Wounding stimulates ALLENE OXIDE SYNTHASE gene and increases the level of jasmonic acid in Ipomoea nil cotyledons

Allene oxide synthase (AOS) encodes the first enzyme in the lipoxygenase pathway, which is responsible for jasmonic acid (JA) formation. In this study we report the molecular cloning and characterization of InAOS from Ipomoea nil. The full-length gene is composed of 1662 bp and encodes for 519 amino acids. The predicted InAOS contains PLN02648 motif, which is evolutionarily conserved and characteristic for functional enzymatic proteins. We have shown that wounding led to a strong stimulation of the examined gene activity in cotyledons and an increase in JA level, which suggest that this compound may be a modulator of stress responses in I. nil.

It was known that LOX and OPR genes encode enzymes crucial for the regulation of JAs biosynthesis.However, the complementation analysis of Arabidopsis thaliana delayed-dehiscence2-2 male-sterility mutant reveals that the damage is caused by interruption of the gene sequence encoding AOS, another of the main JA biosynthesis pathway enzymes [3].AOS genes were also identified in many other plant species [4][5][6].The AOS mRNA level depends on both internal and external factors such as phytohormones, pest attack and wounding.
In this paper we identified InAOS cDNA encoding a member of the CYP74 P450 gene family, involved in JA biosynthesis.The expression pattern in cotyledons after wounding of this gene was measured.We also examined the correlation between changes in AOS mRNA level and endogenous JA in cotyledons after wounding.

Plant materials and growing conditions
Preparation of plant material (Ipomoea nil, Chois 'Violet'; Marutane Seed Co., Kyoto, Japan) was made according to Wilmowicz et al. [7].The seedlings were grown in a growth chamber under the conditions described by Wilmowicz et al. [7] .Cotyledons of plants were perforated at regular intervals 5 × 5 mm.

Plant material for cloning InAOS cDNA and determination of JA level
For experiment examining the wounding effect on the level of endogenous JA and expression of InAOS the cotyledons were collected 20, 30, 40, 50, 120, 240, 360, 480, and 1440 minutes after the beginning of the stress stimulus action, immediately frozen in liquid nitrogen and stored at −80°C.Each experiment was repeated at least three times.All data are presented as mean ± standard error (SE).

Quantitative real-time RT-PCR analysis of InAOS gene expression
The gene expression analyses were performed by real-time PCR (RT-qPCR) with a LightCycler 2.0 Carousel-Based System (ROCHE Diagnostics GmbH, Germany) and the LightCycler TaqMan Master Kit (ROCHE Diagnostics GmbH, Germany).All expression procedures were performed according to Frankowski et al. [8].
Gene-specific primers and UPL probe were designed using the Universal ProbeLibrary Assay Design Center (http://www.roche-applied-science.com/sis/rtpcr/upl).Actin (InACT4) was used as a reference endogenous control for normalization purposes.qPCR analyses were performed with the gene specific primers: InAOS (60 bp) 5'-CGGAGATGTTTGAGGGTTTG-3' (forward) and 5'-CATTCAAGCCGACTT-TACCC-3' (reverse) with hydrolysis probe UPL 134, InACT4 (65 bp) with the gene specific primers 5'-GGAAATACAGTGTCTGGATTGGA-3' (forward) and 5'-CCA-CATCTGTTGGAATGTGC-3' (reverse) with hydrolysis probe UPL 139 (ROCHE Diagnostics GmbH, Germany).Relative quantification was performed using standard curves from serial dilutions of cDNA.The efficiency tested was >99%.The computer application used for the analysis was LCS4.0 (ROCHE Diagnostics GmbH, Germany) and for the calculations and graphs -MS Office Excel (Microsoft).PCR reactions were performed in triplicate for each RNA sample.All data are presented as mean ± standard error (SE).further details such as certification time and a signing reason in case any alterations made to the final content.If the certificate is missing or invalid it is recommended to verify the article on the journal website.

Determination of endogenous JA
The method described by Wilmowicz et al. [9] for endogenous JA analyses was used.d 5 -JA (100 ng) were added to the crude extract as internal standards.SIM GC/MS was performed by monitoring m/z 193, 198, 224, 229.

Isolation of cDNA for the allene oxide synthase
The coding sequence of InAOS was obtained using 3'RACE-PCR (GeneBank accession number HM357792.2).The full-length InAOS cDNA containing start and stop was obtained using 5'RACE-PCR and is composed of 1662 bp and encodes for 519 amino acids (Fig. 1a 1b).
Predicted amino acid InAOS sequence analysis and alignment with others showed that there were four conserved domains characteristic of cytochrome P450 proteins, which are common for all the AOS family.Because InAOS contains motifs that are evolutionarily conserved and characteristic of AOS it can be suspected that this gene encodes for a functional enzymatic protein.

Expression analysis of InAOS and endogenous jasmonic acid level in wounded cotyledons of I. nil
As shown in Fig. 2a wounding plants resulted in a strong increase in InAOS expression level in the cotyledons of   [4,6,11].The heme-binding domain preserved among cytochrome P450s and strongly conserved cysteine residue (in boldface type) is marked grey [4,6].The characteristic KIFF motif within the oxygen-binding domain is in italics and boldface type [13].b The phylogenetic relationship of InAOS compared with the AOS from A. thaliana and other plant species.A phylogram tree was generated using ClustalW.Percentages placed in the column were generated in BLAST and deducted InAOS amino acid sequence identity when compared with A. thaliana and other species of AOS.5-d-old seedlings of I. nil during the first hour after stimulus action and the reaction is accompanied by an increase in the endogenous JA level (Fig. 2b).The highest InAOS transcript accumulation in reference to the control plants (0 h) was observed 20 minutes after the wounding.The expression of this gene remained on a similar, high level 30, 40, and 50 minutes after stress and after that it decreased (120 min) and was similar to that in non-wounded plants.
We also showed that JA content directly after wounding cotyledons of I. nil was 6-fold higher than in the control plants (Fig. 2b).The level of endogenous JA increases over 50 minutes and reaches a maximum value of about 30 ng/g fresh weight in 30 min, which was 10 times higher than for non-wounded cotyledons.Beginning from 120 minutes after the stimulus action, the level of JA gradually decreases and although it slightly oscillates, the results were not significantly different from the control value (0 h).

Discussion
Maintenance of a JAs level appropriate for the control various processes is possible by multilevel regulation of their biosynthesis, in which a key role is played by, inter alia, LOX, AOS, AOC, and OPR3.AOS, the first enzyme of the octadecanoid biosynthesis pathway, is a major site of pathway control, which proceeds through feedback amplification of its biosynthesis from downstream metabolites.The expression of this gene is tissue-specific and correlates with endogenous JA level.
In this study we obtained the AOS cDNA from cotyledons of I. nil (InAOS) and analyzed its sequence (Fig. 1a).The InAOS gene encodes for a protein similar to that of allene oxide synthases identified in other plant species [4,5,10] (Fig. 1b).The InAOS contains a conserved domain with heme-binding cysteine (Cys-470), which is characteristic for cytochrome P450s [4].This suggests that InAOS encodes for a functional enzymatic protein, which may have similar functions as in other plant species.
Expression of AOS was shown to be tightly linked with elevated JA content during the wound response in A. thaliana [11].In wounded cotyledons of I. nil, a transient increase in AOS mRNA level was observed, with a maximum between 20 and 50 min after stimulus (Fig. 2a).The corresponding level of JA during the first 50 min is shown in Fig. 2. Wound-induced expression of the AOS gene was also observed in leaves of several plants, e.g., tomato, barley [5,11].The JAs content in plants influences the level and specificity of the defence responses.These hormones are some of the main messengers in the cell (local response) or at the systemic level.The rate of changes in the level of endogenous jasmonates is different for individual plant species, ranging from several seconds to several minutes after the stress stimulus [12].
In this paper we characterized InAOS gene encoding the first allene oxide synthase identified in Ipomoea nil involved in JAs biosynthesis, which may play an substantial role in JA systemic accumulation in response to wounding.
S S L A V H F Q I P S Q K S S L T 20 61 TTGAAGCCTTCTAGCCGGCGTTTCAAGATTTGCCCTGTTTCTGCCACCGTGTCGGATACT L K P S S R R F K I C P V S A T V S D T 40 121 CCGCCGTCGGTTTCCTTGTCCCCGGTCCCGGAGAAACTCCCAAAAAGGAAAATACCCGGC P P S V S L S P V P E K L P K R K I P G 60 181 GACTATGGGCTGCCGTTGATCGGGCCGTGGAAAGATCGGCTGGACTATTTCTACAATCAA D Y G L P L I G P W K D R L D Y F Y N Q 80 241 GGAAGAGAGGAGTTTTTCCGGTCTCGGGTTCAGAAATACGGGTCGACGGTTTTCAGGACG G R E E F F R S R V Q K Y G S T V F R T 100 301 AACATGCCACCCGGCCCGTTCATTTCCTTCAGCCCTAACGTCGTGGTTTTGCTCGACGGC N M P P G P F I S F S P N V V V L L D G 120 361 AAGAGTTTTCCGACGCTGTTTGATCCGGGAAAGGTCGAGAAACGGGATCTCTTCACCGGA K S F P T L F D P G K V E K R D L F T G 140 421 ACTTTCATGCCCTCCACGGAACTCACCGGCGGGTACCGGATCCTCTCGTATCTGGACCCG T F M P S T E L T G G Y R I L S Y L D P 160 481 TCGGAGCCGAAGCACGCGCAACTGAAGCAGCTGATGTTCTTTCTCCTCTCCTCCCGGCGC S

Fig. 1 a
Fig. 1 a Coding sequence of InAOS cDNA and the deduced amino acid sequence.Subsequent nucleotide positions are marked on the left side of the figure, and amino acid positions on the right.The translation initiation point (the start codon) and termination point (the stop codon) are in boldface type.Small letters are used for regions not subject to translation.Four conserved domains characteristic of cytochrome P450 proteins, which are common for all the AOS family are underlined.Light grey denotes the typical transit peptide for chloroplast targeting within deduced N-terminal sequences [4,6,11].The heme-binding domain preserved among cytochrome P450s and strongly conserved cysteine residue (in boldface type) is marked grey [4,6].The characteristic KIFF motif within the oxygen-binding domain is in italics and boldface type [13].b The phylogenetic relationship of InAOS compared with the AOS from A. thaliana and other plant species.A phylogram tree was generated using ClustalW.Percentages placed in the column were generated in BLAST and deducted InAOS amino acid sequence identity when compared with A. thaliana and other species of AOS.GeneBank accession numbers from top to bottom: BAJ78216.1,NP_001236432.1,XP_002302453.1,CAD29735.1,NP_001234833.1,ABC17856.1,HM357792.2,CAC82911.1,BAM76723.1,ABS50433.1,AAY27751.1,NP_199079.1.
Fig. 1 a Coding sequence of InAOS cDNA and the deduced amino acid sequence.Subsequent nucleotide positions are marked on the left side of the figure, and amino acid positions on the right.The translation initiation point (the start codon) and termination point (the stop codon) are in boldface type.Small letters are used for regions not subject to translation.Four conserved domains characteristic of cytochrome P450 proteins, which are common for all the AOS family are underlined.Light grey denotes the typical transit peptide for chloroplast targeting within deduced N-terminal sequences [4,6,11].The heme-binding domain preserved among cytochrome P450s and strongly conserved cysteine residue (in boldface type) is marked grey [4,6].The characteristic KIFF motif within the oxygen-binding domain is in italics and boldface type [13].b The phylogenetic relationship of InAOS compared with the AOS from A. thaliana and other plant species.A phylogram tree was generated using ClustalW.Percentages placed in the column were generated in BLAST and deducted InAOS amino acid sequence identity when compared with A. thaliana and other species of AOS.GeneBank accession numbers from top to bottom: BAJ78216.1,NP_001236432.1,XP_002302453.1,CAD29735.1,NP_001234833.1,ABC17856.1,HM357792.2,CAC82911.1,BAM76723.1,ABS50433.1,AAY27751.1,NP_199079.1.

Fig. 2 a
Fig. 2 a The expression level of InAOS transcript related to InACT4 in wounded and control cotyledons of Ipomoea nil.The expression activity was measured in three independent replicates.SE is marked on the bars.b Changes in JA content (ng/g fresh weight) in mechanically wounded cotyledons of Ipomoea nil.The control was non-wounded plants.Values are means of three separate samples with two replications for each sample.