© 2014 Scandinavian Plant Physiology Society, ISSN 0031-9317
Physiologia Plantarum 152: 59–69. 2014
The 9-lipoxygenase Osr9-LOX1 interacts with the 13-lipoxygenase-mediated pathway to regulate resistance to chewing and piercing-sucking herbivores in rice Guoxin Zhoua,b , Nan Rena , Jingfeng Qia , Jing Lua , Caiyu Xianga , Hongping Jua , Jiaan Chenga and Yonggen Loua,∗ a
State Key Laboratory of Rice Biology, Institute of Insect Sciences, Zhejiang University, Hangzhou, 310058, China Key Laboratory for Quality Improvement of Agriculture Products of Zhejiang Province, Department of Plant Protection, Zhejiang Agriculture & Forestry University, Lin’an, 311300, China b
Correspondence *Corresponding author, e-mail: [email protected] Received 28 November 2013 doi:10.1111/ppl.12148
Oxylipins produced by the 13-lipoxygenase (LOX) have been reported to play an important role in plant defense responses to herbivores. Yet, the role of oxylipins produced by the 9-LOX pathway in this process remains largely unknown. Here we cloned a gene encoding a chloroplast-localized 9LOX, Osr9-LOX1, from rice. Transcriptional analysis revealed that herbivore infestation, mechanical wounding and jasmonic acid (JA) treatment either repressed or did not enhance the level of Osr9-LOX1 transcripts at early stages but did at later stages, whereas salicylic acid (SA) treatment quickly increased the transcript level of Osr9-LOX1. Antisense expression of Osr9-lox1 (asr9lox1) decreased the amount of wound-induced (Z )-3-hexenal but increased levels of striped stem borer (SSB)-induced linolenic acid, JA, SA and trypsin protease inhibitors. These changes were associated with increased resistance in rice to the larvae of the SSB Chilo suppressalis. In contrast, although no significant differences were observed in the duration of the nymph stage or the number of eggs laid by female adults between the brown planthopper (BPH) Nilaparvata lugens that fed on as-r9lox1 lines and BPH that fed on wild-type (WT) rice plants, the survival rate of BPH nymphs that fed on as-r9lox1 lines was higher than that of nymphs that fed on WT plants, possibly because of a higher JA level. The results demonstrate that Osr9-LOX1 plays an important role in regulating an herbivore-induced JA burst and cross-talk between JA and SA, and in controlling resistance in rice to chewing and phloem-feeding herbivores.
Introduction Plants have evolved complex defense systems to protect themselves against different challenges. Such systems can recruit and coordinate various defense pathways to respond to various abiotic and biotic environmental stresses. Among others, the oxylipin-, ethylene (ET)-, salicylic acid (SA)-, brassinosteroid- and reactive oxygen species-mediated signaling pathways are involved in
plant defense responses. Oxylipins are lipid-derived signaling molecules that play roles in mediating defense responses to biotic and abiotic stress in plants, and participate in diverse developmental processes (Farmer et al. 2003, Demmig-Adams et al. 2013, Wasternack and Hause 2013). Plant messengers derived from oxidatively modified polyunsaturated fatty acids are collectively known
Abbreviations – BPH, brown planthopper; ET, ethylene; GLVs, green leaf volatiles; HPL, hydroperoxide lyase; JA, jasmonic acid; LeA, linolenic acid; LOX, lipoxygenase; qRT-PCR, quantitative real-time polymerase chain reaction; SA, salicylic acid; SSB, striped stem borer; TrypPIs, trypsin protease inhibitors; WT, wild type.
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as oxylipins. This class of compounds includes acid hydroperoxides, hydroxyl-, oxo-, or keto-fatty acids, divinyl ethers and volatile aldehydes such as the important plant stress hormone jasmonic acid (JA), its precursor, 12-oxo-phytodienoic acid, and its derivative, methyl jasmonate (MeJA) (Mosblech et al. 2009, Demmig-Adams et al. 2013). The first step in oxylipin biosynthesis is the oxidation of polyunsaturated fatty acids to form fatty acid hydroperoxides. This can occur non-enzymatically but mainly occurs via enzymatic peroxidation catalyzed by lipoxygenases (LOXs) (Demmig-Adams et al. 2013). In plants, LOXs are the key enzymes involved in generating fatty acid derivatives during oxylipin metabolism (Feussner and Wasternack 2002, Mosblech et al. 2009). Plant LOXs can be classified into two groups based on their mechanism of action, that is, on whether they add oxygen to the 9- (9-LOX) or the 13- (13-LOX) position of the substrate carbon chain to generate 9- or 13hydroperoxides, respectively (Feussner and Wasternack 2002). The most well-known bioactive oxylipin, JA, is biosynthesized from the fatty acid substrate α-linolenic acid (α-LeA) via the 13-LOX pathway. Genetic and biochemical methods have been used to show that the 13-LOX pathway and its products, such as JA and green leaf volatiles (GLVs) produced by the hydroperoxide lyase (HPL) pathway play important roles in direct and indirect induced defenses against herbivores in diverse dicot species, including tobacco (Halitschke and Baldwin 2003, Kessler et al. 2004), Arabidopsis (Chauvin et al. 2013, Garcia-Marcos et al. 2013) and potato (Royo et al. 1999), and in monocots, including rice (Wang et al. 2008, Zhou et al. 2009) and maize (Cho et al. 2012, Christensen et al. 2013). However, little is known about the role of the 9-LOX pathway in plant defenses against herbivores, although there is increasing evidence that the pathway influences plant development (Vellosillo et al. 2007, Gao et al. 2008), stomatal closure (Montillet et al. 2013), defense responses to pathogens (Gao et al. 2007, Montillet et al. 2013, Vellosillo et al. 2013) and plant cell death (Sayegh-Alhamdia et al. 2008, Hwang and Hwang 2010, Garcia-Marcos et al. 2013). Rice, one of the most important staple foods worldwide, is severely affected by chewing herbivores and phloem feeders; these cause substantial losses in rice crops in Asia (Cheng and He 1996). Previous studies have shown that the oxylipin pathway, especially the one mediated by 13-LOX oxylipin, plays an important role in mediating resistance to both types of herbivores in rice. For example, the JA signaling pathway was found to positively regulate resistance in rice to chewing herbivores, including rice striped stem borer (SSB, Chilo suppressalis) and leaf folder (Cnaphalocrocis 60
medinalis), but negatively modulate resistance to the piercing-sucking herbivore, rice brown planthopper (BPH, Nilaparvata lugens) (Zhou et al. 2009, Tong et al. 2012). GLVs were also reported to affect the performance of SSB larvae and BPHs (Qi et al. 2011, Tong et al. 2012). Wang et al. (2008) reported that OsLOX1, showing 9- and 13-LOX dual positional specificity, positively mediates levels of endogenous JA, (Z )-3-hexenal and LeA and affects the resistance of rice to BPH (Wang et al. 2008). Rice varieties lacking LOX-3 (9-LOX) showed decreased insect damage and their grain was more insect-resistant, while those with LOX-3 showed increased insect damage because volatiles attractive to insects were released (Zhang et al. 2007, Tang and Zhou 2012). Previously, we used microarray technology to detect genome-wide transcriptional changes in rice in response to infestation by SSBs. Our results revealed that Osr9-LOX1 transcript levels increased in SSB-treated rice plants (Zhou et al. 2011). However, there is still much to learn about the roles of the 9-LOX pathway in the rice defense response to herbivores. In this study, we isolated a rice 9-LOX gene, Osr9-LOX1, and analyzed its expression patterns and subcellular localization. Using a reverse genetics approach, we obtained rice lines with antisense expression of Osr9-LOX1 (as-r9lox1). We determined the role of Osr9-LOX1 in herbivore-induced defense by measuring the levels of JA and SA and defense-related chemicals such as trypsin protease inhibitors (TrypPIs) and GLVs. We also evaluated resistance to a chewing herbivore, SSB, and a phloem feeder, BPH. Our results provide evidence that Osr9-LOX1 is involved in the herbivore defense response of rice.
Materials and methods Plant growth and insects The rice (Oryza sativa) genotypes used in this study were Xiushui 11 wild type (WT, untransformed) and three as-r9lox1 transgenic lines (see below) in the same background. Pre-germinated seeds were cultured in plastic bottles (diameter, 8 cm; height, 10 cm) in a greenhouse at 28 ± 2◦ C, under a 14-h light/10-h dark photoperiod. After 10 days, seedlings were transplanted into 50-l communal hydroponic boxes containing a rice nutrient solution (Qi et al. 2011). After 30–35 days, seedlings were transferred to plastic pots (8 cm in diameter × 10 cm high), each with one or two plants (one WT, the other an as-r9-lox transgenic plant). Plants were used for experiments 4–5 days after transplanting. Colonies of SSB and BPH were originally obtained from rice fields in Hangzhou. Both herbivores were
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maintained on seedlings of Shanyou 63 (a variety susceptible to SSB and BPH) in a controlled climate room at 26 ± 2◦ C under a 12-h light/12-h dark photoperiod and 80% relative humidity. Generation and characterization of as-r9lox1 transgenic lines To construct the plant transformation vector, a 957bp portion (Fig. S1) of the Osr9-LOX1 cDNA in the plasmid pr9-LOX (see below) was PCR-amplified using the primers 5 -GCC AGACACTCATCAACG-3 (F) and 5 -CAGCCACAAACCTACACC-3 (R). The obtained 0.96-kb PCR fragment was cloned into pCAMBIA1301, yielding the transformation vector pCAMBIA-as-r9LOX (Fig. S2B) that contained the hygromycin resistance gene hph and the reporter gene gus as selectable markers. The vector was used to transform the rice variety Xiushui 11 via an Agrobacterium tumefaciens-mediated transformation procedure (Hiei et al. 1994). The methods used to screen the homozygous T 2 plants and to identify the number of inserts were as described elsewhere (Zhou et al. 2009). For most of the experiments, two T 2 homozygous lines L182 and L219, each harboring a single insertion (Fig. S3), and WT plants were used.
described by Zhou et al. (2009). Plants were individually sprayed with 2 ml JA (100 μg ml−1 ) or SA (70 μg ml−1 ) (Sigma, St. Louis, MO) in 50 mM sodium phosphate buffer (adjusted with 1 M citric acid to pH 8, with 0.01% Tween). Control plants were sprayed with 2 ml buffer (BUF). Isolation of the full-length cDNA of Osr9-LOX1 Total RNA was isolated from WT plants infested by SSB larvae for 24 h, and then Osr9-LOX1 cDNA fragments were obtained by reverse transcription-polymerase chain reaction (RT-PCR) using the primer pair r9-LOX1-F (5 -ACCACCAACATAGCTTCTTTTG-3 ) and r9-LOX1R (5 -TTAACAGCCACAAACCTACAC-3 ). The primers were designed based on the sequence of the rice LOX gene Osr9-LOX1 (accession number AB099850), which shows high homology to the partial sequence of r9-LOX1-like gene that was cloned by suppression subtractive hybridization and found to be elicited by SSB larvae feeding (Zhou GX and Lou YG, unpublished data). The PCR product was cloned into the pGEM-T easy vector (Promega, Madison, WI) and sequenced. Analysis of Osr9-LOX1 transcript levels
Plant treatments
SSB treatment Pots containing only one plant were used for this experiment. Plants were individually infested by a thirdinstar larva of SSB that had been starved for 2 h (SSB). Control plants (Con) were left untreated.
BPH treatment Plants (one plant per pot) were individually infested with 15 gravid BPH females that were confined in a glass cylinder (diameter, 4 cm; height, 8 cm, with 48 small holes each 0.8 mm in diameter). One empty cylinder was attached to each of the control plants (non-infested).
Mechanical wounding treatment Plants (one plant per pot) were individually damaged with a needle at a low position on the stem. The damaged region was approximately 2 cm long and contained 200 needle pricks (W). Control plants (Con) were left undamaged.
To analyze the effects of various stresses on transcript levels of Osr9-LOX1, WT plants were randomly assigned to one of five treatments, W, SSB, BPH, JA and SA, and their corresponding controls, Con, non-infested and BUF. The stems (SSB treatment and its corresponding control) or leaf sheaths (BPH, W, JA and SA treatments and their corresponding controls) were harvested at various times after the start of treatment (Fig. 2). Each treatment at each time interval was replicated five times. The transcript levels of Osr9-LOX1 were investigated using quantitative real-time PCR (qRT-PCR). Total RNA was isolated using the SV Total RNA Isolation System (Promega). A 10 ng sample of total RNA was reversetranscribed using the PrimeScript RT-PCR kit (TaKaRa, Dalian, China). qRT-PCR was performed on a CFX96TM Real-Time system (Bio-RAD, Hercules, CA). A rice actin gene OsACT (TIGR ID Os03g50885) was used as an internal standard to normalize cDNA concentrations. The primers and TaqMAN probe sequences used for TaqMAN qRT-PCR (Premix Ex Taq Kit; TaKaRa) are shown in Table S1. Subcellular localization of Osr9-LOX1
JA and SA treatments Pots with one plant were used for these experiments. The method for JA and SA treatment was the same as
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A 270-bp sequence of Osr9-LOX1 that included the start codon (ATG) and encoded the N-terminal part of the Osr9-LOX1 protein was cloned by PCR using the primers 61
LOX-F: 5 -GTCGACCACCACCAACATAGCTTC-3 and LOX-R: 5 -AAGGTACCAGATTCGCCTCTGAT-3 . The partial sequence was fused to GFP (accession number U87973) and the fusion gene was inserted into pCAMBIA1301, yielding the transformation vector pr9LOX1-GFP. The methods for transient transformation of Nicotiana tabacum leaves using pr9LOX1-GFP and imaging the subcellular localization of Osr9-LOX1 under a confocal laser-scanning microscope (LEICA TCS SP5DMI6000; Leica, Wetzlar, Germany) were as described elsewhere (Zhou et al. 2009). Analyses of SA, JA and GLVs Plants of WT and three as-r9lox1 transgenic lines L54, L182 and L219 were randomly assigned to SSB and control treatments. Stems were harvested at 0, 1.5, 3 and 5 h after SSB treatment, and JA and SA were analyzed by GC-MS with labeled internal standards as described by (Lou and Baldwin 2003). Each treatment at each time interval was replicated four to five times. GLV [(Z )-3-hexenal] emissions were analyzed with a portable gas analyzer (zNose 4200; Electronic Sensor Technology; http://www.estcal.com) as described by (Zhou et al. 2009). At least seven replicates were analyzed for each genotype (WT, L182 and L219). Analysis of TrypPIs Plants (one plant per pot) from each line (WT, L182 and L219) were infested by SSBs. After 0, 3 and 5 days, stem samples (0.2–0.3 g per sample) were harvested and immediately frozen in liquid nitrogen before being stored at −80◦ C. The TrypPI concentration was measured by the radial diffusion assay as described by van Dam et al. (2001) and is expressed as nmol per mg total protein. At least four individual samples were analyzed in each treatment at each time interval (van Dam et al. 2001). Linolenic acid analysis We determined the LeA levels in two as-r9lox1 and one WT line at 0, 0.5 and 1 h after SSB treatment. For each treatment and each time interval, stems (0.15 g) of five plants were sampled. The extraction and measurement of LeA were as described by Qi et al. (2011). Herbivory experiments
SSB performance measurement Seven day old SSB larvae, which had been weighed and starved for 2 h, were allowed to infest stems of the two as-r9lox1 lines (L182 and L219) and WT plants, each 62
plant with one larva. We evaluated 25–29 replicate plants from each line. Larval mass was measured (to an accuracy of 0.1 mg) 7 days after the start of the experiment, and the increased percentage of larval mass on each line was calculated.
BPH performance measurement The survival rates of BPH nymphs on WT and asr9lox1 plants were recorded 11 days after introducing the herbivore, as described by (Zhou et al. 2009). The experiment was repeated eight or nine times, depending on the line. We also calculated the average duration of the BPH nymph stage. To test the effect of JA and SA on the survival rates of BPH nymphs, using a similar method as described in Baldwin et al. (1998) and Yuan et al. (2007), JA and SA were added to the rice nutrient solution to a final concentration of 50 μM and 10 mM, respectively. Non-manipulated plants grown in nutrient solution were used as controls (Con). The experiments were repeated nine times. Data analysis Differences in herbivore performance, expression levels of genes and herbivore-induced JA, SA, LeA, TrypPI and GLV levels on different treatments, lines or treatment times were determined by one-way ANOVA (Student’s t tests for comparing two treatments). Datasets that were not normally distributed or had unequal variance were arcsin- or log-transformed before analysis. If the ANOVA was significant (P < 0.05), Duncan’s multiple range test was used to detect significant differences between groups. Data were analyzed with STATISTICA 6.1 software (StatSoft, Inc. Tulsa, OK; http://www.statsoft.com).
Results Isolation and characterization of Osr9-LOX1 We screened rice plants infested by SSB for herbivoreinduced transcripts. One of the sequenced clones showed similarity to Osr9-LOX1 (Mizuno et al. 2003). Using RT-PCR, we cloned an approximately 2.7-kb sequence of this gene, which included a full open reading frame of 2592 bp (Fig. S1). Nucleotide BLAST analysis showed that the sequence was completely identical (100%) to that of the gene Osr9-LOX1 (Mizuno et al. 2003), which was isolated from rice seeds and encoded an enzyme with C9-specific LOX activity. To determine the subcellular localization of Osr9LOX1, we generated an r9-LOX1::EGFP fusion gene, driven by the CaMV35S promoter (Fig. S2B), and transiently expressed the construct in leaves of N.
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Fig. 1. Localization of Osr9-LOX1 (A) and relative expression levels (±SE, n = 3–5) of Osr9-LOX1 in different tissues of rice (B). I and Ia (magnification of I), Osr9-LOX1: GFP fluorescence (green) chloroplast; II and IIa (magnification of II), autofluorescence (red); III and IIIa (magnification of III), merged (yellow). L, leaf; R, root; S, stem; SP, spikelet. Arrows indicate chloroplasts.
tabacum. Confocal laser-scanning microscopy analysis showed that fluorescence emitted from the fusion protein (Fig. 1A–I, green) completely overlapped (Fig. 1A–III, yellow) with the autofluorescence emitted from chloroplasts (Fig. 1A–II, red), suggesting that Osr9-LOX1 was localized in the chloroplasts. qRT-PCR analysis revealed low constitutive expression of Osr9LOX1 in rice leaves, stems, and spikelets, but high constitutive expression in roots. Its expression level in the roots was approximately 12-fold, 20-fold and 27fold that in rice leaves, stems and spikelets, respectively (Fig. 1B). Herbivore infestation, wounding and JA and SA treatments affect expression levels of Osr9-LOX1 We mechanically wounded stems of rice plants and monitored transcript levels of Osr9-LOX1 by qRTPCR analysis. The transcript levels of Osr9-LOX1 had markedly decreased by 1 h after wounding, but its transcript levels increased between 2 and 48 h after
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Fig. 2. Expression levels of Osr9-LOX1 in rice plants under various treatments. (A–E), Relative transcript levels of Osr9-LOX1 in leaf sheaths of plants treated by wounding (W, A), infestation with brown planthopper (BPH, B), jasmonic acid (JA, D) and salicylic acid (SA, E), and in stems of plants treated by infestation with SSB (C); Con, nonmanipulated; Buf, buffer treatment. Rice OsACT (Os03g50885) was used as an internal control. Values are means ±SE (n = 3–5) and were obtained in one experiment with at least three biological replicates. Similar results were obtained in two independent experiments. Asterisks indicate significant differences between treatments and respective controls at each time point (*P < 0.05; **P < 0.01, Student’s t-test).
wounding (Fig. 2A). Similarly, BPH feeding initially decreased transcript levels at 0.5 and 1 h but by 4 and 8 h after feeding, a rapid increase was seen (Fig. 2B). The feeding activity of the SSB caterpillar resulted in a significant increase in Osr9-LOX1 transcript levels between 1 h and 24 h but not 0.5 h after feeding (Fig. 2C). Interestingly, both JA and SA treatments increased the transcript levels of Osr9-LOX1; however, unlike JA treatment, which enhanced the transcript levels of Osr9-LOX1 later (4 and 8 h after feeding), SA treatment immediately increased the levels of Osr9LOX1 transcripts (0.5 and 1 h after treatment) (Fig. 2D, E). Silencing Osr9-LOX1 results in increased JA, SA and GLV levels We constructed an antisense transgenic vector (Fig. S2A) and generated transgenic rice plants via A. tumefaciens-mediated transformation. Three independently transformed lines (L54, L182 and L219) containing a single insertion by Southern blot were used in this experiment (Fig. S3). We used qRT-PCR to estimate the levels of Osr9-LOX1 transcripts in antisense transgenic lines and found that the transcript levels of Osr9-LOX1 in L54, L182 and L219 were significantly decreased to 63
1.4%, 0.5%, and 26.9% of levels in WT plants (Fig. 3A). Roots of 7-day-old as-r9lox1 seedlings were shorter than those of WT, but there was no obvious difference in shoot development between WT and transformed lines (Fig. S4). Basal JA levels in the as-r9lox1 lines did not differ from those in WT plants (Fig. 3B). The JA levels in WT plants and as-r9lox1 lines had significantly increased by 1.5 h after infestation with SSB (Fig. 3B). Compared with levels in WT plants, the herbivore-induced JA levels in the three as-r9lox1 lines had significantly increased by 3 h after the start of herbivore attack (Fig. 3B). We quantified LeA, which serves as a substrate for the biosynthesis of JA and GLVs, in the herbivore-infested WT plants and in two as-r9lox1 lines. The levels of LeA were significantly and marginally significantly (P = 0.1135) higher in the two as-r9lox1 lines, L219 and L182, respectively, than in WT plants at 0.5 h after SSB infestation (Fig. 3B, insets). We analyzed the emissions of GLVs from as-r9lox1 lines and WT plants. The level of constitutive emission of (Z )-3-hexenal did not differ between as-r9lox1 lines and WT plants, but wounding-induced emission of (Z )3-hexenal was significantly lower from as-r9lox1 lines, L182 and L219, than from WT plants (Fig. 3C). After SSB treatment, SA levels were higher in transgenic line L54 than in WT plants at 3 h, and higher in transgenic lines, L182 and L219, than in WT plants at 5 h (Fig. 3D). Silencing Osr9-LOX1 increases elicited TrypPI levels and resistance to SSB In rice, it has been reported that JA signaling pathway positively regulates the production of TrypPIs; these in turn affect the performance of SSB larvae (Zhou et al. 2009). Therefore, we analyzed the production of TrypPIs in WT plants and as-r9lox1 lines after SSB infestation for 0, 3 and 5 days, and the effects of silencing Osr9-LOX1 on resistance to SSB. As expected, both constitutive and herbivore-induced TrypPI levels were higher in asr9lox1 lines, L182 (at 0 and 5 days after SSB infestation) and L219 (at 0 and 3 days after SSB infestation), than in WT plants (Fig. 4B). Moreover, the performance of SSB larvae was significantly decreased on transgenic lines, L182 and L219, compared with that on WT plants. After 7 days of feeding, the SSB caterpillars that fed on L182 and L219 lines were smaller than those that fed on WT plants (Fig. 4A); the masses of larvae that fed on L182 and L219 lines were 80.8 and 78.7%, respectively, of the masses of larvae that fed on WT plants. Consistent with these findings, the as-r9lox1 lines were less damaged than were WT plants by SSB larvae (Fig. 4C). 64
Silencing Osr9-LOX1 decreases resistance to BPH Because silencing OsHI-LOX with 13-special LOX activity enhanced resistance to BPH (Zhou et al. 2009), we also assessed the performance of BPH on as-r9lox1 lines. To determine the effect of silencing Osr9-LOX1 on BPH nymph development, we calculated the average duration of the BPH nymph stage and found that the duration of this stage was the same, regardless of whether they fed on as-r9lox1 lines or WT plants (Fig. 5A). Also, there was no difference in the fecundity between adult female BPH feeding on as-r9lox1 lines and those feeding on WT plants (Fig. 5C). However, BPH nymphs that fed on as-r9lox1 lines showed higher survival rates compared to those that fed on WT plants (Fig. 5B). To determine whether the increased survival rate of BPH nymphs that fed on as-r9lox1 lines is because of the increase in levels of JA or SA, we investigated the effect of exogenous application of JA and SA on the survival rate of BPH nymphs. The results revealed that JA treatment enhanced the survival rate of BPH nymphs, whereas SA treatment did not (Fig. 5D).
Discussion In this study, we cloned a LOX gene in rice. Nucleotide BLAST analysis showed that the sequence of this gene was identical to that of Osr9-LOX1, which was first isolated from developing rice seeds that showed C9-specific LOX activity (Mizuno et al. 2003). Intriguingly, like 13LOX OsHI-LOX (Zhou et al. 2009) and RCI-1 (Schaffrath et al. 2000, Zabbai et al. 2004) and the dual positional LOX OsLOX1 (Wang et al. 2008) in rice, Osr9-LOX1 is also located in the chloroplast (Fig. 1). Transcriptional analysis revealed that herbivore infestation, mechanical wounding and JA treatment repressed or did not enhance the level of Osr9-LOX1 transcripts at early stages but elicited it at late stages, whereas SA treatment only increased the transcript level of Osr9-LOX1 at early stages (Fig. 2). Moreover, the antisense expression of Osr9-LOX1 enhanced the levels of LeA, JA and SA but decreased the level of (Z )-3-hexenal in rice, which subsequently increased resistance in rice to SSBs but decreased resistance to BPHs (Fig. 5). These findings, taken together, demonstrate that Osr9-LOX1 plays an important role in defense against herbivores in rice. Analyses of the constitutive transcription patterns of Osr9-LOX1 in different rice organs revealed higher transcript levels in roots than in leaves, stems, or spikelets. The roots of as-r9lox1 lines were significantly shorter than those of WT plants (Fig. S4). Together, these findings suggested that the r9-LOX1-mediated 9-LOX pathway may have an important role in root
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Fig. 3. Levels of Osr9-LOX1 transcripts (A), jasmonic acid (JA, B), (Z)-3-hexenal (C) and salicylic acid (SA, D) in as-r9lox1 and WT plants after infestation by striped stem borer (SSB). Mean expression levels (±SE, n = 3) of Osr9-LOX1 in as-r9lox1 and WT plants 24 h after infestation by SSB; (B, D) Mean levels (±SE, n = 4–5) of JA (B) and SA (D) in as-r9lox1 and WT plants at 0, 1.5, 3 and 5 h after infestation by SSB; (C) Mean levels (±SE, n = 7–10) of (Z)-3-hexenal in leaves of as-r9lox1 lines and WT plants before (Con) and immediately after cutting leaves into small pieces (W). Insert: mean levels (±SE, n = 5) of LeA in as-r9lox1 and WT plants at 0, 0.5 and 1 h after infestation by SSB. Letters indicate significant differences among as-r9lox1 lines and WT plants at each time point (B, D).or among treatments (A, C) (P < 0.05, Duncan’s multiple-range test).
development. Reduced shoot and root lengths were also observed in the maize mutant lox3-4; this mutant carries a true null allele of ZmLOX3, which encodes an enzyme with 9-LOX-specific catalytic activity (Gao et al. 2008). In Arabidopsis, 9-LOX pathway-derived oxylipins such as 9-hydroxyoctadecatrienoid acid (9-HOT) were shown to play roles in cell wall modification and also in lateral root development (Vellosillo et al. 2007, Vellosillo et al. 2013). We monitored changes in Osr9-LOX1 transcript levels after various treatments by qRT-PCR analysis and found that wounding, BPH infestation, SSB damage and JA and SA treatments affected transcript levels (Fig. 2). Interestingly, all of these treatments except for SA repressed or did not induce the transcription of Osr9LOX1 at early stages but induced transcription at late stages (Fig. 2). Given an induction of mechanical wounding, herbivore infestation and JA treatment on JA production (McConn et al. 1997, Zhou et al. 2011) and a negative regulation of Osr9-LOX1 on JA biosynthesis (Fig. 3) in rice, the induction of these treatments on Osr9-LOX1 might be related to the JA burst in rice: repression or non-induction of Osr9-LOX1 at an early stage will trigger JA production, whereas the induction
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of Osr9-LOX1 at a late stage will inhibit JA biosynthesis; this process will keep JA in rice at an appropriate level and thus result in an appropriate defense response that is dependent on damage level. This hypothesis is supported by the result from SA treatment. In rice, it has been reported that there is an antagonistic role between SA and JA (Yuan et al. 2007). Here we found that SA treatment could quickly enhance the levels of Osr9LOX1 transcripts (Fig. 2). This result suggests that the upregulation of Osr9-LOX1 might be one of pathways that SA uses to inhibit JA biosynthesis. Previously, we reported that OsNPR1 plays an important role in regulating the herbivore-induced JA burst in rice (Li et al. 2013), suggesting there is a set of genes responsible for the herbivore-induced JA burst in plants. When attacked by an herbivore, plants perceive the signals from herbivores and modulate when they express certain genes; these genes form the damage-dependent JA burst. Antisense expression of Osr9-LOX1 in rice markedly increased the herbivore-induced levels of LeA, JA and SA but decreased GLV levels (Fig. 3). Considering that Osr9-LOX1 is also located in the chloroplast (Fig. 2), as is OsHI-LOX (Zhou et al. 2009), we can infer that in vivo, OsHI-LOX and Osr9-LOX1 are located in the same 65
Fig. 4. Resistance of as-r9lox1 lines and WT plants to striped stem borer (SSB). (A), Mean growth rates (% ±SE, n = 27–30) of individual SSB larvae fed on as-r9lox1 and WT plants; (B), Mean TrypPI levels (±SE, n = 4–6) in stems of two as-r9lox1 lines and WT plants 0, 3 and 5 days after plants were individually infested by a third-instar SSB larva; (C) Damage phenotypes of as-r9lox1 lines L182 and L219 and WT plants that were individually infested by an SSB third-instar larva for 7 days. Letters indicate significant differences among as-r9lox1 lines and WT plants (A) or among as-r9lox1 lines and WT plants at each time point (B) (P < 0.05, Duncan’s multiple-range test).
sub-organelle of the chloroplast and compete for the substrate, LeA. Thus, it is understandable that antisense expression of Osr9-LOX1 enhanced the level of LeA, which then allowed more LeA to enter the 13-LOX pathway and be converted into JA (Fig. 3). A similar result was also reported in maize: the inactivation of the 9-LOX ZmLOX3 resulted in increased LeA and JA levels in Aspergillus flavus-infected lox3-4 mutants (Gao et al. 2009). Unexpectedly, silencing Osr9-LOX1 in rice decreased the wounding-elicited production of GLVs, other products of the 13-LOX pathway. This suggests that Osr9-LOX1 affects the 13-LOX pathway 66
not only by influencing the substrate LeA but also by affecting the activity of HPL. As stated above, there is antagonism between SA and JA in rice (Yuan et al. 2007). However, here we found that the antisense expression of Osr9-LOX1 enhanced the elicited levels of both of JA and SA (Fig. 3). This may further confirm the above hypothesis, namely, that Osr9-LOX1 is one of important nodes where the interaction between JA and SA is regulated; the antisense expression of Osr9-LOX1 impairs the antagonistic role of SA and JA and thus triggers an increase in both. Further research should elucidate how Osr9-LOX1 regulates the production of JA, SA and GLV. In rice, it has been well documented that the JA signaling pathway positively regulates the production of TrypPIs; these in turn affect the performance of SSB larvae (Zhou et al. 2009). Moreover, GLVs, (Z )-3hexenal and (Z )-3-hexen-1-ol, have a negative effect on the performance of SSB larvae (Qi et al. 2011). Here we observed that as-r9lox1 plants compared to WT plants accumulated a higher level of herbivoreinduced TrypPIs and a lower level of (Z )-3-hexenal, and enhanced resistance and tolerance to SSB (Fig. 4). Therefore, the increase in resistance and tolerance of asr9lox1 lines to SSB probably resulted from their higher levels of JA and TrypPIs compared to WT plants. We also evaluated the performance of BPH on asr9lox1 lines. The antisense expression of Osr9-LOX1 did not affect the fecundity of adult BPH or the duration of the nymph stage, but BPH nymphs showed a higher survival rate on as-r9lox1 plants than on WT plants (Fig. 5). Because silencing Osr9-LOX1 increased the levels of herbivore-induced JA and SA (Fig. 3), we speculated that elevated JA and SA levels may affect BPH nymph survival. The BPH nymphs that fed on rice plants supplied with a JA-containing nutrient solution showed a higher survival rate than those that fed on control plants. However, the presence of SA in the nutrient solution did not affect the survival of BPH nymphs (Fig. 5). These results are consistent with those of our previous study (Zhou et al. 2009), which clearly indicated that the JA pathway negatively affects rice resistance to BPH. Du et al. (2009) also found that the transcript levels of genes encoding components of the JA pathway were substantially lower in plants with the resistance gene Bph14 than in susceptible WT plants (Du et al. 2009). Although (Z )-3-hexenal, a GLV, was reported to have no effect on the feeding and oviposition preference of female BPH adults (Qi et al. 2011, Tong et al. 2012), it is not clear whether (Z )-3-hexenal affects the survival rate of BPH nymphs. Thus, the higher survival rates of BPH nymphs on as-r9lox1 lines compared to on WT plants might be related to the higher JA levels in these
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Fig. 5. Performance of brown planthopper (BPH) on as-r9lox1 lines and WT plants. (A) Mean developmental duration (+SE, n = 23–58) of nymph stage of BPH fed on two as-r9lox1 lines and WT plants; (B) Mean survival rates (±SE, n = 7–10) of newly hatched BPH nymphs at different time after they were placed on as-r9lox1 lines and WT plants; (C) Mean number of eggs (±SE, n = 20) laid by individual female BPH adults 7 days after they were placed on as-r9lox1 lines and WT plants; (D) Mean survival rates (±SE, n = 7–10) of newly hatched BPH nymphs at different times after they were placed on WT plants whose roots had been treated with JA at a concentration of 50 μM, SA at a concentration of 10 mM or kept non-manipulated (Con). Letters indicate significant differences among treatments (A), or among lines (B) or treatments (D) at each time point (P < 0.05, Duncan’s multiple-range test).
transgenic lines, suggesting that the resistance in asr9lox1 lines to BPH is mediated by JA. Ye et al. (2012) reported that silencing OsCOI1 did not affect the feeding behavior of female BPH adults or the survival rate of BPH nymphs. This result suggests that in rice, the JA pathway that regulates resistance to BPH is independent of the JA-Ile-COI1-JAZ system. JA, in rice, has been reported to negatively regulate the production of H2 O2 , an important signal or chemical defense against BPH (Zhou et al. 2009, Lu et al. 2011). Thus, the Osr9-LOX1mediated resistance in rice to BPH may be related to the production of H2 O2 , an issue that needs to be elucidated in the future. In summary, we demonstrate that Osr9-LOX1 is a chloroplast-located C9 position-specific LOX and could regulate the levels of LeA, JA, SA and GLVs in rice by interacting with the 13-LOX pathway; in addition, Osr9LOX1 mediates root development and resistance in rice to herbivores. We propose that Osr9-LOX1 may be an important node that mediates the JA burst, cross-talk between JA and SA, and trade-offs between resistance
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to chewing herbivores and to piercing and sucking herbivores in rice. Further research will help determine the mechanism underlying the effect of Osr9-LOX1 on the production of JA, SA and GLVs in rice. Acknowledgements – We thank Ran Li, Chunkai Kong and Xiaopeng Wang for their invaluable assistance with laboratory work, and Emily Wheeler for editorial assistance. The study was jointly sponsored by the National Basic Research Program of China (2010CB126200), the National Natural Science Foundation of China (31101451), the National Program of Transgenic Variety Development of China (2011ZX08009-003-001) and the Outstanding Young Scholars Fund of Zhejiang Agriculture & Forestry University (2034070001).
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Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1: Primers and probes used for qRT-PCR analyses of target genes. Fig. S1: cDNA and deduced amino acid sequence of isolated rice Osr9-LOX1 gene. Region used for antisense transformation is underlined. Fig. S2: Rice transformation vector pCAMBIA-as-r9LOX (13.4 kb) with hgh and gus as plant selectable marker genes (A) and transformation vector pr9LOX1-GFP (B) used to determine subcellular localization of Osr9LOX1. Fig. S3: DNA gel-blot analysis of as-r9lox1 T2 lines (L54, L182 and L219) and one WT plant. Genomic DNA was digested with XbaI and EcoRI. Blots were hybridized with a probe specific for the gus reporter gene. All three asr9lox1 lines, L54, L182 and L219, have a single T-DNA insertion. Fig. S4: Growth phenotypes of as-r9lox1 lines and WT plants (5-day-old seedlings).
Edited by T. Berberich
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Physiologia Plantarum 152: 59–69. 2014
The 9-lipoxygenase Osr9-LOX1 interacts with the 13-lipoxygenase-mediated pathway to regulate resistance to chewing and piercing-sucking herbivores in rice Guoxin Zhoua,b , Nan Rena , Jingfeng Qia , Jing Lua , Caiyu Xianga , Hongping Jua , Jiaan Chenga and Yonggen Loua,∗ a
State Key Laboratory of Rice Biology, Institute of Insect Sciences, Zhejiang University, Hangzhou, 310058, China Key Laboratory for Quality Improvement of Agriculture Products of Zhejiang Province, Department of Plant Protection, Zhejiang Agriculture & Forestry University, Lin’an, 311300, China b
Correspondence *Corresponding author, e-mail: [email protected] Received 28 November 2013 doi:10.1111/ppl.12148
Oxylipins produced by the 13-lipoxygenase (LOX) have been reported to play an important role in plant defense responses to herbivores. Yet, the role of oxylipins produced by the 9-LOX pathway in this process remains largely unknown. Here we cloned a gene encoding a chloroplast-localized 9LOX, Osr9-LOX1, from rice. Transcriptional analysis revealed that herbivore infestation, mechanical wounding and jasmonic acid (JA) treatment either repressed or did not enhance the level of Osr9-LOX1 transcripts at early stages but did at later stages, whereas salicylic acid (SA) treatment quickly increased the transcript level of Osr9-LOX1. Antisense expression of Osr9-lox1 (asr9lox1) decreased the amount of wound-induced (Z )-3-hexenal but increased levels of striped stem borer (SSB)-induced linolenic acid, JA, SA and trypsin protease inhibitors. These changes were associated with increased resistance in rice to the larvae of the SSB Chilo suppressalis. In contrast, although no significant differences were observed in the duration of the nymph stage or the number of eggs laid by female adults between the brown planthopper (BPH) Nilaparvata lugens that fed on as-r9lox1 lines and BPH that fed on wild-type (WT) rice plants, the survival rate of BPH nymphs that fed on as-r9lox1 lines was higher than that of nymphs that fed on WT plants, possibly because of a higher JA level. The results demonstrate that Osr9-LOX1 plays an important role in regulating an herbivore-induced JA burst and cross-talk between JA and SA, and in controlling resistance in rice to chewing and phloem-feeding herbivores.
Introduction Plants have evolved complex defense systems to protect themselves against different challenges. Such systems can recruit and coordinate various defense pathways to respond to various abiotic and biotic environmental stresses. Among others, the oxylipin-, ethylene (ET)-, salicylic acid (SA)-, brassinosteroid- and reactive oxygen species-mediated signaling pathways are involved in
plant defense responses. Oxylipins are lipid-derived signaling molecules that play roles in mediating defense responses to biotic and abiotic stress in plants, and participate in diverse developmental processes (Farmer et al. 2003, Demmig-Adams et al. 2013, Wasternack and Hause 2013). Plant messengers derived from oxidatively modified polyunsaturated fatty acids are collectively known
Abbreviations – BPH, brown planthopper; ET, ethylene; GLVs, green leaf volatiles; HPL, hydroperoxide lyase; JA, jasmonic acid; LeA, linolenic acid; LOX, lipoxygenase; qRT-PCR, quantitative real-time polymerase chain reaction; SA, salicylic acid; SSB, striped stem borer; TrypPIs, trypsin protease inhibitors; WT, wild type.
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as oxylipins. This class of compounds includes acid hydroperoxides, hydroxyl-, oxo-, or keto-fatty acids, divinyl ethers and volatile aldehydes such as the important plant stress hormone jasmonic acid (JA), its precursor, 12-oxo-phytodienoic acid, and its derivative, methyl jasmonate (MeJA) (Mosblech et al. 2009, Demmig-Adams et al. 2013). The first step in oxylipin biosynthesis is the oxidation of polyunsaturated fatty acids to form fatty acid hydroperoxides. This can occur non-enzymatically but mainly occurs via enzymatic peroxidation catalyzed by lipoxygenases (LOXs) (Demmig-Adams et al. 2013). In plants, LOXs are the key enzymes involved in generating fatty acid derivatives during oxylipin metabolism (Feussner and Wasternack 2002, Mosblech et al. 2009). Plant LOXs can be classified into two groups based on their mechanism of action, that is, on whether they add oxygen to the 9- (9-LOX) or the 13- (13-LOX) position of the substrate carbon chain to generate 9- or 13hydroperoxides, respectively (Feussner and Wasternack 2002). The most well-known bioactive oxylipin, JA, is biosynthesized from the fatty acid substrate α-linolenic acid (α-LeA) via the 13-LOX pathway. Genetic and biochemical methods have been used to show that the 13-LOX pathway and its products, such as JA and green leaf volatiles (GLVs) produced by the hydroperoxide lyase (HPL) pathway play important roles in direct and indirect induced defenses against herbivores in diverse dicot species, including tobacco (Halitschke and Baldwin 2003, Kessler et al. 2004), Arabidopsis (Chauvin et al. 2013, Garcia-Marcos et al. 2013) and potato (Royo et al. 1999), and in monocots, including rice (Wang et al. 2008, Zhou et al. 2009) and maize (Cho et al. 2012, Christensen et al. 2013). However, little is known about the role of the 9-LOX pathway in plant defenses against herbivores, although there is increasing evidence that the pathway influences plant development (Vellosillo et al. 2007, Gao et al. 2008), stomatal closure (Montillet et al. 2013), defense responses to pathogens (Gao et al. 2007, Montillet et al. 2013, Vellosillo et al. 2013) and plant cell death (Sayegh-Alhamdia et al. 2008, Hwang and Hwang 2010, Garcia-Marcos et al. 2013). Rice, one of the most important staple foods worldwide, is severely affected by chewing herbivores and phloem feeders; these cause substantial losses in rice crops in Asia (Cheng and He 1996). Previous studies have shown that the oxylipin pathway, especially the one mediated by 13-LOX oxylipin, plays an important role in mediating resistance to both types of herbivores in rice. For example, the JA signaling pathway was found to positively regulate resistance in rice to chewing herbivores, including rice striped stem borer (SSB, Chilo suppressalis) and leaf folder (Cnaphalocrocis 60
medinalis), but negatively modulate resistance to the piercing-sucking herbivore, rice brown planthopper (BPH, Nilaparvata lugens) (Zhou et al. 2009, Tong et al. 2012). GLVs were also reported to affect the performance of SSB larvae and BPHs (Qi et al. 2011, Tong et al. 2012). Wang et al. (2008) reported that OsLOX1, showing 9- and 13-LOX dual positional specificity, positively mediates levels of endogenous JA, (Z )-3-hexenal and LeA and affects the resistance of rice to BPH (Wang et al. 2008). Rice varieties lacking LOX-3 (9-LOX) showed decreased insect damage and their grain was more insect-resistant, while those with LOX-3 showed increased insect damage because volatiles attractive to insects were released (Zhang et al. 2007, Tang and Zhou 2012). Previously, we used microarray technology to detect genome-wide transcriptional changes in rice in response to infestation by SSBs. Our results revealed that Osr9-LOX1 transcript levels increased in SSB-treated rice plants (Zhou et al. 2011). However, there is still much to learn about the roles of the 9-LOX pathway in the rice defense response to herbivores. In this study, we isolated a rice 9-LOX gene, Osr9-LOX1, and analyzed its expression patterns and subcellular localization. Using a reverse genetics approach, we obtained rice lines with antisense expression of Osr9-LOX1 (as-r9lox1). We determined the role of Osr9-LOX1 in herbivore-induced defense by measuring the levels of JA and SA and defense-related chemicals such as trypsin protease inhibitors (TrypPIs) and GLVs. We also evaluated resistance to a chewing herbivore, SSB, and a phloem feeder, BPH. Our results provide evidence that Osr9-LOX1 is involved in the herbivore defense response of rice.
Materials and methods Plant growth and insects The rice (Oryza sativa) genotypes used in this study were Xiushui 11 wild type (WT, untransformed) and three as-r9lox1 transgenic lines (see below) in the same background. Pre-germinated seeds were cultured in plastic bottles (diameter, 8 cm; height, 10 cm) in a greenhouse at 28 ± 2◦ C, under a 14-h light/10-h dark photoperiod. After 10 days, seedlings were transplanted into 50-l communal hydroponic boxes containing a rice nutrient solution (Qi et al. 2011). After 30–35 days, seedlings were transferred to plastic pots (8 cm in diameter × 10 cm high), each with one or two plants (one WT, the other an as-r9-lox transgenic plant). Plants were used for experiments 4–5 days after transplanting. Colonies of SSB and BPH were originally obtained from rice fields in Hangzhou. Both herbivores were
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maintained on seedlings of Shanyou 63 (a variety susceptible to SSB and BPH) in a controlled climate room at 26 ± 2◦ C under a 12-h light/12-h dark photoperiod and 80% relative humidity. Generation and characterization of as-r9lox1 transgenic lines To construct the plant transformation vector, a 957bp portion (Fig. S1) of the Osr9-LOX1 cDNA in the plasmid pr9-LOX (see below) was PCR-amplified using the primers 5 -GCC AGACACTCATCAACG-3 (F) and 5 -CAGCCACAAACCTACACC-3 (R). The obtained 0.96-kb PCR fragment was cloned into pCAMBIA1301, yielding the transformation vector pCAMBIA-as-r9LOX (Fig. S2B) that contained the hygromycin resistance gene hph and the reporter gene gus as selectable markers. The vector was used to transform the rice variety Xiushui 11 via an Agrobacterium tumefaciens-mediated transformation procedure (Hiei et al. 1994). The methods used to screen the homozygous T 2 plants and to identify the number of inserts were as described elsewhere (Zhou et al. 2009). For most of the experiments, two T 2 homozygous lines L182 and L219, each harboring a single insertion (Fig. S3), and WT plants were used.
described by Zhou et al. (2009). Plants were individually sprayed with 2 ml JA (100 μg ml−1 ) or SA (70 μg ml−1 ) (Sigma, St. Louis, MO) in 50 mM sodium phosphate buffer (adjusted with 1 M citric acid to pH 8, with 0.01% Tween). Control plants were sprayed with 2 ml buffer (BUF). Isolation of the full-length cDNA of Osr9-LOX1 Total RNA was isolated from WT plants infested by SSB larvae for 24 h, and then Osr9-LOX1 cDNA fragments were obtained by reverse transcription-polymerase chain reaction (RT-PCR) using the primer pair r9-LOX1-F (5 -ACCACCAACATAGCTTCTTTTG-3 ) and r9-LOX1R (5 -TTAACAGCCACAAACCTACAC-3 ). The primers were designed based on the sequence of the rice LOX gene Osr9-LOX1 (accession number AB099850), which shows high homology to the partial sequence of r9-LOX1-like gene that was cloned by suppression subtractive hybridization and found to be elicited by SSB larvae feeding (Zhou GX and Lou YG, unpublished data). The PCR product was cloned into the pGEM-T easy vector (Promega, Madison, WI) and sequenced. Analysis of Osr9-LOX1 transcript levels
Plant treatments
SSB treatment Pots containing only one plant were used for this experiment. Plants were individually infested by a thirdinstar larva of SSB that had been starved for 2 h (SSB). Control plants (Con) were left untreated.
BPH treatment Plants (one plant per pot) were individually infested with 15 gravid BPH females that were confined in a glass cylinder (diameter, 4 cm; height, 8 cm, with 48 small holes each 0.8 mm in diameter). One empty cylinder was attached to each of the control plants (non-infested).
Mechanical wounding treatment Plants (one plant per pot) were individually damaged with a needle at a low position on the stem. The damaged region was approximately 2 cm long and contained 200 needle pricks (W). Control plants (Con) were left undamaged.
To analyze the effects of various stresses on transcript levels of Osr9-LOX1, WT plants were randomly assigned to one of five treatments, W, SSB, BPH, JA and SA, and their corresponding controls, Con, non-infested and BUF. The stems (SSB treatment and its corresponding control) or leaf sheaths (BPH, W, JA and SA treatments and their corresponding controls) were harvested at various times after the start of treatment (Fig. 2). Each treatment at each time interval was replicated five times. The transcript levels of Osr9-LOX1 were investigated using quantitative real-time PCR (qRT-PCR). Total RNA was isolated using the SV Total RNA Isolation System (Promega). A 10 ng sample of total RNA was reversetranscribed using the PrimeScript RT-PCR kit (TaKaRa, Dalian, China). qRT-PCR was performed on a CFX96TM Real-Time system (Bio-RAD, Hercules, CA). A rice actin gene OsACT (TIGR ID Os03g50885) was used as an internal standard to normalize cDNA concentrations. The primers and TaqMAN probe sequences used for TaqMAN qRT-PCR (Premix Ex Taq Kit; TaKaRa) are shown in Table S1. Subcellular localization of Osr9-LOX1
JA and SA treatments Pots with one plant were used for these experiments. The method for JA and SA treatment was the same as
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A 270-bp sequence of Osr9-LOX1 that included the start codon (ATG) and encoded the N-terminal part of the Osr9-LOX1 protein was cloned by PCR using the primers 61
LOX-F: 5 -GTCGACCACCACCAACATAGCTTC-3 and LOX-R: 5 -AAGGTACCAGATTCGCCTCTGAT-3 . The partial sequence was fused to GFP (accession number U87973) and the fusion gene was inserted into pCAMBIA1301, yielding the transformation vector pr9LOX1-GFP. The methods for transient transformation of Nicotiana tabacum leaves using pr9LOX1-GFP and imaging the subcellular localization of Osr9-LOX1 under a confocal laser-scanning microscope (LEICA TCS SP5DMI6000; Leica, Wetzlar, Germany) were as described elsewhere (Zhou et al. 2009). Analyses of SA, JA and GLVs Plants of WT and three as-r9lox1 transgenic lines L54, L182 and L219 were randomly assigned to SSB and control treatments. Stems were harvested at 0, 1.5, 3 and 5 h after SSB treatment, and JA and SA were analyzed by GC-MS with labeled internal standards as described by (Lou and Baldwin 2003). Each treatment at each time interval was replicated four to five times. GLV [(Z )-3-hexenal] emissions were analyzed with a portable gas analyzer (zNose 4200; Electronic Sensor Technology; http://www.estcal.com) as described by (Zhou et al. 2009). At least seven replicates were analyzed for each genotype (WT, L182 and L219). Analysis of TrypPIs Plants (one plant per pot) from each line (WT, L182 and L219) were infested by SSBs. After 0, 3 and 5 days, stem samples (0.2–0.3 g per sample) were harvested and immediately frozen in liquid nitrogen before being stored at −80◦ C. The TrypPI concentration was measured by the radial diffusion assay as described by van Dam et al. (2001) and is expressed as nmol per mg total protein. At least four individual samples were analyzed in each treatment at each time interval (van Dam et al. 2001). Linolenic acid analysis We determined the LeA levels in two as-r9lox1 and one WT line at 0, 0.5 and 1 h after SSB treatment. For each treatment and each time interval, stems (0.15 g) of five plants were sampled. The extraction and measurement of LeA were as described by Qi et al. (2011). Herbivory experiments
SSB performance measurement Seven day old SSB larvae, which had been weighed and starved for 2 h, were allowed to infest stems of the two as-r9lox1 lines (L182 and L219) and WT plants, each 62
plant with one larva. We evaluated 25–29 replicate plants from each line. Larval mass was measured (to an accuracy of 0.1 mg) 7 days after the start of the experiment, and the increased percentage of larval mass on each line was calculated.
BPH performance measurement The survival rates of BPH nymphs on WT and asr9lox1 plants were recorded 11 days after introducing the herbivore, as described by (Zhou et al. 2009). The experiment was repeated eight or nine times, depending on the line. We also calculated the average duration of the BPH nymph stage. To test the effect of JA and SA on the survival rates of BPH nymphs, using a similar method as described in Baldwin et al. (1998) and Yuan et al. (2007), JA and SA were added to the rice nutrient solution to a final concentration of 50 μM and 10 mM, respectively. Non-manipulated plants grown in nutrient solution were used as controls (Con). The experiments were repeated nine times. Data analysis Differences in herbivore performance, expression levels of genes and herbivore-induced JA, SA, LeA, TrypPI and GLV levels on different treatments, lines or treatment times were determined by one-way ANOVA (Student’s t tests for comparing two treatments). Datasets that were not normally distributed or had unequal variance were arcsin- or log-transformed before analysis. If the ANOVA was significant (P < 0.05), Duncan’s multiple range test was used to detect significant differences between groups. Data were analyzed with STATISTICA 6.1 software (StatSoft, Inc. Tulsa, OK; http://www.statsoft.com).
Results Isolation and characterization of Osr9-LOX1 We screened rice plants infested by SSB for herbivoreinduced transcripts. One of the sequenced clones showed similarity to Osr9-LOX1 (Mizuno et al. 2003). Using RT-PCR, we cloned an approximately 2.7-kb sequence of this gene, which included a full open reading frame of 2592 bp (Fig. S1). Nucleotide BLAST analysis showed that the sequence was completely identical (100%) to that of the gene Osr9-LOX1 (Mizuno et al. 2003), which was isolated from rice seeds and encoded an enzyme with C9-specific LOX activity. To determine the subcellular localization of Osr9LOX1, we generated an r9-LOX1::EGFP fusion gene, driven by the CaMV35S promoter (Fig. S2B), and transiently expressed the construct in leaves of N.
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Fig. 1. Localization of Osr9-LOX1 (A) and relative expression levels (±SE, n = 3–5) of Osr9-LOX1 in different tissues of rice (B). I and Ia (magnification of I), Osr9-LOX1: GFP fluorescence (green) chloroplast; II and IIa (magnification of II), autofluorescence (red); III and IIIa (magnification of III), merged (yellow). L, leaf; R, root; S, stem; SP, spikelet. Arrows indicate chloroplasts.
tabacum. Confocal laser-scanning microscopy analysis showed that fluorescence emitted from the fusion protein (Fig. 1A–I, green) completely overlapped (Fig. 1A–III, yellow) with the autofluorescence emitted from chloroplasts (Fig. 1A–II, red), suggesting that Osr9-LOX1 was localized in the chloroplasts. qRT-PCR analysis revealed low constitutive expression of Osr9LOX1 in rice leaves, stems, and spikelets, but high constitutive expression in roots. Its expression level in the roots was approximately 12-fold, 20-fold and 27fold that in rice leaves, stems and spikelets, respectively (Fig. 1B). Herbivore infestation, wounding and JA and SA treatments affect expression levels of Osr9-LOX1 We mechanically wounded stems of rice plants and monitored transcript levels of Osr9-LOX1 by qRTPCR analysis. The transcript levels of Osr9-LOX1 had markedly decreased by 1 h after wounding, but its transcript levels increased between 2 and 48 h after
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Fig. 2. Expression levels of Osr9-LOX1 in rice plants under various treatments. (A–E), Relative transcript levels of Osr9-LOX1 in leaf sheaths of plants treated by wounding (W, A), infestation with brown planthopper (BPH, B), jasmonic acid (JA, D) and salicylic acid (SA, E), and in stems of plants treated by infestation with SSB (C); Con, nonmanipulated; Buf, buffer treatment. Rice OsACT (Os03g50885) was used as an internal control. Values are means ±SE (n = 3–5) and were obtained in one experiment with at least three biological replicates. Similar results were obtained in two independent experiments. Asterisks indicate significant differences between treatments and respective controls at each time point (*P < 0.05; **P < 0.01, Student’s t-test).
wounding (Fig. 2A). Similarly, BPH feeding initially decreased transcript levels at 0.5 and 1 h but by 4 and 8 h after feeding, a rapid increase was seen (Fig. 2B). The feeding activity of the SSB caterpillar resulted in a significant increase in Osr9-LOX1 transcript levels between 1 h and 24 h but not 0.5 h after feeding (Fig. 2C). Interestingly, both JA and SA treatments increased the transcript levels of Osr9-LOX1; however, unlike JA treatment, which enhanced the transcript levels of Osr9-LOX1 later (4 and 8 h after feeding), SA treatment immediately increased the levels of Osr9LOX1 transcripts (0.5 and 1 h after treatment) (Fig. 2D, E). Silencing Osr9-LOX1 results in increased JA, SA and GLV levels We constructed an antisense transgenic vector (Fig. S2A) and generated transgenic rice plants via A. tumefaciens-mediated transformation. Three independently transformed lines (L54, L182 and L219) containing a single insertion by Southern blot were used in this experiment (Fig. S3). We used qRT-PCR to estimate the levels of Osr9-LOX1 transcripts in antisense transgenic lines and found that the transcript levels of Osr9-LOX1 in L54, L182 and L219 were significantly decreased to 63
1.4%, 0.5%, and 26.9% of levels in WT plants (Fig. 3A). Roots of 7-day-old as-r9lox1 seedlings were shorter than those of WT, but there was no obvious difference in shoot development between WT and transformed lines (Fig. S4). Basal JA levels in the as-r9lox1 lines did not differ from those in WT plants (Fig. 3B). The JA levels in WT plants and as-r9lox1 lines had significantly increased by 1.5 h after infestation with SSB (Fig. 3B). Compared with levels in WT plants, the herbivore-induced JA levels in the three as-r9lox1 lines had significantly increased by 3 h after the start of herbivore attack (Fig. 3B). We quantified LeA, which serves as a substrate for the biosynthesis of JA and GLVs, in the herbivore-infested WT plants and in two as-r9lox1 lines. The levels of LeA were significantly and marginally significantly (P = 0.1135) higher in the two as-r9lox1 lines, L219 and L182, respectively, than in WT plants at 0.5 h after SSB infestation (Fig. 3B, insets). We analyzed the emissions of GLVs from as-r9lox1 lines and WT plants. The level of constitutive emission of (Z )-3-hexenal did not differ between as-r9lox1 lines and WT plants, but wounding-induced emission of (Z )3-hexenal was significantly lower from as-r9lox1 lines, L182 and L219, than from WT plants (Fig. 3C). After SSB treatment, SA levels were higher in transgenic line L54 than in WT plants at 3 h, and higher in transgenic lines, L182 and L219, than in WT plants at 5 h (Fig. 3D). Silencing Osr9-LOX1 increases elicited TrypPI levels and resistance to SSB In rice, it has been reported that JA signaling pathway positively regulates the production of TrypPIs; these in turn affect the performance of SSB larvae (Zhou et al. 2009). Therefore, we analyzed the production of TrypPIs in WT plants and as-r9lox1 lines after SSB infestation for 0, 3 and 5 days, and the effects of silencing Osr9-LOX1 on resistance to SSB. As expected, both constitutive and herbivore-induced TrypPI levels were higher in asr9lox1 lines, L182 (at 0 and 5 days after SSB infestation) and L219 (at 0 and 3 days after SSB infestation), than in WT plants (Fig. 4B). Moreover, the performance of SSB larvae was significantly decreased on transgenic lines, L182 and L219, compared with that on WT plants. After 7 days of feeding, the SSB caterpillars that fed on L182 and L219 lines were smaller than those that fed on WT plants (Fig. 4A); the masses of larvae that fed on L182 and L219 lines were 80.8 and 78.7%, respectively, of the masses of larvae that fed on WT plants. Consistent with these findings, the as-r9lox1 lines were less damaged than were WT plants by SSB larvae (Fig. 4C). 64
Silencing Osr9-LOX1 decreases resistance to BPH Because silencing OsHI-LOX with 13-special LOX activity enhanced resistance to BPH (Zhou et al. 2009), we also assessed the performance of BPH on as-r9lox1 lines. To determine the effect of silencing Osr9-LOX1 on BPH nymph development, we calculated the average duration of the BPH nymph stage and found that the duration of this stage was the same, regardless of whether they fed on as-r9lox1 lines or WT plants (Fig. 5A). Also, there was no difference in the fecundity between adult female BPH feeding on as-r9lox1 lines and those feeding on WT plants (Fig. 5C). However, BPH nymphs that fed on as-r9lox1 lines showed higher survival rates compared to those that fed on WT plants (Fig. 5B). To determine whether the increased survival rate of BPH nymphs that fed on as-r9lox1 lines is because of the increase in levels of JA or SA, we investigated the effect of exogenous application of JA and SA on the survival rate of BPH nymphs. The results revealed that JA treatment enhanced the survival rate of BPH nymphs, whereas SA treatment did not (Fig. 5D).
Discussion In this study, we cloned a LOX gene in rice. Nucleotide BLAST analysis showed that the sequence of this gene was identical to that of Osr9-LOX1, which was first isolated from developing rice seeds that showed C9-specific LOX activity (Mizuno et al. 2003). Intriguingly, like 13LOX OsHI-LOX (Zhou et al. 2009) and RCI-1 (Schaffrath et al. 2000, Zabbai et al. 2004) and the dual positional LOX OsLOX1 (Wang et al. 2008) in rice, Osr9-LOX1 is also located in the chloroplast (Fig. 1). Transcriptional analysis revealed that herbivore infestation, mechanical wounding and JA treatment repressed or did not enhance the level of Osr9-LOX1 transcripts at early stages but elicited it at late stages, whereas SA treatment only increased the transcript level of Osr9-LOX1 at early stages (Fig. 2). Moreover, the antisense expression of Osr9-LOX1 enhanced the levels of LeA, JA and SA but decreased the level of (Z )-3-hexenal in rice, which subsequently increased resistance in rice to SSBs but decreased resistance to BPHs (Fig. 5). These findings, taken together, demonstrate that Osr9-LOX1 plays an important role in defense against herbivores in rice. Analyses of the constitutive transcription patterns of Osr9-LOX1 in different rice organs revealed higher transcript levels in roots than in leaves, stems, or spikelets. The roots of as-r9lox1 lines were significantly shorter than those of WT plants (Fig. S4). Together, these findings suggested that the r9-LOX1-mediated 9-LOX pathway may have an important role in root
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Fig. 3. Levels of Osr9-LOX1 transcripts (A), jasmonic acid (JA, B), (Z)-3-hexenal (C) and salicylic acid (SA, D) in as-r9lox1 and WT plants after infestation by striped stem borer (SSB). Mean expression levels (±SE, n = 3) of Osr9-LOX1 in as-r9lox1 and WT plants 24 h after infestation by SSB; (B, D) Mean levels (±SE, n = 4–5) of JA (B) and SA (D) in as-r9lox1 and WT plants at 0, 1.5, 3 and 5 h after infestation by SSB; (C) Mean levels (±SE, n = 7–10) of (Z)-3-hexenal in leaves of as-r9lox1 lines and WT plants before (Con) and immediately after cutting leaves into small pieces (W). Insert: mean levels (±SE, n = 5) of LeA in as-r9lox1 and WT plants at 0, 0.5 and 1 h after infestation by SSB. Letters indicate significant differences among as-r9lox1 lines and WT plants at each time point (B, D).or among treatments (A, C) (P < 0.05, Duncan’s multiple-range test).
development. Reduced shoot and root lengths were also observed in the maize mutant lox3-4; this mutant carries a true null allele of ZmLOX3, which encodes an enzyme with 9-LOX-specific catalytic activity (Gao et al. 2008). In Arabidopsis, 9-LOX pathway-derived oxylipins such as 9-hydroxyoctadecatrienoid acid (9-HOT) were shown to play roles in cell wall modification and also in lateral root development (Vellosillo et al. 2007, Vellosillo et al. 2013). We monitored changes in Osr9-LOX1 transcript levels after various treatments by qRT-PCR analysis and found that wounding, BPH infestation, SSB damage and JA and SA treatments affected transcript levels (Fig. 2). Interestingly, all of these treatments except for SA repressed or did not induce the transcription of Osr9LOX1 at early stages but induced transcription at late stages (Fig. 2). Given an induction of mechanical wounding, herbivore infestation and JA treatment on JA production (McConn et al. 1997, Zhou et al. 2011) and a negative regulation of Osr9-LOX1 on JA biosynthesis (Fig. 3) in rice, the induction of these treatments on Osr9-LOX1 might be related to the JA burst in rice: repression or non-induction of Osr9-LOX1 at an early stage will trigger JA production, whereas the induction
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of Osr9-LOX1 at a late stage will inhibit JA biosynthesis; this process will keep JA in rice at an appropriate level and thus result in an appropriate defense response that is dependent on damage level. This hypothesis is supported by the result from SA treatment. In rice, it has been reported that there is an antagonistic role between SA and JA (Yuan et al. 2007). Here we found that SA treatment could quickly enhance the levels of Osr9LOX1 transcripts (Fig. 2). This result suggests that the upregulation of Osr9-LOX1 might be one of pathways that SA uses to inhibit JA biosynthesis. Previously, we reported that OsNPR1 plays an important role in regulating the herbivore-induced JA burst in rice (Li et al. 2013), suggesting there is a set of genes responsible for the herbivore-induced JA burst in plants. When attacked by an herbivore, plants perceive the signals from herbivores and modulate when they express certain genes; these genes form the damage-dependent JA burst. Antisense expression of Osr9-LOX1 in rice markedly increased the herbivore-induced levels of LeA, JA and SA but decreased GLV levels (Fig. 3). Considering that Osr9-LOX1 is also located in the chloroplast (Fig. 2), as is OsHI-LOX (Zhou et al. 2009), we can infer that in vivo, OsHI-LOX and Osr9-LOX1 are located in the same 65
Fig. 4. Resistance of as-r9lox1 lines and WT plants to striped stem borer (SSB). (A), Mean growth rates (% ±SE, n = 27–30) of individual SSB larvae fed on as-r9lox1 and WT plants; (B), Mean TrypPI levels (±SE, n = 4–6) in stems of two as-r9lox1 lines and WT plants 0, 3 and 5 days after plants were individually infested by a third-instar SSB larva; (C) Damage phenotypes of as-r9lox1 lines L182 and L219 and WT plants that were individually infested by an SSB third-instar larva for 7 days. Letters indicate significant differences among as-r9lox1 lines and WT plants (A) or among as-r9lox1 lines and WT plants at each time point (B) (P < 0.05, Duncan’s multiple-range test).
sub-organelle of the chloroplast and compete for the substrate, LeA. Thus, it is understandable that antisense expression of Osr9-LOX1 enhanced the level of LeA, which then allowed more LeA to enter the 13-LOX pathway and be converted into JA (Fig. 3). A similar result was also reported in maize: the inactivation of the 9-LOX ZmLOX3 resulted in increased LeA and JA levels in Aspergillus flavus-infected lox3-4 mutants (Gao et al. 2009). Unexpectedly, silencing Osr9-LOX1 in rice decreased the wounding-elicited production of GLVs, other products of the 13-LOX pathway. This suggests that Osr9-LOX1 affects the 13-LOX pathway 66
not only by influencing the substrate LeA but also by affecting the activity of HPL. As stated above, there is antagonism between SA and JA in rice (Yuan et al. 2007). However, here we found that the antisense expression of Osr9-LOX1 enhanced the elicited levels of both of JA and SA (Fig. 3). This may further confirm the above hypothesis, namely, that Osr9-LOX1 is one of important nodes where the interaction between JA and SA is regulated; the antisense expression of Osr9-LOX1 impairs the antagonistic role of SA and JA and thus triggers an increase in both. Further research should elucidate how Osr9-LOX1 regulates the production of JA, SA and GLV. In rice, it has been well documented that the JA signaling pathway positively regulates the production of TrypPIs; these in turn affect the performance of SSB larvae (Zhou et al. 2009). Moreover, GLVs, (Z )-3hexenal and (Z )-3-hexen-1-ol, have a negative effect on the performance of SSB larvae (Qi et al. 2011). Here we observed that as-r9lox1 plants compared to WT plants accumulated a higher level of herbivoreinduced TrypPIs and a lower level of (Z )-3-hexenal, and enhanced resistance and tolerance to SSB (Fig. 4). Therefore, the increase in resistance and tolerance of asr9lox1 lines to SSB probably resulted from their higher levels of JA and TrypPIs compared to WT plants. We also evaluated the performance of BPH on asr9lox1 lines. The antisense expression of Osr9-LOX1 did not affect the fecundity of adult BPH or the duration of the nymph stage, but BPH nymphs showed a higher survival rate on as-r9lox1 plants than on WT plants (Fig. 5). Because silencing Osr9-LOX1 increased the levels of herbivore-induced JA and SA (Fig. 3), we speculated that elevated JA and SA levels may affect BPH nymph survival. The BPH nymphs that fed on rice plants supplied with a JA-containing nutrient solution showed a higher survival rate than those that fed on control plants. However, the presence of SA in the nutrient solution did not affect the survival of BPH nymphs (Fig. 5). These results are consistent with those of our previous study (Zhou et al. 2009), which clearly indicated that the JA pathway negatively affects rice resistance to BPH. Du et al. (2009) also found that the transcript levels of genes encoding components of the JA pathway were substantially lower in plants with the resistance gene Bph14 than in susceptible WT plants (Du et al. 2009). Although (Z )-3-hexenal, a GLV, was reported to have no effect on the feeding and oviposition preference of female BPH adults (Qi et al. 2011, Tong et al. 2012), it is not clear whether (Z )-3-hexenal affects the survival rate of BPH nymphs. Thus, the higher survival rates of BPH nymphs on as-r9lox1 lines compared to on WT plants might be related to the higher JA levels in these
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Fig. 5. Performance of brown planthopper (BPH) on as-r9lox1 lines and WT plants. (A) Mean developmental duration (+SE, n = 23–58) of nymph stage of BPH fed on two as-r9lox1 lines and WT plants; (B) Mean survival rates (±SE, n = 7–10) of newly hatched BPH nymphs at different time after they were placed on as-r9lox1 lines and WT plants; (C) Mean number of eggs (±SE, n = 20) laid by individual female BPH adults 7 days after they were placed on as-r9lox1 lines and WT plants; (D) Mean survival rates (±SE, n = 7–10) of newly hatched BPH nymphs at different times after they were placed on WT plants whose roots had been treated with JA at a concentration of 50 μM, SA at a concentration of 10 mM or kept non-manipulated (Con). Letters indicate significant differences among treatments (A), or among lines (B) or treatments (D) at each time point (P < 0.05, Duncan’s multiple-range test).
transgenic lines, suggesting that the resistance in asr9lox1 lines to BPH is mediated by JA. Ye et al. (2012) reported that silencing OsCOI1 did not affect the feeding behavior of female BPH adults or the survival rate of BPH nymphs. This result suggests that in rice, the JA pathway that regulates resistance to BPH is independent of the JA-Ile-COI1-JAZ system. JA, in rice, has been reported to negatively regulate the production of H2 O2 , an important signal or chemical defense against BPH (Zhou et al. 2009, Lu et al. 2011). Thus, the Osr9-LOX1mediated resistance in rice to BPH may be related to the production of H2 O2 , an issue that needs to be elucidated in the future. In summary, we demonstrate that Osr9-LOX1 is a chloroplast-located C9 position-specific LOX and could regulate the levels of LeA, JA, SA and GLVs in rice by interacting with the 13-LOX pathway; in addition, Osr9LOX1 mediates root development and resistance in rice to herbivores. We propose that Osr9-LOX1 may be an important node that mediates the JA burst, cross-talk between JA and SA, and trade-offs between resistance
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to chewing herbivores and to piercing and sucking herbivores in rice. Further research will help determine the mechanism underlying the effect of Osr9-LOX1 on the production of JA, SA and GLVs in rice. Acknowledgements – We thank Ran Li, Chunkai Kong and Xiaopeng Wang for their invaluable assistance with laboratory work, and Emily Wheeler for editorial assistance. The study was jointly sponsored by the National Basic Research Program of China (2010CB126200), the National Natural Science Foundation of China (31101451), the National Program of Transgenic Variety Development of China (2011ZX08009-003-001) and the Outstanding Young Scholars Fund of Zhejiang Agriculture & Forestry University (2034070001).
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Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1: Primers and probes used for qRT-PCR analyses of target genes. Fig. S1: cDNA and deduced amino acid sequence of isolated rice Osr9-LOX1 gene. Region used for antisense transformation is underlined. Fig. S2: Rice transformation vector pCAMBIA-as-r9LOX (13.4 kb) with hgh and gus as plant selectable marker genes (A) and transformation vector pr9LOX1-GFP (B) used to determine subcellular localization of Osr9LOX1. Fig. S3: DNA gel-blot analysis of as-r9lox1 T2 lines (L54, L182 and L219) and one WT plant. Genomic DNA was digested with XbaI and EcoRI. Blots were hybridized with a probe specific for the gus reporter gene. All three asr9lox1 lines, L54, L182 and L219, have a single T-DNA insertion. Fig. S4: Growth phenotypes of as-r9lox1 lines and WT plants (5-day-old seedlings).
Edited by T. Berberich
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