Título artículo / Títol article: Metabolic and Regulatory Responses in Citrus rootstock in Response to Adverse Environmental Conditions Autores / Autors Argamasilla, Rosa ; Gómez Cadenas, Aurelio ; Arbona Mengual, Vicent Revista: Journal of Plant Growth Regulation June 2014, Volume 33 Versión / Versió: Postprint de l’autor Cita bibliográfica / Cita ARGAMASILLA, Rosa; GÓMEZ-CADENAS, Aurelio; bibliogràfica (ISO 690): ARBONA, Vicent. Metabolic and regulatory responses in citrus rootstocks in response to adverse environmental conditions. Journal of Plant Growth Regulation, 2014, 33.2: 169-180. url Repositori UJI: http://hdl.handle.net/10234/127405 Journal of Plant Growth Regulation Metabolic and regulatory responses in citrus rootstocks in response to adverse environmental conditions Journal: Journal of Plant Growth Regulation F Manuscript ID: JPGR-13-0044.R1 o Manuscript Type: Original Manuscripts r Date Submitted by the Author: n /a Complete List of Authors: Argamasilla, Rosa; Universitat Jaume I (UJI), Dept. Ciències Agràries i del P Medi Natural Gómez-Cadenas, Aurelio; Universitat Jaume I (UJI), Dept. Ciències e Agràries i del Medi Natural Arbona, Vicent; Universitat Jaume I (UJI), Dept. Ciències Agràries i del e Medi Natural r abiotic stress, drou ght, metabolomics, plant hormones, phenylpropanoids, Keywords: soil flooding R e v i e w Springer Page 1 of 80 Journal of Plant Growth Regulation 1 2 3 Title: Metabolic and regulatory responses in citrus rootstocks in response to adverse 4 5 environmental conditions 6 7 8 Running title: Metabolomics of abiotic stress in citrus 9 10 11 Authors: Rosa Argamasilla, Aurelio Gómez-Cadenas, Vicent Arbona* 12 13 14 Affiliation: Ecofisiologia i Biotecnologia. Departament de Ciències Agràries i del Medi 15 16 Natural. Universitat Jaume I. Castelló de laPlana, Spain. 17 18 F 19 Corresponding Author: 20 o 21 22 r Vicent Arbona 23 24 25 P Dept. Ciències Agràries i del Medi Natural 26 27 e 28 Universitat Jaume I. e 29 30 r 31 E-12071 Castelló de la Plana 32 33 R 34 35 Spain e 36 v 37 e-mail: [email protected] i 38 39 e 40 Ph. +34 964 72 8101 41 42 w 43 Fax. +34 964 72 8216 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 1 Springer Journal of Plant Growth Regulation Page 2 of 80 1 2 3 Abstract 4 5 6 In response to adverse environmental conditions, plants modify their metabolism in 7 8 order to adapt to the new conditions. To differentiate common responses to abiotic 9 10 stress from specific adaptation to a certain stress condition, two citrus rootstocks 11 12 (Carrizo citrange and Cleopatra mandarin) with a different ability to tolerate stress were 13 14 15 subjected to soil flooding and drought, two water stress conditions. In response to these 16 17 conditions, both genotypes showed altered root proline and phenylpropanoid levels, 18 F 19 especially cinnamic acid that was a common feature to Carrizo and Cleopatra. This was 20 o 21 correlated with alterations in the levels of phenylpropanoid derivatives likely involved 22 r 23 in lignin biosynthesis. In the regulatory part, levels of both stress hormones ABA and 24 25 P 26 JA decreased in response to soil flooding irrespective of the genotype relative flooding 27 e 28 tolerance but, on the contrary, concentration of both metabolites increased in response e 29 30 r to drought, showing JA a transient accumulation after a few days and ABA a 31 32 progressive pattern of increase. These responses are probably associated to different 33 R 34 35 regulatory processes under soil flooding and derought. In addition, alterations in IAA 36 v 37 levels in citrus roots seemed to be associated to particular stress tolerance. Moreover, i 38 39 both genotypes exhibited a low degree of overlapping ine the metabolites induced under 40 41 similar stress conditions, indicating a specific mechanism to cope with stress in plant 42 w 43 species. Results also indicated a different metabolic basal status in both genotypes that 44 45 46 could contribute to stress tolerance. 47 48 49 Keywords: abiotic stress, drought, metabolomics, plant hormones, phenylpropanoids, 50 51 soil flooding, 52 53 54 55 56 57 58 59 60 2 Springer Page 3 of 80 Journal of Plant Growth Regulation 1 2 3 Introduction 4 5 6 Environmental variables such as temperature, water availability, irradiance or soil 7 8 osmolality affect plants in different ways depending on their ability to tolerate a specific 9 10 adverse situation (Des Marais and Juenger 2010, Qin and others 2011). The most 11 12 damaging situation is probably water shortage that dramatically affects plant 13 14 15 performance and, ultimately, survival. This water deprivation is mainly due to a 16 17 limitation in the availability of capillary water or liquid water trapped between the soil 18 F 19 particles which can be efficiently absorbed by plant roots. Plants respond to this 20 o 21 situation by accumulating compatible solutes or soil salts thus decreasing their water 22 r 23 potential and closing stomata to avoid dehydration (Munns 2011). A paradigmatic 24 25 P 26 situation is salt stress that has a double component: a first phase of osmotic stress and a 27 e 28 second phase of ionic stress that occurs after over-accumulation of toxic ions, such as e 29 30 Na+ and Cl-, in photosynthetic organs (rMunns 2011). 31 32 33 R 34 35 e 36 In citrus, the accumulation of Cl- in leaves vinduces down-regulation of the 37 i 38 photosynthetic system and reduction in gas exchange parameters, ultimately leading to 39 e 40 41 the overproduction of reactive oxygen species and oxidative stress (Arbona and others 42 w 43 2003, López-Climent and others 2008). As well as in salt stress, the intensity of the 44 45 response to water deprivation is related to the ability of the plant to regulate water 46 47 relations (Moya and others 2003). Those genotypes with a lower transpiration rate and a 48 49 higher ability to rapidly close stomata (for example, Cleopatra mandarin) have 50 51 52 improved performance under drought conditions (López-Climent and others 2008). 53 54 Citrus responses to abiotic stress also include accumulation of jasmonic acid (JA) and 55 56 abscisic acid (ABA) in roots and leaves (de Ollas and others 2012, Gómez-Cadenas and 57 58 59 60 3 Springer Journal of Plant Growth Regulation Page 4 of 80 1 2 3 others 1996). In addition, there is an accumulation of 1-aminocyclopropane-1- 4 5 carboxylic acid in water-stressed roots that can be eventually transported to the aerial 6 7 part and oxidized to ethylene triggering leaf and organ drop (Gómez-Cadenas and others 8 9 1996). 10 11 12 On the contrary, when subjected to other situations that induce water shortage such as 13 14 15 soil flooding, plants develop different strategies to cope with stress (Arbona and others 16 17 2008, Arbona and Gómez-Cadenas 2008, Arbona and others 2009b). In citrus, tolerance 18 F 19 to soil flooding seems to be associated to higher transpiration rates and root hydraulic 20 o 21 conductivity. In a previous work, Arbona and others (2009b) found that under 22 r 23 continuous soil flooding Carrizo citrange plants did not show any alteration in gas 24 25 P 26 exchange or chlorophyll fluorescence parameters for thirty days whereas both 27 e 28 parameters rapidly decreased in plants of the sensitive genotype Cleopatra mandarin e 29 30 r that also exhibited leaf mid-vein yellowing and curling symptoms. Soil flooding 31 32 tolerance was also correlated to the ability to delay JA and ABA accumulation in leaves. 33 R 34 35 However, no variations in the hormonal profilee that could be linked to tolerance were 36 v 37 found in roots since both JA and ABA levels importantly decreased right after stress i 38 39 imposition (Arbona and Gómez-Cadenas 2008). e 40 41 42 w 43 44 45 All these results, taken together, indicate that specific hormonal signalling profiles 46 47 might have evolved associated to particular stress situations. Nevertheless, stressed 48 49 plants exhibit similar physiological responses. Then, the question whether different 50 51 52 signalling events regulate similar biochemical responses or not, seems of particular 53 54 relevance in this context. 55 56 57 58 59 60 4 Springer Page 5 of 80 Journal of Plant Growth Regulation 1 2 3 Based on our knowledge, citrus genotypes with varying ability to tolerate different 4 5 water shortage situations show similar physiological responses (stomatal closure, down- 6 7 regulation of photosynthesis and production of reactive oxygen species) when subjected 8 9 to abiotic stress situations (Arbona and others 2003, 2008). However, under identical 10 11 stress conditions, model plants alter their metabolism in different ways, causing diverse 12 13 14 secondary metabolite profiles (Arbona and others 2010). This different response could 15 16 be associated to a particular basal secondary metabolite composition but also to a 17 18 different regulation of the metabolism. In a previous publication, Arbona and others F 19 20 (2010) found that oeven close-related plant genotypes showed very little overlapping in 21 22 r 23 the metabolites altered by a specific stress condition. 24 25 P 26 Phenolic derivatives constitute the most diverse array of secondary metabolites found in 27 e 28 plants. In particular, phenylpropanoids (cinnamic acid, coumaric acid, caffeic acid and e 29 30 r ferulic acid) are synthesized from phenylanaline via phenylalanine ammonia lyase 31 32 (PAL), a enzyme that catalyzes its deamination rendering cinnamic acid, the first 33 R 34 35 precursor of flavonoid and lignin biosyntheseis. The increase in PAL activity and 36 v 37 phenylpropanoid content under different adverse environmental conditions has been i 38 39 reported (Cabane and others 2012, Moura and others 20e10, Vincent and others 2005). 40 41 Phenylpropanoids are precursors of lignins, which constitute an important stress defense 42 w 43 mechanism, especially at the root level where these compounds are involved in cell wall 44 45 46 composition and stiffness (Cabane and others 2012, Vincent and others 2005). Besides 47 48 the structural function of phenylpropanoids, a role as antioxidants has been proposed 49 50 (Moura and others 2010). As a response to soil flooding, citrus increase their 51 52 antioxidant capacity in terms of enzyme activity and soluble antioxidants (Arbona and 53 54 55 others 2008). Along with this response, flavonoid levels in tolerant genotypes were less 56 57 58 59 60 5 Springer Journal of Plant Growth Regulation Page 6 of 80 1 2 3 affected than in sensitive ones, suggesting that flavonoids might be also part of their 4 5 tolerance mechanism (Djoukeng and others 2008). 6 7 8 Other secondary metabolites known to have a functional role in response to abiotic 9 10 stresses are carotenoids and xantophylls. These compounds are lipophilic compounds 11 12 synthesized in plants from isopentenyl pyrophosphate (IPP) via the plastidial methyl 13 14 15 erythritol phosphate (MEP) pathway. Xantophylls are synthesized from β-carotene via 16 17 its conversion to zeaxanthin and sequentially to violaxanthin by epoxidation. Finally, an 18 F 19 arrangement in one epoxy ring of violaxanthin to form an allenic bond forms 20 o 21 neoxanthin , the precursor of ABA in plants. In this sense, overexpression of carotenoid 22 r 23 24 biosynthetic genes in transgenic tobacco plants improved osmotic and salt stress 25 P 26 tolerance by channelling carotenoid flux to ABA biosynthesis leading to increased 27 e 28 levels of this phytohormone (Cidade and others 2012). e 29 30 r 31 To investigate hormonal and secondary metabolite responses and their relationship with 32 33 R abiotic stress tolerance, two stress conditions: progressive drought and soil flooding 34 35 e were assayed in two citrus genotypes used as rootstocks in modern citriculture: Carrizo 36 v 37 i 38 citrange and Cleopatra mandarin. These rootstocks were chosen because of their 39 e 40 different tolerance to the stress conditions assayed. The study focuses on roots as the 41 42 w first organ sensing the stress derived from soil water perturbation. Proline accumulation, 43 44 hormonal and secondary metabolite profiles were analyzed in the two rootstock species 45 46 47 under the two stress conditions mentioned above. 48 49 50 Materials and methods 51 52 53 Plant material, treatments and sample collection 54 55 56 57 58 59 60 6 Springer Page 7 of 80 Journal of Plant Growth Regulation 1 2 3 Four-month-old horticulturally true-to-type seedlings of Cleopatra mandarin (Citrus 4 5 reshni Hort. ex Tan.) and Carrizo citrange (Citrus sinensis L. Osb. × Poncirus trifoliata 6 7 L. Raf.) were used in soil flooding and drought experiments. Plants were purchased 8 9 from a commercial nursery and immediately transplanted to 2-L plastic pots with 10 11 different substrates depending on the kind of experiments (see below). Before the onset 12 13 14 of the experiments, all plants were watered three times a week as described in (Arbona 15 16 and others 2006) and allowed to acclimate for 2 months. During plant acclimation and 17 18 experiments, plants were kept in a greenhouse under the following conditions: 26 ± 4.0 F 19 20 ºC day temperaturoe, 18 ± 3.0 ºC night temperature, relative humidity between 70 and 21 22 r 23 90%, and a 16-h photoper iod. 24 25 P 26 27 e 28 e 29 Flooding stress 30 r 31 32 To carry out flooding experiments a mixture of peat moss:perlite:vermiculite in an 8:1:1 33 R 34 ratio was used as a substrate. At the end of the acclimation period, two groups of 12 35 e 36 plants each were selected based on the uniforvmity in appearance and state of 37 i 38 development: one was set as control and watered three times a week as described in in 39 e 40 41 Arbona and others (2006) and the other group was subjected to soil waterlogging. To 42 w 43 impose stress, pots were placed in opaque plastic bags and then into pots of higher 44 45 capacity (4 L) and filled with tap water until complete saturation of the soil field 46 47 capacity, adding more water when needed. Root samples of three plants per treatment 48 49 group were collected after 1, 3, 6 and 8 days of treatment. Young roots were selected 50 51 52 and immediately frozen in liquid nitrogen. The samples were stored at -80 °C until 53 54 analyses. 55 56 57 Drought stress 58 59 60 7 Springer Journal of Plant Growth Regulation Page 8 of 80 1 2 3 Before experiments, a total of 30 citrus seedlings were transplanted to plastic pots using 4 5 perlite as a substrate that allows a tight control of the water content due to its low 6 7 moisture-retaining capacity. Half of the plants were used as controls and watered three 8 9 times a week as described in Arbona et al. (2006) and the other was subjected to 10 11 drought by simply stop watering. The treatment lasted for 14 days when leaf symptoms 12 13 14 of dehydration were apparent. Throughout this period, young root samples from three 15 16 plants per treatment were collected at days 3, 5, 7, 12 and 14, frozen immediately in 17 18 liquid nitrogen and stored at -80 °C for further analyses. F 19 20 o 21 Proline analysis 22 r 23 24 Ground frozen leaf tissue (0.05 g) was extracted in 5 ml of 3% sulfosalicylic acid 25 P 26 27 (Panreac, Barcelona, Spain) useing a homogenizer (Ultra-Turrax, IKA-Werke, Staufen, 28 e 29 Germany), at maximum speed. After centrifugation at 4,000×g for 35 min at 4°C, 30 r 31 proline was determined as described by Bates and others (1973). Briefly, 1 ml of the 32 33 R supernatant was combined with 2 ml of a mixture of glacial acetic acid and ninhydrin 34 35 e reagent (Panreac) in a 1:1 (v:v) ratio. The reaction mixture was incubated in a water 36 v 37 i 38 bath at 100 ° C for 1 h and then partitioned against 2 ml of toluene. Absorbance was 39 e 40 read in the organic layer at 520 nm. A standard curve was performed with standard 41 42 w proline (Sigma-Aldrich, Madrid, Spain). 43 44 45 Phytohormone analyses 46 47 48 Plant hormone and different phenylpropanoids (cinnamic, caffeic, coumaric and ferulic 49 50 acids) were extracted and analyzed essentially as described in Durgbanshi and others 51 52 53 (2005) with slight modifications. Briefly, 0.5 g of ground frozen plant material was 54 55 extracted in 5 ml of distilled water after spiking with 100 ng of d -ABA, prepared as in 6 56 57 Gómez-Cadenas and others (2002); dihydrojasmonic acid (100 ng), synthesized in the 58 59 60 8 Springer