JXB Advance Access originally published online on December 23, 2004
Journal of Experimental Botany 2005 56(412):713-723; doi:10.1093/jxb/eri038
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RESEARCH PAPER |
Stress-responsive 
-dioxygenase expression in tomato roots
Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, BC, Canada V5A 1S6
* To whom correspondence should be addressed. Fax: +1 604 291 3496. E-mail: aplant{at}sfu.ca
Received 9 June 2004; Accepted 23 September 2004
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Abstract |
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Alpha-dioxygenase (
-DOX) enzymes catalyse the oxygenation of fatty acids to yield a newly identified group of oxylipins that play a role in protecting tissues from oxidative damage and cell death. In tomato (Lycopersicon esculentum Mill.) -DOX was identified as salt-regulated by differential display of mRNA, and is represented by a small gene family comprising at least three members: LE-DOX1, -2, and -3 of which only LE-DOX1 was salt-responsive. The enhancement of LE-DOX1 expression in roots by salt, wounding and challenge with Pythium aphanidermatum (Edson) Fitzp. suggests that -DOX-generated oxylipins may mediate the response of roots to these environmental stresses. In roots, LE-DOX1 was abscisic acid (ABA)-responsive. However, in the ABA-deficient mutant flacca salt-responsive expression was equivalent to that in the wild type. Similarly, in roots exposed to fluridone (FLU) salt up-regulated expression; however, in this case salt-responsive LE-DOX1 expression was greater than that in roots that were not exposed to FLU. A possible explanation for this is provided by the role of ABA in suppressing ethylene accumulation in osmotically stressed roots. The ethylene-generating agent ethephon and precursor 1-aminocyclopropane-1-carboxylic acid markedly elevated LE-DOX1 expression, and this enhanced expression was suppressed by ABA. LE-DOX1 expression in salt-stressed roots was not markedly affected by AVG indicating that ABA may be responsible for enhanced -DOX expression in salt-treated roots.
Key words:
Abscisic acid, -dioxygenase, ethylene, Lycopersicon esculentum, oxylipin, roots, salt stress
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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A significant proportion of the world's arable land is covered with various salt-affected soils that limit productivity (Szabolcs, 1994
). Irrigated lands, which are the most productive in terms of crop yield, are most at risk from salinization. Plants growing in saline soils are exposed to two types of stresses. The first of these is an osmotic stress arising from the high level of dissolved ions and the resulting lowered water potential of the soil solution. The second stress is ionic in nature and a consequence of dissolved ions that enter the tissues of the root and, ultimately, the shoot system of the plant, and exert toxic effects. Roots are the first and most critical organ to experience salinity. Such exposure reduces their growth and development thereby reducing their capacity to explore the soil environment for mineral and water uptake. Plants exposed to high salinity are also affected by the generation of reactive oxygen species (ROS) resulting in the up-regulation of antioxidative systems (Shalata et al., 2001; Mittova et al., 2004). In addition, it was recently demonstrated that salt ions induce programmed cell death (PCD) in roots. This may allow the development of new roots following the death of salt-affected roots (Katsuhara and Shibasaka, 2000; Huh et al., 2002) thus permitting the exploration of new soil environments to facilitate water uptake. In this regard PCD may be an important aspect of salt tolerance.
The physiological and metabolic changes elicited in plants by salt stress are underpinned by changes in the expression of a large number of genes (Kawasaki et al., 2001), many of which are salt-regulated in roots. These genes encode products involved in co-ordinating changes in plant metabolism, re-establishing ion homeostasis, protection against the damaging effects of excess ions, dehydration and ROS, repair of damaged cellular components, as well as signal transduction and gene regulation. In addition, a significant number of genes encode proteins for which the function in salt-stressed plants is not known or understood. In an effort to isolate novel salt-responsive genes that are expressed in roots, differential display of mRNA in salt- versus non-treated tomato roots was performed (Wei et al., 2000). One of the partial salt-responsive cDNAs, JWS-20, shared considerable similarity to the C-terminus of a pathogen-induced oxygenase (PIOX) from tobacco.
In tobacco, PIOX gene expression is responsive to infection of leaves with Erwinia amylovora, caterpillar herbivory, wounding, UV-B, and the application of jasmonic acid (JA), salicylic acid (SA), and chemicals that elicit the production of ROS (Sanz et al., 1998; Hermsmeier et al., 2001; Izaguirre et al., 2003). In Arabidopsis, PIOX expression is also responsive to pathogen challenge, salicylic acid, and chemicals that promote ROS; however, JA was ineffective at altering expression levels (Ponce de Leon et al., 2002). The PIOX polypeptide shares significant identity with cyclo-oxygenases of animals that direct the biosynthesis of prostaglandins. Subsequently, it was demonstrated that PIOX is an -dioxygenase enzyme that catalyses the conversion of linolenic (18:3) acid to a 2-R-hydroperoxide derivative (Hamberg et al., 1999). Thus, PIOX may be involved in the generation of lipid-derived (oxylipin) signals in pathogen-challenged and wounded plants. The PIOX protein was recently renamed to reflect its enzymatic function as -dioxygenase or -DOX (Ponce de Leon et al., 2002).
This paper is the first, to the authors' knowledge, to describe the salt-responsive nature of -DOX expression, a finding that suggests that lipid-derived signals may co-ordinate the response of roots to salt stress. There are at least three genes that encode -DOX-like polypeptides in the tomato genome. Differential regulation of -DOX isoforms in salt-treated, and mechanically wounded roots was detected. To determine the signals that regulate -DOX expression in salt-stressed roots, the role played by abscisic acid (ABA) was explored. Data presented indicate that, in salt-treated roots, ABA plays a role in regulating -DOX expression. Furthermore, as a result of manipulations to reduce endogenous root ABA content, ethylene emerged as a positive regulator of -DOX expression. The evidence presented suggests that ethylene and ABA can interact to regulate -DOX expression in roots, substantiating a role recently ascribed to ABA in suppressing ethylene evolution in osmotically stressed roots (Spollen et al., 2000). Taken together, these data suggest that -DOX-generated oxylipin signals may co-ordinate responses to environmental stress in roots and that ABA, and possibly ethylene, regulate their production.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Materials
Seeds of tomato (Lycopersicon esculentum Mill. cv. Ailsa Craig) and the near-isogenic mutant flacca (flc) were grown and maintained as described in Wei et al. (2000)
. Surface-sterilized seeds were germinated in moistened vermiculite contained within a plastic grid lined with a plastic mesh. Upon germination the roots grew through the mesh into the 2/3 strength Murashige and Skoog (MS) nutrient solution. The MS solution was aerated and changed at least once per week. Plants were maintained in a growth chamber (Conviron 125L incubator) for 16/8 h at 25/21 °C in the light/dark. Mature plants were maintained in a greenhouse under conditions typical for a spring to summer season in Burnaby, BC, Canada.
Experimental treatments
Two to three-month-old greenhouse-grown plants were used for spatial expression studies. Six-week-old plants were used for all other experiments. Salt treatments were imposed by the addition of NaCl to the nutrient solution to a final concentration of 170 mM. Wounding was conducted by crushing along the whole length of the roots with forceps. Pathogen challenge was carried out by adding a single inoculum of mycelial fragments of Pythium aphanidermatum (Edson) Fitzp. (400 000 l–1) to the nutrient solution. ABA (mixed isomers, ±cis/trans ABA; Sigma), ethephon, 1-aminoethoxyvinylglycine (AVG), and 1-aminocyclopropane-1-carboxylic acid (ACC) were added to the nutrient media either alone or in combination with a salt treatment. Control treatments were conducted by transferring plants to fresh nutrient solution for the duration of the experimental period. Fluridone (FLU) treatments were applied by exposing plants to FLU (SePRO Corporation, Carmel, IN) in nutrient solution for 24 h prior to transfer to salt or MS solution lacking FLU for a further 24 h. Following each treatment root tissues were harvested and frozen in liquid N2. Each treatment was performed independently at least three times, and a pooled root sample derived from approximately 35 plants was used.
Isolation of RNA
Total RNA for northern blot hybridization and RT-PCR analyses was extracted using the LiCl-phenol method described by Prescott and Martin (1987).
Northern hybridization analyses
Twenty µg total RNA was size separated on a formaldehyde denaturing 1.2% agarose gel and blotted onto a positively-charged nylon membrane (Boehringer Mannheim, Biomedicals Laval, Quebec, Canada) according to Sambrook et al. (1989). RNA was fixed by UV-crosslinking (UV Stratalinker 2400, Stratagene Inc., La Jolla, CA, USA) followed by baking at 80 °C for 30 min. Even loading of RNA samples was established by inspecting the ethidium bromide stained gel for the major ribosomal RNAs and probing the blot with a tomato 18S rRNA probe. Membranes were pre-hybridized at 65 °C for 2 h in 100 mM NaH2PO4, 50 mM Na2P2O7, 1 mM EDTA, 7% SDS, and 100 µg ml–1 sheared salmon sperm DNA. Hybridization proceeded in the presence of the radiolabelled probe at 65 °C overnight. Following hybridization, the membrane was washed three times in 2x SSC, 0.1% SDS at room temperature followed by two washes in 1x SSC, 0.1% SDS at 65 °C and one wash in 0.5x SSC, 0.1% SDS at 65 °C and exposed to X-ray film (X-Omat blue XB-1, Kodak, Toronto, Canada) with an intensifying screen at –80 °C. The average exposure time for LE-DOX1 was 16 h, LE-DOX1-5' 1–3 d, LE-DOX2-5' 3 d, and LE-DOX3 16 h. Band intensity was determined using Scion image version 1.62c (macrofunction gel plot 2) and subsequently normalized by dividing the hybridization signal obtained for the -DOX probe by that of the rRNA probe. 5' probes for LE-DOX1 and LE-DOX2 were generated utilizing the following forward and reverse primers: 5'-TAT CTT GGA GCA CGG CGG AG-3' and 5'-CTA AAG GAC TTG AGT GGG-3' or 5'-CAA AAT GAA TCT CCG CGA CA-3' and 5'-TCC GGT AGG AGT TTC TTT TGA T-3', respectively.
Nucleotide sequencing and analyses
Nucleotide sequence determination was carried out by the NAPS Unit (Biotechnology Laboratory, University of British Columbia, Canada) on a Perkin Elmer 377 (ABI Prism) DNA analyser. Nucleotide sequences were submitted to the NCBI BLAST server for BLASTN and BLASTX searches against the non-redundant and EST databases (Altschul et al., 1997). Multiple alignments were performed using CLUSTALW (http:/www2.ebi.ac.uk/clustalw/) and MacVector 7.1.1 using a 10.0 open gap penalty, 40% delay divergent and Blosum similarity matrix. Maximum parsimony (Eck and Dayhoff, 1966; Fitch, 1977) and Neighbor–Joining (NJ) (Saitou and Nei, 1987) unrooted trees were generated in PAUP* (Swofford, 2000) using full-length amino acid sequences. The NJ tree was generated using pairwise distances calculated using absolute distances. Maximum parsimony of equally weighted and unordered character state transformations were used to generate the most parsimonious trees based on a branch and bound search. Initial tree(s) were constructed using random stepwise addition. Branch swapping was implemented through tree bisection and reconnection (TBR) and steepest descent. For both types of trees, data were resampled using either 1000 branch and bound or NJ bootstrap replicates.
Genomic Southern hybridization
Genomic DNA was extracted according to Dellaporta et al. (1983). Genomic DNA (20 µg) was digested with EcoRI, HindIII, XhoI, or BamHI and analysed by Southern hybridization as described in Sambrook et al. (1989). The membrane was washed twice each in: 2x SSC, 0.1% SDS, 1x SSC, 0.1% SDS, and 0.5x SSC, 0.1% SDS at 65 °C, and finally in 0.1x SSC, 0.1% SDS at 68 °C. Following the washes the membrane was exposed to X-ray film at –80 °C with an intensifying screen.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Isolation of three
-DOX cDNAs from tomatoBLASTN searches against dbEST with the salt-responsive partial cDNA JWS-20 (GenBank accession: AW062238; Wei et al., 2000
) revealed the existence of several tomato ESTs with significant similarity to JWS-20. Closer inspection of these ESTs revealed that they defined three tomato -DOX gene loci.
cDNA clones corresponding to three ESTs (341283, cLEW8G12; 428893, cLEW26H11; and 554653, cTOD20F16; GenBank accession: AW979675, BF098372, BI935764) were obtained from Clemson University Genomics Institute (CUGI). The cLEW8G12 cDNA insert was 2115 nucleotides long and contained a single open reading frame (ORF) that encodes a polypeptide of 639 amino acids, with a predicted molecular weight of 86 kDa. The cLEW26H11 clone contained an insert of 2078 nucleotides, the 3'-end of which was identical to JWS-20. However, the predicted ORF possessed a 40 amino acid deletion at the N-terminus, and examination of the nucleotide sequence revealed the presence of an intron. Primers were designed and utilized in RT-PCR to generate a product that spanned both the deletion and intron. The nucleotide sequence of the resulting amplicon lacked both the deletion and the intron indicating that the JWS-20-related -DOX gene produced a viable transcript. The ORF corresponding to JWS-20 was 642 amino acids with a predicted molecular weight of 87 kDa. The nucleotide sequence of the third -DOX gene was constructed from alignment of ESTs 355349, 554655, 247591, 470139, 261869, and the nucleotide sequence for the FEEBLY gene (U35643
[GenBank]
; van der Biezen et al., 1996). The nucleotide sequence was completed by sequencing portions of the cTOD20F16 cDNA that corresponded to gaps or unverifiable nucleotide sequences in the alignment. The complete sequence was 2007 nucleotides long and contained a single ORF that encodes a polypeptide of 632 amino acids with a predicted molecular weight of 85 kDa. The nucleotide sequences of cLEW8G12 and cLEW26H11 were very similar to each other (85% identity) whereas cTOD20F16 was less similar (63% and 66% identity to cLEW8G12 and cLEW26H11, respectively). Hereafter cLEW8G12 will be referred to as LE-DOX1 (GenBank accession AY344539), cLEW26H11 as LE-DOX2 (AY344540
[GenBank]
) and cTOD20F16 as LE-DOX3 (BK001477
[GenBank]
). LE-DOX1 corresponds to TC126269 in the TIGR Tomato Gene Index (http://www.tigr.org/tigr-scripts/tgi/T_index.cgi?species=tomato); LE-DOX2 to TC127979, TC122317, and the singleton BF098372
[GenBank]
; LE-DOX3 corresponds to TC119265, TC119649, and BG124577
[GenBank]
. A potential fourth -DOX gene is represented by BE432966
[GenBank]
, and shares 85% nucleotide sequence identity to LE-DOX1.
Sequence similarity BLASTX searches with the nucleotide sequence of LE-DOX1 against the non-redundant databases revealed significant similarity to plant -DOX sequences (Fig. 1). The polypeptide encoded by LE-DOX1 shares high similarity with -DOX from Nicotiana attenuata (85% identity), N. tabacum (84% identity), Arabidopsis thaliana (At-DOX1; 73% identity) and Oryza sativa (64% identity), a cyclo-oxygenase-like protein from Capsicum annuum (82% identity) and the feebly-like protein from A. thaliana (At-DOX2; 62% identity). LE-DOX3 corresponds to the FEEBLY gene isolated as a result of insertional mutagenesis of tomato (van der Biezen et al., 1996; Meissner et al., 2000). It is more similar to the Arabidopsis feebly-like (At-DOX2) protein than to LE-DOX1 (71% identity versus 63% identity, respectively) and probably represents an -DOX isoform as indicated by the phylogenetic tree in Fig. 2B. Alpha-dioxygenases are haem enzymes that incorporate dioxygen into fatty acids and share structural similarity with mammalian prostaglandin-H synthases (PGHS). Amino acid residues involved in haem binding (His-165 and His-389 of LE-DOX1), and initiating the oxygenation reaction (Tyr-386) are conserved in plant -DOX, whereas a Ser residue involved in substrate binding (Ser-564) is not conserved in the LE-DOX1 or Arabidopsis -DOX1 polypeptides (Fig. 1).
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Genomic southern analyses for each of the three tomato
-DOX genes revealed a similar banding pattern when LE-DOX1 or LE-DOX2 were utilized as probes (Fig. 2A), whereas a distinct banding pattern was detected by the LE-DOX3 probe. This is consistent with the lower nucleotide sequence similarity shared between LE-DOX1 or LE-DOX2 and LE-DOX3. The multiple hybridizing DNA fragments detected by the LE-DOX1 and LE-DOX2 probes are consistent with known restriction enzyme sites in the cDNA inserts.
-DOX is responsive to salt stress in roots
JWS-20 corresponds to a salt-responsive gene and northern analyses using this partial cDNA revealed up-regulation by salt (Wei et al., 2000). To ascertain whether LE-DOX1, LE-DOX2, and LE-DOX3 are salt-responsive, it was necessary to distinguish gene-specific transcripts. Due to the high degree of nucleotide sequence identity between LE-DOX1 and LE-DOX2, probes were prepared from the 5'-end of the cDNA for northern hybridization analyses. To assess the extent of cross-hybridization between the less similar LE-DOX1 and LE-DOX3 sequences, full-length cDNAs were blotted and hybridized against LE-DOX1. This revealed approximately 12% cross-hybridization (Fig. 3). Subsequent northern analyses used either the LE-DOX1 full-length or 5'-probe, both of which detected a similar pattern of transcript accumulation that was distinct from that obtained using the LE-DOX2 5'-probe or the LE-DOX3 full-length probes (Figs 4, 5).
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Salt treatment up-regulated LE
-DOX1 expression in roots at 8 h and 24 h (Fig. 4). LE-DOX2 expression was not affected by salt at these times, whereas LE-DOX3 was marginally up-regulated at 8 h, but not 24 h, after the application of salt. Salt had a negative effect on the LE-DOX transcript level for all isoforms at the earliest time intervals following salt exposure (Fig. 4). LE-DOX1 was down-regulated 30 min after salt application, LE-DOX2 and -3 were down-regulated 30 min and 2 h after the salt treatment commenced. This may be due to an osmotic shock associated with the salt treatment.
In control roots, expression of LE-DOX1 and LE-DOX2 peaked at 2 h then fell to lower levels 24 h after the plants were transferred to fresh nutrient media. To establish whether this was due to circadian regulation of gene expression, non-treated root tissue was collected at intervals during a 26 h period. Expression peaked after 2 h, coincident with the 2 h sampling for time-course experiments (not shown). A smaller peak was detected 12 h later. The expression level detected after 24 h was lower than that at 0 h; however, it increased 3-fold 2 h later. As such, circadian or diurnal regulation of LE-DOX1 and -2 may account for the expression detected in non-treated roots.
-DOX is responsive to wounding and pathogen challenge of roots
-DOX expression is responsive to biotic stress imposed by pathogen infection or caterpillar feeding in the leaves of tobacco, hot pepper, and Arabidopsis (Sanz et al., 1998; Hermsmeier et al., 2001; Kim et al., 2002; Ponce de Leon et al., 2002). To determine whether -DOX was responsive to mechanical wounding, roots were wounded by pinching with forceps. Wounding elicited strong up-regulation of LE-DOX1 at 8 h and 24 h after wounding (Fig. 5). Expression of LE-DOX2 was also responsive to wounding at 8 h and 24 h whereas LE-DOX3 was not wound-responsive. Exposure to Pythium aphanidermatum elicited an increase in LE-DOX1 expression (Fig. 5). Due to the salt- and wound-responsive nature of LE-DOX1, this isoform was the focus for further study.
Spatial expression analyses of LE-DOX1 expression
To determine whether LE-DOX1 expression occurred in organs other than the roots, RNA was extracted from young, mature, and senescent leaves, roots, open and closed flowers, green and red fruit, and the seeds extracted from green and red fruit of greenhouse-grown plants. LE-DOX1 transcripts were detected in the roots and were absent in all other organs (Fig. 6).
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Does ABA play a role in regulating LE
-DOX1 expression?Previously LE
-DOX expression was shown to be responsive to exogenous ABA (Wei et al., 2000). Application of ABA up-regulated the expression of LE-DOX1 in roots (Fig. 7A) confirming the ABA-responsive nature of LE-DOX1 and suggesting a role for ABA in gene regulation. Evidence for such a role was further explored using flacca (flc), an ABA-deficient mutant with a reduced accumulation of ABA in salt-treated roots (Chen and Plant, 1999). Relative to the transcript level in non-treated flc, salt increased the LE-DOX1 transcript level in flc roots 24 h after the application of salt (Fig. 7B). The extent of the salt-induced up-regulation of LE-DOX1 expression in flc at 24 h was similar to that observed in roots of wild-type plants and, on occasion, was higher (not shown). At 8 h following salt application no salt-specific induction was observed in flc due to high levels of expression in control roots; overall, expression in non-treated flc roots was higher than in the wild type (not shown).
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Because flacca accumulates some ABA in roots (Chen and Plant, 1999
; Sagi et al., 1999), fluridone (FLU), an inhibitor of carotenoid biosynthesis was used to prevent the accumulation of ABA. FLU was applied as a pretreatment and is effective at lowering ABA levels in the roots of tomato seedlings (Chen and Plant, 1999; Jin et al., 2000). In the roots of FLU-pretreated plants LE-DOX1 expression was marginally higher than in non-treated roots. A subsequent salt treatment effected an increase in LE-DOX1 transcript level that was greater than that in FLU pretreated control roots (Fig. 7c). Interestingly, the LE-DOX1 transcript level in salt-treated roots following a FLU pretreatment was greater than that present in salt-treated roots that had not previously been exposed to FLU.
Does ethylene play a role in regulating LE-DOX1 expression?
It was recently demonstrated that ABA prevents excess production of ethylene in osmotically stressed seedling roots (Spollen et al., 2000). It has previously been shown that root ABA levels are reduced substantially in FLU-pretreated plants (Chen and Plant, 1999). As such, the enhanced LE-DOX1 expression in roots of FLU-pretreated plants may be caused by enhanced ethylene evolution that is itself a consequence of the low level of ABA. To establish whether ethylene plays any role in regulating LE-DOX1, plants were exposed to the ethylene-generating agent ethephon and the precursor of ethylene synthesis, 1-aminocyclopropane-1-carboxylic acid (ACC). In the roots of ACC- and ethephon-treated plants, LE-DOX1 expression was markedly enhanced (Fig. 8A). Application of 1-aminoethoxyvinylglycine (AVG), an inhibitor of ethylene biosynthesis, did not have a major effect on the LE-DOX1 expression level in salt-treated roots, although at the higher concentrations expression was somewhat elevated (Fig. 8B, C).
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Do ABA and ethylene interact to regulate LE
-DOX1 expression in roots?LE
-DOX1 expression is responsive to ABA, but markedly more so to ethylene. In response to salt stress, ABA levels increase in roots of tomato (Chen and Plant, 1999; Jin et al., 2000) and may suppress the effects of ethylene as reported by Spollen et al. (2000). In support of this, when ACC and salt were applied together LE-DOX1 expression was reduced to a level approaching that elicited by salt alone (Fig. 8A, C). Likewise, the LE-DOX1 transcript level in roots exposed to ACC together with ABA was lower than that in ACC-treated roots (Fig. 8C) providing further evidence for an interaction between ethylene and ABA in regulating LE-DOX1 expression in tomato roots. |
Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Alpha-dioxygenases catalyse the primary oxygenation of fatty acids at the
-carbon to yield unstable 2-hydroperoxy fatty acids that represent a new class of oxylipins (Hamberg et al., 1999). This is the first report, with the authors' knowledge, to describe salt stress-responsive -DOX expression suggesting that oxylipins may mediate the responses of roots to salt stress.
In tomato, -DOX-related ESTs define a small gene family comprised of at least three members. Of the three members that were the focus of this study, only one, LE-DOX1 was responsive to salt. Genome-wide analyses of stress-responsive expression revealed that -DOX transcript levels increase in drought-stressed roots of barley and Arabidopsis (Ozturk et al., 2002; Seki et al., 2002). Low levels of LE-DOX1 expression were detected in shoot tissue of non-stressed plants; however, neither consistent nor substantial salt-responsive expression in shoots has been found thus far (not shown). Spatial analyses affirm the root-specific expression of LE-DOX1.
LE-DOX1 and LE-DOX2 are similar to each other at the nucleotide sequence level, whereas LE-DOX3 is less similar and likely represents a distinct isoform. Arabidopsis possesses two -DOX genes; At-DOX1 is closely related to LE-DOX1 and -2, whereas At-DOX2 is more similar to LE-DOX3 (Figs 1, 2). The LE-DOX3 gene has been disrupted by insertional mutagenesis resulting in a pronounced phenotype termed feebly (van der Biezen et al., 1996; Meissner et al., 2000). Feebly contains high anthocyanin levels during seedling development and subsequently develops into weak plants with pale green leaves and deformed fruit. ESTs that define LE-DOX3 are represented in cDNA libraries from flower buds, ovaries, and developing fruit. LE-DOX3 was expressed in roots but was not responsive to salt or wounding injuries. Van der Biezen et al. (1996) speculated that FEEBLY is involved in a metabolic pathway giving rise to physiologically disturbed plants when absent. This implicates -DOX function in normal development in tomato.
Direct wounding of roots up-regulated expression of LE-DOX2, whereas wounding or challenge with the necrotrophic pathogen, Pythium aphanidermatum up-regulated expression of LE-DOX1 (Fig. 4). This suggests a general role for -DOX in the protection of roots against a range of stresses. Thus far, -DOX expression has been associated with biotic stress arising from pathogen-challenge and mechanical- or caterpillar-induced wounding to leaves. -DOX expression was also responsive to signalling molecules associated with these environmental insults as well as to chemicals that elicit the generation of ROS (Sanz et al., 1998; Hermsmeier et al., 2001; Ponce de Leon et al., 2002; Weber et al., 2004). Ponce de Leon et al. (2002) demonstrated that Arabidopsis with reduced -DOX were more susceptible to pathogen challenge and went on to suggest that -DOX protects plants from oxidative stress. As such, -DOX-generated oxylipins may be involved in this process (Hamberg et al., 2003). In this regard, reports describing salt stress-induced oxidative stress and PCD in roots are noteworthy (Katsuhara, 1997; Katsuhara and Shibasaka, 2000; Huh et al., 2002; Shalata et al., 2001). It is tempting to speculate that -DOX-generated oxylipins may play a similar role and protect roots from oxidative damage and cell death associated with environmental stress.
Investigation into endogenous signals that regulate LE-DOX1 expression in salt-stressed roots focused on ABA, a hormone associated with co-ordinating osmotic stress responses (Plant and Bray, 1999). Expression of LE-DOX1 was responsive to exogenous ABA; however, salt-responsive expression in flc is inconsistent with a major role for ABA in regulating LE-DOX1 expression (Fig. 7B). Flacca can accumulate some ABA in roots and is impaired in the transport of ABA from roots to the shoot (Chen and Plant, 1999; Sagi et al., 1999). Therefore, it is possible that the salt-responsive LE-DOX1 expression detected in the roots of this mutant reflect its ability to accumulate some ABA. To resolve this issue FLU was used to reduce ABA levels. Enhanced salt-responsive LE-DOX1 expression in FLU-treated roots relative to that in roots that were not treated with FLU was unexpected (Fig. 7C). A logical explanation is that ethylene may positively regulate LE-DOX1 since elevated ABA levels restrict ethylene production in roots of osmotically-stressed plants (Spollen et al., 2000).
The dramatic enhancement of LE-DOX1 expression following application of ACC or ethephon supports a role for ethylene in regulating LE-DOX1 (Fig. 8A). Ethylene mediates responses to pathogenic (Diaz et al., 2002) and non-pathogenic organisms (Knoester et al., 1999), to soil compaction (Hussain et al., 1999), and to flood-induced anoxia that gives rise to aeranchyma formation via PCD (Drew et al., 2000). Hyperosmotic stress has been shown to induce ACC synthase and presumably therefore ethylene production in cultured tomato cells (Felix et al., 2000); however, it is not clear whether osmotic stress induces ethylene evolution in intact plants (Morgan et al., 1990; Narayana et al., 1991). The reduced expression level detected when ACC was applied together with either salt or ABA suggests that ABA and ethylene can interact to influence LE-DOX1 expression in roots. However, in salt-stressed roots enhanced expression of LE-DOX1 may not be influenced substantially by ethylene since AVG had a minimal effect on expression levels. Therefore, salt-responsive LE-DOX1 expression may be mediated by the elevated level of ABA present in salt-stressed roots, which simultaneously suppresses ethylene evolution and/or its effects. Ongoing research is addressing the role of -DOX in salt-stressed roots as well as the nature of the upstream signalling pathways that regulate -DOX expression and thereby the production of -DOX-generated oxylipins.
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Acknowledgements |
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Our sincere thanks are owed to Agnes Tsui and Scott DiGuistini for excellent technical assistance. Special thanks to Raymond Yip for providing and assisting us with the Pythium aphanidermatum cultures. Funding for this work was provided by a Discovery grant to ALP from NSERC.
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Footnotes |
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Abbreviations: ABA, abscisic acid;
-DOX, -dioxygenase; FLU, fluridone; AVG, 1-aminoethoxyvinylglycine; ACC, 1-aminocyclopropane-1-carboxylic acid; AC, Ailsa Craig; flc, flacca. |
References |
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