JXB Advance Access originally published online on March 7, 2005
Journal of Experimental Botany 2005 56(414):1229-1237; doi:10.1093/jxb/eri117
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Published by Oxford University Press [2005] on behalf of the Society for Experimental Biology.
RESEARCH PAPER |
Application of Bruchin B to pea pods results in the up-regulation of CYP93C18, a putative isoflavone synthase gene, and an increase in the level of pisatin, an isoflavone phytoalexin*
1Horticultural Crops Research Unit, USDA-ARS, Corvallis, OR 97330, USA
2Department of Horticulture, Oregon State University, Corvallis, OR 97331, USA
3USDA-ARS, Chemicals Affecting Insect Behavior Laboratory, Building 007, BARC-W, Beltsville, MA 20705, USA
Present address and to whom correspondence should be sent: Department of Crop and Soil Science, Oregon State University, Corvallis, OR 97331, USA. Fax: +1 541 737 0909. E-mail: lol.cooper{at}oregonstate.edu
Received 20 September 2004; Accepted 14 January 2005
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResults and discussionReferences |
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Bruchins, mono and bis (3-hydroxypropanoate) esters of long chain
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-diols, are a recently discovered class of insect elicitors that stimulate cell division and neoplasm formation when applied to pods of peas and certain other legumes. Differential display analysis resulted in the identification of an mRNA whose level was increased by the application of Bruchin B to pea pods. The corresponding amplification product was cloned and sequenced and a full length cDNA sequence was obtained. This cDNA and the gene from which it was derived were assigned the name CYP93C18 based upon sequence similarities to the cytochrome P450 mono-oxygenase CYP93C subfamily, which contains isoflavone synthase genes from legumes. RNA gel blots and quantitative RT-PCR demonstrated that expression of CYP93C18 increased within 8 h of bruchin treatment to a maximum of 100–200-fold of the level in untreated pods, and then declined. The up-regulation of CYP93C18 was followed by an increase in the level of the isoflavone phytoalexin, pisatin. Pisatin was detectable in the bruchin-treated pods after 16 h and reached a maximum between 32 h and 64 h. This, the first report of induction of phytoalexin biosynthesis by an insect elicitor, suggests that Bruchin B not only stimulates neoplasm formation, but also activates other plant defence responses. Key words: Bruchin, CYP93C18, cytochrome P450, insect elicitor, Neoplastic pod, phytoalexin, pisatin, Pisum sativum
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Bruchins, mono and bis (3-hydroxypropanoate) esters of long chain
,-diols, are a new class of insect-derived natural products that stimulate cell division and neoplasm formation when applied to pods of peas and certain other legumes (Doss et al., 2000
; Oliver et al., 2000). The isolation and characterization of the bruchins followed the observation that oviposition by the pea weevil Bruchus pisorum L. (Coleoptera: Bruchidae), an important pest of pea, on pods of peas carrying the wild-type allele of the Neoplastic pod (Np) gene resulted in browning followed by cell division and callus formation at the site of oviposition (Berdnikov et al., 1992; Hardie, 1993; Doss et al., 1995). The calli formed in response to oviposition reduce weevil infestation of the pea seed via both indirect and direct mechanisms (Berdnikov et al., 1992; Hardie, 1993; Doss et al., 2000) and the bruchins have been referred to as ‘herbivore-specific elicitors’ (Kessler and Baldwin, 2002).
Direct application of a bruchin to both Np/Np and np/np pods stimulates browning and swelling and ultimately results in callus formation. The calli formed on np/np pods are much smaller than those seen on Np/Np pods and much of their mass results from cell enlargement rather than cell division; however, the minimum dose required to elicit a response on pods of either genotype appears to be about the same (Doss et al., 2000). Interestingly, the Np gene was described 30 years ago, long before its role in insect resistance was noted, because its presence causes pods grown under greenhouse conditions to develop patches of callus (neoplasms) on their surface (Dodds and Matthews, 1966; Snoad and Matthews, 1969). Such neoplasms do not form under natural sunlight, as their formation is stimulated by the attenuation of ultraviolet wavelengths (UV) by greenhouse coverings (Snoad and Matthews, 1969), nor do they form spontaneously in the greenhouse in peas homozygous for the recessive allele (np).
The work reported here was undertaken as part of a larger project to evaluate the changes in gene expression that are stimulated by bruchin application to pea pods. Among the genes up-regulated was CYP93C18, a gene in the CYP93C subfamily of cytochrome P450 mono-oxygenases. This subfamily comprises several (legume) isoflavone synthase (IFS) genes. Isoflavone synthases catalyse the first specific step in the biosynthesis of the isoflavone phytoalexins of legumes (Steele et al., 1999). Phytoalexins are antimicrobial compounds produced by plants in response to infection by plant pathogens (Hammerschmidt and Nicholson, 1999). Bruchin B application also led to the biosynthesis of pisatin, an isoflavone phytoalexin of pea (Cruickshank and Perrin, 1962; Perrin and Bottomley, 1962). It has been hypothesized that phytoalexins play a role in disease resistance, but despite considerable study such a role has not been definitively demonstrated (Hammerschmidt, 1999, 2003). The up-regulation of CYP93C18, and the increase in pisatin content would appear to be, along with neoplasm formation, part of the defence response brought about by application of a bruchin to pods of pea.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Plant material and bruchin treatment
The pea (Pisum sativum L.) lines used in these studies were derived from a cross between the lines C887–332 (Np/Np) and I3 (np/np) (Doss et al., 1995
, 2000) and were homozygous for either the Np or np allele. Near-isogenic lines of the F12 generation were used throughout the study, with the exception of the differential display analysis (F9 near-isogenic lines). Pea plants were either grown outdoors under natural light or in a greenhouse supplemented with UV light (UVA-340, O-Panel Co. USA) to prevent spontaneous callus formation (Doss et al., 2000). Pods in the late flat pod stage (Meicenheimer and Muehlbauer, 1982), approximately 3.5–5 cm in length, were treated on one side with 6 ng bruchin B [(Z)-9-Docosen-1,22-diol bis-(3-hydroxypropanoate) ester, C28H52O6] (Doss et al., 2000; Oliver et al., 2000) in 60 µl 50% ethanol, while on the opposite side, 50% ethanol was applied or it was left untreated. An untreated sample of pod tissue was collected at the time of treatment. After the appropriate treatment period, the pods were removed from the plant, placed onto ice, and split along the suture. The seeds and the untreated portions of the pod were removed. The pod samples were frozen immediately in liquid nitrogen and stored at –80 °C.
RNA extraction, poly(A)+ RNA selection and cDNA synthesis
Total RNA was isolated from the pod tissue samples (10 g for the differential display (DD) study and 0.5 g for the time-course experiments) using a guanidine thiocyanate–phenol extraction solution based upon the method of Chomczynski (1993). Poly(A)+ RNA was prepared from total RNA (in the DD study) using the PolyATract® system (Promega Corp., Madison, WI), following the manufacturer's protocol. First-strand cDNA synthesis (primed with an oligo (dT)12-18 primer) was performed using Dnase I-treated total RNA or poly(A)+ RNA, and Superscript II reverse transcriptase (all from Life Technologies, now Invitrogen Corp., Carlsbad, CA), following the manufacturer's protocol. The cDNA samples were quantified using either ethidium bromide dot quantitation (Sambrook and Russell, 2001) or with a Dyna Quant 200 fluorometer (Amersham Biosciences, Piscataway, NJ), following the manufacturer's protocol.
Differential display analysis, cloning and sequencing
Np/Np and np/np pea plants (F9 near-isogenic lines) were grown in a greenhouse, under supplemental ultraviolet light (to prevent spontaneous callus formation) with controlled temperatures. Pods were treated with Bruchin B or ethanol for 24 h, collected, and poly(A)+ RNA was isolated (all steps as described above). A modified, non-radioactive version (Doss, 1999) of DD (Liang and Pardee, 1992) analysis was performed using cDNA synthesized from poly(A)+ RNA using pairwise combinations of 12 anchored primers T11MN (M=A, C or G and N=A, C, G, T) and a set of arbitrary ten base primers, OPA-01 through OPA-20 (all from Operon Technologies, now Qiagen Operon, Alameda, CA). The amplification products were fractionated using 6% non-denaturing PAGE and visualized by silver staining. The differentially displayed band (confirmed using an independent set of pod tissues), was excised from the polyacrylamide gel, reamplified and cloned using the pGem®-T Easy Vector System (Promega Corp, Madison, WI) and sequenced at the Center for Gene Research and Biotechnology (CGRB), Oregon State University. Sequences were analysed using BioEdit Sequence Alignment Editor (Hall, 1999).
5' Rapid Amplification of cDNA Ends (RACE)
5' RACE (GeneRacer®, Invitrogen Corp, Carlsbad, CA) was performed following the manufacturer's protocol using poly(A)+ RNA isolated from bruchin-treated Np/Np pods (F9 near-isogenic lines). Nested gene-specific primers were designed based upon the sequence of the fragment obtained from DD (GT13R1: 5'-TGG GTC TCT TCC CAC TGC CCA TAC A-3' and GT13R2: 5'-CAC ATC CTC CTT CCT GAC CCA AAC G-3'). A single amplification product was obtained and was cloned using the TOPO TA® cloning vector pCR®4-TOPO® (Invitrogen Corp, Carlsbad, CA), sequenced and analysed as above.
RNA gel blot analysis of changes in CYP93C18 expression
Total RNA was isolated as above from bruchin- and ethanol-treated pod tissue (Np/Np and np/np F12 near-isogenic lines) over a 32 h time-course and was denatured with glyoxal (10 µg per treatment). After fractionation by electrophoresis (1.5% (w/v) agarose gel) in the presence of ethidium bromide, the RNA was transferred (downward capillary transfer with an alkaline buffer) and fixed to a positively charged nylon membrane (Zeta Probe®, Bio-Rad), following standard procedures for RNA gel blots (Sambrook and Russell, 2001). A radiolabelled PCR product (amplified using the primers: GT13F3 (5'-CAT GCC CAC CAT TGT TGT ATC C-3') and GT13R3 (5'-TGC TCA CTT TCT TCG ATT TCT CC-3')) was prepared by random primer labelling (Feinberg and Vogelstein, 1983, 1984) (RadPrime® DNA Labeling System, Life Technologies, now Invitrogen) using 50 µCi [-32P]dCTP (New England Nuclear, now Perkin Elmer) in a 50 µl reaction. The probe was passed over a gel filtration column (Micro Bio-Spin® P-30, Bio-Rad) before addition to the prehybridization solution. Hybridization of the probe to the membrane, washing, and autoradiography were carried out using standard procedures (Sambrook and Russell, 2001). The images shown in Fig. 3 were prepared using an AlphaImager® System (Alpha Innotech) and Adobe Photoshop®.
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Quantitative reverse transcriptase-PCR (QRT-PCR)
Total RNA was isolated from bruchin- and ethanol-treated pod tissue over a 32 h time-course, DNase I-treated and reverse transcribed as above. Quantitative reverse transcriptase-PCR (QRT-PCR) was performed using the SYBR® green master mix (Applied Biosystems Inc, Foster City, CA), as recommended by the manufacturer, except that the reactions were done in 25 µl volumes and the final concentration of the primers used was 500 nM. Primers for QRT-PCR (GT13 315F 5'-GGA GAA CGT TAT GGC CCT TTG-3' and GT13 388R 5'-ATG AGT TTG AAG GAA GAG GTT GAA GA-3') were designed using Primer Express software (Applied Biosystems, Inc) and synthesized by Qiagen Operon. Primers (PsActF2 5'-CAC AAT TGG CGC TGA AAG AAT-3' and PsActR2 5'-TAA TTG AGT TAA ATG TCG TCT CAT GGA T-3'), based upon the sequence of a highly conserved region of pea ß-actin (PEAc9, GenBank Accession no. PSU81047), were used to prepare an amplification product that served as an endogenous control for the QRT-PCR assays.
Quantitative RT-PCR was performed on a PE Biosystems ABI Prism® 7700 Sequence Detection System (Applied Biosystems Inc) using the default program. After amplification, the PCR products were subjected to a heat dissociation program consisting of a 35 °C temperature gradient from 60 °C to 95 °C over approximately 20 min and analysed using the ABI Prism® Dissociation Analysis software (Applied Biosystems, Inc) to distinguish the target amplicon from non-specific PCR products. For the initial runs, the real-time RT-PCR products were also analysed by agarose gel electrophoresis to verify that a single amplicon of the appropriate size had been produced.
Estimations of relative changes in expression of CYP93C18 in bruchin- and ethanol-treated pods was carried out using the Comparative CT method (Applied Biosystems Inc, 2001). Each data point is an average of the 2–
CT values calculated for three independently performed QRT-PCR measurements (i.e. three individual plates), while on each plate, each reaction was replicated three times. Relative quantitation of gene expression depends upon the assumption that the efficiencies of amplification of the target and reference sequences are approximately equal. The absolute value of the slope of log input versus CT should be less than ±0.1 (Applied Biosystems Inc, 2001). That this was the case was verified by amplification using each of the primer sets; CYP93C18, the target gene, and pea ß-actin, the endogenous control gene, using standard curves of pod cDNA from 1 pg to 10 ng and plotting the Ct (CtCYP93C18–CTPsAct) versus the log quantity of cDNA (data not shown).
Pisatin determinations
Pisatin was extracted from Bruchin B-treated pod tissue (Np/Np and np/np F12 near-isogenic lines) over an 80 h time-course, based on a procedure described by Schwochau and Hadwiger (1969). A 0.5 g FW portion of frozen pod tissue was ground to a fine powder in a mortar and pestle cooled with liquid nitrogen. After the powder was extracted three times with 4 ml portions of 95% ethanol, the ethanolic extracts were filtered, combined and taken to dryness in vacuo. The residue was taken up in 5 ml water and partitioned five times against equal volumes of hexanes. The combined hexanes fractions were taken to dryness in vacuo, and the residue was dissolved in 4 ml absolute ethanol. The ethanolic solution was taken to dryness under nitrogen, and the residue was redissolved in 50–200 µl of absolute ethanol. Three independent extractions were performed for each time point, the pisatin values (µg pisatin g–1 FW of pod tissue) are presented with the standard error of the mean of the three determinations.
Pisatin concentration was determined using HPLC with a 250x4.6 mm column packed with Adsorbosil® C-18 (5 µm) (Alltech). A flow rate of 1 ml min–1 with a linear gradient of 70–100% methanol was used. The gradient was preceded by 5 min at 70% methanol, and followed by 15 min at 100% methanol. Under these conditions pisatin had a retention time of approximately 8 min. Spectrophotometric (309 nm) detection was used, and estimates of pisatin concentration were based on peak area. The identity of the pisatin peak was confirmed using liquid chromatography-mass spectrometry (LC-MS), and a set of spiked samples allowed an estimate of recovery (87.5±4.9%, mean ±standard error of the mean for n=5), which was used in the estimates of pisatin level. A sample of authentic pisatin (recrystallized from heptane) was prepared from naturally infected pea seedlings using solvent partitioning, column, and thin-layer chromatography (Van Etten et al., 1975). The identity of this sample, which was used for development of HPLC methods, and as a pisatin standard for HPLC determinations, was confirmed using MS (Kobayashi et al., 1993).
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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CYP93C18, a cytochrome P450, was isolated by differential display
Differential display, performed with cDNA populations from pea pods treated either with Bruchin B or ethanol, yielded 19 amplification products whose appearance was either bruchin treatment-specific or genotype-specific (data not shown). One of these, designated GT13, obtained with the primer combination T11GT/OPA-13, appeared to have been derived from a gene that was up-regulated by bruchin treatment, with stronger up-regulation in Np/Np pods than in np/np pods. After cloning and sequencing, the length of the GT13 clone was 710 bp, which corresponded to the size of the band observed on the DD gels.
The sequence of the GT13 cDNA clone (boxed region in Fig. 1) extended from the open reading frame of the gene into the 3' untranslated region (UTR). The full-length cDNA sequence was compiled from the sequence of the GT13 clone and that of the 5' RACE product. The full-length cDNA, and the gene from which it was derived, were assigned the name CYP93C18 by Dr David Nelson (personal communication) based upon motifs indicative of a cytochrome P450 mono-oxygenase and sequence similarity to isoflavone synthase genes from other members of the legume family.
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CYP93C18 contains the characteristic haem-binding domain, highlighted in Fig. 1, FgsGrRmCpG, where the capital letters represent the conserved amino acids common to all classic cytochrome P450 mono-oxygenases (Schuler, 1996
). Cytochrome P450 mono-oxygenases, which often catalyse NADPH and oxygen-dependent hydroxylations (Schuler, 1996; Chapple, 1998) are represented by more than 1000 enzymes in 62 families in plants (cytochrome P450 website http://drnelson.utmem.edu/CytochromeP450.html). A recent study of the available rice and Arabidopsis genome sequences revealed more than 600 P450 genes in those two species alone (Nelson et al., 2004). The predicted amino acid start codon (ATG) is at nucleotide 101 and the open reading frame runs to a stop codon (TAA) at position 1673, encoding a predicted protein of 524 amino acids. The full-length cDNA sequence has been deposited in the GenBank database under the Accession no. AF532999.
CYP93C18 is a putative isoflavone synthase (IFS)
The isoflavone synthases (also referred to as 2-hydroxyisoflavanone synthase) of legumes are members of a small subfamily within the cytochrome P450s. Isoflavone synthases catalyse the first committed step in the biosynthesis of the isoflavone phytoalexins, hydroxylation at carbon-2 and the 2,3-aryl migration, giving rise to the isoflavonoid skeleton (Dixon and Paiva, 1995; Akashi et al., 1999; Overkamp et al., 2000).
A PSI-BLAST (Altschul et al., 1997) search of the GenBank non-redundant database carried out with the deduced amino acid sequence of CYP93C18 revealed sequence similarity with other isoflavone synthase genes from legumes. The highest similarities at the amino acid level were to isoflavone synthases from barrel medic (Medicago truncatula) 88%, chickpea (Cicer arietinum) CYP93C3 (Overkamp et al., 2000) 86%, licorice (Glycyrrhiza echinata) CYP93C2 (Akashi et al., 1999) 83%, and soybean (Glycine max) GmIFS1 (Jung et al., 2000) 82%. Another pea isoflavone synthase, PsIFS1 (Jung et al., 2000) is only 80% similar at the amino acid level to the deduced amino acid sequence of CYP93C18. The alignment of the amino acid sequences of CYP93C18 with isoflavone synthases from other Fabaceae (Fig. 2) shows where the similarities and differences lie.
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The function of CYP93C18 was inferred based upon a strong similarity to the sequences of legume isoflavone synthase genes already characterized. CYP93C3 was identified after suspension-cultured cells of chickpea were treated with fungal elicitors (Overkamp et al., 2000
). CYP93C2 from licorice was expressed in recombinant yeast microsomes and shown to encode 2-hydroxyisoflavanone synthase (Akashi et al., 1999). Similarly, in assays of the proteins encoded by PsIFS1 and GmIFS1 and GmIFS2 in engineered yeast strains, the flavanone naringenin was converted to the isoflavone genistein (Jung et al., 2000).
CYP93C18 expression increases within 4 h of Bruchin B application
RNA gel blots and QRT-PCR were used to assess the changes in the level of CYP93C18 mRNA over a 32 h time-course of bruchin treatment. With RNA gel blots the amount of CYP93C18 mRNA (Fig. 3A, C) increased from an undetectable level at the time of treatment (time <1.5 h) to a maximum at 8 h, then declined again to an undetectable level in the bruchin-treated pods of both lines. The treatment effect appeared to be more pronounced in the pods of the np/np peas. No effect of ethanol treatment could be detected in either line. The observed size of the transcript on the RNA gel blot (
1.6 kb) matched the length of the open reading frame, 1572 bp.
This pattern of expression was confirmed using quantitative RT-PCR (Fig. 3B, D). There was a 30-fold increase in the amount of CYP93C18 mRNA in the Np/Np pods within 4 h after bruchin treatment with a further increase to almost a 100-fold by 8 h, relative to the untreated control pod tissue. In the np/np pods there was an even greater up-regulation of CYP93C18. Four h after the application of bruchin to the pods, the level of CYP93C18 mRNA was more than 50 times greater than that in the untreated control pods, and by 8 h it had increased to a level about 230-fold higher.
This pattern is similar to what was seen in the RNA gel blot analysis of CYP93C2 from licorice, which revealed that the IFS mRNA accumulated transiently upon elicitation of the cells. Intense signals were observed in preparations from the cells 3–6 h post-elicitation, prior to the accumulation of the end-product, medicarpin, after 24 h (Akashi et al., 1999). In chickpea suspension cultures CYPC93C3 transcript levels reached a maximum 4–6 h after elicitor treatment, but the transcript was also expressed constitutively, and only a slight increase was noted upon elicitation (Overkamp et al., 2000).
Recently, the expression patterns of the genes encoding the two IFS isoforms in soybean were characterized. Isoflavone synthase transcripts were found primarily in the roots and seeds and the expression pattern of GmIFS1 was found to be consistent with the physiological roles of isoflavonoids as defence compounds against pathogens and as signal molecules to symbiotic bacteria in soybean (Subramanian et al., 2004).
The pattern of expression inferred from examination of band intensity with differential display suggested that CYP93C18 expression 24 h after application of a bruchin was greater in NP/NP pods than np/np pods. By contrast, in the time-course experiment (Fig. 3), greater up-regulation was seen in the np/np pods than in the NP/NP pods at all the time points where significant amounts of transcript could be detected. This discrepancy may be partially explained by the differences in the growing conditions used for the two experiments. The pea plants used for the differential display study were grown inside a greenhouse, under supplemental ultraviolet light (to prevent spontaneous callus formation) and contolled temperatures, while the samples for the time-course study were taken from pea plants grown outside under ambient temperatures and natural sunlight. It may also be significant that differential display was carried out using poly(A)+ RNA isolated from F9 near-isogenic lines; whereas the time-course experiment was carried out using F12 near-isogenic lines.
Changes in level of the phytoalexin pisatin result from Bruchin B application and follow up-regulation of CYP93C18
The biosynthesis of pisatin, the well-studied phytoalexin of pea, is induced by infection with fungal pathogens as well as by exposure to a number of abiotic stimuli (Cruickshank and Perrin, 1962). Pisatin is an isoflavone, as are the phytoalexins of a number of other legumes (Jung et al., 2000), and isoflavone synthases catalyse the first committed step in their biosynthesis.
Given the up-regulation of an isoflavone synthase by treatment of pea pods with a bruchin, it was reasonable to investigate the effect of bruchin treatment on pisatin biosynthesis. Pisatin was barely detectible in untreated pods, but increased about 11-fold in both np/np and Np/Np pods after 16 h of treatment (Fig. 4). Np/Np pods accumulated more pisatin than did np/np pods. This pattern contrasted with that seen with CYP93C18 expression suggesting that factors other than CYP93C18 transcription levels are rate-limiting for pisatin production.
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Bruchins and plant defence
It is reported here that the application of Bruchin B to pea pods results in an increased expression of a putative isoflavone synthase gene CYP93C18, which encodes an enzyme involved in phytoalexin synthesis in the Fabaceae. Moreover, it is demonstrated that pisatin, the well-studied isoflavone phytoalexin from pea, increases in concentration in pea pods that have been treated with a bruchin. An obvious question is, why does an insect elicitor such as bruchin induce the biosynthesis of a compound usually thought to be involved in induced resistance to fungal attack (Cruickshank and Perrin, 1962
; George and Van Etten, 2001). While the answer to this question must await additional study, there are several possible explanations. First, it is possible that bruchins, or compounds that induce the same reaction by the plant as the bruchins, are not restricted to insects. Indeed, bruchin-like activity in a pea pod bioassay (Doss et al., 2000) has been shown in chromatographic fractions prepared from a number of organisms (RP Doss, JE Oliver, and WM Proebsting, unpublished results), including Botrytis cinerea Pers:Fr., a plant pathogenic fungus.
However, a more probable explanation for the ability of Bruchin B to induce phytoalexin production follows from a consideration of recent reports that show that insect attack can result in resistance against various plant pathogens (Hatcher, 1995). This probably occurs because of the lack of specificity in the stimuli that induce resistance (Karban and Kuc, 1999). This is advantageous to the plant because many of the defensive substances produced are active against a range of organisms. In particular, Berenbaum (1988) has pointed out that phytoalexins ‘...are non-specific in terms of their mode of action and ... are broadly biocidal.’ Within the legumes, Sutherland (1980) demonstrated that a number of isoflavonoid phytoalexins, including pisatin, can deter feeding of Costelytra zealandica (White) (Coleoptera: Scarabaeidae) larvae, subterranean pests of pasture plants in New Zealand. Similarly, Hart (1983) found that the addition of glyceolin, the isoflavone phytoalexin of soybean, to artificial diet, could reduce the efficiency of food utilization by the soybean looper, Pseudoplusia includens (Walker), (Lepidoptera: Noctuidae), and that feeding by the Mexican bean beetle Epilachna varivestis Mulsant, (Coleoptera: Coccinellidae) was strongly deterred on soybean cotyledons in which high glyceolin levels were present.
Pea pods are susceptible to infection by a number of fungal diseases in nature, among which are downy mildew, powdery mildew, and Botrytis mould (Hagedorn, 1991). The prior induction of pisatin accumulation by bruchin application or, indirectly by pea weevil oviposition might have an impact on the development of subsequent disease symptoms.
Conversely, Hammerschmidt (1999), noted that preformed antimicrobial compounds played a minor role in resistance compared with defences activated after infection. In addition, many fungi have the ability to demethylate and thus detoxify the pea phytoalexin pisatin (George and Van Etten, 2001). Further experimentation will be necessary to determine if bruchin-induced pisatin would play a role in protecting peas against fungal infection.
Bruchin B, an insect-derived compound, stimulates cell division and neoplasm formation when applied to pods of peas homozygous for Np and np (Doss et al., 2000; Oliver et al., 2000). The application of Bruchin B also resulted in the up-regulation of CYP93C18, a putative isoflavone synthase gene, and a subsequent increase in the level of the phytoalexin, pisatin. The responses observed in the near-isogenic Np/Np and np/np lines varied, indicating that there are, most likely, other influencing factors. These factors may be abiotic, such as the exposure of the pea plants to natural sunlight or supplemental UV light or there may be uncharacterized genetic differences that account for the variation in responses. Nevertheless, it appears that the induction of CYP93C18 and the increase in pisatin content, along with neoplasm formation, form part of the defence response brought about by application of Bruchin B to pods of pea.
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Acknowledgements |
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The authors wish to thank Dr David R Nelson (University of Tennessee, Memphis) for the assignment of the P450 sequence to CYP93C18 and Dr William W Proebsting (Oregon State University) for supplying near-isogenic lines of pea, homozygous for either the Np or np allele.
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Footnotes |
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* The nucleic acid sequence data reported in this paper have been deposited in the GenBank database under the Accession no. AF532999.
Abbreviations: N, Neoplastic pod; IFS, isoflavone synthase; RACE, Rapid Amplification of cDNA Ends; QRT-PCR, quantitative reverse transcriptase-PCR; UV, ultraviolet.
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