). At 500 mM NaCl, leaf Na+ concentration (ca. 4 mmol g−1 DW) was comparable in both C. maritima and T. salsuginea, but the growth activity of the latter species (assessed on the RGR basis) was much more inhibited relative to C. maritima (−77% versus −28% compared to the control, respectively) (Debez et al., 2008; Ghars et al., 2008). In addition, in C. maritima no salt-related toxicity symptoms were noted, except on the oldest leaves (personal observation). High leaf Na+ concentration was associated with increased leaf thickness and succulence, even at 500 mM NaCl (Debez et al., 2006). Increased leaf thickness or succulence has been interpreted as an adaptation of halophytes in terms of conservation of internal water and dilution of accumulated salts (Koyro and Lieth, 2008). Concomitant increases in leaf Na+ accumulation and leaf succulence in salt-treated plants suggested that this halophyte may use the osmotic strategy when dealing with salinity. Control of intracellular sodium concentration is vital for plants coping with salinity and lacking salt removal structures at the leaf surface. This is partly achieved by excluding and/or compartmentalizing Na+ , thereby preventing its excessive cytosolic buildup (Apse and Blumwald, 2002). Both these processes are mediated by Na+ /H+ antiporters, which use the pH gradient generated by the plasma membrane (PM) and vacuolar (V) H+-ATPases (localized at the plasmalemma and the tonoplast, respectively), to actively transport Na+ against its electrochemical gradient toward the vacuole and/or through the plasma membrane (Zhu, 2001). The bi-directional transport of sodium contributes not only to ion homeostasis and cell turgor, it also protects the metabolic enzymes from salt toxicity (Aharon et al., 2003). In C. maritima, there are complementary mechanisms involving both vacuolar (V H+ -ATPase) and plasma membrane (PM H+ -ATPase), respectively at moderate (100–300 mM NaCl) and severe salinities (300–500 mM NaCl) (Debez et al., 2006). 5.2.2.2. K+ /Na+ selectivity. K+ homeostasis at the whole plant and cellular levels plays a crucial role in salt adaptation of plants. The increased Na+ accumulation in C. maritima leaves was associated with reduction in K+ uptake and consequently reduction in the K+ /Na+ ratios. However, restriction of K+ uptake by roots was compensated by an increase in the K+ use efficiency (i.e., the quantity of biomass produced per unit of absorbed K+ ) (Debez et al., 2008). Hence, C. maritima can use K+ parsimoniously for specific functions when challenged with salinity, and K+ could be replaced by Na+ for osmotic adjustment by accumulation in the vacuoles. In addition, C. maritima exhibited a strong selectivity for K+ over Na+ , so that the SK+, Na+ ratio, calculated from ion concentrations in the leaves and in the culture medium (SK+, Na+ = (K+ /(K+ + Na+ )]leaves /[K+ /(K+ + Na+ )]medium ) remained higher in C. maritima leaves than in the culture medium, even at high external NaCl concentration (500 mM NaCl) (Debez et al., 2004). 5.2.2.3. Photosynthesis. The photosynthetic activity, assessed by net photosynthetic rate (A), stomatal conductance (gs ) and transpiration rate (E), was significantly improved in C. maritima at the optimal salinity (100 mM NaCl) followed by a decline at higher salinities (Debez et al., 2008). The reduction of stomatal aperture in

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Table 2 Salt-responses of C. maritima compared with T. salsuginea, A. thaliana, and B. napus based on the plant relative growth rate (RGR). Species

C. maritima T. salsuginea A. thaliana B. napus

Relative growth rate Salinity (mM)

Source

0

50

100

200

0.09 0.09 0.07 0.07

0.12 0.09 0.04 –

0.11 0.09 0.04 0.06

0.1 0.07 0.02 0.04

response to salt stress is a characteristic response of salt-adapted plants and may prevent water loss through transpiration. Hence, the limitation in stomatal conductance (gs ) and subsequently transpiration rate (E) may be considered as an adaptive mechanism to cope with excessive salt, rather than merely a negative consequence of it (Megdiche et al., 2008). At supra-optimal salinities a marked decrease in the relative quantum yield of photosystem II (ФPSII ) was observed, along with a significant increase in nonphotochemical quenching (NPQ) and almost constant maximum quantum yield of photochemistry (Fv /Fm ). Hence, the performance of C. maritima when salt challenged could be partly ascribed to its ability to maintain PSII functional integrity. It is also noteworthy that the chlorophyll concentration was unaffected up to 500 mM NaCl. These ecophysiological data were recently confirmed used proteomic experiments that indicated a significantly higher abundance of several proteins involved in photosynthesis upon salt-exposure of C. maritima to 100 mM NaCl for one month (Debez et al., 2012). 5.2.2.4. Antioxidant responses. Available literature suggests that the capacity to limit oxidative damage is an important determinant of C. maritima performance when salt-challenged. Ben Amor et al. (2006) investigated salt (100, 200, and 400 mM NaCl) effect on antioxidant activities in two Tunisian accessions of C. maritima collected from Tabarka (north of Tunisia, humid bioclimatic stage) and Jerba (south of Tunisia, arid bioclimatic stage). Interestingly, growth of the Jerba accession was improved after 100 mM NaCl treatment for 20 days as compared to that of control whereas ‘Tabarka’ appeared to be salt-sensitive. The relative salt tolerance of ‘Jerba’ was found to be partly due to higher antioxidant enzyme (superoxide dismutase, catalase, peroxidase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase) activities and glutathione content, together with lower malondialdehyde content and H2 O2 concentration than in ‘Tabarka’. Higher polyphenol content and antioxidant activity (smaller IC50 values for both 1,1diphenyl-2-picrylhydrazyl and superoxide scavenging) also partly explained the better salt-tolerance of Jerba accession (Ksouri et al., 2007). In a recent comparative study, Ellouzi et al. (2011) compared early changes in growth and antioxidant defence of C. maritima with those of A. thaliana when challenged with 400 mM and 100 mM NaCl, respectively. Reduction in growth at supra-optimal salinity was less pronounced in C. maritima than in A. thaliana. Hydrogen peroxide (H2 O2 ) concentration and antioxidant enzyme (superoxide dismutase, catalase and peroxidase) activities were maximal in leaves of both species 4 h after salt treatment, but decreased rapidly in C. maritima whereas they remained high in A. thaliana. Maximum values of malondialdehyde concentration were registered after 24 h of salt stress in both species before decreasing but only in C. maritima. Hence, unlike in A. thaliana, antioxidant mechanisms took place early following salt exposure in C. maritima enabling the latter to cope with salt stress and cope with salt-induced oxidative stress. This may explain, at least partially, the difference in salt tolerance between halophytes and glycophytes.

Debez et al. (2008) Ghars et al. (2008) Ghars et al. (2008) Naeem et al. (2011)

5.2.2.5. Genetic and molecular insights into salt response of C. maritima. C. maritima shows considerable variation, within and between sub-species, especially in fruit morphology and leaf shape (Gandour et al., 2008). Leaf shape is variable, becoming more pinnatified among populations originating from arid climate in comparison to those originating from humid climates, which can be correlated with adaptation strategies to heat, high light intensity and drought (Ciccarelli et al., 2010). Isozymes analysis using 13 polymorphic loci encoding eight enzyme systems allowed identification of the variability within and among nine geographically distant Tunisian C. maritima populations (Gandour et al., 2008). Moreover, the genetic distance between populations was correlated with sea water stream speed rather than to their geographic distance. A significant variability in salt response was observed in some Tunisian C. maritima accessions. The better salt tolerance of Jerba (arid climatic area) accession compared to Tabarka (humid climatic area) was related to its capacity to maintain leaf water status even at 400 mM NaCl. The salt tolerant accession had particularly high proline content, no doubt part of an osmotic adjustment process (Megdiche et al., 2007). Molecular responses of C. maritima toward salinity are poorly documented. So far, only the expression of cysteine protease inhibitor (cystatin) genes in leaves of C. maritima plants exposed to water deficit stress or salinity has been investigated (Megdiche et al., 2009). Cystatins show different expression patterns not only during plant development but also in response to abiotic constraints, including heat shock, water-deficit stress and salinity (Pernas et al., 2000; Gaddour et al., 2001; Wang et al., 2003). Moreover, cystatins may play a role in the regulation of protein turnover during seed development (Corre-Menguy et al., 2002) and plant defence against insect predation and pathogens (Oppert et al., 2003). Megdiche et al. (2009) isolated a new cystatin cDNA (CmC) encoding a 221 amino acid protein with a calculated molecular mass of 25 kDa. This protein was constitutively expressed in nonstressed plants of different accessions. Augmented expression was observed under severe salinity except in the ecotype from the arid region (Jerba). Water deficit also increased CmC expression in two ecotypes, with the highest value observed in the ecotype from the humid region (Tabarka). 5.2.3. Seed production Several yield traits such as the harvest index, the silique number per plant, and the seed number per plant were maximal at 100 mM NaCl. C. maritima was efficient in protecting seeds from the accumulation of salt, but salinities above 200 mM NaCl were harmful for plant fertility (seed viability dramatically affected concomitant with empty siliques). Individual seed mass was also salt-sensitive, probably due to the impairment of leaf initiation and photosynthetic activity. The salt-induced reduction of seed mass and viability did not parallel the quantity of Na+ present in seeds, which was almost the same for all treatments. This observation strengthens the hypothesis that the adverse effects of salinity on seed development resulted from the salt-induced restriction of seed nutrition and filling. Interestingly, high ovule abortion and embryo senescence leading to reduced fertility in A. thaliana under salt-stress

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A

Seed oil content (% / DW)

35 30 25 20 15 10 50

0

100

200

300

500

NaCl (mM)

Seed oil composition (% of total fatty acids)

40

C18:1 C16:0

C22:1 C18:0

C18:3 C16:1

C18:2 C20:0

C20:1 C22:0

B

30

20

10

0 0

100

200

300

400

500

NaCl (mM) Fig. 1. Salt-induced quantitative (A) and qualitative (B) changes in the seed oil of C. maritima (n = 3 per treatment). C16:0 : palmitic acid; C16:1 : palmitoleic acid; C18:0 : stearic acid; C18:1 : oleic acid; C18:2 : linoleic acid; C18:3 : linolenic acid; C20:0 : arachidic acid; C20:1 : gadoleic acid; C22:0 : behenic acid; C22:1 : erucic acid.

have been proposed to represent an adaptive strategy enabling the plant to conserve resources useful for its acclimation to stressful environmental conditions (Sun et al., 2004). 5.3. Economical potential Alternative uses of crops for non-food purposes may be an interesting source of profit for farmers, as is happening for high erucic acid oils. The current demand for these oils is still limited: at world level, it is nearly 20,000 tons of erucic acid, corresponding to about 57,000 tons of oils. The actual world need for seeds containing erucic acid is not very large, about 100,000–120,000 tons, but the positive trend observed in the last few years should allow significant extension of cultivation of erucic-acid-rich crops. This implies that studies on the adaptation of species containing erucic acid in various environments are essential (Zanetti et al., 2012). Erucic acid is an unsaturated fatty acid (C22:1) with a large number of applications in the chemical industry because it confers desirable technological characteristics, such as high lubricity, cold stability and fire resistance, on oils and derived compounds (Zanetti et al., 2012). The use of erucic acid in industry (green chemistry) has been increasing considerably in the last 10 years (Zanetti et al., 2009). Erucic acid is mainly used to produce erucamide, a slip agent for polyethylene and polypropylene films. The world market for slip agents derived from erucic acid is now estimated at approximately 30,000 tons per year, and the consumption trend is expected to increase at a rate of 3–5% per annum. Erucic acid also has other

interesting industrial uses, such as the production of behenic acid, behenic alcohol and their derived compounds, which are used as pour point depressants and to produce caprenin (Gunstone, 2004). Members of the Brassicaceae are plants with the highest potential in terms of production of erucic acid per hectare and percentages of this fatty acid in their oil: particularly important, worldwide, are B. napus var. oleifera and, in warmer climates, B. carinata and Crambe abyssinica Hochst. ex R.E. Fries (Zanetti et al., 2006; Meakin, 2007). Besides its ecological interest for beach dune fixation due to a deep root system (40 cm length) with extensive lateral roots (Davy et al., 2006), C. maritima shows a potential as an oilseed crop (Zarrouk et al., 2003). Seed-oil (40% on dry weight basis) of C. maritima contained a high proportion of erucic acid (25–35% of total fatty acids) (Ghars et al., 2006). Seed oil content (on a dry weight basis) was unaffected by salinity (Fig. 1A) whereas erucic acid level increased markedly, reaching 26% at 500 mM NaCl (two-fold higher than the control value). This was concomitant with a significant decrease in oleic acid, which was the major fatty acid in seed oil of the control (Fig. 1B). The abovementioned data are of practical significance in the context of biosaline agriculture using domesticated halophytes, since they point the economic potential and the feasibility of cultivating C. maritima. However, one important trait that needs attention is the fact that yield in terms of seed production may be reduced by at least 50%, as a consequence of the fruit morphology of C. maritima. This is characterized by the presence of two segments, an upper very fragile segment which dehisces easily at full

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Table 3 Comparison of botanical, physiological and genomic traits in T. salsuginea (Shandong) and C. maritima.

Plant family Chromosome number (2n) 1C genome sizea (Mb) 2C DNA contenta (pg) Life cycle (month) Height at maturity (cm) Vernalization/stratification Seed yield plant−1 Germination Behavior Photosynthetic activityb Photosystem II integrity Threshold Na+ accumulation Na+ vacuolar compartmentalization Osmotic effect K+ /Na+ selectivity Compatible solute Economic interests Ecological interest Transformation efficiency a b

T. salsuginea

C. maritima

Brassicaceae 14 328 0.67 3 ∼20–30 Required 4000–8000 Sensitive Absence of ion toxicity Facultative halophyte Affected Preserved ca. 4 mmol g−1 DW Limited Decreased water content Efficient selectivity K+ /Na+ Proline – – Yes

Brassicaceae 18 675 1.4 3–4 ∼100–120 Not required 10,000 Tolerant up to 200 mM NaCl Absence of ion toxicity Obligate halophyte Enhanced, affected at 500 mM NaCl Preserved >4 mmol g−1 DW Effective up to 300 mM NaCl Succulence Efficient selectivity K+/ Na+ Proline, Glycine betaine Oilseed cash crop Dune fixation Not yet determined

Our evaluations by cytometry. Assessed by gas exchange parameters.

maturation, and a lower segment that remains attached to the mother plant. According to Gandour et al. (2011), harvesting siliques 10–15 days before yellowing (full maturity stage) not only avoids the loss of the upper segment of siliques, but would also result in a two-fold increase of the seed (though immature) production per plant, despite the loss observed in both seed-oil content and the erucic acid content after direct extraction from fruits (−14% and −30%, respectively). The authors estimated the gain in erucic acid production when harvesting immature seeds to 40% as compared to the amount obtained by harvesting mature seeds; this was due to the augmentation of the yield in seed-oil amount per plant by avoiding seed loss. There are also several indications for the medicinal potential of this species as a source of natural antioxidants like phenols, ascorbic acid, flavonoids, coumarins, alkaloids, triterpenes, sterols and sulfur glycosides (Ben Amor et al., 2006; Ksouri et al., 2012; Radwan et al., 2008). These compounds are known to prevent cardiovascular diseases and different types of cancer. Halophytes are potentially interesting for production of secondary metabolites useful for food and medicinal applications especially when originating from biotopes characterized by extreme climates. Interestingly, an antifungal activity of phytoalexins and glucosinolates was reported in C. maritima (Sellam et al., 2007) and C. maritima extract has been used in cosmetic composition, especially a moisturizing composition for skin, due to an active principle of the glucosinolates (Fabre and Dussert, 2000). 6. Conclusions and perspectives Progress in the last decade in our understanding on regulatory networks of plant response to salt stress are mostly centered around studies on genomic, proteomic and metabolomic comparison between A. thaliana and its halophytic relative, T. salsuginea. However, as a facultative halophyte, the salt tolerance of T. salsuginea is based mainly on its ability to avoid Na+ translocation to the leaves. Moreover, the lack of vacuolar compartmentalization capacity of Na+ to ensure osmotic adjustment limits the use T. salsuginea as a model for succulent halophytes (Table 3). Among the Brassicaceae, C. maritima is worthy of attention, because it meets all the criteria for being a good genetic model system (small genome, diploid, self-compatible) (Table 3). However it remains to

be established whether this species can be easily transformed using Agrobacterium tumefaciens. To cope with salt stress, C. maritima has developed several tolerance mechanisms, including: (i) maintenance of water status; (ii) ability to use Na+ for osmotic adjustment; (iii) efficiency of selective K+ uptake, in spite of high Na+ concentration in the medium; and (iv) preservation of the functional integrity of the photosynthetic apparatus. So, in light of these advantageous criteria, we propose C. martima as a suitable halophytic model to identify molecular mechanisms that regulate tolerance to salt stress in plants. Acknowledgements Cytometry was done on the Imagif Facility (CNRS, Gif-surYvette, France) and we thank Nicolas Maunoury for his aid. This work was supported by the Tunisian Ministry of Higher Education, Scientific Research and Technology (LR02CB02) and was performed in the framework of the COST action FA0901 “Putting Halophytes to Work – From Genes to Ecosystems”. The precious collaboration of Professors Mokhtar Zarrouk, Bernhard Huchzermeyer, HansWerner Koyro, Helmut Lieth, Francesca Sévilla, Sergi Munné-Bosch, and Christian Magné is gratefully acknowledged. References Aharon, R., Shahak, Y., Wininger, S., Bendov, R., Kapulnik, Y., Galili, G., 2003. Overexpression of a plasma membrane aquaporin in transgenic tobacco improves plant vigor under favorable growth conditions but not under drought or salt stress. The Plant Cell 15, 439–447. Alemán, F., Manuel Nieves-Cordones, M., Vicente Martinez, V., Francisco Rubio, F., 2008. Differential regulation of the HAK5 genes encoding the high-affinity K+ transporters of Thellungiella halophila and Arabidopsis thaliana. Environmental and Experimental Botany 65, 263–269. Amtmann, A., 2009. Learning from evolution: Thellungiella generates new knowledge on essential and critical components of abiotic stress tolerance in plants. Molecular Plant 1, 3–12. Apse, M.P., Blumwald, E., 2002. Engineering salt tolerance. Current Opinion in Biotechnology 13, 146–150. Aronson, J.A., 1989. HALOPH a data base of salt tolerant plants of the world. Office of Arid Land Studies, University of Arizona, Tucson, AZ, USA. Bailey, C.D., Koch, M.A., Mayer, M., Mummenhoff, K., O’Kane Jr., S.L., Warwick, S.I., Windham, M.D., Al-Shehbaz, I.A., 2006. Toward a global phylogeny of the Brassicaceae. Molecular Biology and Evolution 23, 2142–2160. Barbour, M.G., Rodman, J.E., 1970. Saga of the west coast sea roket, Cakile edentula ssp. California and Cakile maritima. Rhodora 72, 370–386.

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