An oriental melon 9-lipoxygenase gene CmLOX09 response to stresses, hormones, and signal substances#

An oriental melon 9-lipoxygenase gene CmLOX09 response to stresses, hormones, and signal substances # # 薄皮甜瓜9-脂氧合酶(9-LOX)类型的 CmLOX09 对逆境、激素和信号类物质的响应 first-author corresp ‡Corresponding Author ‡Corresponding Author †E-mail:hyqiaaa@126.com †E-mail:hyqiaaa@126.com hyqiaaa@126.com Key Laboratory of Protected Horticulture of Education Ministry and Liaoning Province, College of Horticulture, Shenyang Agricultural University, Shenyang 110866, China Key Laboratory of Protected Horticulture of Education Ministry and Liaoning Province, College of Horticulture, Shenyang Agricultural University, Shenyang 110866, China In plants, lipoxygenases (LOXs) play a crucial role in biotic and abiotic stresses. In our previous study, five 13-LOX genes of oriental melon were regulated by abiotic stress but it is unclear whether the 9-LOX is involved in biotic and abiotic stresses. The promoter analysis revealed that CmLOX09 (type of 9-LOX) has hormone elements, signal substances, and stress elements. We analyzed the expression of CmLOX09 and its downstream genes— CmHPL and CmAOS —in the leaves of four-leaf stage seedlings of the oriental melon cultivar “Yumeiren” under wound, hormone, and signal substances. CmLOX09 , CmHPL , and CmAOS were all induced by wounding. CmLOX09 was induced by auxin (indole acetic acid, IAA) and gibberellins (GA 3 ); however, CmHPL and CmAOS showed differential responses to IAA and GA 3 . CmLOX09 , CmHPL , and CmAOS were all induced by hydrogen peroxide (H 2 O 2 ) and methyl jasmonate (MeJA), while being inhibited by abscisic acid (ABA) and salicylic acid (SA). CmLOX09 , CmHPL , and CmAOS were all induced by the powdery mildew pathogen Podosphaera xanthii . The content of 2-hexynol and 2-hexenal in leaves after MeJA treatment was significantly higher than that in the control. After infection with P . xanthii , the diseased leaves of the oriental melon were divided into four levels—levels 1, 2, 3, and 4. The content of jasmonic acid (JA) in the leaves of levels 1 and 3 was significantly higher than that in the level 0 leaves. In summary, the results suggested that CmLOX09 might play a positive role in the response to MeJA through the hydroperoxide lyase (HPL) pathway to produce C6 alcohols and aldehydes, and in the response to P . xanthii through the allene oxide synthase (AOS) pathway to form JA. 目的 2 研究 CmLOX09 及其下游基因 CmHPL 和 CmAOS 对逆境、激素和信号类物质的响应,进一步测定茉莉酸甲酯(MeJA)处理后叶片中绿叶挥发物的含量以及接种白粉病菌后叶片中茉莉酸含量的变化,探讨脂氧合酶(LOX)响应这两种胁迫的可能途径. 创新点 2 通过对 CmLOX09 启动子中顺式作用原件的分析预测,首次研究薄皮甜瓜9-LOX类型的 CmLOX09 对机械损伤、激素、信号类物质以及生物胁迫的响应. 方法 2 利用Plant CARE软件对 CmLOX09 启动子响应元件进行预测分析(图S1);利用荧光定量聚合酶链反应(qRT-PCR)技术分析甜瓜在机械损伤、激素、信号类物质以及生物胁迫处理后叶片中 CmLOX09 、 CmHPL 和 CmAOS 的表达模式;利用气相色谱-质谱连用仪(GC-MS)测定叶片中绿叶挥发物的含量(图4);利用高效液相色谱-串联质谱法(HPLC-MS/MS)分析和测定叶片中茉莉酸的含量(图6). 结论 2 本研究结果显示: CmLOX09 参与机械损伤、激素、信号类物质及白粉病菌的防御反应(图1~3,5).9-LOX类型的 CmLOX09 可能通过氢过氧化物裂解酶(HPL)途径产生的绿叶挥发物(GLV)来响应MeJA(图4),并通过丙二烯合酶(AOS)途径产生的茉莉酸来响应真菌胁迫(图6).综上所述,9-LOX类型的 CmLOX09 可能在生物和非生物胁迫反应中起重要作用. 9-Lipoxygenase (9-LOX) Hydroperoxide lyase (HPL) Allene oxide synthase (AOS) Green leaf volatile Jasmonic acid 9-脂氧合酶 氢过氧化物裂解酶 丙二烯合酶 绿叶挥发物 茉莉酸 Introduction 1 Lipoxygenases (LOXs, EC 1.13.11.12) are a group of non-heme iron-containing enzymes, which are widely distributed throughout animals, plants, and fungi (Liavonchanka and Feussner, 2006 2006 ; Ivanov et al., 2010 2010 ; Christensen and Kolomiets, 2011 2011 ). In plants, LOXs are related to many biological processes, including growth and development (Keereetaweep et al., 2015 2015 ), synthesis of aroma compounds (Shen et al., 2014 2014 ), synthesis of ethylene (Griffiths et al., 1999 1999 ), ripening and senescence (Hou et al., 2015 2015 ), and they especially play a crucial role in defense against biotic and abiotic stresses (Wang et al., 2008 2008 ; Maschietto et al., 2015 2015 ). LOXs can catalyze the oxygenation of polyunsaturated fatty acids (PUFAs) with a Z , Z -1,4-pentadiene structure to form fatty acid hydroperoxides (Brash, 1999 1999 ). These hydroperoxides act as substrates for seven downstream branches of the LOX pathway to form a wide variety of active compounds, collectively called oxylipins (Feussner and Wasternack, 2002 2002 ). The hydroperoxide lyase (HPL) and allene oxide synthase (AOS) pathways responsible for green leaf volatiles (GLVs) and jasmonic acid (JA) production, respectively, are the predominant and comprehensible pathways activated in the stress response (Birkett et al., 2000 2000 ). Overexpression of CsiHPL1 greatly enhances the resistance of tomatoes to fungal Aleuria aurantia lectin (AAL), which indicates that CsiHPL1 is involved in plant defense against fungal attack (Xin et al., 2014 2014 ). The expression of AOS is greatly induced by wounding treatment and the level of JA in Arabidopsis wounding plants is limited by AOS expression (Park et al., 2002 2002 ). There are many studies suggesting that LOX gene expression and LOX activity are regulated by different stresses such as wounding, low and high temperatures, hormones, signaling substances, and biotic stress (Maccarrone et al., 2000 2000 ; Porta et al., 2008 2008 ; Bhardwaj et al., 2011 2011 ; Christensen et al., 2013 2013 ; Hu et al., 2013 2013 , 2015 2015 ). In tomato plants, the expression of TomloxD has been rapidly induced by wounding (Heitz et al., 1997 1997 ). ZmLOX10 in maize was up-regulated by wounding and cold stresses (Nemchenko et al., 2006 2006 ). Drought stress could increase LOX activity dramatically in brassica seedlings (Alam et al., 2014 2014 ). Salt stress could also significantly increase LOX activity in rice (Mostofa et al., 2015 2015 ). In Arabidopsis , LOX3 was dramatically induced under salt treatment (Ding et al., 2016 2016 ). The LOX activity of soybean zygotic embryos was increased significantly after indole acetic acid (IAA) treatment (Liu et al., 1991 1991 ). CjLOX from Caragana jubata could be regulated by signal substances such as abscisic acid (ABA), salicylic acid (SA), and methyl jasmonate (MeJA) (Bhardwaj et al., 2011 2011 ). The LOX activity of oriental melon seedlings was increased after treatment with ABA, SA, hydrogen peroxide (H 2 O 2 ), and MeJA (Liu et al., 2016 2016 ). Cucumber mosaic virus (CMV) infection led to the up-regulated expression of LOX genes in Arabidopsis thaliana leaves (la Camera et al., 2009 2009 ). DkLOX3 -overexpression ( DkLOX3-OX ) in Arabidopsis showed more tolerance to Botrytis cinerea (Hou et al., 2018 2018 ). In particular, the 9-LOX gene CaLOX1 was distinctively induced in pepper leaves ( Capsicum annuum L.) that had been inoculated with Xanthomonas campestris pv. vesicatoria (Hwang and Hwang, 2010 2010 ). Also, after X. campestris pv. malvacearum infection, high LOX activity supported cell death in cotton (Sayegh-Alhamdia et al., 2008 2008 ). Plant LOXs are encoded by multigene families (Andreou and Feussner, 2009 2009 ) and classified as 9-LOXs and 13-LOXs, according to the position, at which oxygen is inserted into linoleic acid or linolenic acid (Feussner and Wasternack, 2002 2002 ). In our previous study, we have identified 18 LOX genes in the melon genome (named CmLOX01 – 18 ) (Zhang et al., 2014 2014 ). In addition, five 13-LOX genes were found to respond to abiotic stress and signal substances (Liu et al., 2016 2016 ). However, the defense-related functions of 9-LOXs in oriental melon are still poorly understood. An oriental melon ( Cucumis melon var. makuwa Makino) 9-LOX gene, CmLOX09 , which encodes a 9-specific LOX, was isolated from melon fruits (Zhang et al., 2015 2015 ). The phylogenetic analysis of CmLOX09 showed that it is closely related to cucumber CsLOX09 , and responds to low temperatures and 1-aminocyclopropane-1-carboxylic acid (ACC) (Yang et al., 2012 2012 ; Zhang et al., 2015 2015 ). Moreover, the pepper 9-LOX gene CaLOX1 also plays a significant role in osmosis, drought, high salinity, and microbial pathogens’ stress responses (Lim et al., 2015 2015 ). The promoter analysis revealed that the CmLOX09 promoter has various elements of abiotic stress, hormones, signal substances, and biotic stress. Therefore, we analyzed the effects of wounding, hormones treatments, signal substances treatments and inoculation with a powdery mildew pathogen Podosphaera xanthii on oriental melon seedlings in order to elucidate the role of CmLOX09 in various stress responses. Furthermore, the expression of two downstream genes CmHPL and CmAOS was measured. The GLVs of HPL-derived oxylipins served as signals to defend against wounding, fungal and insect attack (Christensen et al., 2013 2013 ; Ameye et al., 2015 2015 ). Based on the high-level expression patterns of CmLOX09 and CmHPL under MeJA treatment compared with other treatments, the levels of GLVs in leaves under MeJA treatment were determined in order to investigate whether CmLOX09 responded to MeJA through the HPL pathway to produce GLVs. Moreover, 13-LOXs have been shown to be associated with the production of JA in resistance to microbial pathogens (Hu et al., 2013 2013 ). To test whether the 9-LOX gene CmLOX09 was involved in pathogen-induced production of JA, the levels of JA were measured after inoculation with P . xanthii . Materials and methods 1 Plant materials and treatments 2 All the treatments were carried out with oriental melon ( Cucumis melon var. makuwa Makino), cultivar “Yumeiren” (Changchun, China). Seedlings were grown in pots (compost:peat:soil=1:1:1) in a greenhouse (temperature 25 °C (day)/22 °C (night), natural light, relative humidity 60%–70%). The melon seedlings with a four-leaf stage were treated as follows. To induce wounding, the four expanded true leaves were wounded with a pair of surgical scissors and the wounded leaves were collected, and the healthy leaves from uninjured seedlings were collected as controls. The third and fourth true leaves were sampled at 0, 0.5, 1.5, 3, 6, 12, and 24 h after treatment. For hormone treatments, 100 μmol/L IAA and 100 μmol/L gibberellins (GA 3 ) were applied to oriental melon seedlings (10 ml for each plant) and the control seedlings were sprayed with 0.1% (v/v) ethanol solution (10 ml for each plant). For all the seedlings, the third and fourth true leaves without veins were harvested at 0, 1, 3, 6, 12, 24, 72, 120, and 168 h. For signal substance treatments, seedlings were sprayed (10 ml for each seedling) with 100 μmol/L ABA, 5 mmol/L SA, 10 mmol/L H 2 O 2 , and 100 μmol/L MeJA, and the control seedlings of ABA, SA, and MeJA treatments were sprayed with 0.1% (v/v) ethanol solution (10 ml for each seedling), while the controls of H 2 O 2 were sprayed with sterile water (10 ml for each seedling). Plants treated with MeJA were tightly sealed in a plastic bag. For all the seedlings, after 0, 1, 3, 6, 12, 24, 72, 120, and 168 h the third and fourth true leaves without veins were harvested. P . xanthii was collected from a naturally infected plant, and then true leaves were sprinkled in order to inoculate the melon seedlings sampled at different disease levels. The disease grade of inoculation with P . xanthii in the leaves of oriental melon was as follow: level 0, no lesions; level 1, a mild disease, lesion area 0%–25%; level 2, the onset of mild, lesion area 25%–50%; level 3, moderate incidence, lesion area 50%–75%; level 4, severe disease, lesion area > 75%. All plant materials were frozen in liquid nitrogen immediately after collecting and stored at −80 °C until used. Promoter analysis 2 Firstly, the 9-LOX gene CmLOX09 (GenBank: MELO3C0014482) and the NCBI program were used to search for the DNA skeleton of the CmLOX09 . Secondly, the translation start site is defined as +1; the promoter sequence was 2000 bp upstream of the translation start site. Finally, the PLANT CARE software ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html http://bioinformatics.psb.ugent.be/webtools/plantcare/html ) was used to analyze the response element of the promoter sequence (Fig. S1 S1 ). RNA extraction and gene expression analysis by qRT-PCR 2 Total RNA was extracted from 0.5 g treated leaves under liquid nitrogen using the RNAprep Pure Plant Total RNA Extraction Kit (Kangwei, Beijing, China) according to the manufacturer’s instructions. The complementary DNA (cDNA) was synthesized from 2 µg of RNA using the M-MLV RTase cDNA Synthesis Kit (TaKaRa, Tokyo, Japan) according to the manufacturer’s instructions. Primer sequences are listed in Table 1 1 . Quantitative real-time polymerase chain reaction (qRT-PCR) was performed in a 20-μl reaction volume using PrimeScript™ RT Master Mix (Perfect Real Time) (TaKaRa, Tokyo, Japan) on an ABI PRISM 7500 Sequence-Detection System according to the manufacturer’s protocol. The PCR program was followed by 45 cycles of 95 °C for 30 s, 95 °C for 5 s and 60 °C for 34 s, 95 °C for 5 s, 60 °C for 1 min, and 95 °C for 30 s. A constitutively expressed 18S RNA gene was used as an internal reference. Three independent biological experiments were used as replicates for the qRT-PCR. The relative gene expression of CmLOX09 , CmHPL , and CmAOS was calculated using the 2 −ΔΔ C T method.  GenBank: CmLOX09 (MELO3C0014482); CmHPL (AF081955); CmAOS (AF081954) Name Oligonucleotide Sequence CmLOX09 CmLOX09-F 5'-CAGATCCATCTTGTGAAC-3' CmLOX09-R 5'-AGTTGGTAGAGTCATTCC-3' CmHPL CmHPL-F 5'-AGCGTTTTACTCCTCTTCTGGC-3' CmHPL-R 5'-CTTCTTCCTTCACGGTTGTCCT-3' CmAOS CmAOS-F 5'-TTCTTCCTCGTCTTCTTCCTCTC-3' CmAOS-R 5'-CAAATACTCTTCCCTCCCCTGAT-3' 18S RNA 18sRNA-F 5'-AAACGGCTACCACATCCA-3' 18sRNA-R 5'-CACCAGACTTGCCCTCCA-3' Green leaf volatile analysis 2 The GLVs of oriental melon leaves were analyzed using gas chromatography-mass spectrometry (GC-MS; Trace GC Ultra-ITQ 900, Thermo Scientific, Waltham, MA, USA). About 3 g of frozen leaves were ground and then transferred to a 20-ml glass vial (Thermo, USA), and 10 ml 20% (0.2 g/ml) NaCl solution was then added to the samples, along with 1-octanol (50 μl, 59.5 mg/L) as an internal standard. The mixture was completely homogenized and the glass vial was sealed with a crimp-top cap with a silicone/aluminium septa seal (20 mm, Thermo, USA). Then, the volatiles were extracted as described by Tang et al. ( 2015 2015 ). Extraction and determination of JA 2 Approximately 0.5–1.0 g fresh leaves were ground in liquid nitrogen to a fine powder, and an extraction buffer composed of isopropanol/hydrochloric acid was then added to the powder. The extract was shaken at 4 °C for 30 min. Dichloromethane was then added and shaken at 4 °C for 30 min and centrifuged at 13 000 r/min for 5 min at 4 °C. The lower organic phase was extracted, then dried with nitrogen and dissolved in 200 μl methanol (0.1% methanoic acid), followed by filtering with a 0.22-μm filter membrane. Finally, the product was analyzed using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Methanol (0.1% methanoic acid) was used to prepare different concentrations of standard solution. JA was analyzed using a ZORBAX SB-C18 (Agilent Technologies, USA) column (2.1 μm×150 μm; 3.5 μm). A volume of 2 μl of sample solution was injected for HPLC analysis. Methanol/0.1% methanoic acid and ultrapure water/0.1% methanoic acid served as mobile phase A and mobile phase B, respectively. The spray voltage was 4500 V, the pressure of the aux gas, nebulizer, and air curtain were 70, 65, and 15 psi (pound per square inch; 1 psi=6895 N/m 2 ), respectively, and the atomizing temperature was 400 °C. Statistical analysis 2 Three replicates of each treatment were carried out. The data were analyzed using the SPSS 13.0 statistics program, and significant differences were compared using the Duncan’s multiple range test for each experiment at P < 0.05 level. The charts were made using Origin 8.0. Results 1 Cis -regulatory element analysis of CmLOX09 promoter 2 The promoter analysis revealed that the CmLOX09 promoter has various cis -regulatory elements as shown in Fig. S1 S1 , such as light responsive elements (3-AF1 binding site, Box 4, ACE, AT1-motif), fungal elicitor responsive elements (Box-W1 element), hormone elements (P-box element), signal substance elements (ABRE element, TCA element), and ci- acting elements involved in defense and stress responsiveness (TC-rich repeats). Effects of wounding on expression of CmLOX09 , CmHPL , and CmAOS in leaves of oriental melon 2 The expression patterns of CmLOX09 , CmHPL , and CmAOS after wounding treatment were examined to verify whether CmLOX09 is related to wounding treatment. After wounding treatment, the expression of CmLOX09 , CmHPL , and CmAOS began to increase at 0.5 h, and all reached the highest level at 1.5 h. The expression of CmLOX09 and CmHPL was both significantly higher than the controls at 0.5, 1.5, 3, and 6 h (Figs. 1a and 1b 1a and 1b ). The expression of CmAOS was significantly higher than the controls at 0.5, 1.5, and 3 h, and then returned to normal levels at 6 h (Fig. 1c 1c ). This finding suggested that CmLOX09 might play a positive role in plants against wounding treatment through the HPL and AOS pathways. (a) Expression pattern of CmLOX09 after wounding treatment. (b) Expression pattern of CmHPL after wounding treatment. (c) Expression pattern of CmAOS after wounding treatment. Data are presented as mean±standard error from three replicates with three biological repeats. * P < 0.05, ** P < 0.01, compared to the respective control (CK) at each point Effects of hormone treatments on expression of CmLOX09, CmHPL, and CmAOS in leaves of oriental melon 2 Since there were cis -acting elements, such as hormone elements (P-box element) in the promoter of CmLOX09 , we wanted to check whether the CmLOX09 was regulated by hormones. The expression of CmLOX09 , CmHPL , and CmAOS in oriental melon leaves at the four-leaf stage was analyzed in response to IAA and GA 3 . After IAA treatment, CmLOX09 expression was significantly higher than the controls at 6, 72, 120, and 168 h, and reached its highest level at 168 h (Fig. 2a 2a ). Basically, the expression trends of CmHPL and CmAOS were similar; the expression was significantly higher than the controls at 6, 72, 120, and 168 h, while it was significantly lower than the controls at 1, 3, and 12 h (Figs. 2b and 2c 2b and 2c ). On the contrary, after GA 3 treatment, the expression of CmLOX09 was down-regulated at 1 and 3 h. However, the expression of CmLOX09 was significantly higher than the controls from 6 to 168 h, and reached the highest level at 168 h (Fig. 2d 2d ). The expression of CmHPL was significantly lower than the controls at all time except at 72 h (Fig. 2e 2e ). The expression of CmAOS was significantly lower than the controls except at 72 and 168 h (Fig. 2f 2f ). Our results implied that CmLOX09 , CmHPL , and CmAOS were differentially induced by hormones, which may be caused by the GA elements (P-box element) in the promoter of CmLOX09 showing a differential response to IAA and GA 3 (a) Expression patterns of CmLOX09 after IAA treatment. (b) Expression patterns of CmHPL after IAA treatment. (c) Expression patterns of CmAOS after IAA treatment. (d) Expression patterns of CmLOX09 after GA 3 treatment. (e) Expression patterns of CmHPL after GA 3 treatment. (f) Expression patterns of CmAOS after GA 3 treatment. Data are presented as mean±standard error from three replicates with three biological repeats. * P < 0.05, ** P < 0.01, compared to the respective control at each point Effects of signal substances on expression of CmLOX09 , CmHPL , and CmAOS in leaves of oriental melon 2 ABA, SA, H 2 O 2 , and MeJA are important signaling molecules that regulate defense against stresses in plants (Yang et al., 2012 2012 ; Liu et al., 2016 2016 ). According to cis -acting elements, ABRE and TCA elements in the promoter of CmLOX09 , to test whether the CmLOX09 was regulated by the signal molecules, the expression of CmLOX09 , CmHPL , and CmAOS in oriental melon leaves at the four-leaf stage in response to ABA, SA, H 2 O 2 , and MeJA was analyzed. After ABA treatment, the expression of CmLOX09 was down-regulated significantly at 1 and 3 h (Fig. 3a 3a ). The expression of CmHPL reached its maximum level at 12 h and was significantly higher than the controls at 12, 24, and 72 h while being significantly lower than the controls at 1 and 3 h (Fig. 3b 3b ). The expression of CmAOS was significantly higher than the control at 72 h and reached the highest level while being significantly lower than the controls at 1 and 3 h (Fig. 3c 3c ). (a) Expression patterns of CmLOX09 after ABA treatment. (b) Expression patterns of CmHPL after ABA treatment. (c) Expression patterns of CmAOS after ABA treatment. (d) Expression patterns of CmLOX09 after SA treatment. (e) Expression patterns of CmHPL after SA treatment. (f) Expression patterns of CmAOS after SA treatment. (g) Expression patterns of CmLOX09 after H 2 O 2 treatment. (h) Expression patterns of CmHPL after H 2 O 2 treatment. (i) Expression patterns of CmAOS after H 2 O 2 treatment. (j) Expression patterns of CmLOX09 after MeJA treatment. (k) Expression patterns of CmHPL after MeJA treatment. (l) Expression patterns of CmAOS after MeJA treatment. Data are presented as mean±standard error from three replicates with three biological repeats. * P < 0.05, ** P < 0.01, compared to the respective control at each point The expression of CmLOX09 , CmHPL , and CmAOS was down-regulated by SA at different degrees. The expression of CmLOX09 was significantly lower than the controls from 1 to 72 h (Fig. 3d 3d ). The expression of CmHPL was significantly lower than the controls at the treatment time except at 6 and 168 h (Fig. 3e 3e ). The expression of CmAOS was significantly lower than the controls at the treatment time except at 6 and 24 h (Fig. 3f 3f ). The expression of CmLOX09 , CmHPL , and CmAOS was largely up-regulated by H 2 O 2 . Basically, the expression trends of CmLOX09 , CmHPL , and CmAOS were similar and significantly higher than the controls at 1 and 3 h, showed its maximal level at 3 h and then declined at 6 h after treatment (Figs. 3g–3i 3g–3i ). The expression of CmLOX09 , CmHPL , and CmAOS was up-regulated by MeJA in different degrees. The expression of CmLOX09 , CmHPL , and CmAOS was significantly higher than the controls at the treatment time, and reached its highest levels at 168, 24, and 3 h, respectively (Figs. 3j–3l 3j–3l ). In summary, the expression of CmLOX09 , CmHPL , and CmAOS was induced by H 2 O 2 and MeJA, while inhibited by SA. The expression of CmLOX09 was down-regulated by ABA. The expression of CmHPL and CmAOS was down-regulated by ABA in the early stage while up-regulated in the late stage. Our results showed that CmLOX09 might respond to stresses through the production of signal substances such as H 2 O 2 and MeJA rather than through ABA and SA. Effect of MeJA treatment on GLV content in leaves of oriental melon 2 Based on the expression patterns of CmLOX09 and CmHPL , we found that CmLOX09 and CmHPL exhibited a high-level expression under MeJA treatment compared with other treatments. We therefore hypothesized that CmLOX09 might be involved in MeJA-induced GLV synthesis. To test this hypothesis, we analyzed the emission of GLVs in oriental melon leaves after MeJA treatment. The treatment time of 6, 72, and 168 h was selected to verify whether the accumulation of GLVs is induced by high-level CmHPL expression. The results of GC-MS showed that 10 alcohols, 3 aldehydes, 3 esters, 2 ketones, 1 other and 10 alcohols, 1 aldehyde, 5 esters, 1 ketone, 4 others were measured in the control and leaves with MeJA treatment, respectively (Table S1 S1 ). However, after MeJA treatment, the content of 2-hexynol and 2-hexenal was significantly higher than that of the controls at 6, 72, and 168 h, with the highest level found at 72 h (Figs. 4a and 4b 4a and 4b ). Taken together, these findings suggested that CmLOX09 response to MeJA might occur through the HPL pathway to produce C6 alcohols and aldehydes. Hence, we speculated that high-level CmHPL expression induced by wounding and H 2 O 2 might promote the accumulation of GLVs to respond to stress. (a) 2-Hexynol release from oriental melon leaves after treatment at 6, 72, and 168 h. (b) 2-Hexenal release from oriental melon leaves after treatment at 6, 72, and 168 h. Data are presented as mean±standard error from three replicates with three biological repeats. ** P < 0.01, compared to the respective control at each point Effects of inoculation with P . xanthii on expression of CmLOX09 , CmHPL , and CmAOS in leaves of oriental melon 2 Similarly, there was a fungal elicitor responsive element in the promoter of CmLOX09 . To investigate whether CmLOX09 was regulated by fungus, the expression of CmLOX09 , CmHPL , and CmAOS in oriental melon leaves after inoculation with P . xanthii was analyzed. As shown in Fig. 5 5 , the expression of CmLOX09 , CmHPL , and CmAOS was significantly induced by P . xanthii . The expression of CmLOX09 and CmHPL was strongly induced at the mild morbidity level and later tended to be stable but also significantly higher than that at level 0 (Figs. 5b and 5c 5b and 5c ). The expression of CmAOS was higher in the incidence levels of 1, 2, and 3 than in level 4; however, the expression of level 4 was also significantly higher than that of level 0 (Fig. 5d 5d ). These findings indicated that the 9-LOX gene CmLOX09 was involved in biotic stress and might respond to this stress through the HPL or AOS pathway. (a) Different disease grades in oriental melon leaves after inoculated with P. xanthii . (b) Expression patterns of CmLOX09 after inoculated with P. xanthii . (c) Expression patterns of CmHPL after inoculated with P. xanthii . (d) Expression patterns of CmAOS after inoculated with P. xanthii . Numbers indicate the different disease grades (0, no lesions; 1, a mild disease, lesion area 0%–25%; 2, the onset of mild, lesion area 25%–50%; 3, moderate incidence, lesion area 50%–75%; 4, severe disease, lesion area > 75%). Data are presented as mean±standard error from three replicates with three biological repeats. ** P < 0.01 compared to the healthy leaves (0) Effect of inoculation with P . xanthii on content of JA in leaves of oriental melon 2 Studies showed that 13-LOXs were involved in JA accumulation in defense against microbial pathogens (Hu et al., 2013 2013 ). In this study, the endogenous JA, after inoculation with P . xanthii , was determined to verify whether the 9-LOX gene CmLOX09 was involved in the fungus-induced production of JA. The level of JA in diseased leaves was analyzed using HPLC-MS/MS (Fig. S2 S2 ). As expected, the content of JA in levels 1 and 3 was significantly higher than that in level 0, and the content of JA in level 4 was significantly lower than that in level 0. However, the content of JA in level 2 did not differ from that in level 0, and the content of JA in diseased leaves was basically consistent with the trend of CmAOS expression (Fig. 6 6 ). Collectively, these findings demonstrated that the CmLOX09 response to P . xanthii might work through the AOS pathway to form JA. Whereas, based on the expression patterns of CmLOX09 and CmHPL , the possibility that the CmLOX09 response to P . xanthii occurs through the HPL pathway to produce GLVs could not be denied. Data are presented as mean±standard error from three replicates with three biological repeats. * P < 0.05, ** P < 0.01, compared to the healthy leaves (0) Discussion 1 The promoter analysis indicated that the CmLOX09 promoter contained stress response element TC-rich repeats, and the expression pattern of CmLOX09 on hormones, signal substances, and fungi stress was consistent with the prediction results of the promoter analysis. The CmLOX09 promoter had a P-box element and the expression was induced by GA 3 . CmLOX09 had ABRE and TCA elements and that the expression was inhibited by ABA and SA may be due to the response elements playing a negative role in the expression of CmLOX09 . CmLOX09 had Box-W1 elements and its expression was induced by P . xanthii . Although there were no wounding or MeJA elements, the results showed that CmLOX09 was induced by wounding and MeJA. To date, the best characterized LOX pathways are the HPL and AOS pathways, which were involved in the stress response. The GLVs produced by the HPL pathway played a vital role in defense against herbivores, and showed a certain antibacterial activity (Kallenbach et al., 2011 2011 ). ZmLOX10 is involved in the wound-induced regulation of GLV biosynthesis (Christensen et al., 2013 2013 ). LOX-H1 co-suppression decreased the release of GLVs (León et al., 2002 2002 ). Similar to GLVs, the synthesis of JA, by the AOS pathway, as a signal substance was also involved in the defense response. Studies showed that the tomato TomloxD was involved in wound-induced JA biosynthesis (Yan et al., 2013 2013 ). The LOX activity increased and the JA content accumulated dramatically in a short time in Arabidopsis thaliana leaves by wounding treatment (Bell et al., 1995 1995 ). Antisense expression of NaLOX3 significantly reduced wound-induced JA accumulation (Halitschke and Baldwin, 2003 2003 ). Ginseng responded to wounding that may be involved in the production of GLVs and JA at the wounded sites (Bae et al., 2016 2016 ). In this study, the expression of CmLOX09 and its downstream genes CmHPL and CmAOS was all induced by wounding, suggesting that CmLOX09 may respond to wounding by both HPL and AOS pathways. LOXs showed a differential response to hormones. Ethylene, IAA, GA 3 , and 6-benzylaminopurine (6-BA) are well-known hormones that participate in plant biological processes. Previous studies reported that CaLOX1 expression was significantly induced by ethylene treatment. In this study, CmLOX09 was up-regulated in the late stage of IAA and GA 3 treatments; interestingly, CmHPL and CmAOS were up-regulated by IAA treatment and down-regulated by GA 3 treatment. The LOX activity of soybean was greatly increased by IAA treatment (Liu et al., 1991 1991 ). After GA 3 treatment, LOX activity was inhibited, the expression of DkLOX01 and DkLOX03 was down-regulated, and then fruit ripening was delayed and fruit firmness was increased (Lv et al., 2014 2014 ). Studies indicated that various LOX member responses to IAA and GA 3 were different, while the specific mechanism still remains unclear. ABA, SA, H 2 O 2 , and MeJA are central signaling molecules in the defense responses to abiotic stresses (Neill et al., 2002 2002 ; Durrant and Dong, 2004 2004 ; Zhang et al., 2006 2006 ; Guo and Stotz, 2007 2007 ). Studies showed that after ABA, SA, H 2 O 2 , and MeJA treatments, CmLOX09 responded rapidly, and the two downstream genes CmHPL and CmAOS actively participated in the stress. The expression of CmLOX09 was inhibited by ABA, but the expression of CmHPL and CmAOS was induced by ABA. Previous studies showed that CmLOX10 and CmLOX12 were up-regulated by ABA; meanwhile, CmLOX13 and CmLOX18 were down-regulated by ABA (Liu et al., 2016 2016 ). ZmLOX6 declined dramatically starting at 1 h and reached its lowest level at 6 h after treatment with ABA (Gao et al., 2008 2008 ). In this study, the expression of CmLOX09 , CmHPL , and CmAOS was down-regulated by SA. The expression of CmLOX09 was significantly inhibited from 1 to 72 h. Our previous study showed that only CmLOX12 was induced by SA among the five 13-LOXs; others were inhibited by SA (Liu et al., 2016 2016 ). However, the 9-LOX gene CaLOX1 was induced by SA treatment (Hwang and Hwang, 2010 2010 ). The specific reasons for the above differences are not clear; it may be caused by the difference between species and genes. The expression of CmLOX09 , CmHPL , and CmAOS was strongly induced by H 2 O 2 at 3 h and declined after this time. H 2 O 2 was added to the medium and the LOX gene expression and activity of lentil in the roots were increased to some extent (Maccarrone et al., 2000 2000 ). The expression of CmLOX09 , CmHPL , and CmAOS was induced by MeJA. The expression of CsLOX2 was rapidly up-regulated with MeJA and reached its maximal level up to 100-fold at 3 h after treatment (Yang et al., 2012 2012 ). CjLOX was strongly induced after spraying with MeJA (Bhardwaj et al., 2011 2011 ). Furthermore, based on the expression patterns of the three genes after signal substance treatment, we also investigated the emission of GLVs at 6, 72, and 168 h after MeJA treatment. The 2-hexynol and 2-hexenal content was significantly higher than that of the controls in oriental melon leaves in response to MeJA treatment; these data suggested that CmLOX09 may contribute to the production of C6 volatiles. After wounding treatment, the levels of Z -3-hexenal and Z -3-hexan-1-ol in the leaves of two LOX lines and wild type (WT) plants were significantly higher than those in the untreated leaves; however, the emission of Z -3-hexenal and Z -3-hexan-1-ol from transgenic plants did not result from levels released by the WT plant—this finding suggested that OsHI-LOX does not supply substrates for the production of GLVs in rice (Zhou et al., 2009 2009 ). The LOX10-2 and LOX10-3 mutants provide substrates for the HPL pathway for GLV production in maize leaves in response to wounding or herbivory by Salix exigua (Christensen et al., 2013 2013 ). The 9-LOX gene NaLOX1 was induced in tobacco leaves infected with Phytophtora parasitica var. nicianae (Rancé et al., 1998 1998 ). The 9-LOX gene GhLOX1 is associated with the hypersensitive reaction of cotton cell death responses to X . campestris pv. malvacearum (Marmey et al., 2007 2007 ). The CaLOX1 of pepper was distinctively induced by X . campestris pv. vesicatoria , the number of cell death reactions decreased, and it was more susceptible to infection by pathogens in CaLOX1 silence pepper plant, which indicated that CaLOX1 was involved in the defense response of pepper to pathogenic microorganisms and cell death response (Hwang and Hwang, 2010 2010 ). In the present study, after inoculation with P . xanthii , the expression of CmLOX09 , CmHPL , and CmAOS at different disease levels was significantly higher than that at level 0, which indicated that CmLOX09 was involved in the response to fungus. Overexpression of TomloxD in transgenic tomatoes increased the content of endogenous JA and enhanced resistance to Cladosporium fulvum compared with non-transformed tomato plants, which suggested that TomloxD was involved in the synthesis of JA (Hu et al., 2013 2013 ). Tomato plants with suppressed expression of the TomloxD gene have diminished LOX activity and the content of endogenous JA, which finally lead to increase in their susceptibility to microbial pathogens (Hu et al., 2015 2015 ). Antisense expression of AtLOX2 decreased wound-induced JA accumulation (Bell et al., 1995 1995 ). CMV inoculation increased the expression of two LOXs genes that participate in JA biosynthesis (la Camera et al., 2009 2009 ). In Arabidopsis , LOX3 and LOX4 contribute to JA synthesis (Chauvin et al., 2016 2016 ). In this study, the expression of CmLOX09 was strongly induced by P . xanthii , JA analysis showed that levels 1 and 3 contain higher endogenous JA compared with the level 0, the trend of JA was similar to the expression pattern of CmAOS , and the results indicated that the disease leaves might response to fungal stress by the 9-LOX-AOS pathway; however, the role of CmLOX09 in responses to biotic and abiotic stress needed to be verified using gene editing or overexpressed technique. Since lipid peroxidation closely related to LOX, we will measure malondialdehyde (MDA) or thiobarbituric acid reactive substances (TBARS) in next work. Conclusions 1 Here we reported the expression of CmLOX09 , CmHPL , and CmAOS in the leaves of melon seedlings that were regulated by wounding, hormones (IAA, GA 3 ), signal substances (ABA, SA, H 2 O 2 , MeJA), and biotic stresses ( P . xanthii ), which indicated that CmLOX09 , CmHPL , and CmAOS were all induced by mechanical wounding and differentially induced by hormones and signal substances. In addition, we analyzed the content of GLVs after MeJA treatment and the content of JA after inoculation with P . xanthii . Taken together, these results implied that the CmLOX09 response to MeJA might occur through the HPL pathway to produce C6 alcohols and aldehydes; and the CmLOX09 response to P . xanthii might occur through the AOS pathway to form JA. Currently, the molecular mechanism of the CmLOX09 -mediated pathway still remains unclear in plant responses. Moreover, the relationship between CmLOX09 and signal molecules is needed to be clarified. 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