Mechanisms of tolerance to salinity in banana: physiological, biochemical, and molecular aspects/Mecanismos de tolerancia a salinidade em bananeira: aspectos fisiologicos, bioquimicos e moleculares.
Salinity is one of the most significant abiotic stresses in the world, constituting a limiting factor that adversely affects the growth of plants in general and the productivity of agricultural crops. In the last decade, more than 900 million hectares worldwide were recorded as having been affected by salinity (FAO 2008).
Plant species, glycophytes or halophytes, that survive in saline environments exhibit adaptation strategies that involve physiological, biochemical and molecular mechanisms. Studies have shown that plants develop a tolerance to osmotic stress, ionic toxicity stress (FLOWERS et al., 2015), and oxidative stress (Foyer; SHIGEOKA, 2011). To alleviate [Na.sup.+] toxicity and re-establish cellular homeostasis is necessary the extrusion of [Na.sup.+] by the salt overly sensitive pathway (SOS) (PARDO, 2010), and this transport across the membrane is dependent of proton ATPase activity (VITART et al., 2004). the salt SOS pathway functionally consists of the [Na.sup.+] transporter, SOS1, the protein kinase SOS2, and the Ca[2.sup.+] sensor SOS3, which constitute a functional model that ensures ionic homeostasis in plants that are adapted or tolerant to saline stress (FEKI et al., 2014). another important mechanism for tolerance is the increase in the activity of antioxidant enzymes, to avoid the effects of oxidative stress (FOYER; SHIGEOKA 2011). This is a mechanism that avoid the accumulation of reactive oxygen species (ROS) that causes damage to nucleic acids, proteins, membrane lipid peroxidation and disrupt the redox homeostasis (AZOOZ et al., 2009).
Tolerance is affected by many different genes involved in these various metabolic pathways, such as ion extrusion, ion compartmentalization, synthesis of compatible solutes and scavenging ROS (SHI et al., 2000; SUN et al., 2010; KUMAR et al., 2009). Genotypes that differ with regard to their tolerance for salinity therefore exhibit qualitative and/or quantitative differences in gene expression. The identification and understanding of the molecular control mechanisms involved in the tolerance to abiotic stresses may result in the use of molecular tools to produce new more tolerant commercial cultivars (GUPTA; HUANG, 2014) In many circumstances, it is the sensitivity of the roots to stress that limits the plant productivity. Despite this direct action of stress on the roots, the majority of studies focus primarily on the leaf tissue in experiments involving the exposure of plants to saline stress. However, the combination of the physiological relevance and structural simplicity of the root tissues makes them an obvious target for functional genomic analysis (KUMAR et al., 2015) Several techniques have been developed to identify genes that are differentially expressed and that are involved in the mechanism of tolerance to abiotic stress in plants (GUPTA; HUANG, 2014). Among these techniques, DDRT-PCR, still managing a lower volume of information than the other techniques, has a relatively low cost and is easy to perform (HANG; PARDEE, 1995). Several plant-related studies have shown that DDRT-PCR can be effectively used to clone genes that are preferentially expressed in plants by abiotic stress (UEDA et al., 2002).
Considering the importance of saline stress as a limiting factor for banana production and given that the physiological and biochemical specific responses to salinity represent a combination of prior molecular events activated by the perception of salt, it is necessary to identify and characterize the genes involved in the response to salinity. The objective of this study was to isolate, sequence, and identify differentially expressed genes in the saline stress-tolerant banana genotype, as well as to associate these genes with their physiological and biochemical mechanisms.
MATERIALS AND METHODS
Micropropagated banana plantlets (Musa sp.) of Tap Maeo and Berlin genotypes, which are tolerant to and sensitive to salinity, respectively, were acclimated for 25 days in a hydroponic system prior to the experiments. The plants were grown in a nutrient solution containing 742.86 mg.L-1 soluble fertilizer (3% N, 11% [P.sub.2][O.sub.5], 38% [K.sub.2]O, 4% MgO, 11% S, 0.025% B, 0.004% Mo, 0.01% Cu-EDTA, 0.025% Zn-EDTA, 0.07% Fe-EDTA, and 0.04% Mn-EDTA) and 840.00 mg.[L.sup.-1] calcium nitrate (15.5% N and 19.0% Ca). The salt treatment began after the acclimation period by adding 50 mol.[m.sup.-3] NaCl. The treatment without NaCl was used as the control.
The plants were analyzed considering the time of the expression of the events to be evaluated.
At the beginning of the saline treatment (time zero) and after 24 and 48 hours, five plants in each treatment group were collected. The roots were frozen in liquid nitrogen to extract the total RNA and for the DDRT-PCR analysis. Seven days after the initiation of the treatments, five plants in each treatment were collected to evaluate membrane lipid peroxidation and the [H.sup.+]ATPase activity in the membrane. After fifteen days of treatment, the leaf area was measured, and five plants were collected to analyze the [Na.sup.+] and [K.sup.+] concentrations.
The experiment was carried out in a completely randomized design with two levels of salt (0 and 50 mol.[m.sup.-3] NaQ), two cultivars (tap Maeo and Berlin) making a total of 4 subplots. Each subplot contained 10 plants of each genotype, with a total of 40 plants. the experiment was repeated 3 times. the data set was sub mitted to analysis of variance (ANOvA) and mean values were compared using Tukey's test (P < 0.05). the leaf area (LA) was determined using a leaf area meter (Li-Cor, Inc., Lincoln, Nebraska, USA). the analysis of the [Na.sup.+] and [K.sup.+] concentrations was performed using flame photometry.
The plasma membrane vesicles were isolated by differential centrifugation of the root primordial. the ATPase activity of the plasma membrane was determined by measuring the Pi liberated during ATP hydrolysis, according to the colorimetric method described by Fiske and Subbarrow, with the modifications (FACANHA; DE MEIS, 1998). Vanadate (membrane ATPase inhibitor) and nitrate (V-ATPase inhibitor) were used as inhibitors. the total RNA was extracted from the roots using TRIZOI. For DDRT-PCR, a 1 [micro]g aliquot of total RNA was used as a standard to obtain cDNA and for the subsequent PCR amplification. The cDNA synthesis reactions were performed at 50[degrees]C for 50 minutes. the PCR reactions were performed using combinations of anchor primers ([A.sub.1], [A.sub.2], [A.sub.3] or [A.sub.4]) with random primers ([B.sub.1], [B.sub.2], or [B.sub.3]). the amplification conditions were as follows: 94[degrees]C for 2 minutes, followed by 40 cycles at 94[degrees]C for 30 seconds, 42[degrees]C for 1 minute, and 72[degrees]C for 1 minute, as well as a final elongation stage at 72[degrees]C for 5 minutes. PCR was performed at least three times for each primer pair. The PCR-amplified products were separated on 1.5% agarose gels. the genes induced by salt were defined when the expression was 1.5 fold greater than that of the control, as quantified by the PCR product. the nucleotide sequences of each clone were determined using an automatic DNA sequencer (ABI PRISM 377; Applied Biosystems, Foster City, California, USA), using the Big Dye Terminator kit (Applied Biosystems). The BlastX program (ALTSCHUL et al. 1997) was used to compare the obtained sequences with homologous sequences in the databases of the National Center for Biotechnology Information (NCBI; http://www. ncbi.nlm.nih.gov/blast), the Institute for Genomic Research (TIGR; http://compbio.dfci.harvard. edu/tgi/cgi-bin/tgi/Blast/index.cgi), and the DNA Data Bank of Japan (DDBJ; http://www.ddbj.nig. ac.jp/search/blast). Multiple sequence alignments were performed using ClustalW software (http:// www.ebi.ac.uk) (THOMPSON et al., 1994), and a phylogenetic tree was constructed using the neighbor-joining method with MEGA 3.1 software (KUMAR et al., 2004). The reliability of the tree was assessed by bootstrap analysis with 1500 simulations and edited using MEGA 3.1 software.
RESULTS AND DISCUSSION
The salinity reduced the leaf area of both genotypes by different magnitudes (Table 1). In the tolerant genotype, Tap Maeo, the reduction in leaf area was 21.8%, whereas the reduction in the sensitive genotype Berlin was 45.2%.
The leaf area reduction that occurs due to the increase of the levels of salt is a common response in banana and has been previously described in 46 genotypes evaluated by different researchers (GOMES et al., 2004; SILVA et al., 2009; FERRAZ, 2008; WILLADINO et al., 2011); these genotypes exhibited reductions in leaf area that varied from 16.3 to 69.2%. Decreased leaf area is related to constrained C[O.sub.2] fixation, which reduces the net assimilation rate and increases the production of ROS through the Mehler reaction (MILLER et al., 2009). The reduction in leaf area is also due to the deviation of energy required for the activation and maintenance of metabolic activities that are associated with salinity tolerance, such as the maintenance of membrane integrity, the synthesis of organic solutes, ion extrusion, and the regulation of ion transport and distribution in various organs and within cells (MUNNS; TESTER, 2010).
The [K.sup.+] concentration in the shoots and roots varied with the addition of NaCl to the nutrient solution. Whereas the sodium concentration increased in the roots with increasing salinity but did not differ between the genotypes. In addition in the shoots, while the Tap Maeo genotype demonstrated a small increase in [Na.sup.+] concentration the Berlin, sensitive genotype, exhibited an increase of more than 300% (Table 1). This [Na.sup.+] distribution pattern, which is characterized by low concentrations of that ion in the shoots is characteristic of salinity tolerance, as observed in 30 banana genotypes exposed to the same conditions (GOMES et al., 2004; GOMES et al., 2005; WILLADINO et al., 2011). The maintenance of ionic homeostasis in the tolerant genotypes requires the extrusion of [Na.sup.+] from the cytosol to the vacuole or apoplast through the [Na.sup.+]/[H.sup.+] antiporter (SHI et al., 2002).
The analysis of total RNA revealed fragments corresponding to rRNA 28S and 18S with optical quality ([A.sub.260/230]) and density ([A.sub.260/280]) ratios of approximately 1.9 and 1.7, suggesting low contamination with polysaccharides and proteins, respectively, and a high yield of total RNA.
Twelve primer combinations were used. The optimal pattern and resolution clarity of the amplified products were obtained with the [A.sub.2][B.sub.2] primer pair 48 hours after stress.
The transcripts of all of the samples ([T.sub.0], [T.sub.1], and [T.sub.2]) generated by the same pair of primers were applied side-by-side. twenty-three differentially expressed amplicons were isolated, cloned, and sequenced. The length of the amplicons varied between 300 and 1900 base pairs (bp). the similarity between the sequences obtained here and previously known gene sequences was determined. Similarity was considered to be present when the E values were less than (1e-05), which would therefore be considered significant.
The tolerant variety, tap Maeo, have the differentially expressed Musa 07 cDNA fragment (table 2) in the roots that have a high homology to SOS1 gene that encoding SOS1, the [Na.sup.+]/[H.sup.+] antiport. this cDNA fragment (approximately 500 bp) have an amino acid sequence with 85% homology to the Salt Overly Sensitive 1 (SOS1) gene of the glycophyte Arabidopsis thaliana (access number, AF256224) (SHI et al., 2000) (Figure 1) and 81% homology to Pisum sativum, Thellungiella halophile, and Brassica napus.
The salt overly sensitive (SOS) pathway functionally consists of the [Na.sup.+] transporter, SOS1, the protein kinase SOS2, and the Ca2+ sensor SOS3, which constitute a functional model that ensures cellular homeostasis in plants that are adapted or tolerant to saline stress (FEKI et al., 2014; KUMAR et al., 2009). The phylogenetic tree of the [Na.sup.+]/[H.sup.+] antiport sequences (SOS1), based on higher plants, animals, fungi, and Escherichia coli, indicated two distinct "clusters", as supported by the high bootstrap value, i.e., a vacuole cluster and a plasma membrane cluster, which indicates that Musa 07 is a fragment of the [Na.sup.+]/[H.sup.+] antiporter gene (Figure 2). this same antiporter is also involved in the distribution of [Na.sup.+] between the shoots and the root (PARDO, 2010) by restricting xylem loading with the [Na.sup.+] ion, thereby preventing its transportation to the shoots (SuN et al., 2010; SHI et al., 2002). this altered distribution of [Na.sup.+] prevents toxic concentrations of this cation in the cytoplasm of shoot cells of the tolerant genotype tap Maeo (table 1), thereby protecting the photosynthetic apparatus. the cDNA clone Musa 23, which is differentially expressed in the tap Maeo genotype, exhibited 73% similarity to the amino acid sequence of the SOS2 gene in Arabidopsis (access number AF237670.1) (table 2). Greater expression of SOS2 is responsible for better salinity tolerance response in different genotypes under salt stress (CHAKRABORTY et al., 2012). the SOS2 gene encodes a serine/threonine protein kinase with either a regulatory or second catalytic domain, with a regulatory-catalytic interaction within the SOS2 gene. The protein kinase SOS2 interacts with a [Ca.sup.2+]-binding protein similar to calcineurin, SOS3, which responds to an increased [Ca.sup.2+] concentration in the cytosol and triggers the interaction between SOS2 and SOS3 (FEKI et al., 2014).
The SOS2/SOS3 complex activated by saline stress is responsible for the phosphorylation and activation of SOS1, the [Na.sup.+]/[H.sup.+] antiporter, which is present in the membrane (Miller et al., 2009). therefore, the SOS2 and SOS3 proteins are components of the SOS pathway. the SOS pathway ensures the ionic homeostasis of the tap Maeo genotype through the extrusion of [Na.sup.+] from the cytoplasm to the apoplast or vacuole. the [Na.sup.+]/[H.sup.+] antiporter is a secondary transporter that depends on the activity of AtPases the primary transporters. ATPases are responsible for the electrochemical gradient that ensures the energization of the [Na.sup.+]/[H.sup.+] antiporter (VITART et al., 2004). the plants of the tap Maeo genotype exhibited an increase of more than 20% in the activity of ATPase and PPase, ensuring sodium extrusion, and the sensitive genotype demonstrated a decrease in the activity of these proton pumps (Figure 3).These significant biological markers indicate that activation of the SOS pathway is a fundamental mechanism for saline stress tolerance of banana.
The cDNA fragment Musa 26 encodes betaine aldehyde protein dehydrogenase (BADH, EC 18.104.22.168), enzyme that catalyzes the oxidation of betaine aldehyde to glycine betaine (GB). GB plays important role in stress tolerance that include the stabilization of complex proteins and membranes in vivo, protection of the transcriptional and translational machinery, reduction of ROS accumulation and peroxidation of membrane lipids, and as a molecular chaperone in the refolding of enzymes (GIRI, 2011; CHEN; MURATA, 2011). The peroxidation of membrane lipids was monitored indirectly through the production of malondialdehyde (MDA), a byproduct of lipid peroxidation. The Tap Maeo genotype did not exhibit variations in the MDA concentration when subjected to the stress, however, in the Berlin genotype, the accumulation of [Na.sup.+] caused an increase in the concentration of MDA (Figure 3).
The ability of tolerant varieties to maintain low concentrations of MDA, as well as MDA accumulation in susceptible varieties, indicates that variations in lipid peroxidation of membrane can be used to evaluate the effect of salt stress on cell, once the membrane is the first site to perceive this stress. another cDNA fragment, the Musa 10 clone, was identified and exhibited 71% similarity to the amino acid sequence of the ascorbate peroxidase (APX) gene of Vigna unguiculata (access number u61379.1). APX is the most important enzyme associated with the elimination of [H.sub.2][O.sub.2] from the cytosol and chloroplasts (BARBOSA et al., 2014), preventing the oxidative damage. The excessive production of [H.sub.2][O.sub.2] and other ROS ([O.sup.-.sub.2], O[H.sup.-], and [sup.1][O.sub.2]) is cytotoxic (FOYER; SHIGEOKA, 2011) and frequently occurs in plants subjected to biotic or abiotic stress (MITTLER, 2006). Tolerant genotypes respond to such stress with an increase in the activity of antioxidant enzymes, preventing and avoiding damage to nucleic acids, proteins and membrane lipid peroxidation, thus maintaining redox homeostasis (AZOOZ et al., 2009). The differences on the biochemistry and molecular markers highlights the importance of the antioxidant system as a mechanism of salt stress tolerance in banana.
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LILIA WILLADINO (2), TEREZINHA RANGEL CAMARA (3), MARTA BARBOSA RIBEIRO (4), DANIEL OLIVEIRA JORDAO DO AMARAL (5), FLAVIA SUASSUNA (6), MARCIA VANUSA DA SILVA (7)
(1) (Paper 232-15). Received on September 29, 2015. Accepted for publication July 08, 2016.
(2) Biologist, PhD. Department of Biology, Federal Rural university of Pernambuco, Avenida Dom Manoel de Medeiros s/n, 52021060, Recife, Pernambuco, Brasil. e-mail: firstname.lastname@example.org (autor para correspondencia);
(3) Agronomist, PhD. Department of chemistry, Federal Rural university of Pernambuco, Avenida Dom Manoel de Medeiros s/n, 52021060, Recife, Pernambuco, Brasil. e-mail: email@example.com
(4) Forestry engineer, Master in chemistry, Department of chemistry, Federal Rural university of Pernambuco, Avenida Dom Manoel de Medeiros s/n, 52021060, Recife, Pernambuco, Brasil. e-mail: firstname.lastname@example.org
(5) Biologist, PhD. National Institute of the Semi-Arid, Avenida Francisco Lopes de Almeida, s/n, 58429970, amcpina Grande, Paraiba, Brasil. e-mail: email@example.com
(6) Biologist, Master in Botany, Department of Biology, Federal Rural university of Pernambuco, Avenida Dom Manoel de Medeiros s/n, 52021060, Recife, Pernambuco, Brasil. E-mail: firstname.lastname@example.org
(7) Agronomist, PhD. Department of Biochemistry, Federal university of Pernambuco, Avenida Prof. Moraes Rego s/n, 50670420, Recife, Pernambuco, Brasil. e-mail: email@example.com
Caption: FIGURE 1--Comparison of amino acid sequence between Musa 07 clone of Musa sp. with the SOS1 gene of A. thaliana (AF256224). Sequences labeled with colons, asterisks, periods, and dashes represent those with similar amino acids, identical amino acids, different amino acids, and a lack of amino acids, respectively. The ClustalW program was used to generate the alignment (THOMPSON et al., 1997).
Caption: FIGURE 2--Musa 07 clone position in the phylogenic tree of Na+/H+ antiporter. Multiple alignments of the sequences were performed with CLUSTAL W, and a phylogenic tree was built using the neighbor-joining method with the MEGA 3.1 program. The accession numbers (in parentheses) and the Na+/H+ antiporter sources are as follows: AgNHX1 (AB038492), Atriplex gmelini; AtNHX1 (AF510074), AtSOS1 (AF256224), and AtSOS1 (AY062746), Arabidopsis thaliana; NHE2 (AAD41635) and NHE5 (AAC98696), Homo sapiens; OsNHA1 (AY328087), Oryza sativa; TaSOS1 (AY326952), Triticum aestivum; PpSOS1 (AJ564258), Physcomitrella patens; PeNhaD1 (AJ561195), Populus euphratica; NhaP (BAA31695), Pseudomonas aeruginosa; NHA1 (NP-013239), Saccharomyces cerevisiae; SOD2 (CAA77796), Schizosaccharomyces pombe; NhaA (P13738), Escherichia coli; and PeSOS1 (DQ517530), Populus euphratica
Caption: FIGURE 3--Hydrolytic activity of P-ATPase and [PP.sub.i]ase and MDA concentration in roots of banana genotypes grown for 7 days with or without 50 mol [m.sup.-3] NaCl in the nutrient solution.
TABLE 1--Leaf area (LA), [Na.sup.+] and [K.sup.+] concentrations (g.[kg.sup.-1]), and [K.sup.+]-Na+ ratio in shoots (S) and roots (R) of banana genotypes grown for 15 days with or without 50 mol [m.sup.-3] NaCl in the nutrient solution Genotype Treatment LA [Na.sup.+] (mol [m.sup.-3] Nacl) ([cm.sup.2]) S R Tap Maeo 0 3.26 a 3.7 c 4.2 b 50 2.53 b 5.9 b 6.2 a 0 2.79 b 3.4 c 3.8 b Berlin 50 1.26 c 16.5 a 6.9 a LA Reduction [Na.sup.+] Increase (%) (%) Tap Maeo 21.8 59.5 47.2 Berlin 45.2 385 81.6 Genotype [K.sup.+] [K.sup.+]/[Na.sup.+] S R S R Tap Maeo 31.2 a 41.4 a 8.4 9.8 29.7 a 40.9 a 5.0 6.6 32.3 a 40.6 a 9.5 10.7 Berlin 30.0 a 39.9 a 1.8 5.8 Reduction (%) K+ [K.sup.+]/[Na.sup.+] Tap Maeo 4.6 0.2 49.5 32.7 Berlin 7.1 1.7 89.5 45.8 Data indicated by different letters within each column exhibit significant difference at the 5 % probability level according to Tukey's test TABLE 2--catalog of cDNA detected by differential display in banana roots *. No. cDNA Primer PCR Fragment Access fragment combinations size (bp) number Musa 07 A2B2 500 AF256224.1 Musa 10 A2B2 390 u61379.1 Musa 23 A2B2 430 AF237670.1 Musa 26 A2B2 450 AF359282.1 No. cDNA Best homology Organism Identity of fragment to database aminoacid (%) Musa 07 putative Na+/H+ Arabidopsis thaliana 85 antiporter SOS1 Musa 10 cytosolic ascorbate Vigna unguiculata 81 peroxidase Musa 23 serine/threonine Arabidopsis thaliana 73 protein kinase SOS2 Musa 26 betaine aldehyde Suaeda liaotungensis 72 dehydrogenase (BADH) * Of the sequences obtained, four exhibited a high degree of similarity to amino acids sequence of genes whose functions are already known, nine displayed low similarity, and ten sequences were not similar