Samuele Moretti
Salt and Drought Stress in Olive
Tree (Olea europaea L.): an
1
Abstract ... 3
1.Introduction ... 5
1.1 Olea europaea L.: taxonomy and history, botanical description and actual oliviculture diffusion ... 5
1.1.1 Taxonomy and History ... 5
1.1.2 Botanical aspects ... 7
1.1.3 Oliviculture diffusion and importance ... 8
1.2 Salinity stress in olive plants ... 10
1.2.1 Salinity problem ... 10
1.2.2 Salinity and olive: physiological, biochemical and molecular adjustment ... 12
1.3 Olive fruit: development, anatomy, metabolomics and salt stress-related traits . 16 1.3.1 Fruit characteristics and development ... 16
1.3.2 Phenolic compounds of olive fruit ... 21
1.3.3 Fatty acid in olive fruit ... 23
1.4 Drought stress in olive plant ... 25
1.4.1 Drought problem... 25
1.4.2 Drought and olive: physiological, biochemical and molecular adjustment .. 28
1.5 Stress combination in plants and in olive ... 32
2. Aims ... 36
3. Results and Discussion ... 38
3.1 Olive fruit physiology under salt stress (Annex I)... 38
3.2 Olive flesh fatty acid pathway under salt stress (Annex II) ... 41
3.3 Effect of salt stress and water regime on different olive cultivar (Annex III) ... 46
2
5. Acknowledgements ... 54
6. References ... 56
7. Publications and conference papers ... 71
7.1 Paper in publication ... 71
7.2 Papers in preparation ... 71
7.3 Conference papers ... 71
3
Abstract
Olive tree is a widespread cultivated tree among Mediterranean area. Despite this, Mediterranean climate is not the best for its cultivation, basically for drought and salinity environmental problem.
The physiological, biochemical and molecular response to salinity in olive fruit it has been studied in salt-sensitive Leccino cultivar, able to translocate Na+ to aerial part. In our experimental conditions, results shown a stress response concentrated in early veraison stage (<50% of purple skin). In fact, irrigating with saline water from pit-hardening to harvest, Na+ concentration reach the highest values in fruit flesh of Maturation group 1 (MG1, <50% of purple skin). Particularly, up to 600 mg Kg -1 when subjected to 60 mM NaCl irrigation water and about 2214 mg kg-1 when is irrigated with 80 mM NaCl water. With the advance of ripening, Na+ concentration in flesh is reduced, suggesting a re-translocation in vegetative portion to permit a suitable accumulation of oil droplets in cells. Also antioxidant response is MG-related, showing an increase of DPPH%, PAL activity, Total Phenols and Anthocyanins content (respectively, +15%, +16%, +58% and around doubled). Anatomical traits under salt stress reveal a thickening in all fruit tissue layers (Cuticle, epidermis, Hypodermis and Outer Mesocarp), that could be useful for biotic agent protection.
Fatty acid profile of Triacyl-glicerols (TAGs) under salt stress record major changes in MG1, showing an increment of oleic acid (+5%), a decrement of linoleic acid (-2.36%) and a consequent increment of oleic/linoleic ratio and a related transcriptional down-regulation of FAD6 genes. Low Electron Transport Rate (ETR) between photosystems recorded in the leaves closest to the infructescence could allow to a more oxidized state of those complexes, causing
4 a reduction in the activity levels of the FAD6 enzyme. Also the up-regulation of
FAD2-2 seems to have a role in salt stress response, mainly for maintaining
plasmatic membrane stability. Anyway, total fatty acid richness fraction is not affected by salt stress.
Simultaneous salt and drought stress response it has been studied in cultivars Leccino and Frantoio, differentially-sensitive to both stress.Water status of simultaneous stressed plants were monitored with non-destructive innovative methodologies (Zim-probes), revealing the typical inversion phenomena of Pp curve shape due to an unfavorable air/water ratio typical of plant‘s osmotic injuries.
Physiological and biochemical data shown that simultaneous drought and salt stress didn‘t impair growth, total plant fresh and dry weight and Na+ concentration in Leccino leaves (6359 mg kg-1), although water loss in lysimeters recorded by wheighing (assumed as effective evapo-transpiration) at the end of experiment is influenced (-62% in Leccino vs -53.5% in Frantoio, respect to control). Anyway, Na+content on a whole-plant basis is reduced by water scarcity (-47.6% in Leccino vs -31.6% in Frantoio), explaining the lack of effect on growth and biomass allocation. Major effects of simultaneous drought and salt stress is focused in leaves: non-structural carbon partitioning toward mannitol (39 mg g-1 in Leccino vs 22 mg g-1in Frantoio on a dry weight basis) and cations stechiometric ratios (K/Na ratio 22 times higher in Frantoio and Ca/Na ratio 11 times higher in Frantoio). PCA analysis help to conclude that in simultaneous drought and salt stress conditions only at leaves level is evident a cultivar related response, mainly for higher K/Na and Ca/Na ratios level in leaves.
5
1.Introduction
1.1 Olea europaea L.: taxonomy and history, botanical description and actual oliviculture diffusion
1.1.1 Taxonomy and History
Olive tree (Olea europaea L.) is an evergreen species (tree or shrub) belonging to the Oleaceae family, that includes about 25 genera and more than 600 species dislocated in temperate and tropical areas (Green, 2004; Besnard et al. 2009). The native areas of this species are the eastern Mediterranean Basin coastal regions as well as the south region of Caspian Sea, in north Iran.
The taxonomical classification of Olea genus and the study of domestication event can be quite tricky, and nowadays is still an hot topic of research (Besnard and Rubio de Casas, 2016). Following one of the most used taxonomy classification proposed by Green (2002), 33 species ofthe Olea genus have been classified, organized into 3 subgenera: Olea, Paniculatae and Tetrapilus. The subgenus Olea is composed by two sections, Olea and Ligustroides, and in the first one is included Olea europaea. Six subspecies characterize the Olea
europaea complex: O. europaea europaea, O. europaea cuspidata, O.europaea laperrinei, O. europaea maroccana, O. europaea cerasiformis, and O. europaea guanchica. The Olea europaea complex extends through Macronesia
(O. cerasiformis and O. guanchica), Africa (O. maroccana), Asia (O.
cuspidata), and Mediterranean (O. europaea) regions. Within the Olea europaea subspecies, Green recognizes two varieties: Olea europaea subsp. europaea var. europaea, and O. europaea subsp. europaea var. sylvestris. Only
the first one (var. europaea) is cultivated. Nowadays, the cultivated olive has a wide genetic patrimony organized in more than 1500 cultivars that are
6 predominantly present in southern European countries such as Italy, Spain, France, and Greece (Bartolini, 2008).
Olive (Olea europaea L.) cultivation has a millennial relationship with Mediterranean area, where started before the third millennium B.C. (Loukas et
al. 1983). The cradle of oliviculture is considered to be the middle east area of
Mediterranean (Syria, Turkey, Lebanon, Israel), for then spread to the west area, reaching Spain in 1000 B.C., Greece in 1500 B.C. and Italy more or less in 600 B.C., the 3 more relevant countries for olives production (Figure1).
Figure 1. Historical and Geographical diffusion of olive tree cultivation. From S. Delamont, Appetites and Identities. London: Routledge, 1995.
7 Figure 2.Botanical characteristics of olive
tree. From PABST, 1887.
Nowadays, olive tree is considered the most iconic species between Mediterranean crops, thanks to its economical, ecological and cultural importance (Carrión et al. 2010; Kaniewski et al. 2012).
1.1.2 Botanical aspects
Concerning the botanical characteristics, olive tree is short and low to ground, and rarely its height exceeds 8–15 meters. Leaves are silvery green, oblong, 4– 10 centimeters long and 1–3 centimeters wide. The trunk is typically gnarled and twisted. Flowers are white, small and feathery, with ten-cleft calyx and corolla, bifid stigma and two stamens, and generally develop on the last year's wood, in racemes springing from leaves axils (Cappelletti, 1976). The fruit is a small drupe 1–3 cm long, and in cultivated plants is bigger and thicker-fleshed than in wild ones. The fleshy drupe enclose a sclerified endocarp called pit, and inside the pit is included 1 (rarely 2) seed (Figure 2).
8
1.1.3 Oliviculture diffusion and importance
Nowadays, olive is the second most relevant oil fruit tree, after oil palm, covering more than ten million hectares of land worldwide (FAOSTAT 2014).
Itscultivation is predominantly concentrated (>95% of the cultivated surface) in the Mediterranean basin, where more than 750000000 of olive plants are cultivated for oil and table olive production and where a large proportion of the olive oil produced is also consumed (Baldoni and Belaj 2009). Currently, the 3 leader of world olive oil production are Spain (43%), Italy (14%) and Greece (10%), followed by Turkey, Syria and Tunisia (6%), Morocco (4%), Portugal and Algeria with a little percentage (Figure3).
Figure 3.World production and consumption of olive oil. From International Olive Council (IOC), 2011/2012 Forecast reports.
9 Furthermore, olive cultivation is in expansion in non-traditional producing countries such as the United States, South-America, Australia, Japan and China. In Australia and Argentina olive is now a well-established production reality with orchards irrigated and highly mechanized (Figure3).
Although olive is considered one of the best landscape marker in the Mediterranean area (Blondel et al. 2010; Carrión et al. 2010) and its cultivation has coevolved with the rise of Early Mediterranean civilizations (Kaniewski et
al. 2012), this does not mean that Mediterranean climate is also the optimal one.
In fact, Mediterranean climate is characterized by hot-dry summers and mild to cool, wet winters. The growth, yield and quality of the Mediterranean crops are significantly affected by drought, high temperatures and irradiation (UVs), that are often combined with salinity. Salinity and drought stress has a major role in oliviculture limitation in the Mediterranean basin, although olive is considered intermediate drought- and salt-tolerant plant, in comparison with other mediterranean fruit woody crop (Bracciet al. 2011; Ghrabet al. 2013).
Despite the relevance of olive resistance to environmental stress (Sebastiani
et al. 2002), research in this field are still limited, especially considering olive
fruit physiology, biochemical and molecular response to salt stress and simultaneous drought and salt conditions.
10 1.2 Salinity stress in olive plants
1.2.1 Salinity problem
Soil salinization includes saline, sodic and alkaline soils (van Beek and Tóth, 2012), respectively defined as (i) high salt concentration, (ii) high sodium cation (Na+) concentration, and (iii) high pH, often due to high CO3 2-concentration, in the soil. Concerning the relevance in term of environmental problem, salinity is a major abiotic stress that severely limits plant productivity worldwide. It is a widespread phenomenon, with saline and sodic soils covering 932.2 Mha globally (Rengasamy, 2006). It is calculated that more than 6% of the world's total land and circa 20% of irrigated land suffer from salinity (Munns et al. 2012). Moreover, global food requirements are expected to increase by 70–110% by 2050 (Tillman et al. 2011) and land degradation, urban spread and seawater intrusion are increasing over time (Munns et al. 2012).
This problem concerns arid and sub-arid climates and is caused by several reasons (Figure 4) as high soil evapotranspiration, seawater intrusion and the use of salty water for irrigation (Rengasamy, 2010).
11 In the Mediterranean basin, characterized by high solar radiation, hot temperature and long period of drought, salinity is a main factors influencing crop productivity (Ghrab et al. 2013). Moreover, considering the actual land degradation and soil erosion, seawater intrusion is becoming more frequent in Europe, especially in Mediterranean countries like Italy, south of Spain and Greece, the 3 countries leader in oliviculture (Figure 5).
Figura 5. Salin soil and areas of seawater intrusion in Europe. Compiled from SGDBE, EEA (1999), Daskalaki and Voudouris (2008) and Fischer et al. (2008).
12
1.2.2 Salinity and olive: physiological, biochemical and molecular adjustment
In comparison with other woody fruit crop (generally salt sensitive), olive is considered a glycophite species but with higher tolerance to salt stress. This is due to several physiological, biochemical and molecular adjustment that olive plants figured out in his coevolution with Mediterranean area (Gupta and Huang 2014).
Genotype, plant age, as well as agro-environmental variables, influence olive response to salinity. In the past years scientist develop a threshold for salt-water concentration and olive cultivation: it has been found that 137mM NaCl represents the highest concentration suitable for olive life (Rugini and Fedeli, 1990). In a review on olive tree and salinity, Chartoulakis et al. (2005) organized several olive cultivars on the basis of tolerance and sensitivity to salinity (Table 1).
Tattini et al. (1992) recorded difference salt response between Leccino and Frantoio, two of the most studied Italian cultivar as a model of salt sensitivity and tolerance, respectively. Recently, Pandolfi et al. (2017) contribute to clarify the salt acclimatation process difference in these two cultivars. More authors contributes to clarify genotypic classification on the basis of salt tolerance (Therios and Misonopolis, 1988; Bartolini et al. 1991; Bennlloch et al. 1994; Marin et al. 1995; Chartzoulakis et al. 2002;Soda et al., 2016; Roussous et al., 2017).
It is widely accepted that olive tree ability to exclude or retain Na+ at root level is related to salt-tolerance mechanism (Gucci and Tattini 1997; Chartzoulakis 2005). This because Na+ loading into the xylem is avoided or restrained, preventing the allocation of toxic ions in the shoot. In fact, is documented that tolerant cultivars can maintain a decreasing gradient of these cytotoxic ions from roots to shoot.
13 Table 1. Olive cultivars classification according to salt tolerance level. From Chartoulakis (2005).
Resistance Cultivar Country Source
Tolerant Megaritiki, Lianolia Kerkiras, Greece Therios and Misopolinos (1988)
Kalamata, Kothreiki Greece Chartzoulakis et
al. (2002a,b)
Frantoio Italy Tattini et al. (1992, 1994)
Arbequina, Picual, Jabaluna
Nevadillo Spain
Benlloch et al. (1994)
Lechin de Sevilla, Canivano,
Esscarabajuelo Spain Marin et al. (1995)
Hamed Egypt El-SayedEmtithal
et al. (1996)
Chemlali Tunisia Bouaziz (1990)
Moderately tolerant Amphissis, Koroneiki, Mastoidis, Greece Therios and Misopolinos (1988)
Valanolia, Adramitini Greece Chartzoulakis et
al. (2002a,b)
Maurino, Coratina, Carolca,
Moraiolo Italy
Briccoli Bati et al. (1994) Tattini et
al. (1994) Bartolini et al. (1991)
Aggezi, Toffahi Egypt El-Sayed Emtithal
et al. (1996)
NabaliMuhassan Jordan Al-Absi et al. (2003) Sensitive Chalkidikis, Throubolia, Aguromanaki Greece Therios and Misopolinos (1988), Chartzoulakis et al. (2002a,b)
Leccino Italy Tattini et al.
(1994)
Bouteillan, Nabal, Egypt El-Sayed Emtithal
et al. (1996)
Pajarero, Chetoui, Calego, Spain Benlloch et al. (1994),
14 Chartzoulakis et al. (2005) reports that probably the K–Na exchanges at plasmatic membran level contribute to regulate the translocation of Na+ to the shoot. In fact, Gucci et al. (2003) report that tolerant Frantoio cultivar have a higher K+/Na+ ratio in aerial parts than the sensitive cv. Leccino. Anyway, this mechanism works well at moderate levels of salinity. At high salinity level, Na+ is transported to the shoot, where it is preferably stored in the cell vacuoles. As a general consideration, Cl- uptake and translocation is generally lower than that of Na+ (Bongi and Loreto, 1989; Gucci and Tattini, 1997) and symptoms of salinity and their severity are more often found to correlate to Na+ tissue concentrations but not to Cl- (Aragues et al. 2005). Another stechiometric ratio that allows olive plants to better tolerate salt stress is Ca/Na ratio: in fact, Rinaldelli and Mancuso (1996) report that high Ca/Na allow to higher stability of plasmatic membran, maintain a more suitable K-Na selectivity.
Recently, apoplastic barriers formation and ion localization in root of Leccino and Frantoio salt stressed (120 mM NaCl) were investigated by Rossi
et al. (2015). In comparison with Frantoio, Leccino shown endodermis
apoplastic barriers formation closer to the root apex, and gradient of Na+ from exodermis to inner stele tissues is genotype- and cell types-dependent. Anyway, reduction of Na+gradient observed in Frantoio (from outer to inner root tissues) in response to root apoplastic adjustments, not completely avoid Na+ translocation.
Concerning effects of salt stress in plant, it is possible to clusterize: i) visible effects (roots, leaves, apex and stem tip chlorosis and necrosis, flowers desiccation, ovary abortion, leaves abscission, reduced growth, shortening of internodes, reduction in leaf area; Gucci et al. 2003; Kchaou et al. 2010); ii) effects at root level (osmotic stress due to the decrease in soil water potential
-15
Ψsoil-, inhibition of the absorption of essential elements and nutrients imbalance,
accumulation of inorganic toxic ions (such as Na+ and Cl−) at cellular level, (Marschner 1995; Chartzoulakis, 2005; Tabataei et al. 2006; Perica et al. 2008; Demiral et al. 2011); iii) effects on plant‘s water relations (reductions in pre-dawn water potential -Ψw- and in leaf relative water content – RWC-,
biosynthesis of compatible osmolites like proline, glycine-betaine or non-structural carbohydrates (Theriosand Misopolinos 1988; Tattini et al. 1996; Gucci et al.1997; Tattini and Traversi, 2008); iv) effects on photosynthetic machinery (reduction of the photosynthetic activity, stomatal limitations to photosynthesis, low concentration of CO2 at the chloroplast level due to low stomatal and mesophillar conductance (Loreto et al. 2003; Chartzoulakis et al. 2005; Ben-ahmed et al. 2008; Kchaou et al. 2013); v) biochemical and molecular effects (ROS formation, Ca2+ related second messenger, increasing of transcriptional products putatively related to superoxide dismutase, glutathione reductase , proline dehydrogenase and modulation of phenols pathway (Tattini
et al. 2009; Petridis et al. 2012; Bazakos et al. 2015). Regarding phenols, a
recent experiment performed by Rossi et al. (2016) showed that phenolic compounds remain stable or are depleted under long-time treatment with sodium in the salt-sensitive cultivar Leccino, determining a strong up-regulation of key genes of the phenylpropanoid pathway. In salt-tolerant cultivar Frantoio the phenolic compounds content was always high and the up-regulation of the phenylpropanoid genes was less intense.
As we can see, research on olive salt stress in these years has been focused more on vegetative portion. Focusing effort in olive fruit salt stress related traits could be very important, considering the lack of information in this field and the economic importance covered by this product in our area.
16 1.3 Olive fruit: development, anatomy, metabolomics and salt stress-related traits
1.3.1 Fruit characteristics and development
From a botanical point of view, olive fruit is considered a drupe, formed of a fleshy pericarp (pulp) and of a woody endocarp (stone) that encloses one – rarely two – seed (Fernández 2014). Recently, Hammami and Rapoport (2012) develop a model for categorize and describe more precisely the fleshy pericarp histological organization (Figure 6).
They describe a ―multiseriate exocarp‖, consisting of an epicarp of a single cell layer epidermis covered by cuticle, overlapping the hypodermal tissue. Below the multiseriate exocarp, an outer and inner mesocarp can be distinguished.
At harvest time for oil production, mesocarp, endocarp and seed represent respectively 70-90%, 9-27% and 2-3% of total fruit weight. The mesocarp composition is constitued by 60% water, 30% oil, 4% sugars, 3% protein, and the remainder is primarily fiber and ash. The endocarp contains 10% water, 30% cellulose, 40% other carbohydrates and about 1% oil (Hammami and Rapoport 2012, Fernández 2014).
The seed is made by 30% of water, 27% of oil, 27% of carbohydrates and 10% of protein (Connor and Fereres, 2005). So the volume of the mesocarp is important for the production of the oil, since this tissue is where up to 98% of the oil is accumulated. The olive drupe development lasts for 4–5months and includes 5 main phases: i) fertilization and fruit set, ii) seed development, iii) pit hardening, iv) mesocarp development, v) ripening. Anyway, not all cultivar shown similar fruit trait variation and growth pattern. For example, as reported by Farinelli et al. (2002), intense and regular dry matter accumulation was
17 recorded for 110-125 days after blooming (dab), but in Carolea, Leccino, Maurino, and Moraiolo dry matter accumulation is longer.
Anyway, most of the endocarp and mesocarp cells are produced between 4 and 10 weeks after blooming (Rallo and Rapoport, 2001; Rapoport, 2010).
Figure 6. Histological organization of olive fleshy pericarp. From Hammami and Rapoport, 2012.
From that point until fruit maturity, high cell expansion occurs, leading to produce an additional 10–40% of mesocarp cells. During the first half of the developmental period the fruits increase their weight at more or less linear rates,
18 so that at ∼25 weeks after blooming they reach final size (Fernández et al. 2014) (Figure 7).
Figure 7. Representation of fruit development into the olive biennial cycle scheme. From Fernández et al.(2014).
Ninety per cent of the endocarp growth occurs by 8 weeks after blooming (Rapoport et al. 2004), and then intense lignification events lead to maximum rate of pit-hardening. Pit hardening coincides to the start of oil accumulation in parenchymatic oil-storing cells of the mesocarp, and moreover to the transition between the evocation and floral differentiation processes. In fact, recently findings by Andreini et al. (2008) in Leccino and Puntino cultivars demostred that great level of Zeatin and RNA signal (usually correlated with floral evocation) was preferentially detected in ‗OFF‘ (with-out fruits) shoot axillary bud meristems, particularly when endocarp sclerification of fruits from the previous flowering is taking place. This can support the two-step theory of floral evocation-induction proposed by Lavee (1996).
19 Concerning oil accumulation can continue untill end of autumn. Famiani et
al. (2002) report that oil from Leccino, Maurino and Frantoio fruits harvested in
November was higher in quality than that obtained from fruit harvested in December, suggesting that as the best harvesting time.
Water deficit during pit-hardening might greatly affect both cell number and cell size of mesocarp (Rapoport et al. 2004; Gucci et al. 2009). If water supplies are not enough in this period of high sensitivity of the olive fruit to water stress, fruit flesh cell number, final fruit fresh weight and volume can be reduced, as well as mesocarp-to-endocarp ratio, an important feature for olive fruit quality for both table consumption and oil production (Hammami et al. 2011, 2013). For these reasons, as well as the fresh water scarcity in Mediterranean hot-dry summer, usage of saline water could be a suitable option (Chartzoulakis, 2005; Ben-Ghal et al., 2017). Up to now, no anatomical and histological studies of development under salinity has been conducted in olive fruit. Oil accumulation begins from ca. 8 weeks after blooming in the pulp, increases slowly and takes 20 weeks or more to reach a plateau (Lavee and Wodner, 1991).
Environmental conditions and agronomical practice can considerably influence the oil accumulation pattern and fruit growth. Bartolini et al. (2014) studied in Coratina 5/19 and Frantoio Millenium clones that shading inside the canopy, where the proportion ofincident PAR intercepted by the crop was 47%, appeared to limit mostly the fruit growth and oil body accumulation in mesocarp cells.
The ripening process, characterized by a change in the color of the fruit from lime green to lighter green/purple, starts about 30 weeks after blooming, when the rate of oil accumulation is reaching a plateau. During ripening, also the
20 texture change, from hard to squash (green stage) to easy to squash (cherry stage), considered by several authors the best moment for harvest for the perfect balance between fatty acid insaturation and phenolic compounds (Rotondi et al. 2004).
Concerning studies on salinity and olive fruit, the majority is focused on yield and fruit traits. Examples of the use of moderate saline water (4.2 dS m-1 EC) for irrigation of twelve olive cultivars coming from Mediterranean region are reported by Weissbein et al. (2008). Despite cultivars showed significant differences in growth and yield parameters, the saline irrigation treatment did not induce any effect on olive yield. Long term responses under field salt treatment (EC=5 and 10 dS m–1) has been monitored in the salt tolerant Picual cultivar and data showed that annual yield were not affected by treatments (Melgar et al. 2009). Moreover, in field experiment with Barnea trees (considerate salt tolerant) under 1.2, 4.2, and EC 7.5 dS m–1, Wiesman et al. (2004) established that, while 7.5 dS m–1 EC retarded growth and fruit production, 4.2 dS m–1 EC retarded growth only in the first year of the study showing more fruit per tree than the 1.2 dS m–1 EC treatment used as control.
Olive fruits quality is a key parameter for reach good quality final product but the studies on fruits physiology under salt stress are actually scarce. Some examples of data about effect on fruits traits reveal that saline water (EC=6.5 dS m–1) can increase the fruits dry weight in salt moderately tolerant Manzanillo and also in Uovo di Piccione (Klein et al. 1994). When saline water (EC=7.5 dS m-1) was applied for irrigation of cv. Chemlali (considerate salt tolerant) a general decrease in weight, volume and diameter in drupes was observed by Ben Ahmed et al. (2009), associated with an olive oil content decrease.
21
1.3.2 Phenolic compounds of olive fruit
Olive (Oleae uropaea L.) produces a wide range of secondary metabolites that strongly influence organolectic and nutritional properties of olive oil and fruit (Figure 8).
Figure 8. Representation of putative biosynthetic pathways of different secondary metabolites of olive fruits. From Alagna et al.(2012).
Between these, phenolic compounds are secondary plant metabolites biosynthetized through the shikimic acid pathway (Conde et al. 2008). Nowadays olive products are largely appreciated in the Mediterranean diet for their antimicrobial, anti-cancer, anti-viral, antiinflammatory, hypo-lipidemic and hypoglycemic effects (Alùdatt et al. 2013, 2014).
The olive drupe contains high concentrations of phenolic compounds that can range between 1 and 3% of the fresh pulp weight (Garrido et al. 1997). The principle classes of phenol compounds in olive fruit are, phenolic alcohols (e.g.tyrosol - p-HPEA - and hydroxytyrosol - 3,4-DHPEA), phenolic acids,
22 flavonoids, hydroxycinnamic acid derivates (e.g. verbascoside), lignans and secoiridoids (e.g. Oleuropein, Demethyloleuropein, Ligstroside, p-HPEA-EDA and 3.4-DHPEA-EDA) (Servili et al. 2004). Secoiridoid represent the most abundant phenolic fraction in olive fruit, that are constituted of monoterpenoids with a 3,4-dihydropyranskeleton.
In 2012, Alagna et al. performed a study in which olive fruits were screened for phenolic profiling during ripening from 12 different cultivars (from high phenols variety, like Coratina, to low phenols variety, Dolce d‘Andria) in the same agronomical conditions. The general trend of the majority of phenols studied is the typical decreasing behaviour expected with the advancing of ripening, except for Demethyleuropein, Ligstroside and Lignans. In the same experiment, 35 olive transcripts (like OeGES,OeGE10H,OeADH, and OeDXS, encoding putative geraniol synthase, geraniol 10-hydroxylase, arogenate dehydrogenase and 1-deoxy-D-xylulose-5-P synthase) with a high % of homology to genes implicated in these pathways were identify, and significantly correlate with phenolic compound concentrations. Recently, Alagna et al. (2016) cloned and functionally characterize the first gene of secoiridoid pathway, OeISY, aterpenecyclase that couples an NAD(P)H-dependent 1,4-reduction step with a subsequent cyclization generates the monoterpene scaffold of oleuropein.
Another variety screening of phenolic pattern has been highlighted by Talhaoui et al. (2015): a total of 57 phenolic compounds were determined in six olive cultivars, and a general decrease of total phenolic compounds was observed, characterized by a prevalence of secoiridoids at the beginning of ripening and by a prevalence of simple phenols and flavonoids in the end. These results, confirm the previous one obtained by Alagna et al. (2012).
23 Regarding salinity stress effects on phenolic molecules, not many experimental evidences are provided, and the majority is performed in olive oil. Wiesman et al. (2004) showed that the high polyphenol contents recorded in oils of saline irrigated plants can be explained by the acceleration of maturation of the olives, which could account for the higher levels of phenols. Behn-Amed
et al. (2009) reported that the use of saline water at 7.5 dSm-1 for Chemlali plants irrigation reinforced phenol accumulation in oil. A recent experiment conducted in cultivar Roghani (Dindarlou et al. 2016), crossing three different levels of salinity and five different irrigation regimes, showed for two consecutive years that highest phenols concentration is achieved with an EC salin water range between 2.2 and 7.7 ds m-1 and a water regime of 1.25 ETc.
1.3.3 Fatty acid in olive fruit
Olive fruit fatty acid composition isrepresented by: i) saturated fatty acids (palmitic - C16:0 - and stearic - C18:0 - acids); ii) monounsaturated fatty acids (palmitoleic - C16:1 - and oleic- C18:1 - acids); iii) polyunsaturated fatty acids (linoleic - C18:2 - and linolenic- C18:3 - acids) (Montedoro et al. 2003).
All fatty acids content increase during fruit growth, C18:1 being the most noticeable, which represents 70–80 % of the totality at the end of ripening, followed by C16:0 (10–15 %), C18:2 (5–10 %), and C18:0 (2–3 %) (Inglese et
al. 2011). Both cultivar and ripening stage could influence fatty acid
composition, as well as the carbon and oxygen isotope composition (δ13C and δ18O) of photoassimilates, that could be reflected in a different isotopic composition cultivar and ripening-related (Portanera et. al., 2015).
Carboxylation of acetyl-CoA to malonyl-CoA, catalyzed by Acetil-CoA carboxylase, is the starting point of fatty acids biosynthesys. Then, several step
24 of elongation proceeds through a series of reactions catalyzed by an enzymatic complex, constituted by several individual units, collectively named Fatty Acid Synthase (Sanchez and Harwood, 2002).
The cycle proceeds by adding two carbons in each turn to the acyl chain, until palmitoyl-ACP is formed (C16:0-ACP), and then elongated to stearoyl-ACP (C:18-stearoyl-ACP). From here, several steps of desaturation involving enzymes with different subcellular localization start to form the mono and poly insatured fatty acid. Regarding genes involved in fatty acid biosynthesis and desaturases, several of these have been cloned and functionally characterized. Especially, enoyl-ACP reductase, plastidialstearoyl-ACP desaturase (SAD), omega 6 microsomial (FAD2-1, FAD2-2) and plastidial (FAD6) desaturase, omega 3 microsomal (FAD3-A, FAD3B) and plastidial desaturase (FAD7-1 e FAD7-2) desaturase, acyl-CoA diacylglycerolacyltransferase (DAGAT), the respective enzimes for electron donor cytochrome b5 reductase for microsomial desaturases and ferredoxin-NAD(P) reductase for plastidials one (Hernandez et
al. 2005, 2009, 2011, 2013, 2016). Moreover, Parvini et al. (2016) recently
identified and characterize 3 different isoforms of stearoyl-ACP desaturase (SAD). Measuring lipid content and gene expression analyses, they showed that
OeSAD2 seems to be the major gene contributing to the oleic acid content of the
olive fruit.
Regarding literature of salinity and fatty acid pathway in olive fruit, up to now no gene expression analysis has been conducted. On the contrary, several data about fatty acid variation and profile under saline irrigation are available. Behn amed et al. (2009) in Chemlali plant irrigated with 7.5 dSm-1 observed an increment of oleic acid coping with a decrement of palmitic acid. Stefanoudaki
25 decrement of oleic acid and an increment of both linoleic and linolenic acid, allowing to a lower oleic/linoleic ratio and an higher poly insatured fatty acid fraction. Recently, Dindarlou et al. (2016) highlighted in cultivar Roghani under several saline conditions (EC from 2.2 to 7.7 dSm-1) and low water regime (1.25 ETc) an high increment of unsatured fraction of fatty acid and a depletion in stearic acid.
As we can see from this overview of salinity response of olive fruit, the data available are scarce, and several aspects of salt stress related fruit traits need to be elucidated.
1.4 Drought stress in olive plant
1.4.1 Drought problem
In Mediterranean agro-ecosystems, drought is another big environmental problem. In fact, considering the climate change of the last decade of this region (Cherubini et al, 2003) and the water demand increment for agriculture, we can claim that Mediterranean region expertise a very high level of water shortage problem (Figure 9).
26 Figure 9. Water stress (above) and volumetric irrigation water demand (below) across the Mediterranean region. From Gassert et al.(2013) and Daccache et al. (2014).
Moreover it is predicted that this area will suffer a decrease of annual precipitations, together with an increment in the mean annual temperature at the end of the 21st century. (IPCC, 2014).
In plants, water is the main factor that influence growth and agricultural production. In fact, transpiration is the driven force that enables gas exchanges with the atmosphere, and water flow from roots to aerial part drive the transport of minerals elements, consequently regulating their allocation in plants
27 (Fernández 2014; Sebastiani et al. 2016). To make possible this continuous water column, the preservation of hydraulic functionality is determinant.
Among the other species cultivated in Mediterranean land, olive tree is considered one of the most resistant fruit trees to water scarcity. In fact, despite its traditional and economic importance in the region, olive orchards account for only 6% of water demand (Figure 10), reflecting the fact that only 8% of olive trees are currently irrigated (Daccache et al. 2014).
Figure 10. Estimated irrigation water demand (Mm3) and CO2 emissions (t CO2e) for selected irrigated crops grown in the Mediterranean region. From Daccache et al. (2014).
This due to the presence of morphological, anatomical and physiological traits that allow it to have an anisohydric character resisting to water-deficit stress (Gucci et al., 2002; Tognetti et al, 2004; Sebastiani, 2011), apart of a
28 better use of water than other fruit trees. Bongi and Palliotti (1994) calculated that the number of grams of fruit dry matter accumulated per kilogram of consumed water was 3.17. In fact, high values of intrinsic water use efficiency (WUEi, measured as ratio of the rate of carbon assimilation photosynthesis to
the rate of transpiration) in olive have been reported by different authors (Angelopoulos et al., 1996; Moriana et al., 2002; Diaz-Espejo et al., 2006).
1.4.2 Drought and olive: physiological, biochemical and molecular adjustment
The numerous adaptations developed by olive tree in response to water scarcity permit it to establish and maintain a suitable Ψw from roots to shoot; in fact,
olive tree can tolerate low Ψw (−6/−8 MPa in leaves and stems) and
consequently can extract water from a soil with water potential (Ψsoil) between
−2.5 and −3.5 MPa (Lo Gullo and Salleo 1988; Rhizopoulou et al. 1991; Dichio
et al. 2003, 2005, 2009).
As for salinity, also for drought problem olive respond in a cultivar-related way. Tognetti et al. (2002 and 2006) report the different drought-tolerance in ―Frantoio‖ and ―Leccino‖, showing that Leccino better withstand more negative
Ψ and generally lower gs mainly for a better leaves WUE respect to Frantoio.
Actually, more researcher are involved in clarify the olive cultivar related response to drought (Lo Bianco and Scalisi, 2017).
Concerning advances in anatomical adaptation studies, Rossi et al. (2013), reported variations in vessels diameter in ring of development of mature olive plants: irrigated olives showna reduction (-4%) in vessels number with a <20 μm diameter, but an increase in vessels with >20μm diameter respect to rainfed olives. Xylem vessels with small diameter shown a lower hydraulic
29 conductivity. This enhance olive resistance to drought, reducing the embolism event in the xylem.
Marchi et al. (2005, 2007) observed that leaf anatomic characteristics can contribute to drought resistance: thicker cuticle, stomata abaxial side location, peltate scales and trichomes reachness, create a favorable conditions to maintain gas exchanges in dry climate (Besnard et al. 2009).
Drought stress determines growth inhibition, mainly because both the Ψw
and the turgor potential (Ψp) rapidly go down (Lo Gullo and Salleo 1988). This
inhibit cell division and expansion process inactive growing organs. Other simptoms could be present with the progression of drought stress: leaves senescence and drop, decrease in photosynthetic activity, turgor loss, chlorosis, bronzing, blade folding, shoot wilting and shriveled fruits (Gucci et al. 2003).
Water loss in olive cause a lot of events of osmotic adjustment, active synthesis and storage of metabolic and osmotically compatible solutes: in fact soluble non-structural carbohydrates, inorganic cations, etc., can confering plants the ability to withstand period of water-scarcity, slowing down the Ψπ of
the cells (Ingram and Bartels 1996).
In drought-stressed olives, non-structural carbohidrates (mainly mannitol and glucose) have a major role in active osmotic adjustments. Dichio et al. (2005) reported in drought-stressed Coratina olives an Active Osmotic Adjustment (AOA) of 0.75–1.42 MPa for roots and of 0.45–0.8 MPa for leaves, mainly due to mannitol. This contribute to maintain cell turgor capability to water extraction at very low Ψsoil.
More recently, Boussadia et al. (2013) show that the type of osmotic adjustment can be cultivar dependent: two years old plant of cv Meski and Koroneiki grown in pots under 30 days of drought stress, shown a total osmotic
30 adjustment of 2.1 MPa for the first one and of 2.8 MPa for the second one. Final Ψw resulted of -4.3 MPa for Meski and -6.0 for Koroneiki, and regression
analysis with other osmolites shown that Meski prefer an AOA strategy (maintaining hydration levels with compatible osmolites), while Koroneiki a Passive Osmotic Adjustment one (reducing water content in leaves).
Water deficit reduce photosynthetic activity, slowing down stomatal conductance (gs) and leaf area in olive plant (Fernández et al. 1999;
Hernandez-Santana et al., 2017). A reduction of net CO2 assimilation rate (An) by 85% was
found by Jorba et al. (1985) concomitantly to RWC decrease of 31%.
Under drought stress, abscisic acid (ABA) act as root-to-shoot signaling hormone. In fact, ABA targets are guard cell, where activation of ion channels and gene expression regulation can induce stomatal closure (Christmann et al. 2006). During different drought stress, Guerfel et al. (2009) observe differences in ABA allocation in root and leaves of Chetoui and Chemlali olive plants, according to level of cultivar drought-tolerance. Chemlali (drought-tolerant) ABA accumulation is lower in its leaves under drought stress than Chetoui (drought-sensitive). Other findings regarding photosynthetic response by Angelopoulos et al. (1996) and Moriana et al. (2002), shown that at high levels of drought stress (-4 MPa) An decrease much faster than gs, and also mesofillar
conductance (gm) is involved in stress response. Light-dependent inhibition of
photosystem II (PSII) is also affected.
Concerning molecular advances in drought-stress response, clonation of putative aquaporins (AQPs), carbonic anhydrase (CA) and their transcriptional regulation respect to gs and gm was studied in these years. Secchi et al. (2007)
found and cloned three sequences (OePIP1.1, OePIP2.1, and OeTIP1.1) of putative Aquaporins in both the plasmatic and tonoplastic membrans. Their
31 expression levels during a drought stress period was studied, showing significantly down-regulation in whole plant, suggesting an involvement in water conservation mechanism. A short-term water stress and recovery experiment conducted by Perez-Martin et al. (2014), allow to hypothize correlation with gene expression of OePIP1.1, OePIP2.1, stromal and stomatal CA with the variations observed for gs and gm.
Also Reactive Oxygen Species (ROS) generated by water stress is controlled by several mechanisms of gene regulation (a part the more common non enzymatic way that involve antioxidants molecules like tocopherols, phenols and ascorbic acid). The localization and gene expression of superoxide dismutase (SOD), an enzyme that catalyze formation of oxygen and hydrogen peroxide from superoxide, it has been studied by Corpas et al. (2006). SOD is present in isoenzymatic forms: Mn-SOD, Fe-SOD, CuZn-SOD, represented by respectively 82%, 17% and 0.8%. Fe-SOD were higher in leaves palisade cells, while in phloem the most abundant is Mn-SOD, also the unique isoform present at xylem level. In Coratina olive simultaneous drought and light stressed, SOD and other antioxidant enzymes contribute it has been studied by Sofo et al. (2004a), showing an increase in polyphenol oxidase activity and a decrease in SOD activity, especially in recovery phase in roots and leaves. Increasing level of membrane lipid peroxidation in olive tree leaves and roots under three different drought stress level (−0.5 to −2.4 MPa; −2.5 to −4.9 MPa; −5.0 to −6.3 MPa) it has been reported by Sofo et al. (2004b),associated with an increases in lipoxygenase activity and malondialdehyde content. Also proline increment were found, mainly for maintaining suitable homeostasis for cellular functions. Anyway, Proline has also a nitrogen-source like role, (Ain-Lhout et al. 2001),
32 acting as electron acceptor to prevent ROS damage to the photosystem (Hare et
al. 1998).
1.5 Stress combination in plants and in olive
In the environment, plants are not generally exposed to a single stress (Figure 11) but to a combination of biotic and abiotic stresses that affect their physiological processes (Ramegowda and Senthil-Kumar, 2015). Only in the past few years, researcher have started studying the interactions of combined stresses (Suzuki et al. 2014).
When plants are subjected to simultaneous stresses, their response depends on the combination of the ordinary reactions of stress tolerance, in addition to some characteristic responses acting only under multiple stress (Mittler et al., 2006; Ramegowda and Senthil-Kumar, 2015).Several studies show that plants tolerant to a particular stress factor also exhibit co-tolerance to other stresses (Pastori and Foyer 2002).
Recently, significant advances has been made in understanding the physiological, metabolic and molecular responses of several plant species to a combination of different abiotic stress conditions (Zandalinas et al., 2017).
For example, testing the effects of simultaneous drought and heat stress in Arabidopsis, Vile et al. (2012) found that their co-occurrence is generally more stressful than their single exposure, suggesting different and separate response of plants to these stresses.
33 Figure 11. Stress Matrix, presenting the putative interaction between simultaneous abiotic stresses. Adapted from Mittler (2006) and modified in Mittler and Blumwald (2010), Suzuki et al. (2014).
Also the simultaneity occurrence of salinity and heat stress is highly toxic for plants, maybe because the higher transpiration due to heat stress could result in a great amount of salt taken-up by plants (Keles and Oncel, 2002). The combination of high light intensity and drought or cold stress have detrimental effects for the plants (Giraud et al. 2008; Haghjou et al. 2009), indeed the low temperatures or the reduced transpiration caused an inhibition of the
34 photosynthesis, but the excessive amount of light energy, caused by the high light intensity, can result in an increased production of ROS (Mittler, 2002).
Boron (B) seems to alleviate the negative effects of salt stress (Del Carmen Martinez-Ballesta et al. 2008, 2013), maybe because of the competition between B and NaCl for plants uptake, hypothesis supported by the consideration that NaCl concentration in leaves decreased with the increased presence of B in soil (Del Carmen Martinez-Ballesta et al. 2008). Finally, the combination of nutrient deficiency with other stresses is critical for plants, because micronutrients are essential for the enzymatic response to plants to stress (Mittler and Lumwald, 2010).
In more recent experiments microarray analysis were utilized to understand the transcriptomic response to combined stresses (Rasmussen et al. 2013), finding differential activation in secondary metabolic pathways, involving the interaction of different signaling pathways. In general, we can claim that both susceptible and tolerant responses were observed in plants simultaneously exposed to a combination of different stresses, but it is not clearly understood why in some cases the simultaneity of different stresses resulted in higher tolerance while in other studies to higher susceptibility, making really hard to predict the final plant response to simultaneous stresses.
Concerning Mediterranean crop-species, it will be very important to clarify the response to simultaneous salinity and drought stress, especially because are two major environmental problem in Mediterranean area that interact one with the other. In fact, water scarcity in soil allow to solute accumulation in soil solution, and salt accumulation can cause a decline of Ψsoil and a consequent
water deficit (Therios and Misonopolis, 1988). Up to now, only a little has been studied regarding this interaction, focusing more on herbaceous crop like
35 rapeseed and corn (Amer, 2010; Shabani et al., 2013; Azizian and Sepaskhah, 2014). Regarding woody crop, in Grenache Grapevine, the combination of partial root-zone drying (PRD) with saline water supply (2.46 dS m-1 of EC) increase the total ions concentration on a whole vine plant basis and reduce the midday leaf water potential respect to control (Degaris et al., 2016). In olive tree, a few studies it has been conducted, focusing the attention on soil measurement, olive yield and fatty acid in oil.Ghrab et al. (2013) found in cv. Chemlali that PRD treatment combined with use of saline water (6.7 dS m-1of EC) reduce the pre-dawn and midday stem water potential, as well as total yield in the first 4 years of the trial (-11%). Anyway, after 9 years no difference in yield and oil content in fruit were not reported. Dindarlou et al. (2016) highlighted in olive cv Roghani under several saline conditions (EC from 2.2 to 7.7 dS m-1) and low water regime (1.25 ETc) an high increment of unsatured fraction of fatty acid and a depletion in stearic acid.
So further research to clarify other aspect (like ions partitioning and carbon balance) to combined salt and drought stress in olive are necessary.
36
2. Aims
The aim of the present Thesis was to elucidate how olive fruits respond to the major abiotic stress salinity, and how olive plants react to simultaneous salt and drought stress,widespread conditions in Mediterranean basin.
The targets were to provide a physiological characterization of fruit (see Annex I), investigate the molecular fatty acid pathway regulation under salinity (see Annex II), and to study physiology, Na uptake and translocation,cations and non-structural carbon balance under simultaneous drought and salinity, in olive leaves, stems and roots (see Annex III).
In particular Na+ translocation and accumulation in different fruit tissues was investigated, as well as the pericarp tissues thickening. Triacylglicerols fatty acids pathway metabolism and regulation was studied, analyzing expression of all cloned desaturases, concomitantly to the fatty acid profile. Cations imbalance and non-structural carbon allocation was studied in different plant tissues, toghether with plants water status.
Moreover, a general physiological characterization of plant was performed in all three experiments, studying photosynthetic efficiency, growth parameters and biomass partitioning.
The objectives were reached conducting the experiments in pots, using Olea
europaea L. Leccino genotype, a salt sensitive cultivar able to efficiently
translocate Na+ from root to shoot (Annex I and II). Leccino and Frantoio genotype (differentially salt and drought-tolerant) in micro-lysimeters were used for the simultaneous stress experiment (Annex III).
Atomic absorbtion methodologies and Cryo-SEM coupled with energy-dispersive X-ray microanalysis techniques were used to study Na accumulation in fruit and tissues thickening in reaction to salt stress. For plant water status
37 monitoring, non-destructive Zim-probes were selected. Liquid and Gas chromatography techniques was chosen for the detection and quantification of non-structural charbohidrates and fatty acid compounds, respectively. For gene expression analysis of fatty acid desaturases, a Real Time PCR approach was followed.
38
3. Results and Discussion
3.1 Olive fruit physiology under salt stress (Annex I)
At the end of experiment (49 days) olives were collected and divided into two Maturation Groups (MG), according to the classification used by Camposeo et
al., (2013): MG1 (olives with green skin and <50% purple skin) and MG2 (olives with ≥50% purple skin).
The total fruits remaining on Leccino plants was not affected by 60 mM NaCl irrigation. In fact, the number of fruits per plant was similar: 64±12.9 vs 57±23.9 in control and 60 mM NaCl plants respectively (P=0.561), confirming data of fruit drop, fruit traits (flesh/pit ratio, FW and DW of flesh, pit and seed, fruit longitudinal and transversal diameter, fruit volume) and yield reported by Melgar et al. (2009) in Picual olive under low salinity (5 and 10 dS m-1).
Considering Na accumulation, is evident that was accumulated in drupes of Leccino in a ―MG-related‖ way. In fact, Na uptake increase significantly only in flesh of MG1 fruits (reaching 559 mg kg-1), suggesting that at MG2 (in which Na concentration was about 397 mg kg-1) Na re-mobilization from fruit to vegetative portions could occur. In olive plants, an active process of Na re-translocation trough the phloem has been demonstrated by Tattini and Traversi (2008) in vegetative organs. In our experimental conditions, Na re-translocation at MG2 could be a consequence of oil droplets accumulation in cells that reduce the vacuole volume were Na and other toxic ions are usually stored (Blumwald 2000; Munns 2002).
Biochemical effect of Na accumulation at MG1 induce a clear increase of PAL activity (+16%), total polyphenols (+58%), anthocyanins (around doubled) and antioxidant power (DPPH) in flesh (+15%). These results are in agreement with data on Chemlali drupes under EC=7.5 dS m-1 saline water (Ben Ahmed et
39
al.2009) confirming the widely accepted role of phenolic compounds on
scavenging reactive oxygen species (ROS), activated under any kind of biotic or abiotic stress (Cheynier et al.2013). Although NaCl increases the phenols and anthocyanins at MG1, these parameters maintain their physiological trends during ripening: in fact, total phenols decrease from MG1 to MG1, while anthocyanins reach the highest values at MG2 when black maturation phases occur (Alagna et al. 2012).
Anatomical analysis of freeze-fractured fruit tissues by Cryo-Scanning Electron microscopy, reveal that the fruit-tissues react to salt stress. According to Hammami and Rapoport (2012), olive fruit consists of an epicarp of a single cell layer epidermis covered by cuticle, overlapping hypodermal and outer mesocarp cells. All these fruit layers become thicker under salt. More specifically resulted more than doubled (around triplicated in Hypodermal cell layers) on thickness at the MG1 when 60 mM NaCl values are compared to control ones. The thickening recorded is not related to change in number of specific cell layers, but to an increase in area. In fact, absence of new cell layers was also observed in mesocarp of Leccino fruit under water stress (Rapoport and Costagli 2004) and Gucci et al.,(2009) claimed that cell expansion was more sensitive to water deficit than cell division.
Thicker hypodermis under high salinity (up to EC=12.61 dS m−1) was found in other fleshy fruits like tomato (Ruiz et al. 2015). The induced increment in the epidermis thickness due to salt treatment could be considered useful to protect fruits against biotic and abiotic stress (Hammami and Rapoport 2012). Up to now no association was established between salinity, hypodermis and outer mesocarp in olive fruit nor in other drupe-type fruits. Anyway, the micrometric increase of the cell area of epidermis, hypodermis and outer mesocarp is not
40 statistically correlated with the development of the size of the fruit as instead reported by Hammami and Rapoport (2012), lead us to suppose that flash tissue layers and woody endocarp compete and interacts for growth and development of whole drupe as also reported by Rapoport and Costagli (2004) on Leccino fruits.
In Leccino olive fruits, outer mesocarp cell wall thickness increased in reaction to the salt treatment in mature olive (MG2) and this could be explained by an increase in primary and secondary cell walls, as generally reported for glycophytes plants by Le Gall et al. (2015). Anyway, interesting to note that thickness of cuticle, epidermis, hypodermis and outer mesocarp tissue and cell wall in the Leccino drupes decrease with ripening, independently to salt stress. Decrease in epicarp thickness in several olives cultivar during ripening has been observed by several authors (Lanza and Di Serio2015; Mafra et al. 2001) and it has been associated to a progressive disruption of the middle lamella parenchyma cells due to an increase of the activity of the pectin methylesterase and polygalacturonase (Langley et al. 1994).
In conclusion, results obtained encourage to apply saline irrigation in mature Leccino trees under field conditions, for a long enough period, in order to verify if saline water irrigation for Leccino trees doesn‘t negatively affect fruit production and quality as observed in our pots trial. Moreover, the additional benefits like increment of epidermis thickness and improvement of scavenging activity in MG1 olives are desirable in field conditions, because are useful characteristics to protect fruits against biotic and abiotic stress.
41 3.2 Olive flesh fatty acid pathway under salt stress (Annex II)
During the experiment (75 days) progressive impairment of photosystem II in leaves near to the infructescence of Leccino plants treated with 80 mM NaCl was recorded. Reduction of Chl a fluorescence in treated plants showed a significant decrease starting to the day 45 (–4%) to –8% after 75 days; the decrease was observed two weeks before the start of reduction of shoot elongation. Moreover, a significant decrement in ETR was recorded starting from 15 days of Na treatment (-20% respect to control), following a decreasing behaviour until the end of the experiment, when ETR in control plant is about double in respect to 80 mM NaCl treated plants. Under salt stress, decrease of ETR have been previously observed in other horticultural crop like tomato plants by Zribi et al. (2009).
At the end of experiment, total fruit number were recorded and olives collected were divided according to Camposeo et al. (2013) into four Maturation Groups (MG), MG0 (with green skin), MG1 (with ≤50% purple skin), MG2 (with >50% purple skin), MG3 (with 100% purple skin).
Na treatments did not affect the average number of olives per plants belong to MG0, MG1 and MG2 but induce a significant increment (more than twice) of olive with 100% purple skin (MG3) compared to control treated plants. The acceleration of veraison process under salinity it has been observed in Barnea threes under two levels of saline irrigation (4.2 and 7.5 dS m−1 EC) by Wiesman
et al. (2004), that describes the acceleration as a possible reaction to a stress. Na
accumulation in flesh reach the maximum of 2214 mg kg-1 in treated olive plants of MG1 stage, while Na accumulation in olive flesh of control plants remain in the range of 186-329 mg kg-1 DW. Na accumulation in flesh of MG1 stage fruits, and successive decrease at MG2 and MG3 stage confirm our previous results in previous experiment and support our hypothesis of
re-42 translocation of Na ions in vegetative portions, probably to facilitate oil droplets accumulation in cells. Na re-translocation processes via phloem it has been observed by Tattini and Traversi (2008).
After 75 days of 0 and 80 mM NaCl treatment the Triacylglycerols (TAGs) fatty acid composition in each MG were analysed in olive mesocarp.
Oleic acid was the main fatty acid found in the mesocarp of the fruits of the cultivar Leccino and represented around 70% of fatty acids, regardless of salt treatment. We detected a significant increase of 5% in oleic acid content at MG1 in treated plants while a decrease of linoleic acid was detected (-2.6% compared to control). Therefore, the oleic/linoleic ratio increase in salt treated plants, while the Poly Insatured Fatty Acid/Mono Insatured Fatty Acid (PUFA/MUFA) ratio was lower in comparison to control plants. These results are coherent with Wiesman et al. (2004), who reported a trend of increase in oleic acid and a parallel decrease in linoleic acid in olive oil from salt tolerant cultivar Barnea under 3 different salt regimes (1.2, 4.2 and 7.5 ds m-1 of EC). On the contrary, Stefanoudaki et al. (2009) report in Koroneiki and Mastoidis cultivar (moderately salt tolerant) under 50, 100 and 150 mM NaCl irrigation, a trend of oleic decrement and linoleic increment in respect to control, led to significant increases in total PUFA and a lower ratio of oleic/linoleic acid. These contrasting evidence could be explained by the fact that each genotype has a different fatty acid regulation pathway under salt stress, and that also NaCl concentration in irrigation water has a relevant role. In fact, Weissbein et al. (2008) reported that in olive oil from drupe of the same cultivar that we studied (Leccino) under two saline regimes (1.2 and 4.2 ds m-1 of EC) the fatty acid profile was not significantly affected.
43 We can affirm that salt concentration in tissue is a discriminant factor for a specific response, even in a salt sensitive cultivar like Leccino. In fact, the increase in oleic acid and the decrease in linoleic acid observed in MG1 fruits coincided with the major concentration of Na reached in fruit mesocarp at this maturation stage, suggesting that Na accumulation could change the fatty acid profile.
Recent studies suggested a direct involvement of free fatty acid in abiotic stress signaling in plants (Tumlinson and Engelberth 2008; Upchurch 2008; Hou et al. 2016). In particular, it has been demonstrated that oleic acid regulates nitric oxide synthesis, that participate to defense signaling in Arabidopsis (Mandal et al. 2012). We can suppose that also in our case oleic acid could be involved in this kind of signaling.
In order to gain further insight on the effect of saline irrigation with NaCl water on the olive mesocarp fatty acid biosynthesis, we decided to analyse the transcript levels of the olive fatty acid desaturase genes (SAD1, SAD2, SAD3,
FAD2-1, FAD2-2, FAD6, FAD7-1, FAD7-2, FAD3-A, FAD3-B) in Leccino
fruits at four different maturation stages, subjected to 80 mM NaCl treatment. The transcript levels found in the control plants are in the same range than that reported in Arbequina, Picual, Koroneiki, Canino, Moraiolo and Frantoio (Poghosyan et al. 1999; Banilas et al. 2007; Hernandez et al. 2009; Matteucci et
al. 2011; Hernandez et al. 2016; Parvini et al. 2016) .
Salt stress affected transcript levels of Leccino fatty acid desaturase genes, confirming that desaturases participates in the response to abiotic stress (Upchurch, 2008).
Under our experimental condition, reagarding oleate desaturases, FAD2-2 gene in MG1 fruits mesocarp resulted up regulated, while FAD6 expression
44 levels decreased in the MG1 salt stressed mesocarp. FAD2 family and FAD6 are documented to be very important for salt stress respons. As reported by Zhang
et al. (2009), FAD6 is very important in salt tolerance of Arabidopsis thaliana
8-day-old seedlings under 300 mM NaCl treatment. FAD6 gene transcription is in fact transient regulated, showing an earlier down-regulation follow by up-regulation. Moreover, in the Arabidopsis fad6 mutant, Na accumulation riched higher level in comparison to wild type, suggesting that FAD6 protein was involved in ions homeostasis. In fact, it has been hypotized that disruption of FAD6 function impaired the integrity of cell membranes at high salinity condition, mostly for the decrement of polyinsaturation level of thylakoidal fatty acid membrane. (Zhang et al. 2009). Subsequent experiments by Zhang et
al. (2012) with 300 mM NaCl treatment in Arabidopsis thaliana seedlings show
an up-regulation in FAD2 starting from 6 hours of the treatment. This because lipid composition and level of fatty acid desaturation in plants membrane largely affect tolerance to salt stress, mostly for the essential role in the biophysical characteristics and proper function of membrane-attached proteins (Deuticke and Haest, 1987; Cooke and Burden, 1990). This suggest that FAD2 relative gene expression is increased for mediate fatty acid desaturation of plasmatic and vacuolar membrane lipids, essential for the correct functioning of Na+/H+ exchangers related to the membrane, and consequentially maintain a low concentration of Na+ at cytosolic level (Zhang et al. 2012). The up regulation of FAD2-2 observed in our experiment it could be and effort by plant to maintain this membrane integrity, whyle the down-regulation observed in
FAD6 expression levels at MG1 could be the responsible for the reduction of linoleic acid detected at this stage.
45 When an osmotic injury like drought or salt stress occur, a decrease in ETR from photosystem II to photosystem I can be recorded (Zhrabi et al. 2009; Perez-Martin et al. 2014), as we have pointed out in our results. FAD6 enzyme have a thylakoidal localization in chloroplast, using NAD(P)H, ferredoxin-NAD(P) reductase and ferredoxin as electron donor system (Hernandez et al., 2011). We hypothized that low ETR recorded in leave closest to infructescence could allow to a more oxidized state of those complexes, causing a reduction in the activity levels of the FAD6 enzyme. In our experimental conditions, alteration of FAD6 structure and function could cope also with TAGs fatty acid composition modification, as we can mention above regarding reduction in linoleic acid in MG1 salt stressed fruit.
Although we detected other changes in olive desaturase genes expression levels in developing mesocarp under saline irrigation conditions, we could not observed a correlation with TAG fatty acids composition. One possible explanation could be that only fatty acid that esterify tryacylglicerols (TAGs) are taken into consideration in these experiments. It could be possible that other lipids, like phospholidips o galactolipids, undergo changes in its fatty acid composition (glycolipid, sphingolipid). This could confirm our hypothesis regarding polyinsaturation impairment in thylakoidal membrans consequently
FAD6 transcript down-regulation under salt stress. In addition, the existence of
post-transcriptional regulatory mechanism of olive desaturase genes cannot be discarded.
More studies are necessary to support our hypothesis on fine regulation of ETR and FAD6 activity and functionality, as well as a field experiment to verify if not controlled conditions allow to same results respect to NaCl water irrigation and gene transcription regulation.
