Effects of abiotic stress and hormones on the expressions ...tion, expression of JA-induced genes,...

Journal of Integrative Agriculture 2016, 15(2): 326–338

RESEARCH ARTICLE

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Effects of abiotic stress and hormones on the expressions of five 13-CmLOXs and enzyme activity in oriental melon (Cucumis melo var. makuwa Makino)

LIU Jie-ying1*, ZHANG Chong1*, SHAO Qi1, TANG Yu-fan1, CAO Song-xiao1, GUO Xiao-ou1, JIN Ya-zhong1, 2, QI Hong-yan1

1 Key Laboratory of Protected Horticulture of Liaoning Province/Key Laboratory of Protected Horticulture, Ministry of Education/College of Horticulture, Shenyang Agricultural University, Shenyang 110866, P.R.China

2 College of Agriculture, Heilongjiang Bayi Agricultural University, Daqing 163319, P.R.China

AbstractLipoxygenases (LOXs) are a group of non-heme, iron-containing enzymes and extensively involved in plant growth and development, ripening and senescence, stress responses, biosynthesis of regulatory molecules and defense reaction. In our previous study, 18 LOXs in melon genome were screened and identified, and five 13-LOX genes (CmLOX08, CmLOX10, CmLOX12, CmLOX13 and CmLOX18) were predicted to locate in chloroplast. Phylogenetic analysis result showed that the five genes have high homology with jasmonic acid (JA) biosynthesis-related LOXs from other plants. In addition, pro-moter analysis revealed that motifs of the five genes participate in gene expression regulated by hormones and stresses. Therefore, we analyzed the expressions of the five genes and LOX activity in leaves of four-leaf stage seedlings of oriental melon cultivar Yumeiren under abiotic stress: wounding, cold, high temperature and hydrogen peroxide (H2O2), and signal molecule treatments: methyl jasmonate (MeJA), abscisic acid (ABA) and salicylic acid (SA). Real time qPCR revealed that wounding and H2O2 induced the expressions of all the five genes. Only CmLOX08 was induced by cold while only CmLOX13 was suppressed by high temperature. ABA induced the expressions of CmLOX10 and CmLOX12 while inhibited CmLOX13 and CmLOX18. MeJA increased the 3 genes expressions except CmLOX08 and CmLOX13, whereas SA decreased the effect, apart from CmLOX12. All the abiotic stresses and signal molecules treatments increased the LOX activity in leaves of oriental melon. In summary, the results suggest that the five genes have diverse functions in abiotic stress and hormone responses, and might participate in defense response. The data generated in this study will be helpful in subcellular local-ization and transgenic experiment to understand their precise roles in plant defense response.

Keywords: oriental melons, lipoxygenase, abiotic stress, signal molecules, gene expression

1. Introduction

Lipoxygenases (LOXs, EC 1.13.11.12) are a group of non-heme, iron-containing enzymes which are ubiquitous in plants and animals. LOXs catalyze the polyunsaturated fatty acids (PUFAs) that contain a (1Z, 4Z)-pentadiene system to yield the corresponding (1S, 2E, 4Z)-hydroperoxides which are

Received 5 January, 2015 Accepted 24 June, 2015LIU Jie-ying, E-mail: [email protected]; Correspondence QI Hong-yan, Tel: +86-24-88342305, E-mail: [email protected], [email protected]* These authors contributed equally to this work.

doi: 10.1016/S2095-3119(15)61135-2

© 2016, CAAS. Published by Elsevier Ltd. This is an open access art ic le under the CC BY-NC-ND l icense (http:/ /creativecommons.org/licenses/by-nc-nd/4.0/).

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327LIU Jie-ying et al. Journal of Integrative Agriculture 2016, 15(2): 326–338

collectively known as oxylipins (Liavonchanka and Feussner 2006). Oxylipins produced by LOXs have been reported to play an important role in plant defense responses (Chris-tensen et al. 2013; Garcia-Marcos et al. 2013). The allene oxide synthase catalyzes the fatty acid hydroperoxides produced by the 13-LOX to form unstable allene oxides which can be further metabolized to chiral (9S, 13S)-12-oxo phytodienoic acid (OPDA) by an allene oxide cyclase (AOC), and then products jasmonates (JAs) such as jasmonic acid (JA) and its derivative, methyl jasmonate (MeJA) (Feussner and Wasternack 2002; Demmig-Adams et al. 2013). JAs are now considered to be key regulators for stress-induced gene expression in plants (Browse 2005; Wasternack 2007; Wasternack and Hause 2013). Hydroperoxide lyases cata-lyzes 13-hydroperoxy octadecatrienoic acid to form green leaf volatile (GLV) which act as signals to regulate the expression of defensive genes (Bate and Rothstein 1998) and plant-plant communication after insect elicitation (Engelberth et al. 2004)

Plant LOXs occur in a gene family and can be classified by two methods: For one method, LOXs are classified into 9-LOXs and 13-LOXs based on the specific site of the ox-ygen addition to linoleic acid (LA) at either the C-9 or C-13 residue in the plant (Feussner and Wasternack 2002). By the other method, LOXs are grouped into type I-LOXs and type II -LOXs according to comparison of the primary struc-ture and overall sequence similarity. Enzymes designated type I have a high sequence similarity (>75%) to one another and lack plastid transit peptide. Type II enzymes show only a moderate overall sequence similarity (

328 LIU Jie-ying et al. Journal of Integrative Agriculture 2016, 15(2): 326–338

which were known as cis-acting element and cis-acting regulatory element. Cis-acting element involved in defense and stress responsiveness and cis-acting regulatory ele-ment involved in the response to various hormones. Cm-LOX08, CmLOX12 and CmLOX13 possess motif involved in response to ABA and CmLOX08, CmLOX10 have MeJA

element. CmLOX08, CmLOX10, CmLOX12 and CmLOX18 possess HSE binding site involved in response to heat (Table 2). CmLOX08 also has other motifs in response to SA and cold.

2.3. LOX activity assay

All the treatments caused the total LOX activity of seedling leaves increase in varying degrees (Fig. 2). The LOX activity of leaves treated with cold increased significantly at 12 h, and increase from 3 to 24 h with high temperature, similarly, wounding increased LOX activity from 1.5 to 12 h. MeJA and SA both increased LOX activity from 3 to 24 h, while ABA treatment only induced at 6 h. H2O2 induced LOX activity

StLOXH1 TomLOXC

NaLOX2 VvLOXA PvLOX6

PdLOX1 PdLOX2 CmLOX10

CmLOX12 CmLOX13

AtLOX2 OsRCI-1

ZmLOX10 ZmLOX11 CmLOX08

AtLOX3 AtLOX4 VvLOXO

CmLOX18

13-LOX

9-LOX

NaLOX3 CaLOX2 StLOXH3 TomLOXD

PtLOXA StLOX-2

PdLOX OsLOX1

HvLOX3 OsR9-LOX1

ZmLOX3 HvLOXA

0.1Fig. 1 Phylogenetic relationship among CmLOXs and LOXs from other plant species. The neighborjoining tree was drawn with TreeView. The five melon LOXs were CmLOX08 (MELO3C0011885), CmLOX10 (MELO3C0014627), CmLOX12 (MELO3C0014629), CmLOX13 (MELO3C0014630) and CmLOX18 (MELO3C0024348). LOXs belong to the following plant species: Arabidopsis thaliana: AtLOX2 (At3g45140), AtLOX3 (At1g17420), AtLOX4 (At1g72520); Hordeum vulgare: HvLOX3 (L37358) and HvLOXA (L35931); Nicotiana attenuata: NaLOX2 (AAP83137) and NaLOX3 (AAP83138); Oryza sativa: OsRCI-1 (AJ270938), OsLOX1 (DQ389164), and OsR9-LOX1 (AB099850); Phaseolus vulgaris: PvLOX6 (EF196866); Populus deltoides: PdLOX1 (DQ131178) and PdLOX2 (DQ131179); Solanum lycopersicum: TomLOXC (AAB65766) and TomLOXD (AAB65767); Solanum tuberosum: StLOXH1 (T07062), StLOXH3 (T07062), PtLOXA (X95513), and StLOX-2 (Y18548); Zea mays: ZmLOX3 (AF329371), ZmLOX10 (DQ335768) and ZmLOX11 (DQ335769); Vitis vinifera: VvLOXA (ACZ17391) and VvLOXO (ACZ17393); Prunus dulcis: PdLOX (AJ404331); Capsicum annuum: CaLOX2 (JQ219046).

Table 1 Full-length of the five genesGene Full-length (bp)CmLOX08 2 748CmLOX10 2 709CmLOX12 2 739CmLOX13 2 721CmLOX18 2 724


at 6 h which peaked at 72 h then declined to the control level at 168 h.

2.4. The five 13-LOX genes expressions

Since there were cis-acting elements and cis-acting reg-ulatory elements in the promoter of the five LOX genes, we checked whether the five genes were regulated by the abiotic stress and signal molecules.

The expression of CmLOX08 was induced by wounding, cold, high temperature and H2O2 while inhibited by MeJA and SA (Fig. 3), and no effect under ABA treatment. A slight

increase of CmLOX08 mRNA was observed at 1.5 h after wounding and reached a maximum at 3 h. The transcripts level reached a maximum at 6 h at both 4 and 45°C, while decreased to the lowest point at 6 h after SA treatment. H2O2 treatment induced steady-levels of CmLOX08 mRNA accumulation starting at 6 h and reached a maximum value at 72 h, and then decreased to the level of control.

CmLOX10 was also induced by wounding, high tem-perature and H2O2, but unlike CmLOX08, the expression was up-regulated by MeJA and SA and decreased by cold (Fig. 4). The mRNA transcripts increased after both wounding and MeJA treatments and peaked at 6 h, and

Table 2 List and description of nucleotide motifs discovered in promoter region of five melon LOX genesMotif name Gene name Motif sequence Motif functionTC-rich repeats CmLOX08 ATTTTCTTCA; GTTTTCTTAC Cis-acting element involved in defense and stress responsiveness

CmLOX10CmLOX12CmLOX13CmLOX18

ARE CmLOX08 TGGTTT Cis-acting element involved in hypoxia stress responsivenessCmLOX10CmLOX18

CGTCA-motif CmLOX08CmLOX10

CGTCA Cis-acting regulatory element involved in the MeJA-responsiveness

TGACG-motif CmLOX08CmLOX10

TGACG Cis-acting regulatory element involved in the MeJA-responsiveness

CCAAT-box CmLOX13CmLOX18

CAACGG MYBHv1 binding site

HSE CmLOX08 AAAAAATTTC Cis-acting element involved in heat stress responsivenessCmLOX10CmLOX12CmLOX18

LTR CmLOX08 CCGAAA Cis-acting element involved in low temperature responsivenessMBS CmLOX08 TAACTG MYB binding site involved in drought-inducibility

CmLOX10 TAACTG; CAACTGCmLOX12 TAACTGCmLOX13 CAACTG

GARE-motif CmLOX08 AAACAGA Cis-acting element involved in the gibberellin responsivenessP-box CmLOX10

CmLOX18CCTTTTG Cis-acting element involved in the gibberellin responsiveness

ABRE CmLOX08 TACGTG Cis-acting element involved in the abscisic acid responsivenessCmLOX12 ACGTGGCCmLOX13 GGACACGTGGC; CACGTG;

CGCACGTGTCERE CmLOX12

CmLOX18ATTTCAAA Ethylene-responsive

TGA CmLOX08 AACGAC Auxin responsiveCmLOX12 AACGA; TGACGTAA; AACGAC

CmLOX13 AACGACAuxRR-core CmLOX10

CmLOX12GGTCCAT Cis-acting regulatory element involved in auxin responsiveness

TCA-element CmLOX12 CCATCTTTTT　 Cis-acting element involved in salicylic acid responsivenessCmLOX13 GAGAAGAATA

GC-motif CmLOX10 CCACGGGG Enhancer-like element involved in anoxic specific inducibilityWUN-motif CmLOX13 TCATTACGAA Wound-responsive element


then decreased to normal level at 24 h. The expression decreased by SA and cold treatments.

CmLOX12 was induced by all the treatments except cold (Fig. 5). The expression level increased immediately after wounding and ABA treatment, whereas SA and MeJA induced its expression, reached a maximum value at 12 and 3 h, respectively.

CmLOX13 was induced by wounding and H2O2 and suppressed by ABA, cold and high temperature treatments, and suppressed after SA treatment, but increased at 24 h and then recovered to the control level (Fig. 6).

Similar to CmLOX10, the expression of CmLOX18 was increased by wounding, high temperature, MeJA and H2O2 and suppressed by cold and SA, but its expression was declined by ABA (Fig. 7).

3. Discussion

There are 18 LOXs identified in melon genome and among them five LOXs (CmLOX08, CmLOX10, CmLOX12, CmLOX13 and CmLOX18) are predicted to locate in the chloroplast and possessed a chloroplast transit peptide (Zhang et al. 2014). To gain knowledge of the possible role of the five LOXs under stress, we analyzed the five gene expression and LOX activity under abiotic stress and hormone treatments.

The phylogenetic tree showed that the five genes exhib-ited a close genetic relationship with Arabidopsis AtLOX2, NaLOX3 from tobacco and TomLOXD from tomato (Fig. 1). Antisense-mediated depletion of AtLOX2 reduced wound-in-duced accumulation of JA and a wound- and JA-inducible

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Fig. 2 Changes in activity of lipoxygenase in melon leaves treated with wounding, cold (4°C), high temperature (45°C), methyl jasmonate (MeJA,100 μmol L–1), abscisic acid (ABA, 100 μmol L–1), salicylic acid (SA, 5 mmol L–1) and hydrogen peroxide (H2O2, 5 mmol L–1) at various time points. Total proteins extracted from treated leaves were used for analyzing LOX activity. Values are means±SD of three replicates. Asterisks indicate significant differences between treatments and respective controls at each time point. *, P


gene (Bell et al. 1995). NaLOX3 and TomLOXD are known to provide linolenic acid hydroperoxide substrates for the synthesis of JA (Halitschke and Baldwin 2003; Hu et al. 2013; Yan et al. 2013). The results suggest that the five melon LOX genes might participate in the biosynthesis of JA and defense response to abiotic stress.

The expressions of the five genes under abiotic stress and hormone treatments were in accordance with their mo-tifs (Table 2, Figs. 3–7). Interestingly, the promoter sequenc-es analysis showed that CmLOX08 contained CGTCA-motif (MeJA) and TCA-element (SA) while the expression of CmLOX08 was declined by MeJA and SA treatments, which suggest the two motifs negative regulate the expression of CmLOX08. On the contrary, the expression of CmLOX10 was positive regulated by CGTCA-motif (MeJA) and HSE (heat) as the accumulation of CmLOX10 mRNA increased upon MeJA and high temperature treatments. CmLOX13

was down regulated by ABRE (ABA) and CmLOX18 was up regulated by HSE (heat).

The analysis of expressions of the five genes showed that they were differently regulated by abiotic stress and hormones. MeJA, ABA, SA and H2O2 are important sig-nal compounds regulating defense response to stress in plants (Jia 2012; Yang et al. 2012). Our results showed that CmLOX10, CmLOX12 and CmLOX18 were inducible by wounding and simultaneously up-regulated with MeJA (Figs. 4, 5 and 7), which are also reported in many plants such as cucumber, Arabidopsis and tomato (Melan et al. 1993; Heitz et al. 1997; Yang et al. 2012), suggesting they might participate in the biosynthesis of wound-induced JAs, which would be verified by transgenic experiments in the near future. All the five genes were induced by both wounding and H2O2, which is a key molecule in response to wounding and pathogens, triggering plant cell death to

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Fig. 3 Expressions of CmLOX08 at various time points under wounding, cold (4°C), high temperature (45°C), MeJA (100 μmol L–1), ABA (100 μmol L–1), SA (5 mmol L–1) and H2O2 (10 mmol L

–1) were analyzed by real time qPCR using the primers specific to the five CmLOX genes.


defense to stress (Montillet et al. 2005). LOXs in common bean (PvLOX6) and pepper (CaLOX2) are also induced by H2O2 (Porta et al. 2008; Bhardwaj et al. 2011)

SA is a key signaling molecule required in the develop-ment of systemic acquired resistance (SAR) (Ryals et al. 1995) and it also mediates defence reactions against biotrophic pathogens (Glazebrook 2005). JA is a key compo-nent of resistance mechanisms against necrotrophic patho-gens and insects (Dong 1998). Numerous researches report that JA and SA act antagonistically in defence reactions (Pena-Cortes et al. 1993; Feys and Parker 2000; Cipollini et al. 2004) because JA inhibits SA-induced expression of SAR (Traw et al. 2003). It is suppressed that the present study reported CmLOX12 was induced by both MeJA and SA (Fig. 5). However, more recent studies suggest that SA and JA had a cooperative effect (Salzman et al. 2005; Mur et al. 2006). In several plants LOX genes induced by both

SA and JA are reported, such as barley, maize and common (Weichert et al. 1999; Nemchenko et al. 2006; Porta et al. 2008). Therefore, CmLOX12 might participate in both JA and SA signal pathways. It would be interesting to confirm the function of CmLOX12 in these two signal pathways in the future.

ABA plays an important role in integrating various stress-es like cold and high temperature and controlling down-stream stress responses (Tuteja 2007). The present work showed that CmLOX13 and CmLOX18 were down-regulated by ABA and cold while CmLOX10 and CmLOX12 were up-regulated by ABA and high temperature. However we observed that the time points at which these genes reached the lowest expression level in response to cold were earlier than in response to ABA (Figs. 6 and 7) , while reached the maximum in response to high temperature latter than in response to ABA (Figs. 4 and 5). That may be the result of

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the more direct effect of cold on membrane deterioration and the tolerance to high temperature of melon. Similarly, the LOX genes reached the maximum in response to cold earlier than in response to ABA in cucumber (Yang et al. 2012).

The total LOX activity of leaves was all increased by the treatments. The expression pattern of the five genes treat-ed with wounding, high temperature, ABA and MeJA were mainly consistent with the change of activity. Most of the five genes were suppressed by cold and SA but the LOX activity was increased by these two treatments. That might be the other genes of CmLOX induced by these two treatments.

The AOS- and HPL-derived signaling compounds exe-cute co-operative defence signaling (Halitchke et al. 2004).C6-volatiles have been suggested to be essential to act wound-related pathways, some of which are JA indepen-dent (Bate and Rothstein 1998). In maize GLVs have been shown to play a strong signaling role for the induction of JA

(Engelberth et al. 2004, 2007). ZmLOX10 is specialized in generating GLVs, furthermore, it has been shown to be required for wound-induced JA biosynthesis. The impaired ability of ZmLOX10 mutants to produce GLVs and JA led to compromised resistance to insect attack, suggesting that ZmLOX10 is important in defense response of maize (Christensen et al. 2013). CmLOX10, CmLOX12 and Cm-LOX13 was close to ZmLOX10 and other LOXs participating GLVs generation. Moreover, CmLOX12 was induced by MeJA, ABA, SA and wounding, which was the same as ZmLOX10 (Nemchenko et al. 2006). CmLOX10 and Cm-LOX13 were also regulated by these treatments. Based on the data above, CmLOX10, CmLOX12 and CmLOX13 might participate in plant defense response to stress and GLVs biosynthesis. Overexpression of tomato TomLOXD increases the resistance to high temperature and generation of endogenous JA. CmLOX08, CmLOX10, CmLOX12 and

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CmLOX18 possess HSE binding site involved in response to heat (Table 2) and were induced by high temperature. So we suggest that the expression of these genes increased the biosynthesis of endogenous JA, and then enhanced the resistance to stresses.

4. Conclusion

Here we report the effect of abiotic stresses (wounding, cold, high temperature and H2O2) and signal molecules (ABA, MeJA and SA) on the expressions of five LOXs (CmLOX08, CmLOX10, CmLOX12, CmLOX13 and CmLOX18) and LOX activity. The expressions of five genes and LOX activity in leaves were differently regulated by these treatments, indicating their diverse functions in stress response. Cm-LOX10 and CmLOX18 might participate in the biosynthesis of JA. CmLOX12 is required in several responses because

it was induced by all the treatments except cold. CmLOX10, CmLOX12 and CmLOX13 might participate in GLVs bio-synthesis and plant defense response to stress. Further studies through genetic engineering are needed to learn more about the function of the five genes in plant defense and signaling compounds biosynthesis.

5. Materials and methods

5.1. Plant materials and treatments

All the experiments were carried out with Yumeiren, orien-tal melons (Cucumis melo var. makuwa Makino), from the Yijianpu Mishijie Melon Research Institution (Changchun, China). All the seedlings were grown in pots (soil:peat:com-post=1:1:1) in a greenhouse after seed germination. They were then moved into a growth chamber kept at 25°C/22°C

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(day/night) with 14 h light/10 h dark photoperiod at a pho-tosynthetic photon flux of 250 μmol m−2 s−1, and the relative humidity was 60–70%. For all of the expression studies, melon seedlings at the four-leaf stage were used.

To induce wounding, the expanded true leaves were clipped with a pair of surgical scissors. Both sides of the leaves were clipped from leaf edge to leaf central without hurting veins and the wounded leaves were collected at indicated time intervals, whereas healthy and intact leaves from other uninjured seedlings were collected as controls.

For cold and high temperature treatments, seedlings were moved into a climate chamber kept at 4°C or 45°C for 24 h with 14 h light/10 h dark photoperiod at a photosynthetic photon flux of 250 μmol m−2 s−1, and the relative humidity was 60–70%, and control plants were kept at 25°C with the same conditions described above.

MeJA (Sigma, W341002, USA), ABA (Sigma, A1049,

USA) or SA were dissolved in ethanol (100 mmol L–1 stock solution) and added to the hydroponics solution to 100 μmol L–1 final concentration while SA added to 5 mmol L–1 final concentration. For MeJA, ABA and SA treatments leaves of seedlings were sprayed (10 mL per seedling) with the configured MeJA, ABA or SA solutions. For H2O2 treatment leaves of seedlings were sprayed (10 mL per seedling) with 10 mmol L–1 H2O2 water solution. Control seedings of MeJA, ABA and SA treatments were sprayed (10 mL per seedling) with 0.1% (v/v) ethanol solution while controls of H2O2 were sprayed with water. Seedlings treated with MeJA and its controls were tightly sealed in plastic bags. For all the treatments and controls, the second and third true leaves (functional leaves) without veins were sampled at various time points after treatment. All the samples were frozen immediately after being collected in liquid N2, and stored at –80°C.

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5.2. RNA Isolation and real time qPCR

Total RNA was isolated from leaves using RNA Kit (Kangwei Biotech, Beijing, China) according to the manufacturer’s instructions. Four micrograms of RNA were pretreated with RQ 1DNAase I (Promega, USA) to remove contaminating genomic DNA. The RNA concentration was measured by the NanoDrop spectrophotometer ND-1000 (NanoDrop, USA) and quality was checked by agarose gel electropho-resis (Biowest, Spain) (28S rRNA/18S rRNA ratios).

Reverse transcription was performed with 1 μg of total RNA using M-MLV RTase cDNA Synthesis Kit following the manufacturer’s instructions (Cat#D6130, TaKaRa, Tokyo, Japan). The gene-specific primers of real-time quantitative PCR (qPCR) were the same with our previous study (Zhang et al. 2014), and 18S rRNA DNA fragment (148 bp) of mel-ons was used as the internal standard for each analyzed gene. Primer sequences are listed in Table 3.

Real time qRT-PCR was performed in a 20-μL reaction volume using SuperReal PreMix Plus (SYBR Green) (Cat.FP205, Tiangen Biotech, Beijing, China) on an ABI PRISM 7500 Sequence-Detection System according to manufac-ture’s instructions. The PCR program was at 95°C for 1 min, followed by 45 cycles at 95°C for 15 s and 60°C for 34 s. No template controls for each primer pair were included in each run. At least three different RNA isolations and cDNA syntheses were used as replicates for the qPCR. Samples were run in triplicate on each 96-well plate. The real time PCR data were calibrated relative to the corresponding gene expression level of control seedlings at zero time for each treatment, which were set to 1, following the 2−ΔΔCt method for relative quantification.

5.3. LOX activity

LOX activity was determined as previously described (Axelrod 1981) with some modifications. Approximately 1 g of fresh leaves was ground to a fine powder in liquid

nitrogen. All the powdered tissue was homogenized in 10.0 mL of ice-cold 0.1 mol L–1 phosphate buffer (pH 6.8). The mixture was centrifuged at 15 000×g for 20 min at 4°C and the supernatant was collected as the enzyme source. LOX activity was determined spectrophotometrically by measuring the formation of conjugated dienes during ox-idiation of polyunsaturated fatty acids at 234 nm at 30°C. Linoleic acid was used as the substrate of the reaction. The reaction was started by addition of 0.2 mL of crude leaves extract to 25 μL of substrate containing 2.775 mL of 0.1 mol L–1 phosphate buffer (pH 6.8) as reaction buffer. One activity unit (U) was defined as the increase in one unit of absorbance per minute and results were expressed as specific activity (mU mg–1 protein). Three biological replicates were included and three readings of absorbance for each replicate were recorded.

5.4. Protein content

Protein concentration of enzyme extracts was determined by using Coomassie brilliant blue G-250 with the method described by Bradford (1976) for protein assay with modi-fications (BioRad Protein Assay Kit) according to manufac-turer’s instructions, using bovine serum albumin (BSA) as a standard.

5.5. Phylogenetic analysis of the five CmLOXs

MEGA 4 (http://www.megasoftware.net/mega.html) was used to construct and align the five CmLOX full-length protein sequences, using the neighbor joining (NJ) with the following parameters: poisson correction, complete deletion, and none-bootstrap (Tamura et al. 2007). TreeView 1.66 (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html) was used to visualize the genes and BioEdit5.06 software was used for gene analysis (Page 1996; Hall 1999). PLANT CARE program was used to carry out promoter analysis.

5.6. Statistical analysis

Experiments were performed in a completely randomized design. The data were analyzed by the analysis of vari-ance (ANOVA) using the SPSS 13.0 statistics program, and significant differences were compared by a one-way ANOVA following Duncan’s multiple range tests for each experiment at P


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