- Email: [email protected]
Hydrogen peroxide–induced gene expression in Arabidopsis thaliana
Free Radical Biology & Medicine, Vol. 28, No. 5, pp. 773–778, 2000 Copyright © 2000 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/00/$–see front matter
PII S0891-5849(00)00157-X
Original Contribution HYDROGEN PEROXIDE–INDUCED GENE EXPRESSION IN ARABIDOPSIS THALIANA RADHIKA DESIKAN, STEVEN J. NEILL,
and JOHN
T. HANCOCK
Department of Biological and Biomedical Sciences, University of the West of England, Bristol, UK (Received 22 October 1999; Revised 21 December 1999; Accepted 28 December 1999)
Abstract—Hydrogen peroxide (H2O2) is generated in plants after exposure to a variety of biotic and abiotic stresses, and has been shown to induce a number of cellular responses. Previously, we showed that H2O2 generated during plant-elicitor interactions acts as a signaling molecule to induce the expression of defense genes and initiate programmed cell death in Arabidopsis thaliana suspension cultures. Here, we report for the first time the identification by RNA differential display of four genes whose expression is induced by H2O2. These include genes that have sequence homology to previously identified Arabidopsis genes encoding a late embryogenesis-abundant protein, a DNA-damage repair protein, and a serine/threonine kinase. Their putative roles in H2O2-induced defense responses are discussed. © 2000 Elsevier Science Inc. Keywords—Arabidopsis thaliana, Differential display, DNA-damage repair protein, Gene expression, Hydrogen peroxide, Protein kinase, Senescence-related protein, Free radical
INTRODUCTION
action of a plant cell with either a pathogen or a pathogen-derived elicitor does result in the generation of H2O2, and H2O2 has been shown to induce gene expression and programmed cell death in such cells [11–15]. There is considerable interest in monitoring the changes in gene expression profile in eukaryotes in response to developmental or environmental signals, so as to identify key genes essential for cell functioning. Differential display of RNA is a powerful technique based on reverse-transcriptase polymerase chain reaction (RTPCR) using random primer combinations, such that large proportions of a population of genes expressed in a specific tissue or cell type can be analyzed. Since its original description [16], numerous studies have used this technique to identify differentially expressed genes. In plants, this technique has been used to identify genes differentially expressed in response to pathogen challenge [17,18] and exposure to ozone [19] as well as during senescence [20]. Previous work in our laboratory has shown that Arabidopsis cells generate H2O2 upon challenge by elicitors [13] (probably via the dismutation of superoxide), and that H2O2 itself induces the expression of defense-related genes as well as initiating programmed cell death [14]. Here, we report the use of RNA differential display to
Reactive oxygen species (ROS) such as superoxide (O2•⫺) and hydrogen peroxide (H2O2) are generated under stress situations by a variety of mechanisms in animal and plant cells. Although ROS were originally thought to be only harmful to cells [1], recent data point to cell signaling roles for ROS in both plants and animals [2– 4]. In plants, ROS are generated in response to both abiotic and biotic stresses such as drought, exposure to ultraviolet light and ozone, extremes of temperature and pathogen challenge [5], whilst exogenous H2O2 can induce tolerance to chilling, high temperatures, and biotic stress [6 –9]. Although the role of ROS in plant-pathogen interactions has been extensively studied in recent years, the source of ROS in these systems still remains controversial. There is evidence in the literature for reduced nicotinamide adenine dinucleotide phosphate oxidases, peroxidases, or amine oxidases acting as potential sources of ROS [10]. Nevertheless, it is clear that interAddress correspondence to: John T. Hancock, Department of Biological and Biomedical Sciences, University of the West of England, Coldharbour Lane, Bristol BS16 1QY, UK; Tel: ⫹(44) 117 965-6261; Fax: ⫹(44) 117 976-3871; E-Mail: [email protected] 773
774
R. DESIKAN et al.
reveal that the expression of several Arabidopsis genes, including ones previously identified as encoding a DNAdamage repair protein, a protein kinase, and a senescence-related protein, is induced by exogenous H2O2. MATERIALS AND METHODS
Arabidopsis species suspension cultures Cell suspension cultures of Arabidopsis thaliana var. Landsberg erecta were maintained as described previously [13]. H2O2 was added to cells at the indicated concentrations and times as stated in Results. For controls, appropriate volumes of sterile distilled water were added to cells. Isolation of RNA Total RNA was isolated from Arabidopsis cell suspension cultures treated with or without 20 mM of H2O2 as described previously [14]. Total RNA was treated with DNAase (10 U; Promega, Southampton, UK) to remove any contaminating DNA before differential display. Messenger RNA (mRNA) was isolated from total RNA using Dynabeads (Dynal, Merseyside, UK) according to the manufacturer’s guidelines. Differential display of RNA Differential display was performed on both total RNA and mRNA using an mRNA differential display kit (GenHunter Corp., Nashville, TN, USA). For reverse transcription, 2 l of RNA (0.1 g/l of total or mRNA) was mixed with 2 l of the anchor primer H-T11M (2 M, where H ⫽ AAGC and M ⫽ G/A/C), 4 l of 5 ⫻ RT buffer (125 mM of Tris-HCl [pH 8.3], 188 mM of KCl, 7.5 mM of MgCl2, 25 mM of dithiothreitol), 1.6 l of deoxy nucleotide triphosphate (dNTP) mix (250 M), and sterile distilled water to make the final volume 19 l. The mixture was heated to 65°C for 5 min, followed by heating at 37°C for 10 min. A total of 1 l of Moloney murine leukemia virus (MMLV) RT (100 U/l) was added to each tube, and the reaction progressed at 37°C for 50 min, followed by heating at 75°C for 5 min and cooling on ice. The reactions were stored at ⫺20°C for further use in PCR. PCR was performed for each primer combination in 20 l final volume reactions containing 2 l of the complementary DNA (cDNA) synthesis reaction, 10 l of sterile distilled water, 2 l of 10⫻ PCR buffer (100 mM of Tris-HCl [pH 8.4], 500 mM of KCl, 15 mM of MgCl2, 0.01% gelatin), 1.6 l of dNTP mix (25 M), 2 l of H-AP primer (2 M), 2 l of anchor primer H-T11M (as for the RT step), 0.2 l of ␣-[33P] deoxy-
adenosine triphosphate (specific activity of 110 TBq/ mmol), 0.2 l of Taq polymerase (5 U/l of Taq Gold; Bio/Gene, Bedfordshire, UK). Amplifications were performed for 40 cycles at 94°C for 30 s, 40°C for 2 min, 72°C for 30 s, and 72°C for 5 min. The H-AP primers used in combination with the anchor primers were: HAP1: 5⬘ AAGCTTGATTGCC 3⬘, H-AP2: 5⬘ AAGCTTCGACTGT 3⬘, and H-AP4: 5⬘ AAGCTTCTCAACG 3⬘. cDNA fragments amplified by PCR were separated on a 6% denaturing polyacrylamide gel in Tris-borate buffer used for DNA sequencing. The gel was blotted onto 3MM Whatman paper without fixing, dried, and exposed to autoradiography. Recovery of bands, reamplification of cDNA, cloning, and sequencing The dried gel was aligned with the autoradiogram, and cDNA bands of interest were excised from the gel. The gel slice (along with the Whatman 3MM paper) was soaked in 100 l of sterile distilled water for 10 min and then boiled for 15 min. cDNA was ethanol-precipitated in the presence of glycogen and redissolved in sterile water. For reamplification, 4 l of the cDNA eluted from the gel was used as a template in a PCR reaction of 40 l performed using the same primer set as that employed in the differential display reaction. PCR conditions were as described previously for RT-PCR, except that 20 M of dNTP mix was used instead of 2 M and no radioisotopes were used. Amplified products were analyzed by electrophoresis on a 1% agarose gel. cDNA was cloned into a pCRII vector (TA cloning kit; Invitrogen, NV Leek, The Netherlands) and used as either probes for Northern blot analysis or templates for sequencing (T7 sequencing kit; Pharmacia-Biotech, Hertfordshire, UK). Sequence analysis was performed using BLAST homology searches on the Arabidopsis database (http://genome-www.stanford.edu/Arabidopsis/). Northern blot analysis Northern blot analysis of Arabidopsis total RNA isolated from cells treated with 20 mM of H2O2 for various lengths of time was performed as described earlier [14], with the following modifications. Total RNA (25 g) blotted onto a nylon membrane was prehybridized and hybridized at 42°C overnight in a formamide buffer containing 5⫻ SSPE (a 20⫻ SSPE solution contains 3.6 M of NaCl, 0.2 M of NaH2PO4, 0.02 M of Na2 ethylene diaminetetraacetate, pH 7.4), 5⫻ Denhardt’s solution (a 50⫻ Denhardt’s solution contains 1% [wt/vol] polyvinylpyrrolidone, 1% [wt/vol] bovine serum albumin fraction V, 1% [wt/vol] Ficoll 400), 1% (wt/vol) sodium
H2O2-induced gene expression
775
dodecyl sulfate (SDS), 50 mM of NaH2PO4 (pH 6.8), 10% dextran sulfate, 100 g/ml of denatured salmon sperm DNA, and 50% formamide. Reamplified cDNA products eluted from the agarose gel were used as hybridization probes. After hybridization, the blots were washed in a solution of 2⫻ SSC (a 20⫻ SSC solution contains 3 M NaCl, 0.3 M trisodium citrate) , 0.1% SDS at 65°C. To use the same blots for reprobing, the membranes were stripped in a solution of boiling 0.1% SDS, and hybridization was performed as before. Equivalent RNA loadings were confirmed by ethidium bromide staining of the gel. RESULTS
Differentially expressed genes in response to H2O2 Previous work in our laboratory has shown that exogenous H2O2 induces the expression of genes encoding glutathione S-transferase, a family of enzymes protective against oxidative stress, and PAL (phenylalanine ammonia-lyase) involved in the phenylpropanoid biosynthetic pathway [14] as well as the homologue of the gp91-phox subunit of the reduced nicotinamide adenine dinucleotide phosphate oxidase [21], a potential source of H2O2. Induction of these genes occurred between 30 min and 2 h, with concentrations of H2O2 ranging from 5 to 20 mM. The concentration of H2O2 used in our studies is well below the high levels (1 M) estimated in other plant systems during elicitor challenge [22]. Therefore, for differential display, Arabidopsis suspension cultures were treated with 20 mM of H2O2 for 1 and 2 h. Subsequent to H2O2 treatment, total RNA and mRNA were isolated from these cells and also from control-treated cells. Differential display was performed on both total RNA (DNAase-treated) and mRNA samples using random primer combinations as described in the Materials and Methods section, and the products were analyzed on a 6% polyacrylamide sequencing gel. As shown in Fig. 1, a number of PCR products were present in abundance on a differential display gel. The expression of most of these bands did not change after treatment of the cells with H2O2, suggesting that these bands represent constitutively expressed genes. The abundance of some bands increased or decreased if the cells had been treated with H2O2, however, suggesting that the expression of these genes is modulated by H2O2 in some way (Fig. 1, highlighted by the arrow). To confirm that the PCR products were genuine and to estimate more accurately the size of the products, the bands were cut from the gel and used for a second round of PCR as described in Materials and Methods. These PCR products were subsequently used as probes for Northern blot analysis and sequencing.
Fig. 1. An autoradiograph of a typical differential display gel. Differential display was performed on cDNA derived from both total RNA and mRNA isolated from A. thaliana suspension culture cells treated with water (C) or 20 mM of H2O2 for either 1 h (1) or 2 h (2). The arrow indicates a cDNA induced by H2O2 in both total RNA and mRNA-derived samples.
Using this procedure, four differential display products were identified as representing genes whose expression was responsive to pretreatment of cells with 20 mM of H2O2. The clones were named according to the primer combinations used in the differential display. For example, the product subsequently referred to as 4G1 was one among those generated using primers H-AP4 and H-T11G. The primers used and product sizes obtained are given in Table 1. Northern blot analysis using the differential display products To confirm that the products identified by differential display did indeed represent genes whose expression is modulated by H2O2, the PCR products from the second round of PCR were used as probes for Northern blot analysis of RNA extracted from Arabidopsis suspension cultures that had been pretreated with 20 mM of H2O2 for varying lengths of time. As shown in Fig. 2, the abundance of mRNA hybridizing to clone 1G1 increased Table 1. Differential Display Products Obtained by Reamplification Product 1G1 4G1 4G2 2A1
Primers used H-AP1; H-AP4; H-AP4; H-AP2;
H-T11G H-T11G H-T11G H-T11A
PCR product size 220 330 320 250
bp bp bp bp
776
R. DESIKAN et al.
Fig. 2. Expression of clone 1G1. Northern blot analysis was performed on total RNA derived from Arabidopsis cells treated with water (C) or 20 mM of H2O2 for either 1 h (1) or 2 h (2) using a [32P]-labelled 1G1 cDNA as a probe. The lower panel indicates ribosomal RNA to demonstrate equal loading on an ethidium bromide–stained gel.
after 1 h of treatment with 20 mM of H2O2, with a further increase after 2 h. The RNA profile demonstrates equal gel loading. The expression profiles of the other genes identified by differential display are shown in Fig. 3. The expression of 4G1 increased after 30 min of treatment with H2O2, peaked at 1 h, and then decreased by 5 h. The expression of 4G2 also increased after 30 min but decreased thereafter. Low levels of expression of this gene were seen in the control cells, and after 5 h of H2O2 treatment, the amount of 4G2 mRNA seemed to be reduced. Expression of 2A1 was also seen to be consti-
Fig. 4. Sequence alignment of differential display clones. Amino acid alignment of predicted translation products of clones 4G1, 4G2, and 2A1 with DRT112 (GenBank Acc. No. P42699), APK2b (Acc. No. D88207), SAG21 (Acc. No. AF053065), and SSP (Acc. No. AF069298) performed using Bestfit analysis (GCG, Wisconsin).
tutive, albeit at low levels, but after H2O2 treatment, the content of 2A1 mRNA increased and peaked at 2 h, followed by a subsequent decrease after 5 h. As a control, the same blot was stripped and probed with PAL; the PAL mRNA content increased with time after H2O2 treatment as reported previously [14]. Sequence analysis of the differential display products The differential display products that increased in abundance after pretreatment with H2O2 were sequenced, and the sequences were analyzed using the BLAST homology search facility in the Arabidopsis database. The product 1G1 showed 100% sequence homology to an A. thaliana genomic DNA sequence (Acc. No. AC002986) to which no function has yet been assigned. The products 4G1, 4G2, and 2A1 possessed sequence homology to an Arabidopsis DNA-damage repair protein, DRT112 (Acc. No. P42699); an Arabidopsis serine/threonine protein kinase gene, APK2b (Acc. No. D88207); and an Arabidopsis late embryogenesis-abundant protein, SAG21 (Acc. No. AF053065), respectively. The sequence alignments are shown in Fig. 4. The gene product designated as 2A1 also had homology to another protein similar to several small proteins (SSP) that are induced by heat, auxin, ethylene, and wounding (Acc. No. AF069298). DISCUSSION
Fig. 3. Expression of differential display clones. Northern blot analysis was performed on total RNA derived from Arabidopsis cells treated with water (C) or 20 mM of H2O2 for various lengths of time (indicated in hours). The blot was probed consecutively with [32P]-labelled 4G1, 4G2, 2A1 cDNA, or a PAL1 genomic clone.
The role of H2O2 as a signaling molecule in plants has now been well established [23,24] in contrast to the traditional role of ROS having relatively nonspecific and harmful effects on cell physiology. The generation of superoxide and H2O2 by plant cells is an early response to many biotic or abiotic stresses [5–10], instrumental in
H2O2-induced gene expression
mediating cellular protection against such environmental stimuli. Previous work in our laboratory has shown that H2O2, generated by the oxidative burst after elicitor challenge of Arabidopsis cells, induces the expression of defenserelated genes such as GST and PAL, as well as inducing programmed cell death [14]. It has also been demonstrated in a soybean model that H2O2 orchestrates defense responses, inducing expression of cellular protectant genes as well as initiating programmed cell death [25]. Characterization of the patterns of gene expression induced by H2O2 in plant cells is essential to the identification of genes involved in regulating programmed cell death, a phenomenon that is important in plant defense and other plant developmental processes, including leaf senescence [26]. Differential display is a powerful tool to identify rare mRNA in a given cell under a defined situation. In this study, differential display was used to identify four genes induced by H2O2 in Arabidopsis suspension cultures. Expression of these genes has not previously been reported to be modulated by H2O2 or other ROS. The first clone described, 1G1, has sequence homology to a genomic sequence in Arabidopsis. At present, there is no assigned function to this sequence, but the fact that an mRNA transcript is present within cells demonstrates that this genomic sequence is indeed a coding sequence. As the amount of this transcript is increased in response to exogenous H2O2, it is likely that it encodes a protein with a cellular function related to oxidative stress. The second clone described, 4G1, is identical to an Arabidopsis cDNA encoding a DNA-damage repair protein, DRT112 [27]. This protein partially restored recombination proficiency and DNA-damage resistance to Escherichia coli mutants lacking DNA recombination activities [27]. ROS are known to induce DNA damage in eukaryotes, resulting in a wide variety of modifications [28]. Highly efficient mechanisms have evolved to resist DNA damage, however, including the induction of DNA-repair proteins such as DRT112. In the Arabidopsis suspension culture system, treatment of cells with H2O2 induces the expression of DRT112 (4G1), potentially as a mechanism to protect cells from DNA damage caused by oxidative stress. Interestingly, a cDNA sequence deposited in the database with similarity to DRT112 (Acc. No. AA389764) is also induced by salt stress, another factor inducing oxidative stress [29], suggesting that different abiotic stresses may lead to the induction of these genes. The third clone, 4G2, is identical to an Arabidopsis serine/threonine protein kinase gene APK2b identified by an in vivo binding procedure using a floral homeotic gene product encoding a putative transcription factor,
777
AGAMOUS [30]. The exact function of APK2b has not yet been identified, although the predicted protein sequence is similar to that of a novel protein kinase (APK1) that phosphorylates tyrosine, threonine, and serine residues [31]. Based on structural and expression pattern analyses, it has been hypothesized that both APK1 and APK2 gene products may play a role in signal transduction processes [30,31]. In previous work in our laboratory, we showed that protein phosphorylation is essential for the stimulation of the oxidative burst in Arabidopsis suspension cultures [13]. Recently, we also identified a mitogen-activated protein kinase activated by H2O2 in these cells [32], and preliminary data indicate a role for mitogen-activied protein kinases in programmed cell death [33]. Thus, it is possible that the induction of a serine/threonine kinase, APK2b, by H2O2 is important for the downstream signaling that leads to programmed cell death and other H2O2-induced responses. The final clone reported here, 2A1, is similar to an Arabidopsis late embryogenesis-abundant protein homologue, SAG21 [34]. Unlike the other clones described here, which are identical to the sizes of mRNA previously reported, the size of the 2A1 transcript (630 base pairs [bp]) is somewhat larger than that of the SAG21 mRNA sequence deposited in the database (412 bp), suggesting that this 2A1 clone may be different from SAG21. Nevertheless, the predicted amino acid translation of this sequence is quite similar to SAG21 within the portion analyzed, suggesting similarities in function. A full-length clone is required to determine the true degree of homology between 2A1 and SAG21. SAG21 is a gene expressed in senescing Arabidopsis leaves [34]; late embryogenesis-abundant proteins are induced in response to dehydration stress in developing seeds as well as during exposure to various stresses such as drought, cold, salt, wounding, and plant hormones [35]. In addition to having homology to the gene SAG21, 2A1 has homology to a protein (deposited as a translated genomic sequence in the Arabidopsis database; Acc. No. AF069298) similar to SSP induced by various abiotic stresses such as heat, auxin, ethylene, and wounding. Interestingly, some of these forms of abiotic stresses are also associated with increased levels of H2O2 [5,8,36]. Thus, it might be expected that H2O2 would induce the expression of such stress-related proteins. Senescence is now increasingly viewed as a form of programmed cell death [26]; therefore, it may not be surprising that SAG homologues are induced by H2O2 during programmed cell death. The data presented here show that previously characterized Arabidopsis genes are induced by H2O2, further emphasizing the involvement of H2O2 in both abiotic and biotic stress responses. Clearly, full-length clones and expression studies are needed to analyze fully the function of the genes identified here. Furthermore, the con-
778
R. DESIKAN et al.
tinued use of differential display using new primer combinations will lead to the characterization of more genes that are induced by H2O2 in A. thaliana. REFERENCES [1] Scandalios, J. G., ed. Oxidative stress and the molecular biology of antioxidant defenses. New York: Cold Spring Harbor Laboratory Press; 1997. [2] Finkel, T. Oxygen radicals and signaling. Curr. Opin. Cell Biol. 10:248 –253; 1998. [3] Finkel, T. Signal transduction by reactive oxygen species in non-phagocytic cells. J. Leuk. Biol. 65:337–340; 1999. [4] Hancock, J. T. Superoxide, hydrogen peroxide and nitric oxide as signalling molecules: their production and role in disease. Br. J. Biomed. Sci. 54:38 – 46; 1997. [5] Bartosz, G. Oxidative stress in plants. Acta Physiol. Plant. 19: 47– 64; 1997. [6] Prasad, T. K.; Anderson, M. D.; Martin, B. A.; Stewart, C. R. Evidence for chilling-induced oxidative stress in maize seedlings and a regulatory role for hydrogen peroxide. Plant Cell 6:65–74; 1994. [7] Foyer, C. H.; Lopez-Delgado, H.; Dat, J. F.; Scott, I. M. Hydrogen peroxide- and glutathione-associated mechanisms of acclimatory stress tolerance and signalling. Physiol. Plant. 100:241–254; 1997. [8] Vallelian-Bindeschendler, L.; Schweizer, P.; Mosinger, E.; Metraux, J.-P. Heat-induced resistance in barley to powdery mildew (Blumeria graminis f. sp. hordei) is associated with a burst of active oxygen species. Physiol. Mol. Plant Pathol. 52:185–199; 1998. [9] Van Camp, W.; Van Montagu, M.; Inze, D. H2O2 and NO: redox signals in disease resistance. Trends Plant Sci. 3:330 –334; 1998. [10] Wojtaszek, P. Oxidative burst: an early plant response to pathogen infection. Biochem. J. 322:681– 692; 1997. [11] Yang, Y.; Shah, J.; Klessig, D. F. Signal perception and transduction in plant defense responses. Genes Dev. 11:1621–1639; 1997. [12] Benhamou, N. Elicitor-induced plant defence pathways. Trends Plant Sci. 1:233–240; 1996. [13] Desikan, R.; Hancock, J. T.; Coffey, M. J.; Neill, S. J. Generation of active oxygen in elicited cells of Arabidopsis thaliana is mediated by a NADPH oxidase-like enzyme. FEBS Lett. 382: 213–217; 1996. [14] Desikan, R.; Reynolds, A.; Hancock, J. T.; Neill, S. J. Harpin and hydrogen peroxide both initiate programmed cell death but have differential effects on defence gene expression in Arabidopsis thaliana suspension cultures. Biochem. J. 330:115–120; 1998. [15] Lamb, C.; Dixon, R. A. The oxidative burst in plant disease resistance. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:251– 275; 1997. [16] Liang, P.; Pardee, A. B. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257:967–971; 1992. [17] Truesdell, G. M.; Dickman, M. B. Isolation of pathogen/stressinducible cDNAs from alfalfa by mRNA differential display. Plant Mol. Biol. 33:737–743; 1997. [18] Seehaus, K.; Tenhaken, R. Cloning of genes by mRNA differential display induced during the hypersensitive reaction of soybean after inoculation with Pseudomonas syringae pv. glycinea. Plant Mol. Biol. 38:1225–1234; 1998. [19] Sharma, Y. K.; Davis, K. R. Isolation of a novel Arabidopsis ozone-induced cDNA by differential display. Plant Mol. Biol. 29:91–98; 1995.
[20] Callard, D.; Axelos, M.; Mazzolini, L. Novel molecular markers for late phases of the growth cycle of Arabidopsis thaliana cell-suspension cultures are expressed during organ senescence. Plant Physiol. 112:705–715; 1996. [21] Desikan, R.; Burnett, E. C.; Hancock, J. T.; Neill, S. J. Harpin and hydrogen peroxide induce the expression of a homologue of gp91-phox in Arabidopsis thaliana suspension cultures. J. Exp. Bot. 49:1767–1771; 1998. [22] Jacks, T. J.; Davidonis, G. H. Superoxide, hydrogen peroxide, and the respiratory burst of fungally infected plant cells. Mol. Cell. Biochem. 158:77–79; 1996. [23] Alvarez, M. E.; Lamb, C. Oxidative burst-mediated defense responses in plant disease resistance. In: Scandalios, J. G., ed. Oxidative stress and the molecular biology of antioxidant defenses. New York: Cold Spring Harbor Laboratory Press; 1997: 815– 839. [24] Doke, N. The oxidative burst: roles in signal transduction and plant stress. In: Scandalios, J. G., ed. Oxidative stress and the molecular biology of antioxidant defenses. New York: Cold Spring Harbor Laboratory Press; 1997:785– 813. [25] Levine, A.; Tenhaken, R.; Dixon, R.; Lamb, C. H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell 79:583–595; 1994. [26] Pennell, R. I.; Lamb, C. Programmed cell death in plants. Plant Cell 9:1157–1168; 1997. [27] Pang, Q.; Hays, J. B.; Rajagopal, I. Two cDNAs from the plant Arabidopsis thaliana that partially restore recombination proficiency and DNA-damage resistance to E. coli mutants lacking recombination-intermediate-resolution activities. Nucleic Acids Res. 21:1647–1653; 1993. [28] Laval, J. Role of DNA repair enzymes in the cellular resistance to oxidative stress. Pathol. Biol. 44:14 –24; 1996. [29] Ben-Hayyim, G.; Holland, D.; Eshdat, Y. Salt-induced proteins related to oxidative stress: PHGPX and other proteins of the Halliwell-Asada cycle. In: Clavert, C.; Smallwood, M.; Bowles, D., eds. Plant responses to environmental stress. Oxford, UK: BIOS Scientific Publishers; 1999:185–189. [30] Ito, T.; Takahashi, N.; Shimura, Y.; Okada, K. A serine/threonine protein kinase gene isolated by an in vivo binding procedure using the Arabidopsis floral homeotic gene product, AGAMOUS. Plant Cell Physiol. 38:248 –258; 1997. [31] Hirayama, T.; Oka, A. Novel protein kinase of Arabidopsis thaliana (APK1) that phosphorylates tyrosine, serine and threonine. Plant Mol. Biol. 20:653– 662; 1992. [32] Desikan, R.; Clarke, A.; Hancock, J. T.; Neill, S. J. Hydrogen peroxide activates a MAP kinase-like enzyme in Arabidopsis thaliana suspension cultures. J. Exp. Bot. 50:1863–1866; 2000. [33] Desikan, R.; Clarke, A.; Atherfold, P.; Hancock, J. T.; Neill, S. J. Harpin induces mitogen-activated protein kinase activity during defence responses in Arabidopsis thaliana suspension cultures. Planta. 210:97–103; 1999. [34] Weaver, L. M.; Gan, S.; Quirino, B.; Amasino, R. M. A comparison of the expression patterns of several senescence-associated genes in response to stress and hormone treatment. Plant Mol. Biol. 37:455– 469; 1998. [35] Bartels, D. Late embryogenesis abundant (LEA) proteins: expression and regulation in the resurrection plant Craterostigma plantagineum. In: Smallwood, M. F.; Calvert, C. M.; Bowles, D. J., eds. Plant responses to environmental stress. Oxford, UK: BIOS Scientific Publishers; 1999:153–158. [36] Orozco-Cardenas, M.; Ryan, C. A. Hydrogen peroxide is generated systemically in plant leaves by wounding and systemin via the octadecanoid pathway. Proc. Natl. Acad. Sci. USA 96:6553– 6557; 1999.
PII S0891-5849(00)00157-X
Original Contribution HYDROGEN PEROXIDE–INDUCED GENE EXPRESSION IN ARABIDOPSIS THALIANA RADHIKA DESIKAN, STEVEN J. NEILL,
and JOHN
T. HANCOCK
Department of Biological and Biomedical Sciences, University of the West of England, Bristol, UK (Received 22 October 1999; Revised 21 December 1999; Accepted 28 December 1999)
Abstract—Hydrogen peroxide (H2O2) is generated in plants after exposure to a variety of biotic and abiotic stresses, and has been shown to induce a number of cellular responses. Previously, we showed that H2O2 generated during plant-elicitor interactions acts as a signaling molecule to induce the expression of defense genes and initiate programmed cell death in Arabidopsis thaliana suspension cultures. Here, we report for the first time the identification by RNA differential display of four genes whose expression is induced by H2O2. These include genes that have sequence homology to previously identified Arabidopsis genes encoding a late embryogenesis-abundant protein, a DNA-damage repair protein, and a serine/threonine kinase. Their putative roles in H2O2-induced defense responses are discussed. © 2000 Elsevier Science Inc. Keywords—Arabidopsis thaliana, Differential display, DNA-damage repair protein, Gene expression, Hydrogen peroxide, Protein kinase, Senescence-related protein, Free radical
INTRODUCTION
action of a plant cell with either a pathogen or a pathogen-derived elicitor does result in the generation of H2O2, and H2O2 has been shown to induce gene expression and programmed cell death in such cells [11–15]. There is considerable interest in monitoring the changes in gene expression profile in eukaryotes in response to developmental or environmental signals, so as to identify key genes essential for cell functioning. Differential display of RNA is a powerful technique based on reverse-transcriptase polymerase chain reaction (RTPCR) using random primer combinations, such that large proportions of a population of genes expressed in a specific tissue or cell type can be analyzed. Since its original description [16], numerous studies have used this technique to identify differentially expressed genes. In plants, this technique has been used to identify genes differentially expressed in response to pathogen challenge [17,18] and exposure to ozone [19] as well as during senescence [20]. Previous work in our laboratory has shown that Arabidopsis cells generate H2O2 upon challenge by elicitors [13] (probably via the dismutation of superoxide), and that H2O2 itself induces the expression of defense-related genes as well as initiating programmed cell death [14]. Here, we report the use of RNA differential display to
Reactive oxygen species (ROS) such as superoxide (O2•⫺) and hydrogen peroxide (H2O2) are generated under stress situations by a variety of mechanisms in animal and plant cells. Although ROS were originally thought to be only harmful to cells [1], recent data point to cell signaling roles for ROS in both plants and animals [2– 4]. In plants, ROS are generated in response to both abiotic and biotic stresses such as drought, exposure to ultraviolet light and ozone, extremes of temperature and pathogen challenge [5], whilst exogenous H2O2 can induce tolerance to chilling, high temperatures, and biotic stress [6 –9]. Although the role of ROS in plant-pathogen interactions has been extensively studied in recent years, the source of ROS in these systems still remains controversial. There is evidence in the literature for reduced nicotinamide adenine dinucleotide phosphate oxidases, peroxidases, or amine oxidases acting as potential sources of ROS [10]. Nevertheless, it is clear that interAddress correspondence to: John T. Hancock, Department of Biological and Biomedical Sciences, University of the West of England, Coldharbour Lane, Bristol BS16 1QY, UK; Tel: ⫹(44) 117 965-6261; Fax: ⫹(44) 117 976-3871; E-Mail: [email protected] 773
774
R. DESIKAN et al.
reveal that the expression of several Arabidopsis genes, including ones previously identified as encoding a DNAdamage repair protein, a protein kinase, and a senescence-related protein, is induced by exogenous H2O2. MATERIALS AND METHODS
Arabidopsis species suspension cultures Cell suspension cultures of Arabidopsis thaliana var. Landsberg erecta were maintained as described previously [13]. H2O2 was added to cells at the indicated concentrations and times as stated in Results. For controls, appropriate volumes of sterile distilled water were added to cells. Isolation of RNA Total RNA was isolated from Arabidopsis cell suspension cultures treated with or without 20 mM of H2O2 as described previously [14]. Total RNA was treated with DNAase (10 U; Promega, Southampton, UK) to remove any contaminating DNA before differential display. Messenger RNA (mRNA) was isolated from total RNA using Dynabeads (Dynal, Merseyside, UK) according to the manufacturer’s guidelines. Differential display of RNA Differential display was performed on both total RNA and mRNA using an mRNA differential display kit (GenHunter Corp., Nashville, TN, USA). For reverse transcription, 2 l of RNA (0.1 g/l of total or mRNA) was mixed with 2 l of the anchor primer H-T11M (2 M, where H ⫽ AAGC and M ⫽ G/A/C), 4 l of 5 ⫻ RT buffer (125 mM of Tris-HCl [pH 8.3], 188 mM of KCl, 7.5 mM of MgCl2, 25 mM of dithiothreitol), 1.6 l of deoxy nucleotide triphosphate (dNTP) mix (250 M), and sterile distilled water to make the final volume 19 l. The mixture was heated to 65°C for 5 min, followed by heating at 37°C for 10 min. A total of 1 l of Moloney murine leukemia virus (MMLV) RT (100 U/l) was added to each tube, and the reaction progressed at 37°C for 50 min, followed by heating at 75°C for 5 min and cooling on ice. The reactions were stored at ⫺20°C for further use in PCR. PCR was performed for each primer combination in 20 l final volume reactions containing 2 l of the complementary DNA (cDNA) synthesis reaction, 10 l of sterile distilled water, 2 l of 10⫻ PCR buffer (100 mM of Tris-HCl [pH 8.4], 500 mM of KCl, 15 mM of MgCl2, 0.01% gelatin), 1.6 l of dNTP mix (25 M), 2 l of H-AP primer (2 M), 2 l of anchor primer H-T11M (as for the RT step), 0.2 l of ␣-[33P] deoxy-
adenosine triphosphate (specific activity of 110 TBq/ mmol), 0.2 l of Taq polymerase (5 U/l of Taq Gold; Bio/Gene, Bedfordshire, UK). Amplifications were performed for 40 cycles at 94°C for 30 s, 40°C for 2 min, 72°C for 30 s, and 72°C for 5 min. The H-AP primers used in combination with the anchor primers were: HAP1: 5⬘ AAGCTTGATTGCC 3⬘, H-AP2: 5⬘ AAGCTTCGACTGT 3⬘, and H-AP4: 5⬘ AAGCTTCTCAACG 3⬘. cDNA fragments amplified by PCR were separated on a 6% denaturing polyacrylamide gel in Tris-borate buffer used for DNA sequencing. The gel was blotted onto 3MM Whatman paper without fixing, dried, and exposed to autoradiography. Recovery of bands, reamplification of cDNA, cloning, and sequencing The dried gel was aligned with the autoradiogram, and cDNA bands of interest were excised from the gel. The gel slice (along with the Whatman 3MM paper) was soaked in 100 l of sterile distilled water for 10 min and then boiled for 15 min. cDNA was ethanol-precipitated in the presence of glycogen and redissolved in sterile water. For reamplification, 4 l of the cDNA eluted from the gel was used as a template in a PCR reaction of 40 l performed using the same primer set as that employed in the differential display reaction. PCR conditions were as described previously for RT-PCR, except that 20 M of dNTP mix was used instead of 2 M and no radioisotopes were used. Amplified products were analyzed by electrophoresis on a 1% agarose gel. cDNA was cloned into a pCRII vector (TA cloning kit; Invitrogen, NV Leek, The Netherlands) and used as either probes for Northern blot analysis or templates for sequencing (T7 sequencing kit; Pharmacia-Biotech, Hertfordshire, UK). Sequence analysis was performed using BLAST homology searches on the Arabidopsis database (http://genome-www.stanford.edu/Arabidopsis/). Northern blot analysis Northern blot analysis of Arabidopsis total RNA isolated from cells treated with 20 mM of H2O2 for various lengths of time was performed as described earlier [14], with the following modifications. Total RNA (25 g) blotted onto a nylon membrane was prehybridized and hybridized at 42°C overnight in a formamide buffer containing 5⫻ SSPE (a 20⫻ SSPE solution contains 3.6 M of NaCl, 0.2 M of NaH2PO4, 0.02 M of Na2 ethylene diaminetetraacetate, pH 7.4), 5⫻ Denhardt’s solution (a 50⫻ Denhardt’s solution contains 1% [wt/vol] polyvinylpyrrolidone, 1% [wt/vol] bovine serum albumin fraction V, 1% [wt/vol] Ficoll 400), 1% (wt/vol) sodium
H2O2-induced gene expression
775
dodecyl sulfate (SDS), 50 mM of NaH2PO4 (pH 6.8), 10% dextran sulfate, 100 g/ml of denatured salmon sperm DNA, and 50% formamide. Reamplified cDNA products eluted from the agarose gel were used as hybridization probes. After hybridization, the blots were washed in a solution of 2⫻ SSC (a 20⫻ SSC solution contains 3 M NaCl, 0.3 M trisodium citrate) , 0.1% SDS at 65°C. To use the same blots for reprobing, the membranes were stripped in a solution of boiling 0.1% SDS, and hybridization was performed as before. Equivalent RNA loadings were confirmed by ethidium bromide staining of the gel. RESULTS
Differentially expressed genes in response to H2O2 Previous work in our laboratory has shown that exogenous H2O2 induces the expression of genes encoding glutathione S-transferase, a family of enzymes protective against oxidative stress, and PAL (phenylalanine ammonia-lyase) involved in the phenylpropanoid biosynthetic pathway [14] as well as the homologue of the gp91-phox subunit of the reduced nicotinamide adenine dinucleotide phosphate oxidase [21], a potential source of H2O2. Induction of these genes occurred between 30 min and 2 h, with concentrations of H2O2 ranging from 5 to 20 mM. The concentration of H2O2 used in our studies is well below the high levels (1 M) estimated in other plant systems during elicitor challenge [22]. Therefore, for differential display, Arabidopsis suspension cultures were treated with 20 mM of H2O2 for 1 and 2 h. Subsequent to H2O2 treatment, total RNA and mRNA were isolated from these cells and also from control-treated cells. Differential display was performed on both total RNA (DNAase-treated) and mRNA samples using random primer combinations as described in the Materials and Methods section, and the products were analyzed on a 6% polyacrylamide sequencing gel. As shown in Fig. 1, a number of PCR products were present in abundance on a differential display gel. The expression of most of these bands did not change after treatment of the cells with H2O2, suggesting that these bands represent constitutively expressed genes. The abundance of some bands increased or decreased if the cells had been treated with H2O2, however, suggesting that the expression of these genes is modulated by H2O2 in some way (Fig. 1, highlighted by the arrow). To confirm that the PCR products were genuine and to estimate more accurately the size of the products, the bands were cut from the gel and used for a second round of PCR as described in Materials and Methods. These PCR products were subsequently used as probes for Northern blot analysis and sequencing.
Fig. 1. An autoradiograph of a typical differential display gel. Differential display was performed on cDNA derived from both total RNA and mRNA isolated from A. thaliana suspension culture cells treated with water (C) or 20 mM of H2O2 for either 1 h (1) or 2 h (2). The arrow indicates a cDNA induced by H2O2 in both total RNA and mRNA-derived samples.
Using this procedure, four differential display products were identified as representing genes whose expression was responsive to pretreatment of cells with 20 mM of H2O2. The clones were named according to the primer combinations used in the differential display. For example, the product subsequently referred to as 4G1 was one among those generated using primers H-AP4 and H-T11G. The primers used and product sizes obtained are given in Table 1. Northern blot analysis using the differential display products To confirm that the products identified by differential display did indeed represent genes whose expression is modulated by H2O2, the PCR products from the second round of PCR were used as probes for Northern blot analysis of RNA extracted from Arabidopsis suspension cultures that had been pretreated with 20 mM of H2O2 for varying lengths of time. As shown in Fig. 2, the abundance of mRNA hybridizing to clone 1G1 increased Table 1. Differential Display Products Obtained by Reamplification Product 1G1 4G1 4G2 2A1
Primers used H-AP1; H-AP4; H-AP4; H-AP2;
H-T11G H-T11G H-T11G H-T11A
PCR product size 220 330 320 250
bp bp bp bp
776
R. DESIKAN et al.
Fig. 2. Expression of clone 1G1. Northern blot analysis was performed on total RNA derived from Arabidopsis cells treated with water (C) or 20 mM of H2O2 for either 1 h (1) or 2 h (2) using a [32P]-labelled 1G1 cDNA as a probe. The lower panel indicates ribosomal RNA to demonstrate equal loading on an ethidium bromide–stained gel.
after 1 h of treatment with 20 mM of H2O2, with a further increase after 2 h. The RNA profile demonstrates equal gel loading. The expression profiles of the other genes identified by differential display are shown in Fig. 3. The expression of 4G1 increased after 30 min of treatment with H2O2, peaked at 1 h, and then decreased by 5 h. The expression of 4G2 also increased after 30 min but decreased thereafter. Low levels of expression of this gene were seen in the control cells, and after 5 h of H2O2 treatment, the amount of 4G2 mRNA seemed to be reduced. Expression of 2A1 was also seen to be consti-
Fig. 4. Sequence alignment of differential display clones. Amino acid alignment of predicted translation products of clones 4G1, 4G2, and 2A1 with DRT112 (GenBank Acc. No. P42699), APK2b (Acc. No. D88207), SAG21 (Acc. No. AF053065), and SSP (Acc. No. AF069298) performed using Bestfit analysis (GCG, Wisconsin).
tutive, albeit at low levels, but after H2O2 treatment, the content of 2A1 mRNA increased and peaked at 2 h, followed by a subsequent decrease after 5 h. As a control, the same blot was stripped and probed with PAL; the PAL mRNA content increased with time after H2O2 treatment as reported previously [14]. Sequence analysis of the differential display products The differential display products that increased in abundance after pretreatment with H2O2 were sequenced, and the sequences were analyzed using the BLAST homology search facility in the Arabidopsis database. The product 1G1 showed 100% sequence homology to an A. thaliana genomic DNA sequence (Acc. No. AC002986) to which no function has yet been assigned. The products 4G1, 4G2, and 2A1 possessed sequence homology to an Arabidopsis DNA-damage repair protein, DRT112 (Acc. No. P42699); an Arabidopsis serine/threonine protein kinase gene, APK2b (Acc. No. D88207); and an Arabidopsis late embryogenesis-abundant protein, SAG21 (Acc. No. AF053065), respectively. The sequence alignments are shown in Fig. 4. The gene product designated as 2A1 also had homology to another protein similar to several small proteins (SSP) that are induced by heat, auxin, ethylene, and wounding (Acc. No. AF069298). DISCUSSION
Fig. 3. Expression of differential display clones. Northern blot analysis was performed on total RNA derived from Arabidopsis cells treated with water (C) or 20 mM of H2O2 for various lengths of time (indicated in hours). The blot was probed consecutively with [32P]-labelled 4G1, 4G2, 2A1 cDNA, or a PAL1 genomic clone.
The role of H2O2 as a signaling molecule in plants has now been well established [23,24] in contrast to the traditional role of ROS having relatively nonspecific and harmful effects on cell physiology. The generation of superoxide and H2O2 by plant cells is an early response to many biotic or abiotic stresses [5–10], instrumental in
H2O2-induced gene expression
mediating cellular protection against such environmental stimuli. Previous work in our laboratory has shown that H2O2, generated by the oxidative burst after elicitor challenge of Arabidopsis cells, induces the expression of defenserelated genes such as GST and PAL, as well as inducing programmed cell death [14]. It has also been demonstrated in a soybean model that H2O2 orchestrates defense responses, inducing expression of cellular protectant genes as well as initiating programmed cell death [25]. Characterization of the patterns of gene expression induced by H2O2 in plant cells is essential to the identification of genes involved in regulating programmed cell death, a phenomenon that is important in plant defense and other plant developmental processes, including leaf senescence [26]. Differential display is a powerful tool to identify rare mRNA in a given cell under a defined situation. In this study, differential display was used to identify four genes induced by H2O2 in Arabidopsis suspension cultures. Expression of these genes has not previously been reported to be modulated by H2O2 or other ROS. The first clone described, 1G1, has sequence homology to a genomic sequence in Arabidopsis. At present, there is no assigned function to this sequence, but the fact that an mRNA transcript is present within cells demonstrates that this genomic sequence is indeed a coding sequence. As the amount of this transcript is increased in response to exogenous H2O2, it is likely that it encodes a protein with a cellular function related to oxidative stress. The second clone described, 4G1, is identical to an Arabidopsis cDNA encoding a DNA-damage repair protein, DRT112 [27]. This protein partially restored recombination proficiency and DNA-damage resistance to Escherichia coli mutants lacking DNA recombination activities [27]. ROS are known to induce DNA damage in eukaryotes, resulting in a wide variety of modifications [28]. Highly efficient mechanisms have evolved to resist DNA damage, however, including the induction of DNA-repair proteins such as DRT112. In the Arabidopsis suspension culture system, treatment of cells with H2O2 induces the expression of DRT112 (4G1), potentially as a mechanism to protect cells from DNA damage caused by oxidative stress. Interestingly, a cDNA sequence deposited in the database with similarity to DRT112 (Acc. No. AA389764) is also induced by salt stress, another factor inducing oxidative stress [29], suggesting that different abiotic stresses may lead to the induction of these genes. The third clone, 4G2, is identical to an Arabidopsis serine/threonine protein kinase gene APK2b identified by an in vivo binding procedure using a floral homeotic gene product encoding a putative transcription factor,
777
AGAMOUS [30]. The exact function of APK2b has not yet been identified, although the predicted protein sequence is similar to that of a novel protein kinase (APK1) that phosphorylates tyrosine, threonine, and serine residues [31]. Based on structural and expression pattern analyses, it has been hypothesized that both APK1 and APK2 gene products may play a role in signal transduction processes [30,31]. In previous work in our laboratory, we showed that protein phosphorylation is essential for the stimulation of the oxidative burst in Arabidopsis suspension cultures [13]. Recently, we also identified a mitogen-activated protein kinase activated by H2O2 in these cells [32], and preliminary data indicate a role for mitogen-activied protein kinases in programmed cell death [33]. Thus, it is possible that the induction of a serine/threonine kinase, APK2b, by H2O2 is important for the downstream signaling that leads to programmed cell death and other H2O2-induced responses. The final clone reported here, 2A1, is similar to an Arabidopsis late embryogenesis-abundant protein homologue, SAG21 [34]. Unlike the other clones described here, which are identical to the sizes of mRNA previously reported, the size of the 2A1 transcript (630 base pairs [bp]) is somewhat larger than that of the SAG21 mRNA sequence deposited in the database (412 bp), suggesting that this 2A1 clone may be different from SAG21. Nevertheless, the predicted amino acid translation of this sequence is quite similar to SAG21 within the portion analyzed, suggesting similarities in function. A full-length clone is required to determine the true degree of homology between 2A1 and SAG21. SAG21 is a gene expressed in senescing Arabidopsis leaves [34]; late embryogenesis-abundant proteins are induced in response to dehydration stress in developing seeds as well as during exposure to various stresses such as drought, cold, salt, wounding, and plant hormones [35]. In addition to having homology to the gene SAG21, 2A1 has homology to a protein (deposited as a translated genomic sequence in the Arabidopsis database; Acc. No. AF069298) similar to SSP induced by various abiotic stresses such as heat, auxin, ethylene, and wounding. Interestingly, some of these forms of abiotic stresses are also associated with increased levels of H2O2 [5,8,36]. Thus, it might be expected that H2O2 would induce the expression of such stress-related proteins. Senescence is now increasingly viewed as a form of programmed cell death [26]; therefore, it may not be surprising that SAG homologues are induced by H2O2 during programmed cell death. The data presented here show that previously characterized Arabidopsis genes are induced by H2O2, further emphasizing the involvement of H2O2 in both abiotic and biotic stress responses. Clearly, full-length clones and expression studies are needed to analyze fully the function of the genes identified here. Furthermore, the con-
778
R. DESIKAN et al.
tinued use of differential display using new primer combinations will lead to the characterization of more genes that are induced by H2O2 in A. thaliana. REFERENCES [1] Scandalios, J. G., ed. Oxidative stress and the molecular biology of antioxidant defenses. New York: Cold Spring Harbor Laboratory Press; 1997. [2] Finkel, T. Oxygen radicals and signaling. Curr. Opin. Cell Biol. 10:248 –253; 1998. [3] Finkel, T. Signal transduction by reactive oxygen species in non-phagocytic cells. J. Leuk. Biol. 65:337–340; 1999. [4] Hancock, J. T. Superoxide, hydrogen peroxide and nitric oxide as signalling molecules: their production and role in disease. Br. J. Biomed. Sci. 54:38 – 46; 1997. [5] Bartosz, G. Oxidative stress in plants. Acta Physiol. Plant. 19: 47– 64; 1997. [6] Prasad, T. K.; Anderson, M. D.; Martin, B. A.; Stewart, C. R. Evidence for chilling-induced oxidative stress in maize seedlings and a regulatory role for hydrogen peroxide. Plant Cell 6:65–74; 1994. [7] Foyer, C. H.; Lopez-Delgado, H.; Dat, J. F.; Scott, I. M. Hydrogen peroxide- and glutathione-associated mechanisms of acclimatory stress tolerance and signalling. Physiol. Plant. 100:241–254; 1997. [8] Vallelian-Bindeschendler, L.; Schweizer, P.; Mosinger, E.; Metraux, J.-P. Heat-induced resistance in barley to powdery mildew (Blumeria graminis f. sp. hordei) is associated with a burst of active oxygen species. Physiol. Mol. Plant Pathol. 52:185–199; 1998. [9] Van Camp, W.; Van Montagu, M.; Inze, D. H2O2 and NO: redox signals in disease resistance. Trends Plant Sci. 3:330 –334; 1998. [10] Wojtaszek, P. Oxidative burst: an early plant response to pathogen infection. Biochem. J. 322:681– 692; 1997. [11] Yang, Y.; Shah, J.; Klessig, D. F. Signal perception and transduction in plant defense responses. Genes Dev. 11:1621–1639; 1997. [12] Benhamou, N. Elicitor-induced plant defence pathways. Trends Plant Sci. 1:233–240; 1996. [13] Desikan, R.; Hancock, J. T.; Coffey, M. J.; Neill, S. J. Generation of active oxygen in elicited cells of Arabidopsis thaliana is mediated by a NADPH oxidase-like enzyme. FEBS Lett. 382: 213–217; 1996. [14] Desikan, R.; Reynolds, A.; Hancock, J. T.; Neill, S. J. Harpin and hydrogen peroxide both initiate programmed cell death but have differential effects on defence gene expression in Arabidopsis thaliana suspension cultures. Biochem. J. 330:115–120; 1998. [15] Lamb, C.; Dixon, R. A. The oxidative burst in plant disease resistance. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:251– 275; 1997. [16] Liang, P.; Pardee, A. B. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257:967–971; 1992. [17] Truesdell, G. M.; Dickman, M. B. Isolation of pathogen/stressinducible cDNAs from alfalfa by mRNA differential display. Plant Mol. Biol. 33:737–743; 1997. [18] Seehaus, K.; Tenhaken, R. Cloning of genes by mRNA differential display induced during the hypersensitive reaction of soybean after inoculation with Pseudomonas syringae pv. glycinea. Plant Mol. Biol. 38:1225–1234; 1998. [19] Sharma, Y. K.; Davis, K. R. Isolation of a novel Arabidopsis ozone-induced cDNA by differential display. Plant Mol. Biol. 29:91–98; 1995.
[20] Callard, D.; Axelos, M.; Mazzolini, L. Novel molecular markers for late phases of the growth cycle of Arabidopsis thaliana cell-suspension cultures are expressed during organ senescence. Plant Physiol. 112:705–715; 1996. [21] Desikan, R.; Burnett, E. C.; Hancock, J. T.; Neill, S. J. Harpin and hydrogen peroxide induce the expression of a homologue of gp91-phox in Arabidopsis thaliana suspension cultures. J. Exp. Bot. 49:1767–1771; 1998. [22] Jacks, T. J.; Davidonis, G. H. Superoxide, hydrogen peroxide, and the respiratory burst of fungally infected plant cells. Mol. Cell. Biochem. 158:77–79; 1996. [23] Alvarez, M. E.; Lamb, C. Oxidative burst-mediated defense responses in plant disease resistance. In: Scandalios, J. G., ed. Oxidative stress and the molecular biology of antioxidant defenses. New York: Cold Spring Harbor Laboratory Press; 1997: 815– 839. [24] Doke, N. The oxidative burst: roles in signal transduction and plant stress. In: Scandalios, J. G., ed. Oxidative stress and the molecular biology of antioxidant defenses. New York: Cold Spring Harbor Laboratory Press; 1997:785– 813. [25] Levine, A.; Tenhaken, R.; Dixon, R.; Lamb, C. H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell 79:583–595; 1994. [26] Pennell, R. I.; Lamb, C. Programmed cell death in plants. Plant Cell 9:1157–1168; 1997. [27] Pang, Q.; Hays, J. B.; Rajagopal, I. Two cDNAs from the plant Arabidopsis thaliana that partially restore recombination proficiency and DNA-damage resistance to E. coli mutants lacking recombination-intermediate-resolution activities. Nucleic Acids Res. 21:1647–1653; 1993. [28] Laval, J. Role of DNA repair enzymes in the cellular resistance to oxidative stress. Pathol. Biol. 44:14 –24; 1996. [29] Ben-Hayyim, G.; Holland, D.; Eshdat, Y. Salt-induced proteins related to oxidative stress: PHGPX and other proteins of the Halliwell-Asada cycle. In: Clavert, C.; Smallwood, M.; Bowles, D., eds. Plant responses to environmental stress. Oxford, UK: BIOS Scientific Publishers; 1999:185–189. [30] Ito, T.; Takahashi, N.; Shimura, Y.; Okada, K. A serine/threonine protein kinase gene isolated by an in vivo binding procedure using the Arabidopsis floral homeotic gene product, AGAMOUS. Plant Cell Physiol. 38:248 –258; 1997. [31] Hirayama, T.; Oka, A. Novel protein kinase of Arabidopsis thaliana (APK1) that phosphorylates tyrosine, serine and threonine. Plant Mol. Biol. 20:653– 662; 1992. [32] Desikan, R.; Clarke, A.; Hancock, J. T.; Neill, S. J. Hydrogen peroxide activates a MAP kinase-like enzyme in Arabidopsis thaliana suspension cultures. J. Exp. Bot. 50:1863–1866; 2000. [33] Desikan, R.; Clarke, A.; Atherfold, P.; Hancock, J. T.; Neill, S. J. Harpin induces mitogen-activated protein kinase activity during defence responses in Arabidopsis thaliana suspension cultures. Planta. 210:97–103; 1999. [34] Weaver, L. M.; Gan, S.; Quirino, B.; Amasino, R. M. A comparison of the expression patterns of several senescence-associated genes in response to stress and hormone treatment. Plant Mol. Biol. 37:455– 469; 1998. [35] Bartels, D. Late embryogenesis abundant (LEA) proteins: expression and regulation in the resurrection plant Craterostigma plantagineum. In: Smallwood, M. F.; Calvert, C. M.; Bowles, D. J., eds. Plant responses to environmental stress. Oxford, UK: BIOS Scientific Publishers; 1999:153–158. [36] Orozco-Cardenas, M.; Ryan, C. A. Hydrogen peroxide is generated systemically in plant leaves by wounding and systemin via the octadecanoid pathway. Proc. Natl. Acad. Sci. USA 96:6553– 6557; 1999.