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In higher plants, copper ions, hydrogen peroxide, and cycloheximide have been recognized as very effective inducers of the transcriptional activity of genes encoding the enzymes of the ethylene biosynthesis pathway. In this report, the transcriptional patterns of genes encoding the 1-aminocyclopropane-1-carboxylate synthases (ACSs), 1-aminocyclopropane-1-carboxylate oxidases (ACOs), ETR1, ETR2, and ERS1 ethylene receptors, phospholipase D (PLD)-α1, -α2, -γ1, and -δ, and respiratory burst oxidase homologue (Rboh)-NADPH oxidase-D and -F in response to these inducers in Brassica oleracea etiolated seedlings are shown. ACS1, ACO1, ETR2, PLD-γ1, and RbohD represent genes whose expression was considerably affected by all of the inducers used. The investigations were performed on the seedlings with (i) ethylene insensitivity and (ii) a reduced level of the PLD-derived phosphatidic acid (PA). The general conclusion is that the expression of ACS1, -3, -4, -5, -7, and -11, ACO1, ETR1, ERS1, and ETR2, PLD-γ 1, and RbohD and F genes is undoubtedly under the reciprocal cross-talk of the ethylene and PAPLD signalling routes; both signals affect it in concerted or opposite ways depending on the gene or the type of stimuli. The results of these studies on broccoli seedlings are in agreement with the hypothesis that PA may directly affect the ethylene signal transduction pathway via an inhibitory effect on CTR1 (constitutive triple response 1) activity.
Ethylene production and perception regulate plant responses to a broad spectrum of various biotic and abiotic stimuli. The immediate precursor of ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC), is a product of the reaction controlled by 1-aminocyclopropane-1-carboxylate synthase activity (S-adenosyl-L-methionine methylthioadenosine-lyase, EC 22.214.171.124; ACS). The next step, conversion of ACC to ethylene, is catalysed by 1-aminocyclopropane-1-carboxylate oxidase (ACO).
Different expression of ACS and ACO isozymes encoded by multigene families in response to external and internal stimuli is controlled at the transcriptional and post-transcriptional level (Rottmann et al., 1991; Liang et al., 1992; Barry et al., 1996; Oetiker et al., 1997; Vogel et al., 1998; Kim et al., 2001; Tatsuki and Mori, 2001; Gallie and Young, 2004; Hernandez-Sebastia et al., 2004; Tsuchisaka and Theologis, 2004; Yoshida et al., 2006; Ralph et al., 2007; El-Sharkawy et al., 2008; McClellan and Chang, 2008; Xue et al., 2008; Christians et al., 2009; Gallie et al., 2009; Lin et al., 2009; Tsuchisaka et al., 2009). In plants, ethylene biosynthesis is controlled by two systems: the ethylene autoinhibitory system 1, which generally operates during normal vegetative growth of plant; and system 2, regulated by a positive feedback mechanism, usually responsible for the rapid increase in ethylene production in senescing ethylene-sensitive plant organs, and in ripening climacteric fruits (Nakatsuka et al., 1998; Barry et al., 2000; Kim et al., 2001).
Ethylene production and formation of reactive oxygen species (ROS) are the first biochemical alterations which participate in the signal transduction events involved in programmed cell death (PCD), playing an essential role in response to different abiotic stressors and in a plant defence reaction against various pathogens (Moeder et al., 2002; Woltering et al., 2003; Iakimova et al., 2008; Steffens and Sauter, 2009). In plants, in response to stress, one of the generators of extracellular ROS is the membrane-bound respiratory burst oxidase homologue (Rboh)-NADPH oxidase, catalysing the stimuli-induced extracellular production of superoxide anion which dismutates to hydrogen peroxide (Frahry and Schopfer, 2001; Beckett et al., 2004; Sgherri et al., 2007). However, Rboh function can be activated by exogenous ROS, and the subsequent oxidative burst can suppress death in cells sourrounding sites of Rboh activation (Torres et al., 2005).
Rapidly diffusing across the cell membranes, H2O2 at low concentrations acts as a messenger molecule triggering tolerance against various stresses, but at high concentrations it orchestrates PCD. It has been thought that selective enzymatic or non-enzymatic oxidation of cysteine residues in sensor proteins is a general H2O2 signalling route which can directly cross-talk or compete with nitric oxide (NO) action (Hancock et al., 2005, 2006; Miller and Mittler, 2006; Forman et al., 2008; Neill et al., 2008; Wang and Song, 2008; Jammes et al., 2009; Forman et al., 2010; Paulsen and Carroll, 2010); for details see Fig. 1B. Nonetheless, in general, a close interplay of H2O2 with the other signalling molecules is realised at the transcriptional level (Dat et al., 2001, 2003; Vandenabeele et al., 2003).
All organisms have to maintain a balance between essential and toxic levels of copper. The enhanced activity of Rboh-NADPH oxidase and also an essential accumulation of H2O2 in the plasma membrane and cell walls in response to copper have been shown in different species (Quartacci et al., 2001; Yu et al., 2008; Zhang et al., 2008; Wu et al., 2009). Copper-induced oxidative stress results, first, from the directly catalysed formation of ROS via a Fenton-like reaction and, secondly, from a significantly decreased glutathione (GSH) level by copper ions. Depletion of intracellular GSH increases the cytotoxic effect of ROS (Mattie and Freedman, 2004). Copper can activate transcription through either oxidative stress- or metal-mediated mechanisms, leading to activation of mitogen-activated protein kinase (MAPK) signalling pathways (Ostrakhovitch et al., 2002; Jonak et al., 2004; Mattie and Freeman, 2004; Gaitanaki et al., 2007; Yeh et al., 2007; Chen et al., 2008; Mattie et al., 2008) (see Fig. 1A for details). Although copper and H2O2 effectively stimulate ethylene biosynthesis in higher plants, the most universal and potent inducer of expression of ACS genes is cycloheximide (CHX) (Yamagami et al., 2003). Its role in this induction has not been elucidated to date. CHX has mostly been considered as a eukaryotic protein synthesis inhibitor, but this seems to be only one aspect of its action in living cells. According to some suggestions (Li et al., 2001), CHX can (i) lead to transcriptional activation via loss of labile negative regulators; (ii) inhibit translation of protein products of autorepressive genes, and thus superinduce their transcription; (iii) prevent the synthesis of labile mRNA-degrading enzymes; (iv) cause RNAs to be trapped on polysomes, thus shielding them from cytoplasmic RNases; (v) induce phosphorylation of proteins usually involved in abscisic acid (ABA)-mediated activation; (vi) lead to direct transcriptional activation via phosphorylation of H3 histones; and (vii) uncouple DNA replication and chromatin assembly preventing the formation of a repressive chromatin structure.
Despite a general agreement that plants produce ethylene in response to exposure to copper ions, the copper-induced transcriptional activity of the ethylene biosynthesis genes has only been studied in a limited number of plants. In Arabidopsis, the only ACS6 gene characterised as multiresponsive responds to copper (Arteca and Arteca, 1999). The greatest stimulation has been observed in inflorescences and the youngest leaves, whereas light had an inhibitory effect (Arteca and Arteca, 2007). The copper-inducible expression of two different ACS genes in potato, two distinct ACS genes in Pelargonium hortorum, and an accumulation of ACS transcripts in different cultivars of tobacco have been reported (Avni et al., 1994; Wang and Arteca, 1995; Schlagnhaufer et al., 1997).
At least three, but more often more than three, members of the ACO family regulated in a gene-specific manner occur in the genomes of all plants investigated to date. Some of them are copper inducible, but there are only scarce data from studies performed on Pelargonium hortorum, Nicotiana tabacum, and Nicotiana glutinosa (Avni et al., 1994; Wang and Arteca, 1995; Clark et al., 1997).
In Arabidopsis, the N-terminal fragments of five ethylene receptors (subfamily 1, ETR1 and ERS1; subfamily 2, ETR2, ERS2, and EIN4) are involved in copper-mediated ethylene binding. Ethylene receptors act as negative regulators which actively repress expression of the ethylene-responsive genes in the absence of ethylene. Binding of ethylene turns the receptor activity off, which results in removal of the downstream block on signal transduction (Fig. 2) (Gao et al., 2008; Zhu and Guo, 2008; Lin et al., 2009). A reduction in their overall number increases the ethylene responsiveness of tissues, whereas an enhancement decreases the ethylene sensitivity. Ethylene binding triggers ubiquitin-dependent receptor degradation, therefore synthesis of new receptors is the only way to turn off the ethylene response (Klee, 2002; Kevany et al., 2007). Moreover, it has been shown that ETR1 mediates stomatal closure in response to H2O2 (Desikan et al., 2005). Thus, it is possible that ETR1 can act as a node mediating cross-talk between ethylene and H2O2.
Generally, phosphatidic acid (PA) represents ~1–2% of total phospholipids and is generated by two routes, either directly by phospholipase D (PLD; EC 126.96.36.199) activity or by the sequential action of phospholipase C and diacylglycerol kinase (PAPLC/DGK) (Arisz et al., 2009; Munnik and Testerink, 2009). The up-regulated activity of both routes occurs at the early step of response to stress (Wang, 2005; Bargmann and Munnik, 2006). Multiple types of PLDs reveal different catalytic and regulatory properties, and generate a distinct PAPLD species (Li et al., 2009). Putatively the PAPLD species, the location and timing of PAPLD formation, and its intracellular level are essential determinants of the functioning of PAPLD as a secondary messenger molecule which can act in opposite ways (Wang, 2000).
It has been assumed that PAPLD can enhance the activity of Rboh-NADPH-oxidase (Yu et al., 2008). A direct promotion of the catalytical activity of Rboh-oxidase by PLD-α1-derived PA species and the Cu2+-induced elevation of both PAPLD content and activation of Rboh-oxidase have been found in Arabidopsis (Zhang et al., 2005) and wheat (Navari-Izzo et al., 2006). Furthermore, Navari-Izzo et al. (2006) reported that the reduced PAPLD level in wheat roots almost completely abolished the production of the superoxide anion. Nevertheless, the other isozyme of PLD, PLD-δ activated by H2O2, does not stimulate H2O2 formation; moreover, PAPLD species generated by PLD-δ lead to a decrease in H2O2-promoted PCD (Zhang et al., 2003).
In mammals, the MAPK kinase kinase Raf-1 represents the best known molecular target of PA thus far. One of the plant homologues of Raf-1, CTR1 (constitutive triple response 1), functions as a negative regulator of the ethylene signalling pathway (Fig. 2). It has been shown for Arabidopsis CTR1 that PA binds to CTR1, inhibits its kinase activity, and blocks interactions with the ethylene receptor ETR1 (Testerink et al., 2007, 2008).
PLDs have the unique ability to transfer the phosphatidyl group of their substrates to a primary alcohol instead of water, which results in formation of phosphatidyl alcohols. The formation of the latter, especially that of inactive phospholipid such as phosphatidylbutanol, blocks PAPLD production. Treatment with butanol-1 is used as a very effective method of inhibition of PAPLD signalling in living cells (Morris et al., 1997; Testerink and Munnik, 2005).
Ethylene receptors function as a dimer with a copper ion located in the hydrophobic pocket, but it has been shown that silver ions are able to replace copper. Despite the fact that the silver ion-occupied receptor binds ethylene, the binding site is apparently perturbed so that it is not able to induce the alterations that are necessary to elicit downstream signalling (Rodriguez et al., 1999; Klee, 2002; Klee and Tieman, 2002; O'Malley et al., 2005; Gao et al., 2008). Thus, treatment of plants with silver represses the ethylene responses (see Fig. 2).
This observation has prompted us to check the effect of a decreased intracellular level of PAPLD or the silver-induced ethylene insensitivity on the transcriptional activity of the ethylene biosynthesis enzymes, ethylene receptors, PLD, and Rboh-NADPH-oxidase genes. The aim of this study was to investigate the time course of expression of the above-mentioned genes at the early step of plant response to copper, exogenously applied H2O2, or CHX in Brassica oleracea etiolated seedlings. This investigation has provided evidence that the level of PAPLD considerably affects the expression of the genes discussed and supports the putative regulatory involvement of PAPLD in the ethylene signal transduction pathway.
Various plant species produce both the shorter and longer transcripts for the same ACS isozyme; moreover, the presence of the stress-induced incomplete spliced transcripts of some ACS isozymes has been reported (Nakagawa et al., 1991; Rottmann et al., 1991; Spanu et al., 1993; Olson et al., 1995; ten Have and Woltering, 1997; Peck and Kende, 1998). The role of these non-functional transcripts is still not recognized. Thus, it was decided to analyse the transcriptional pattern of the genes discussed considering the longer amplicons of their cDNA (462–718bp) generated by a semi-quantitative RT-PCR method.
Brassica oleracea var. alboglabra A12 DH seeds used in the experiments were sterilized and sown onto MS medium containing 0.8% agar and 3% sucrose. They were grown for 6d in the dark at 23°C and then were finally transferred to fresh MS liquid medium and placed for an additional 20h in darkness before the treatments. For treatment with different inducers of ethylene biosynthesis, appropriate amounts of CHX, CuCl2, H2O2, AgNO3, butanol-1, and isobutanol were added to liquid MS medium to a predicted final concentration. The sets of experiments were carried out after 0, 0.5, 1, 1.5, 2, 3, 4, and 6h. After treatment, whole seedlings were collected, frozen in liquid nitrogen, and stored at –80°C.
The final concentrations of agents were as follows: 0.005mM CHX, 2.5mM CuCl2, 0.1mM AgNO3, 0.25% H2O2, 0.1% butanol-1, and 0.1% isobutanol.
The seedlings were treated at different time intervals with: (i) 2.5mM CuCl2; (ii) 0.1% butanol-1 added 16h prior to addition of 2.5mM CuCl2; (iii) 0.1mM AgNO3 added 16h prior to addition of 2.5mM CuCl2; (iv) 0.25% H2O2; (v) 0.1% butanol-1 added 16h prior to addition of 0.25% H2O2; (vi) 0.005mM CHX; (vii) 0.1% butanol-1 added 16h prior to addition of 0.005mM CHX; (viii) 0.1% isobutanol added 16h prior to addition of 0.005mM CHX; and (ix) 0.1% butanol alone.
Total RNA was extracted from 7-d-old etiolated B. oleracea seedlings using the Trizol method (Invitrogen) according to the manufacturer's procedure. Total RNA was quantified by UV spectrophotometry and its integrity was assessed on a 1.2% ethidium bromide-stained formaldehyde agarose gel.
The following primers were used for specific amplification: BO-ACS1, F, 5′-TTCAGGATTATCATGGCTTGCCTG-3′ and R, 5′-CTTCTGCGACGCTGATAAACTCG-3′ (X82273); BO-ACS3, F, 5′-GAGTTCAGACAGGCAATTGCAC-3′ and R, 5′-AGTCCCATGTCTTTCGAGAGAC-3′ (AF338652); BO-ACS4, F, 5′-GAATCCTCACGGCATTATCCAG-3′ and R, 5′-AAGTTCGGTTTGGGTTGCTGTC-3′ (AB086353); BO-ACS5, F, 5′-CTAGTCTCAAGAGGAACGGTCAG-3′ and R, 5′-TTCTTGGAAGCGATGAAGTCAACG-3′ (AF074930); BO-ACS7, F, 5′-TGGGTTCAGAGAAAACGCACTG-3′ and R, 5′-CTGACACGTCATCGATGTTCTC-3′ (AF338651); BO-ACS9, F, 5′-AGCTAAGAATCCGGACGCAGCAG-3′ and R, 5′-CATGAACTCGTTTGGAGACTTCAC-3′ (AF074929); BO-ACS11, F, 5′-GTTCCAAGATTACTATGGCTTGCC-3′ and R, 5′-CACTTCTAGAACGCTGGTGAACTC-3' (AF074928); BO-ACO1, F, 5′-GACAAGGTCAGTGGTCTCCAGCTTC-3′ and R, 5′-CCATTGACCAACAATTAACCACCAG-3′ (X81628); BO-ETR1, F, 5′-GCTCAAACACAGTCTTTAGCGAC-3′ and R, 5′-ATCACACTAAACCTCGCACCAG-3′ (AF047476); BO-ERS1, F, 5′-CTATAGGCGATGAGAAACGTCTG-3′ and R, 5′-GTGATTTGGCTGCAAGACGTAGC-3′ (AF047477); BO-ETR2, F, 5′-GGTTTCGGTTTACGGTTGATGC- 3′ and R, 5′-CTGTTCCATGGACTGATATGGAC-3′ (AB078598); BO-PLDα1, F, 5′-CAAGCTATATTGGATTGGCAGAG-3′ and R, 5′-AAATCCGGGAAACTCAGTGACG-3′ (AF090445); BO-PLD α2, F, 5′-CAAGCTATATTGGATTGGCAGAG-3′ and R, 5′-TGATATTACCTTCATTGTCCACAC-3′ (AF090444); BO-PLDγ1, F, 5′-TTTGCGTGTAGAGCTGTTGCAC-3′ and R, 5′-CAGCACTTCCCATGTCTATACTG-3′ (EU591736); BO-PLDδ, F, 5′-GGAAGGTGTTCGAGTTTTGCTAC-3′ and R, 5′-CACGGATCCTGAGTCGATAGAAC-3′ (AY113045); BO-RbohD, F, 5′-GGCACTGATAATGGAAGAGTTGG-3′ and R, 5′-CCATCCTTCCACTCCTTTCAC-3′ (AF424625); BO-RbohF, F, 5′-GCGGTAAAAGCGGACTTCTCAG-3′ and R, 5′-CGGTACTCCGCAATAAAACACTC-3′ (AB008111); and BO-ACT1, F, 5′-GCTATCCAAGCTGTCCTCTC-3′ and R, 5′-GAGAGCTTCTCCTTGATGTC-3′ (AF044573).
Primers for BO-ACS5, 9, and 11 genes and for BO-PLDδ, BO-RbohD, and BO-RbohF genes were designed using the appropriate genes from Sinapis arvensis and Arabidopsis thaliana, respectively. The products of amplification were sequenced to confirm that these primers amplified fragments of the predicted genes from B. oleracea var. alboglabra A12 DH (see Supplementary data available at JXB online). The numbering of the broccoli ACS isozymes BO-ACS4, 5, 7, 9, and 11 was carried out according to their well-characterized counterparts from Arabidopsis, with the exception of BO-ACS1 and BO-ACS3 which correspond to AT-ACS6 and AT-ACS2, respectively.
First-strand cDNA synthesis was performed in a 20μl reaction mixture containing 1μg of total RNA, 0.2μg of random hexamers, four dNTPs, RNase inhibitor, buffer, and M-MLV reverse transcriptase according to the manufacturer's instructions (Promega).
To determine the temporal expression patterns of ACS, ACO, ethylene receptors, PLD, and Rboh-NADPH oxidase D and F genes during the various stresses, a semi-quantitative analysis of steady-state transcript levels using an RT-PCR with gene-specific primers was performed. Reaction mixtures contained 2.5μl of 10× Mg-free buffer (Fermentas), MgCl2, dNTPs, and forward and reverse primers to the final concentration 1.5mM, 0.25mM, and 250nM, respectively. Each reaction mixture contained 1.5μl of appropriate 4-fold diluted cDNA mixture and 1U of Taq polymerase in a final volume of 25μl, which was overlaid with 30μl of mineral oil.
The reaction mixture was maintained at 95°C for 5min and then cycled 28, 30, 31, or 33 times at 95°C for 30s, 54°C for 30s, and 72°C for 90s, with a final extension of 5min at 72°C. The numbers of cycles were determined experimentally for each analysed gene to detect the RT-PCR products in the linear range. To ensure internal control of the reaction, the actin housekeeping gene was amplified simultaneously in one tube with the gene of interest with actin-specific primers. The concentrations of primers were selected to obtain sufficient amounts of both amplicons and to ensure that the primers would not limit the reactions. The number of cycles was chosen to ensure that both products were clearly visible on the agarose gel but stayed in the exponential phase of amplification (28–33 cycles).
The primers were added in such a manner that the final concentration of gene-specific primers was 250nM, in contrast to the concentration of the actin-specific primers which was lower and most often equal to 200nM for 28 cycles, 160nM for 30 and 31 cycles, and 125nM for 33 cycles. Moreover, the RT-PCRs for each gene of interest and the control actin gene have been carried out in independent thermocycler runs from the same cDNA stocks. RT-PCR products were analysed by 1.7% agarose gel electrophoresis and stained with ethidium bromide. Gels were visualized under UV light; images were taken using a gel documentation system.
The steady-state transcript levels of genes of the ethylene synthesis pathway and perception, PLDD-α1, -α2, -γ1, -δ, and NADPH oxidase RbohD and F in 7-d-old etiolated seedlings of B. oleracea var. alboglabra ADH12 in time course experiments using semi-quantitative RT-PCR have been investigated.
To determine the possible influence of the PAPLD signalling on the expression of the above-mentioned genes, the study was performed on seedlings pre-treated or not with butanol-1 before addition of copper, H2O2, or CHX. The suggested effect of treatment with butanol-1 on the ethylene signal transduction pathway is shown in Fig. 2.
Among primary alcohols functioning as transphosphatidylation substrates, butanol-1 has been known to be the most potent; however, it has been shown that plant PLD isozymes differ in their transphosphatidylation potentials (Morris et al., 1997; Mansfeld et al., 2009). In algae, isobutanol functions as a transphosphatidylation substrate but it is significantly less effective than butanol-1, while animal PLDs do not use it (Munnik et al., 1995; Shen et al., 2001). Supplementary Fig. S1 at JXB online shows the effect of treatment of the dark- and light-grown broccoli seedlings with butanol-1 and isobutanol. In contrast to butanol-1, treatment of seedlings with isobutanol did not seem to visibly affect their growth and development. Treatment of seedlings with butanol-3 (a tertiary alcohol, which is neither a transphosphatidylation substrate nor an activator of PLD and is often used as a control in experiments involving butanol-1 action) caused abnormalities in root development (not presented). Thus, pre-treatment with isobutanol was preferred in control experiments of the effect of butanol-1 on the expression of the genes studied. The control experiment involved pre-treatment of seedlings with isobutanol prior to their treatment with the most potent inducer used, CHX (Fig. 5).
Ethylene regulates its own synthesis in a positive or negative feedback manner. To investigate the functioning of such a regulatory system, the response to copper was studied on seedlings pre-treated or not with silver ions. The silver treatment reduced the ethylene sensitivity in these seedlings. The effect of silver on ethylene signalling is illustrated in Fig. 2.
In broccoli after harvest, expression of one ACS and three ACO genes was characterised in florets, sepals, and yellowing leaves, and there are no data regarding expression of ACS and ACO genes in response to other stimuli (Pogson et al., 1995; Yang et al., 2003).
The detailed comparative analysis of the transcriptional pattern of ACS1, -3, -4, -5, -7, and -11 genes in response to copper, H2O2, and CHX is presented in Figs 3, 4, and 5. In contrast to the other ACS genes, the transcripts of the BO-ACS9 gene were not detected under any type of stimulation. Among all the investigated ACS genes, ACS1 showed the highest accumulation of expressed mRNAs (detection of its amplicons at 28 cycles) in response to the earlier mentioned stimuli. The accumulation of transcripts of the other ACS genes was lower and thus they were amplified in a linear range using 31 or 33 cycles.
The ACS1 gene was the only expressed in the same manner rapidly and explicitly in response to different concentrations of copper, in contrast to the other genes whose expression was strongly dependent on the concentration of the stressor (the results of treatment with 0.5mM copper are not presented). Therefore, the conclusion is that the transcriptional induction of these genes observed in seedlings treated with 2.5mM copper may result from the following events occurring in plants at its higher concentration (Fig. 3).
The pre-treatment of seedlings with silver prior to addition of copper hastened the beginning of up-regulation of the ACS1, -3, -7, and -11 genes and/or affected the level of transcripts, relative to their expression in seedlings treated with copper alone. Thus, the suggestion has been made that ethylene controls the start of up-regulation and/or the level of mRNA of these genes via negative feedback. The ACS3 was the only one of the ACS genes investigated whose copper-enhanced expression was slightly down-regulated in the absence of ethylene signalling (Fig. 3). In seedlings treated with copper alone, the low constitutive expression of the ACS4 and ACS5 genes was down-regulated after 30min and 90min of the stressor action, whereas in seedlings pre-treated with silver such a down-regulation of ACS5 appeared later after 6h of treatment with copper, but in the case of ACS4 it did not occur by this time. It seems that the constitutive expression of both genes is negatively controlled by the copper-enhanced ethylene production.
In broccoli, during the time course of the experiment, a low level of transcripts of constitutively expressed ACS1 and 5 genes was not essentially affected by treatment with butanol-1 alone, whereas the expression of other ACS genes was not detected (Fig. 3D). Nonetheless, the interruption of the PAPLD signalling prior to the treatment of plants with copper, H2O2, and CHX visibly affected the expression of all ACS genes studied to a different degree and in different manners in response to them.
In seedlings pre-treated with butanol-1 prior to the addition of copper the pattern of expression of the ACS1, -3, -5, and -7 genes resembled that observed in seedlings pre-treated with silver (Fig. 3B, C). In contrast, the ACS4 and ACS11 genes did not respond in a similar way to both pre-treating agents and the observed differences can suggest that they require the presence of PAPLD or of an appropriately high level of intracellular PA to be effectively transcribed.
The ACS1 gene seems to be the only gene whose expression was rapidly, significantly, and directly up-regulated by H2O2 itself. An increase in the expression of the ACS3 and ACS7 genes followed much later and could be generated by other events in seedlings concomitant with the action of the inducer rather than directly by H2O2. In contrast, the very labile low expression of ACS4, -5, and -11 genes was down-regulated and hardly perceptible after 2h of treatment with H2O2 (Fig 4).
The pre-treatment with butanol-1 prior to addition of H2O2 did not considerably affect the expression of the ACS1 gene, essentially delayed the beginning of up-regulation of the ACS3 gene, enhanced the constitutive level of ACS7, and hastened the start of its up-regulation in comparison with the response of these genes to H2O2 alone. In seedlings pre-treated with butanol-1 prior to addition of H2O2, the accumulation of transcripts of ACS4, -5, and -11 was below the level of detection (detection at 33 cycles) (Fig. 4).
Considerably increased expression of all the ACS genes discussed, except ACS5, in response to the treatment with CHX was observed (Fig. 5). In comparison with the response to the earlier described inducers, the detection of transcripts of the ACS1, -4, and -5 genes occurred at the same number of cycles (28, 33, and 33, respectively), ACS7 and ACS11 at a lower number of cyles (30 cycles), and ACS3 at a higher number (33 cycles). In seedlings pre-treated with butanol-1 and subsequently treated with CHX, the transcripts of ACS3, -4, and -11 genes were below the limit of detection, whereas those of ACS1, -7, and -5 only slightly altered the pattern of expression in comparison with that in seedlings treated with CHX alone (Fig. 5). The level of constitutive expression of ACS5 was labile and dependent on the set of seedling used; however, it always declined in response to CHX.
In seedlings pre-treated with isobutanol and subsequently treated with CHX, the transcriptional patterns of all ACS genes were very similar to those observed in the seedlings treated with CHX alone, except ACS5. Surprisingly, pre-treament with isobutanol prior to the addition of CHX results in the increased expression of the ACS5 gene in response to this latter stimulus (Fig. 5).
The presence of transcripts of ACO2 and ACO3 genes in stimulated and non-stimulated seedlings was controversial (data not presented). In contrast to these genes, ACO1 revealed a rather high constitutive level of expression (detection at 28 cycles) and was up-regulated in response to the inducers used (Figs 3, 4, and 5). The ACO1 gene was highly expressed in response to butanol-1 alone, which implies a significant role for PAPLD signalling in negative regulation of this gene (Fig. 3). The addition of copper to the seedlings previously pre-treated with butanol-1 for the next 3h only slightly increased the enhanced earlier expression of the ACO1 gene, but after 4h of the treatment it led to a decrease (Fig. 3).
The pre-treatment with silver did not considerably affect the constitutive expression of the ACO1 gene but in seedlings pre-treated with silver its up-regulation in response to copper was lowered and remained shorter in comparison with that in response to copper alone. This implies that ethylene controls the level of transcripts of the ACO1 gene by positive feedback (Fig. 3).
H2O2 or CHX alone enhanced the level of transcripts of ACO1 after 1h or 3h of treatments, respectively (Figs 4, ,5).5). An increased expression of the ACO1 gene caused by pre-treatment with butanol-1 was temporarily down-regulated after the subsequent addition of H2O2 or CHX, but in both cases it rapidly returned to its up-regulated level. Nevertheless, for the seedlings treated with butanol-1 and subsequently with CHX after 4h of treatment with this latter stimulus, the enhanced expression of ACO1 decreased and stabilized at a constitutive level.
In plants pre-treated with isobutanol and subsequently treated with CHX, the timing of expression of the ACO1 gene was similar to that observed in the seedlings treated with CHX alone, but the level of its transcripts was visibly higher (Fig. 5).
ETR1 and ERS1 genes displayed moderate constitutive expression which allowed the detection of amplicons of their transcripts at 30 cycles. In contrast, the transcripts of ETR2 were detected at 33 cycles and this gene was the only one whose expression was significantly transitorily up-regulated in response to copper, whereas the accumulation of ETR1 and ERS1 mRNAs fluctuated about the level of the control, with a temporary increase in the latter.
In the plants treated with butanol-1 alone, the ETR1 gene was expressed at similar levels to untreated plants. In contrast, the expression of ERS1 and ETR2 decreased; however, after 21h of treatment with butanol-1 it returned to the initial levels (Fig. 3).
The pre-treatment of seedlings with butanol-1 prior to the addition of copper resulted in a lowered expression of ETR2 and ERS1, and in a slight increase in the expression of ETR1, which nevertheless was down-regulated later, remaining at a constitutive level after 4h of copper action (Fig. 3). The pre-treatment of seedlings with silver prior to addition of copper affected the expression of ETR1, ERS1, and ETR2 in a similar manner to that described for the seedlings pre-treated with butanol-1 (Fig. 3).
In response to H2O2 the expression of all of the ethylene receptor genes investigated was transiently down-regulated; nevertheless, it returned to control levels or was even slightly up-regulated after 4–6h of treatment with H2O2 (Fig. 4). Moreover, the pre-treatment of seedlings with butanol-1 prior to addition of H2O2 reduced the expression of all these genes much more (Fig. 4).
The treatment of seedlings with CHX only slightly enhanced the expression of ETR1, in contrast to ERS1 and ETR2 which were up-regulated to a greater degree. In the seedlings pre-treated with butanol-1, the constitutive expression of ETR1 increased and only slightly fluctuated throughout the whole time course of the subsequent action of CHX. In contrast, the expression of ERS1 was decreased and remained at a low level during the whole treatment with CHX. The abundance of the ETR2 transcripts distinctly decreased in comparison with that in the seedlings treated with CHX alone (Fig. 5).
In seedlings pre-treated with isobutanol and subsequently treated with CHX, the patterns of expression of ETR1, ERS1, and ETR2 genes did not differ significantly from those observed in the seedlings treated with CHX alone (Fig. 5).
It has been established that in B. oleracea the PLD-α2 isozyme is somewhat more active than -α1, and they both slightly differ in their preference for substrates of hydrolysis (Dippe and Ulbrich-Hofmann, 2009); and at least two PLD-γ isoforms occur (Novotna et al., 2003).
In the present study, the expression of the PLD-α1, -α2, -γ1, and -δ genes has been investigated. The abundance of the PLD-α1 and -α2 transcripts in control and stressed seedlings allowed detection of amplicons of their transcripts at 31 cycles, while these of the PLD-γ1 and –δ genes were detected at 33 cycles. PLD-α1, -α2, and PLD-δ did not respond significantly to copper throughout the time of treatment, whereas the accumulation of the PLD-γ1 mRNAs was essentially increased (Fig. 3).
In seedlings treated with butanol-1 alone, the constitutive levels of PLD-α2 declined somewhat, in contrast to PLD-γ1 and -δ which showed barely perceptible up-regulation (Fig. 3).
The pre-treatment of seedlings with butanol-1 prior to the subsequent addition of copper did not considerably affect the level of PLD-α1 and -α2 transcripts during 4h of treatment with this latter stimulus, but strongly down-regulated the expression of the genes after this time. The same treatment slightly lowered the constitutive expression and the copper-induced up-regulation of the PLD-γ1 gene; however, after 4h it was essentially down-regulated in a manner similar to the case of expression of PLD-α1 and -α2 genes. The pre-treatment of seedlings with silver did not significantly influence the expression of PLD-α1 and -α2 genes but, after the addition of copper, a visible decline in the abundance of the PLD-α1 transcripts occurred, and after 3h the accumulation of PLD-α2 mRNA decreased somewhat. In seedlings pre-treated with silver, the copper-induced up-regulation of the PLD-γ1 gene started earlier but the level of transcripts was lower in comparison with that was observed in response to copper alone. The expression of PLD-δ was rather unaffected by the treatments discussed above (Fig. 3).
The treatment with H2O2 transiently lowered the transcriptional expression of the PLD-α1 and -α2 genes. In contrast, in response to this stimulus the accumulation of transcripts of the PLD-γ1 gene were greatly increased. In seedlings pre-treated with butanol-1, the addition of H2O2 essentially temporarily down-regulated the expression of both -α1 and -α2, whereas the timing of expression of PLD-γ1 only was somewhat changed in comparison with that observed in the seedlings treated with H2O2 alone. Constitutive expression of the PLD-α1 and -α2 genes did not alter throughout the 6h of treatment of the seedlings with CHX, whereas the expression of PLD-γ1 clearly increased. In plants pre-treated with butanol-1, the timing of expression of PLD-α1, PLD-α2, and PLD-γ1 genes did not change considerably in comparison with that in the seedlings treated with CHX alone (Fig. 5).
H2O2 and CHX, when used alone, down-regulated the low expression of the PLD-δ gene after 6h of treatment of seedlings. In both cases, the pre-treatment with butanol-1 increased the level of PLD-δ mRNA but only when followed by treatment with H2O2 did it prevent the down-regulation of expression of the PLD-δ gene (Figs 4, ,55).
In seedlings pre-treated with isobutanol and subsequently treated with CHX, the expression of PLD-α1, PLD-α2, and PLD-γ1 genes did not differ significantly from that observed in the seedlings treated with CHX alone; however, the level of transcripts of the PLD-γ1 gene was somewhat elevated (Fig. 5).
After 6h of treatment with CHX alone, the expression of the PLD-δ gene was decreased, but when pre-treated with isobutanol prior to addition of CHX it was even slightly up-regulated after this time (Fig. 5).
The respiratory burst oxidase homologue (Rboh) gene family encodes the key enzymatic subunit of the plant Rboh-NADPH oxidase. The PA elevated activity of Rboh-NADPH oxidase can cause collapse of antioxidant systems that scavenge ROS (Yu et al., 2008). In Arabidopsis, there are 10 different Rboh genes whose expression is mainly transcriptionally controlled in a tissue-specific manner, but RbohD and F genes belong to the group expressed throughout the whole plant (Torres et al., 2002; Kwak et al., 2003; Torres and Dangl, 2005).
In broccoli, in response to the earlier discussed stimuli, the expression of both genes increased, but in different ways. Generally, the abundance of transcripts of the RbohF gene was lower than that of the RbohD gene (both detected at 33 cycles). In response to copper, an increase in the expression of RbohD significantly preceded the expression of RbohF. The up-regulation of expression of RbohD occurred earlier in the seedlings pre-treated with silver in comparison with those treated only with copper. In seedlings pre-treated with butanol-1 the hastened and enhanced up-regulation of the RbohD gene stopped after 6h of treatment with copper. Neither pre-treatment affected the abundance of transcripts of the RbohF gene but only hastened its slight up-regulation (Fig. 3).
The treatment of plants with butanol-1 alone did not considerably affect the expression of RbohD and F genes, causing low transitory expression of RbohD after 21h and similarly low expression of RbohF after 20h of stimulation (Fig. 3).
The expression of RbohF in response to copper and in response to H2O2 was very similar (Figs 3, ,4).4). In contrast to RbohF, the expression of RbohD in response to H2O2 increased significantly later, after 6h of treatment. Therefore, it should be considered whether the high concentration of H2O2 could maintain the expression of RbohD at a constitutive level blocking its up-regulation for at least 4h. The pre-treatment with butanol-1 diminished the level of transcripts of RbohD and F but generally did not alter the time course of their response to H2O2.
Both genes discussed were up-regulated in a similar manner in response to the treatment with CHX and in both of them the pre-treatment of seedlings with butanol-1 only somewhat decreased the accumulation of their transcripts (Fig. 4).
In seedlings pre-treated with isobutanol and subsequently treated with CHX, the timing of expression of RbohD and F genes resembled that observed in the seedlings treated with CHX alone; however, the expression of RbohD and F genes was visibly higher after 4–6h of treatment with CHX in plants pre-treated with isobutanol (Fig. 5).
It has been assumed that two systems, the ethylene autoinhibitory system 1 and system 2, regulated by a positive feedback mechanism control the transcriptional activity of genes encoding ACS and ACO isozymes (Nakatsuka et al., 1998; Barry et al., 2000; Kim et al., 2001). In the absence of ethylene, its receptors operate via the Raf-1-like kinase, CTR1, whose activity suppresses the ethylene responses. In the presence of ethylene, receptors do not stimulate CTR1, which results in its inactivation and induction of a response to ethylene (Zhu and Guo, 2008) (Fig. 2). Testerink et al. (2007, 2008) reported that PA can be a negative regulator of CTR1 via its binding to CTR1’s kinase domain and through reduction of the binding of CTR1’s kinase domain to the ethylene receptor, ETR1. A direct effect of PA on the activity of CTR1 suggests the possibility of turning on of ethylene signalling in the absence of ethylene (Fig. 2D). PA, present in the endoplasmic reticulum all the time, may be involved in a complicated mechanism of CTR1 regulation.
In plants, PA signalling is associated with a broad spectrum of biotic and abiotic stimuli. The host–pathogen interactions stimulate a biphasic PA response. The first, rapid phase following a few minutes of stress generally involves PAPLC/DGK, while the second one involves a specific PAPLD (Wang, 2000; Laxalt and Muunik, 2002; den Hartog et al., 2003; Meijer and Munnik, 2003; Bargmann et al., 2006; Navari-Izzo et al., 2006). Downstream from PA formation, the concomitant biphasic accumulation of ROS takes place (van der Luit et al., 2000; de Jong et al., 2004; Andersson et al., 2006; Arisz et al., 2009). It has been believed that the stress-induced PAPLD can elevate the activity of Rboh-NADPH-oxidase (Yu et al., 2008). In the present experiments, treatment with exogenously added H2O2 can mimic the first initial phase of the oxidative burst.
A comparative analysis of expression of the genes under discussion in (i) ethylene-sensitive seedlings; (ii) seedlings whose sensitivity to ethylene was reduced at the level of receptors; and (iii) seedlings with a lowered level of PAPLD has been made in a previous section (Figs. 3, 4 and 5). Below, the inter-relationships concluded to occur between the action of inducers and the ethylene and PAPLD signalling pathways occurring in the seedlings treated with copper (Fig. 6A), H2O2 (Fig. 6C), and CHX (Fig. 6B) are summarized.
In conclusion, the following phenomena were observed:
Supplementary data are available at JXB online.
Figure S1. Illustration of the effect of the long-term treatment of broccoli seedlings with 0.1% butanol-1 and 0.1% isobutanol. Seedlings were germinated in the dark on MS medium with addition of 3% sucrose, and 1d after germination (denoted as time 0) 10 randomly chosen seedlings were transferred to the same medium without supplements (control seedlings); to medium with 0.1% butanol-1; and to medium with 0.1% isobutanol, and were kept in dark (A) or light (16h light/8h dark, B) conditions throughout the next 7d.
Figure S2. Sequence alignment of partial cDNAs of BO-ACS5, 9, and 11, BO-PLDδ, and BO-RbohD and F (GU942464, GU942465, GU942466, GU942467, GU942468, and GU942463, respectively) with their counterparts from Arabidopsis thaliana and Sinapis arvensis (SA-ACS2, 3 and 4, AT-ACS5, 9 and 11; AF074928, AF074929, and AF074930, NM_125977, NM_114830, and NM_116873, respectively; AT-PLDδ, AT-RbohD and F; NM_179170, NM_124165 and NM_105079, respectively)
We are grateful to Hanna Korcz-Szatkowska for help in preparing plant materials.