versión On-line ISSN 0717-3458
Electron. J. Biotechnol. vol.15 no.1 Valparaíso ene. 2012
Molecular cloning and expression analysis of 12-oxophytodienoate reductase cDNA by wounding in Solanum tuberosum
Mauricio Díaz*1 · Victor Polanco2 · Ingrid Ramírez3 · Hugo Peña-Cortés3
1 Pontificia Universidad Católica de Valparaíso, Escuela de Ingeniería Bioquímica, Laboratorio de Biología Molecular, Valparaíso, Chile
*Corresponding author: firstname.lastname@example.org
Financial support: This work was supported by project FONDECYT 1000221 and FONDEF D00I1027.
Keywords: 12-oxophytodienoate reductase, jasmonic acid, OPDA, Solanum tuberosum, stress, wounding.
Jasmonic acid (JA) and 12-oxophytodienoic acid (OPDA) are signal molecules involved in the stress and defense responses in plants. A full-length cDNA clon of OPR3 encoding 12-oxophytodienoate reductase 3, key enzyme involved in the biosynthesis of JA from linolenic acid was obtained from a Solanum tuberosum cDNA library. Sequence analysis showed that OPR3 encoded a polypeptide of 400 amino acids with a predicted molecular mass of 43.9 kDa and pI of 7.72. The deduced amino acid sequence of OPR3 showed high similarities to other 12-oxophytodienoate reductases. A peroxisomal signal sequence indicates OPR3 probable location in peroxisome. Levels of OPR3 mRNA accumulated in potato leaves reaching maximum levels within 1 hr of mechanical wounding. Elevated levels of JA were correlated to expression of the OPR3 gene.
The response of plants to abiotic and biotic stresses involves changes in gene expression patterns. These changes largely depend on signal molecules such as abscicic acid (ABA), salicylic acid (SA) as well as jasmonic acid (JA) and its biosynthetic precursors (Bari and Jones, 2009; Leon-Reyes et al. 2010). The role that JA plays as signal molecule has been shown by evidence through molecular and biochemical studies. Plants increase JA levels when they are subjected to wounding or attack from insects and pathogens (Schilmiller and Howe, 2005). JA and OPDA induce the expression of pathogenesis related genes like PIN2, THI2.1 and PDF1.2 (Farmer et al. 2003; Taki et al. 2005). Molecular mechanisms of JA signalling have been dissected by discovering protein factors like JAR1 and COI1 from Arabidopsis mutants that are deficient in JA synthesis or unable to respond to JA (Tani et al. 2008). The biosynthesis of JA through the octadecanoic pathway (Vick and Zimmerman, 1984) is initiated from α-linolenic acid (LA), which is thought to be released from chloroplast membrane lipids by specific lipases. This is followed by the action of lipoxygenase (LOX), allene oxide synthase (AOS) and allene oxide cyclase (AOC) to form 12-oxophytodienoic acid (OPDA). Subsequently OPDA is transported from chloroplast to the peroxisome where the 12-oxophytodienoate reductase (OPR) reduces it to 12 oxophytoenoic acid (OPC-8:0). Three cycles of β-oxidation are then required for the shortening of the OPC-8:0 side chain leading to JA (Li et al. 2005). Evidence support the existence of three isoforms: OPR1, OPR2 and OPR3 (Schaller et al. 2000). Different studies have shown that OPR3 catalyzes the reduction of the 9S,13S-OPDA, the precursor physiologically relevant to synthesize JA (Schaller et al. 2000). Since OPDA and JA appear to be different signalling molecules (Stinzi et al. 2001) OPR3 seems to play a key role in controlling the pool of OPDA and JA to respond to different stress. Solanum tuberosum is a major carbohydrate source in human and animal diets around the world. Monoculture and the lack of genetic diversity have helped the emergence and threat to potato production caused by pathogens. Current disease control relies on the use of pesticides that are both environmentally and economically undesirable and have a negative impact on the sustainability of potato production (Savenkov et al. 2003). This infection process can be suppressed by secretion of effectors like protease inhibitors (Odeny et al. 2010). Mechanical damage or infection of potatoes with Phytophthora infestans causes an accumulation of protease inhibitors in exudates of potato tubers, and the treatment with JA intensified the accumulation of these inhibitors in response to the wound stress (Valueva et al. 2003). Inducing systemic resistance responses in crop plants is a promising way of disease management. In this work the aim was to clone the OPR3 cDNA from Solanum tuberosum and evaluate the expression response of the gene and its correlation with JA levels and the systemic response of the plant involving the proteinase inhibitor Pin2, when the plant was subject to mechanical wounding. In this way our work contributes to the knowledge of the presence and importance of the OPR3 gene in the JA pathway and the possibility of inducing systemic resistance responses in potato plants.
Screening of cDNA library
A cDNA library from potato leaves cloned in phage λ ZAP (Stratagene), was screened through phage hybridization. A solution of the cDNA library was laid over a lawn of E. coli XL1-Blue. Some of the cells were then transferred to a membrane of nylon HyBond N+ (Porablot, Stratagene). This membrane was subject to hybridization with a cDNA probe of OPR3 from A. thaliana (kindness to Mussig C), radioactively marked with 32P using the kit RadPrime (GIBCO/BRL) to isolate putative clones of OPR3 with complementary sequences.
Membranes were subject to prehybridization at 42ºC in hybridization buffer (25 mM sodium phosphate bufffer pH 7.2, 25 mM NaCl, 10 mM EDTA, 7% SDS, 30% formamide, 10% PEG 6000 and 0.25 mg/ml salmon sperm DNA) for 2 hrs without probe and then 12 hrs with the probe. Membranes were washed with wash solution (0.5% SDS, 2x SSC) for 10 min and exposed to autoradiography X-OMAT LS (Kodak) at-80ºC. Clones were later propagated in E. coli SOLR allowing the isolation of clones in phagemid Bluescript (pBS). Selected clones were subject to endonuclease restriction analysis and then subject to sequencing through the DNA sequencer ABI3700 at the Centro de Biotecnología e Ingeniería Genética from the Universidad Estatal de Campinas SP Brasil.
Predictions of open reading frames (ORFs) were made using ORF finder from NCBI (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) and Translate Tool from ExPASy (http://us.expasy.org/tools/dna.html). Searches for homology of nucleotide sequences and deduced amino acid sequences to cDNA clones were identified by NCBI BLAST against GenBank database (http://www.ncbi.nlm.nih.gov/BLAST/). Sequence comparison was conducted using the ClustalW program from EMBnet (http://www.ch.embnet.org/software/ClustalW.html). Theoretical molecular weights of deduced polypeptides were made using the Protein property calculator (http://web.expasy.org/compute_pi/). From this bioinformatics analysis the selected cDNA clone was then used for the expression analysis of OPR3 through Northern blot.
Potato plants (Solanum tuberosum, cultivar Désirée) were cultivated in vitro in MS media supplemented with 2% sacarose and solidified in 0.8% agar in a cultivation chamber with controlled temperature (22ºC), relative humidity (70-80%) and photoperiod (16 hrs light/8 hrs dark). Once the adequate size was reached (1 month) they were moved to earth in a greenhouse with the same controlled conditions. After 2 months of growth they were subject to mechanical damage stress. Samples recollected after the treatment were immediately frozen in liquid nitrogen and stored at -80ºC until RNA extraction.
Mechanical damage treatment
Potato plants were damaged by incisions on two leaves per plant and recollected at different times (0.5, 1, 3, 6 and 12 hrs). Control samples from plants that were not damaged were also recollected.
Expression analysis of OPR3 through Northern blot
Total RNA was extracted from potato leaves from different treatments using guanidine hydrochloride-containing buffer followed by direct extraction with phenol/chloroform. The RNA was precipitated from the aqueous phase, washed with 3 M sodium acetate and 70% ethanol, and finally dissolved in water. 20 μg of total RNA were fractioned through electrophoresis gel in 1.1% agarose/formaldehyde and transferred to a membrane of nylon HyBond N+ (Porablot). This membrane was subject to hybridization with different probes: cDNA from OPR3 isolated previously, AOS from tomato and Pin2 from potato radioactively marked with 32P using RadPrime (GIBCO/BRL). Membranes were subject to prehybridization at 42ºC in hybridization buffer for 2 hrs without probe and then for 12 hrs with probe. Membranes were washed at 42ºC with wash solution for 10 min and subject to autoradiography X-OMAT LS (Kodak) at -80ºC.
Quantification of JA
Endogenous JA was extracted from potato plants with mechanical damage. For the quantification of JA levels leaves were homogenized together with an internal standard (dihidro jasmonic acid). An extraction was made with a mixture of acetone/citric acid and was evaporated for 12 hrs. The mixture was decanted and supernatant was recovered. This was followed by an ether extraction and evaporation. The ether phase was loaded on a solid phase column of amino propel, pre equilibrated with ether and washed with chloroform/IPA. An extraction with a mixture of ether/acetic acid was used to recover the fixed product in the column and completely evaporated. The product was passed to a chromatography vial, washed with dichloromethane and completely evaporated. Then the product was subject to derivatization, and the product was resuspended in absolute methanol and derivatization reagent (DMF, dimetilacetal) at 60ºC. Samples were subject to analysis with a GC-MS (HEWLETT PACKARD 5973 Mass Selective Detector), measuring the ions of endogenous JA m/z = 224 and 151 and from the internal standard (dihidro jasmonic acid) m/z = 226 and 153. The concentration of JA was determined with the relation between endogenous JA and the internal standard.
Nucleotide and amino acid sequences
OPR3 cDNA coding for 12-oxophytodienoate reductase from Solanum tuberosum is available at GenBank accession JN241968.
Cloning and sequence analysis
In this study using screening of cDNA library we managed to clone the 12-oxophytodienoate reductase cDNA, GenBank accession JN241968. A full length clone revealed a putative 1203 bp open reading frame encoding a predicted polypeptide of 400 amino acid residues with an estimated molecular weight of 43.9 kDa and pI of 7.72. BLAST searches showed high similarities of the OPR3 from S. tuberosum with other OPR available in the GenBank database. The highest identity of OPR3 from S. tuberosum was with other OPR3, like A. thaliana with 75% and L. esculentum with 99%. OPR3 from potato also showed an identity of 51 and 55% with OPR1 and OPR2 from A. thaliana and 54 and 44% with OPR1 and OPR2 from tomato respectively. The amino acid sequence of OPR3 from S. tuberosum was aligned with OPR3 from A. thaliana and S. lycopersicum (Figure 1). The analysis of the amino acid sequence of OPR3 from S. tuberosum showed the peroxisomal signal peptide (SRL) at the C-terminus of the protein, a feature shared with OPR3 from A. thaliana and S. lycopersicum (Strassner et al. 2002) whereas OPR1 and OPR2 do not show the presence of the peroxisomal signal. This reinforces the hypothesis octadecanoic pathway involves the plastid and peroxisomal compartment. The comparison of the sequence with other OPR and OYE, all oxo-reductases dependent of the cofactor flavin mononucleotide (FMN), shows that the majority of the residues responsible for the binding of FMN and NADPH are conserved; situation observed in other OPR described (Costa et al. 2000). The amino acid sequence comparison of OPR3 from S. tuberosum with that of OPR1 and OPR3 from S. lycopersicum, with known crystallographic structure (Breithaupt et al. 2001; Breithaupt et al. 2009) showed conserved residues at the catalytic site, but different residues at the selectivity site with OPR1, probably because OPR3 should recognize 9S,13S-OPDA instead of 9R,13R-OPDA recognized by OPR1 (data not shown).
Analysis of the expression of OPR3 by mechanical wounding
Transcripts of OPR3 in leaves wounded directly (local response) began to accumulate already at 30 min, reaching the maximum at 1 hr after treatment, beginning to decline after 3 hrs and disappearing after 12 hrs (Figure 2a). In leaves not wounded directly (systemic response) no transcript of OPR3 accumulated (Figure 2b). This is in accordance with evidence that OPR3 transcript accumulate after wounding in A. thaliana (Schaller et al. 2000) and tomato (Strassner et al. 2002). To verify the adequate mechanical wounding the expression of AOS and Pin2 were analyzed. Transcript of AOS and Pin2 accumulated both local (in mayor proportion) and systemically (in less proportion) in response to mechanical wounding confirming the adequate treatment. Evidence that support this is that AOS transcript accumulates both locally and systemically in response to mechanical damage (Lulai et al. 2011). Pin2 transcripts also accumulate during mechanical damage (Peña-Cortés et al. 1995). To relate the expression of OPR3 with the biosynthesis of JA, endogenous levels of JA were quantified. Not damaged leaves (control) presented levels of 420.90 ng gr-1fw, while locally damaged leaves started to accumulate rapidly at 30 min, reaching a maximum of 1530.24 ng gr-1fw after 1 hr, to later start to decline after 3 hrs, reaching control levels after 12 hrs (Figure 3). These results clearly shows that OPR3 gene expression from S. tuberosum is inducible by mechanical damage and that its expression in related with an increase in endogenous JA levels.
These patterns of OPR3 gene expression and JA levels are consistent with a function of OPR3 in the octadecanoid metabolism in S. tuberosum. The cloning of the OPR3 gene opens the way for future work involving transgenic potato plants carrying constructs for overexpression of the OPR3 cDNA and for antisense inhibition of OPR3 gene expression. These will help clarify the specific function of OPR3 and octadecanoids in stress responses.
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