AOC (allene oxide cyclase; EC 126.96.36.199), an essential enzyme in jasmonic acid and its methyl ester biosynthesis, was cloned from Camptotheca acuminata (named as CaAOC), a native medicinal plant species in China. CaAOC had significant similarity at the amino-acid level with AOCs from other plant species. Comparison between the sequences of the full-length cDNA and genomic DNA of CaAOC revealed that the genomic DNA of CaAOC contained an 89-bp intron and a 240-bp intron. Southern-blot analysis indicated that CaAOC was a multiple-copy gene, and real-time quantitative PCR analysis showed that CaAOC was expressed constitutively in all organs tested, with the highest expression level in leaves. The results from treatment experiments using different signalling components, including methyl jasmonate, abscisic acid, salicylic acid and H2O2, revealed that expression of CaAOC had a prominent diversity. Heavy metal (copper) significantly enhanced CaAOC expression, whereas wounding (induced by UV-B) was not so effective.
JA (jasmonic acid) and its methyl ester MeJA (methyl JA), commonly known as jasmonates, have been identified as signals of altered gene expression in various plants when responding to biotic and abiotic stresses, as well as in distinct stages of plant development [1,2]. There are at least five groups of jasmonate-induced plant defence proteins: proteinase inhibitors, thionins, proline-rich proteins, enzymes involved in phenyl propanoid metabolism and ribosome-inactivating proteins . Jasmonates seem to be the most prominent among the diverse lipid-derived signals.
In plants, JA is the key terminal product of the octadecanoid pathway . The biosynthesis of JA proceeds via a series of steps, starting with the release of LA (linolenic acid) and its conversion into 13-hydroperoxide by the lipoxygenase-catalysed insertion of molecular oxygen into position 13 of LA. The sequential action of AOS (allene oxide synthase; EC 188.8.131.52) catalyses the formation of an unstable allene oxide. This allene oxide is then cyclized by AOC (allene oxide cyclase; EC 184.108.40.206) into the ultimate precursor of JA, OPDA (12-oxo-phytodienoic acid) [5,6] (Figure 1). AOC seems to be the preferential target in the regulation of JA in a biosynthetic capacity. It was first cloned from tomato as a single-copy gene [5,7]. Some signalling components [SA (salicylic acid), ABA (abscisic acid) and H2O2], JA and heavy metals strongly and transiently up-regulate the expression of AOC transcript. There are dramatic changes at the levels of JA, of OPDA, their methyl esters (MeJA and methyl OPDA), and of dinor-OPDA in most tomato flower organs following AOC overexpression .
Jasmonate biosynthetic pathway
To date, for AOC, much biochemical data for the purified enzyme are available, including substrate specificity [9,10], but only a few molecular sequences and characteristics have been studied, and only a few studies regarding the Chinese traditional medicinal plant have been carried out. In order to analyse the physiological importance of this step of JA biosynthesis, Camptotheca acuminata, known as the Chinese Happy Tree, which has pharmaceutical uses, was chosen as the model tree species in our plant research programme. Various organs of this species contain a type of alkaloid camptothecin and its derivatives, which have interesting biological activities, showing strong anti-neoplastic activity [11,12]. It has been reported that camptothecin and 10-hydroxycamptothecin could also be detected in callus, which is a good material for research . On the basis of sequence homology, a full-length cDNA was isolated from C. acuminata which coded for a protein of 26.8 kDa. ChloroP 1.1 analysis (http://www.cbs.dtu.dk/services/ChloroP/) showed that CaAOC (C. acuminata AOC) contained an N-terminal cTP (chloroplast transit peptide) , suggesting that CaAOC could be localized in chloroplasts.
MATERIALS AND METHODS
Plant materials and RNA extraction
C. acuminata cultured cell lines, initiated from young leaves and maintained in MS (Murashige and Skoog) solid medium supplemented with 5 mg/l naphthalene acetic acid, 0.5 mg/l 6-benzyladenine, 0.3 mg/l 2,4-dichlorophenoxyacetic acid and 30 g/l sucrose, were used for total RNA isolation with TRIzol™ reagent (Invitrogen) according to the manufacturer's instruction. After isolation, the total RNA was stored in −80°C prior to use for cDNA cloning and various treatments. Different tissues, including roots, stems, leaves, young flower buds, opening flowers, fading flowers and seeds from the C. acuminata plant, which were collected from the Fudan University Campus (Shanghai, People's Republic of China), were also used for total RNA isolation.
To study the changes of gene expression under different treatments, 15-day-old C. acuminata calli, subcultured on fresh medium, were soaked in solutions of 100 μM ABA, 100 μM MeJA, 100 μM SA, 10 mM H2O2, 100 μM CuCl2, 100 μM PbSO4 or 100 μM MnCl2, or exposed to 5 μmol·m−2·s−1 UV-B. Concurrently, a set of control calli was similarly treated with distilled water. The calli were then grown at 25°C under light for 60 min, frozen in liquid N2 and stored at −80°C. Different concentrations of SA, ABA, MeJA and H2O2 were used in the treatment of calli in order to find the most suitable concentrations for the optimal experiments. The result showed that SA (100 μM), ABA (100 μM), MeJA (100 μM) and H2O2 (10 mM) showed their maximal effects (results not shown).
CaAOC full-length cDNA by RACE (rapid amplification of cDNA ends)
The 3′-RACE system was used for 3′-cDNA end amplification of CaAOC. First, AP [5′-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT-3′; reverse transcriptase primer], AUAP (5′-GGCCACGCGTCGACTAGTAC-3′; universal amplification primer) and two degenerate conservative primers, CaAOC3-1 [5′-CTCGG(A/C)GATCT(T/C)GT(G/C)CC-3′, as 3′-RACE first amplification primer] and CaAOC3-2 [5′-AGCTT(T/C)TA(T/C)TTCGG(A/T/C)G(A/G)(T/C)TA(T/C)GG-3′, as 3′-RACE nested amplification primer], which were designed on the basis of two highly conserved amino-acid sequences (LGDLVP and SFYFGDYG) of other AOCs, were used for similarity-based isolation of the 3′-conserved fragment of CaAOC by standard gradient PCR amplification. The amplified PCR product was purified and cloned into pMD18-T vector (TaKaRa), and the sequence was verified by DNA sequencing. A 615-bp fragment was amplified and BLAST analysis showed that the 615-bp fragment had high similarity with AOCs from other plant species. This fragment was subsequently used to design gene-specific primers for the cloning of 5′-cDNA and full-length cDNA of CaAOC by RACE.
5′-RACE, including reverse transcription, dC tailing and PCR amplifications, were carried out according to the manufacturer's instructions (Clontech). Two gene-specific primers, CaAOC5-1 (5′-CGTAGTCTCCGAAGTAGAAGCTGTAG-3′, as 5′-RACE first amplification primer) and CaAOC5-2 (5′-CGTACACGTCACCTTTCTTCTCCGG-3′, as 5′-RACE nested amplification primer), UPM (long: 5′-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3′; Short: 5′-CTAATACGACTCACTATAGGGC-3′, as the first amplification primer) and NUP (5′-AAGCAGTGGTATCAACGCAGAGT-3′, as the nested amplification primer) were used for 5′-RACE. By aligning and assembling the products of 3′-RACE and 5′-RACE, the full-length CaAOC was deduced and subsequently amplified by proof-reading reverse transcriptase PCR amplification with primers CaAOCFull5 (5′-TCACCCATTACATCCATCCTCACCAAAC-3′) and AP. PCR was carried out under the following conditions: 3 min at 94°C, 28 cycles (35 s at 94°C, 35 s at 60°C, 2 min at 72°C) and 8 min at 72°C. The amplified PCR product was purified and cloned into the pMD18-T vector, and the sequence verified by DNA sequencing. In total, three independent positive clones were sequenced to check for errors introduced by PCR.
Cloning of the genomic sequence corresponding to the full-length
In order to investigate if any intron(s) is present in the region of the CaAOC gene corresponding to the full-length cDNA, PCR amplification was carried out by using 1.5 μg of total genomic DNA, extracted from C. acuminata calli by a CTAB (cetyltrimethylammonium bromide)-based method , as a template with the primers CaAOCFull5 and CaAOCFull3 (5′-GACCATCATCATTCTCGAGCATCCTC-3′) under the following conditions: 3 min at 94°C, 28 cycles (50 s at 94°C, 50 s at 60°C, 3 min at 72°C) and 8 min at 72°C. The PCR product was purified and cloned into the pMD18-T vector, and the sequence was verified by DNA sequencing.
Bioinformatic analysis of CaAOC was carried out online at http://www.ncbi.nlm.nih.gov and http://cn.expasy.org. VNTI Suite 6 was used for multiple alignment analysis of plant AOC proteins. The cTP analysis was carried out at http://www.cbs.dtu.dk/services/ChloroP/ . Secondary structure prediction was carried out by hierarchical neural network analysis .
Total genomic DNA was isolated from 1 g of fresh C. acuminata leaf material using the CTAB-based method . Aliquots of DNA (20 μg/sample) were digested overnight at 37°C with BamHI, BglII, EcoRI, HindIII and XbaI, which did not cut within the probe region, fractionated by 0.85% agarose gel electrophoresis and transferred on to a positively charged Hybond-N+ nylon membrane (Amersham Pharmacia). The 534-bp long 5′-cDNA end of CaAOC was used as a template for probe labelling using the primers CaAOCFull5 and CaAOC5-1. The PCR conditions and amplification procedure were the same as those used for 5′-RACE PCR. Probe labelling (with biotin), hybridization and signal detection were performed using Gene Images™ random prime labelling module and CDP-Star detection module following the manufacturer's instructions (Amersham Pharmacia). The filter was washed under high-stringency conditions (65°C) and the hybridized signals were visualized by exposure to X-ray film (Fuji) at room temperature (25°C) for 1.5 h.
RT-QPCR (real-time quantitative PCR)
RT-QPCR was carried out to investigate the expression profiles of CaAOC in different organs of C. acuminata during rhythmic growth of the callus and under different treatments. All RNA samples were digested with DNase I (RNase-free) prior to use. Aliquots of 0.4 μg of total RNA were employed in the RT-QPCR reaction using random hexamer primers. RT-QPCR was performed on an iCycler iQTM Real Time PCR machine (Bio-Rad) with gene-specific primers CaAOC-RT-F (5′-AAACCTCAGACTCACCACCACA-3′) and CaAOC-RT-R (5′-TTCCACGGTCTCGTTCGTT-3′), 18S rRNA primers 18SF (5′-ATGATAACTCGACGGATCGC-3′) and 18SR (5′-CTTGGATGTGGTAGCCGTTT-3′), and the SYBR ExScript RT-PCR kit (TaKaRa) protocol was used to confirm changes in gene expression. The target messages 18S rRNA and CaAOC in unknown samples were quantified three times by measuring the Ct (threshold cycle) value and extrapolation to standard curves constructed with serial dilution templates of known concentrations. For each reaction, 2 μl of cDNA (corresponding to 50 ng of total RNA), diluted 1:9 with EASY dilution (TaKaRa), 10 μl of 2× SYBR Premix Ex Taq™, 0.2 μM forward primer, 0.2 μM reverse primer and nuclease-free water were made up to a final volume of 20 μl. The thermal cycle conditions used were: 10 s at 94°C, followed by 40 cycles of amplification (5 s at 95°C and 25 s at 62°C). Melting curve analysis and agarose gel electrophoresis were performed following each RT-QPCR to assess product specificity. A comparative method for quantification and ΔCt were adopted. The Ct values corresponding to 18S rRNA endogenous house-keeping control gene expression were subtracted from the Ct values for the expression of CaAOC in order to calculate ΔCt. These values were measured for three samples. The products of RT-QPCR were resolved on 1% agarose gel electrophoresis and produced bands of 140 bp for CaAOC and 108 bp for 18S rRNA, as predicted by template sequences.
RESULTS AND DISCUSSION
Cloning of the full-length cDNA and genomic DNA of
The full-length cDNA and genomic DNA sequences of CaAOC were cloned, as described in the Materials and methods section. The full-length cDNA of CaAOC was 1130 bp with 5′- and 3′-untranslated regions, and a poly(A) tail, and contained a 738-bp ORF (open reading frame) encoding a 246-amino-acid protein (GenBank® accession no. AY863428). The comparison of the full-length cDNA and the genomic DNA of CaAOC revealed that the genomic DNA contained two introns, one of 89 bp and the other of 240 bp.
The deduced CaAOC had a calculated molecular mass of 26.8 kDa and a pI of 8.71 (http://cn.expasy.org/cgi-bin/protparam). Protein–protein BLAST analysis showed that CaAOC has high homology with other AOCs (Figure 2). The deduced CaAOC amino-acid sequence showed very broad and high local identities and similarities to AOCs from species of broad plant classes, such as Solanum tuberosum (Entrez Protein database accession no. AAN37418: with 69% identity and 84% similarity), Lycopersicon esculentum (CAB95731, with 71% identity and 85% similarity), Bruguiera sexangula (BAB21610, with 67% identity and 78% similarity), Oryza sativa (CAD38519, with 73% identity and 86% similarity), Arabidopsis thaliana (CAC83761, with 62% identity and 80% similarity; CAC83762, with 62% identity and 79% similarity; CAC83763, with 65% identity and 81% similarity; CAC83764, with 59% identity and 76% similarity), Physcomitrella patens (CAD48752, with 65% identity and 74% similarity; CAD48753, with 64% identity and 74% similarity). ChloroP1.1 analysis showed that CaAOC contained an N-terminal cTP, with the most probable cleavage site being between Lys61 and Cys62 . On the basis of hierarchical neural network analysis , the CaAOC protein was shown to be composed of approx. 24.32% α-helix, 22.16% extended strand and 53.51% random coil. Random coils were the most abundant structural elements of CaAOC, penetrating through most parts of the CaAOC protein, whereas α-helices and extended strands were intermittently distributed in the protein.
Multi-alignment of the deduced CaAOC protein with 10 most homologous plant AOC proteins
All of the bioinformatic analysis results strongly suggested that CaAOC should be a functional plant AOC protein involved in JA biosynthesis.
The number of genes encoding AOC was different among various plant species. AOC was encoded by a single gene in tomato and barley [5,8], by two in the genome of rice and by five closely related genes in A. thaliana [7,17]. In order to investigate whether CaAOC in C. acuminata was a single- or multiple-copy gene, Southern-blot analysis was performed under high-stringency conditions and the result showed that a few hybridizing bands were present in each lane, indicating that CaAOC was a multiple-copy gene (Figure 3).
Expression profile of
CaAOC in different organs
To investigate the CaAOC expression pattern in different organs of C. acuminata, total RNAs extracted from roots, stems, leaves, young flower buds, opening flowers, fading flowers and seeds were used in RT-QPCR analysis. The result showed that CaAOC is expressed in a constitutive manner in organs, but at different levels; the highest expression was found in leaves (Figure 4). Previous immunoblot analysis showed that AOC in A. thaliana accumulated preferentially in leaves and carried high levels of AOC protein . Among plant developmental processes, pollen maturation and seedling growth were JA-dependent [19,20]. Our present and previous studies [18–20] suggested the role of JA in senescence for C. acuminata leaves by transcriptional up-regulation of genes encoding AOC etc. According to the RT-QPCR result, the transcription level of CaAOC in seed and opening flower was much higher, which may be attributed to the effect of JA on seed germination and flower development.
Expression profiling analysis of
CaAOC in different organs of C. acuminata
Growth curve and rhythmic expression of
RNAs were extracted from C. acuminata calli subcultured on fresh medium for various durations, including 0, 5, 10, 15, 20, 25 and 30 days, and subjected to RT-QPCR analysis to study the expression of CaAOC. Growth curves were determined by inoculating 1.5–2.0 g (fresh mass) of C. acuminata calli on to fresh medium. The fresh mass was measured after each 5-day-interval in triplicate experiments. The result showed that CaAOC was expressed in a fluctuating manner, with the highest expression found after 15 days of culture, coinciding with the callus growth curve (Figure 5).
Callus growth curve of
C. acuminata and rhythmic expression analysis of CaAOC
Wounding, heavy metal and signalling molecules differently regulate
The transcription on the octadecanoid pathway nearly reached higher levels at the time of 60 min under different treatments [21–23]. It has been reported that under certain stresses, rice OsAOC expression was transient, reaching a maximum at 60 min . To examine the changes of gene expression under different treatments, the 15-day-old C. acuminata calli were used for RNA isolation after 60 min of individual treatment. It was interesting to note that wounding by UV-B plus water (UV1) and UV-B alone (UV2) did not induce higher CaAOC expression compared with the water control (CON1) and air control (CON2) (Figure 6A). In dicots, such as tomato and tobacco, it has been verified that wounding causes the increase in JA levels and then the accumulation of the AOC transcript , and, in rice, it was observed that wounding by cutting induced OsAOC expression within 30 min. All of these results suggest that the mechanisms of JA accumulation by cut-wounding and by UV-B wounding are different in plants.
Expression profiling analysis of
CaAOC under various treatments
It was demonstrated earlier that SA blocked the wound-induced JA biosynthesis upstream of OPDA in tomato leaves , and acted downstream of JA in tomato . However, SA did not inhibit OsAOC gene expression in rice . Our finding that the accumulation of CaAOC transcript induced by SA was lower compared with the water control suggests that SA inhibits CaAOC expression in C. acuminata, which is similar to the result in tomato. ABA did not up-regulate AOS transcript levels in the monocot barley and other dicot species examined to date , but there have been no reports on the effects of ABA and H2O2 on AOC expression in the short-term (such as several hours). In a long-term experiment, ABA strongly suppressed rice OsAOC expression, whereas H2O2 enhanced OsAOC mRNA levels . In the present study, RT-QPCR analysis revealed that ABA and MeJA significantly enhanced CaAOC transcript expression, but SA and H2O2 suppressed its expression compared with the water control and air control in C. acuminata (Figure 6A). The AOC transcript from C. acuminata that is regulated by signalling molecules appears to be different from rice, suggesting that, although there is conservation in JA signalling in dicots and monocots, the transcriptional regulation of AOC appears to be different.
It was reported that there was a significant increase in endogenous JA levels in rice leaves treated with heavy metals and environmental pollutants [17,27]. It is speculated that the expression of the CaAOC gene would be affected by heavy metals, which would provide additional proof for the induction of JA synthesis by heavy metals. Our present study confirmed that the heavy metal copper did significantly enhance CaAOC expression compared with both the water control and air control, whereas other metals (manganese and lead) did not show a prominent difference compared with water control (Figure 6A).
Synergistic effect of wounding and signalling molecules on
Wounding can induce expression of the genes encoding enzymes specific for JA biosynthesis and promote the synthesis of some signalling molecules in various plant species. Cross-communication between signalling components, leading to defence responses, is well known [17,22,28–30]. In the present study, UV-B was applied to examine its co-effect with ABA, MeJA, SA and H2O2 on the CaAOC transcript. The expressions of CaAOC induced by ABA dropped down with UV-B plus, and UV-B enhanced the inducing effect of MeJA. However, the suppression effects of SA and H2O2 on CaAOC transcript were diluted by UV-B (Figure 6). It indicated that UV-B might play a role in blocking the effects of these four signalling molecules on CaAOC transcript, and the synergistic treatment with MeJA and SA led to the nearly equal expression of CaAOC compared with the water control.
A common feature of all plant species analysed to date is their abilities to respond to biotic or abiotic stresses with an endogenous rise of JA, which appears within the first few hours upon the onset of stress . As for other plant species, isolation of cDNAs coding for enzymes of JA biosynthesis is the main tool to analyse this rise in JA in C. acuminata. The cloning and characterization of CaAOC will be helpful for further understanding of the role of AOC in the JA biosynthetic pathway in the medicinal plant, the Chinese Happy Tree.
allene oxide cyclase
allene oxide synthase
Camptotheca acuminata AOC
chloroplast transit peptide
rapid amplification of cDNA ends
real-time quantitative PCR
This work was funded by the National Basic Research Program of China [973 Program, grant number 2007CB108805]; the China National ‘863’ High-Tech Program [grant number 2007AA10Z189]; the Chinese Ministry of Education [grant number C04N]; and the Shanghai Science and Technology Committee [grant number 08391911800].
The nucleotide sequence data reported will appear in GenBank®, EMBL, DDBJ and GSDB Nucleotide Sequence Databases under the accession number AY863428.