Local and systemic wound-induction of RNase and nuclease activities in Arabidopsis: RNS1 as a marker for a JA-independent systemic signaling pathway

Authors


* For correspondence (fax +1 517 353 9168; e-mail green@msu.edu).

Summary

Induction of defense-related genes is one way in which plants respond to mechanical injury. We investigated whether RNases are involved in the wound response in Arabidopsis thaliana. As in other plant systems, several activities are induced with various timings in damaged leaves, stems and seedlings in Arabidopsis, including at least three bifunctional nucleases, capable of degrading both RNA and DNA, as well as RNS1, a member of the ubiquitous RNase T2 family of RNases. The strong induction of RNS1 is particularly interesting because it occurs both locally and systemically following wounding. The systemic induction of this RNase indicates that members of this family may be involved in defense mechanisms in addition to their previously hypothesized functions in nutrient recycling and remobilization. Additionally, the systemic induction appears to be controlled independently of jasmonic acid, and the local induction of RNS1 and the nuclease activities are independent of both JA and oligosaccharide elicitors. Consequently, a novel systemic pathway, likely involving a third signal, appears to exist in Arabidopsis.

Introduction

One of the mechanisms by which plants respond to wounding, either mechanical or feeding-related, is the activation of transcription of a variety of genes. The products of these genes include defensive proteins such as proteinase inhibitors (Boulter, 1993; Ryan, 1990), components of signal transduction pathways, for example, systemin in tomato and potato (Ryan and Pearce, 1998), proteins that may be involved in wound healing (Bowles, 1990), and other proteins whose functions in the wound response are as yet unknown.

While the expression of some of these genes is proximal to the injury site, others are expressed at greater distances and constitute the systemic response (Green and Ryan, 1972). In tomato, multiple signals appear to be involved in activating gene expression. Local responses are thought to be controlled by carbohydrate signals released from injured plant cell walls (Farmer and Ryan, 1992). Long-distance effects are initiated by the small peptide systemin (Ryan and Pearce, 1998). Both oligosaccharides and systemin initiate the de novo production of abscisic acid (ABA) and jasmonic acid (JA), which leads to accumulation of the transcripts of wound-responsive (WR) genes (León et al., 2001). Ethylene also appears to be involved in the amplification of systemin-activated signaling (O'Donnell et al., 1996). In addition, electrical signals have been implicated in the systemic response (Wildon et al., 1992). In general, wound signals in tomato are thought to constitute a unified reaction that mounts both local and systemic defense and repair responses (León et al., 2001), although a JA-independent WR gene has been identified (O'Donnell, 1998).

In Arabidopsis, the emerging picture has striking differences compared with the tomato system. In this case, two distinct signaling pathways can be identified. Some genes whose transcripts accumulate upon wounding are independent of JA, while the expression of others requires jasmonate synthesis and perception (Titarenko et al., 1997). Recently, the JA-independent response was shown to be activated by oligosaccharides (Rojo et al., 1999). It was suggested that the two pathways work in an antagonistic manner, so that the inhibition of the JA-dependent pathway occurs in local wounded leaves through the induction of ethylene by the oligosaccharide elicitors. Conversely, only JA-dependent genes would be activated systemically, due to the limited diffusion range of the oligosaccharides (Rojo et al., 1999). However, a JA-dependent WR gene has been found that is strongly expressed both locally and systemically in Arabidopsis (Kubigsteltig et al., 1999).

In other systems, RNases have been shown to accumulate in response to mechanical wounding or pathogen attack. In tomato, for example, the transcript for RNase LE accumulates in wounded leaves (Lers et al., 1998). In tobacco, extracellular RNase activity increases in response to fungal invasion, and the RNase NE transcript accumulates in correlation with pathogen infection (Galiana et al., 1997). RNase NW, another tobacco activity with high sequence similarity to RNase NE, is induced in wounded leaves (Kariu et al., 1998). Interestingly, exogenous application of RNase A on tobacco leaves inhibits growth of a fungal as well as a viral pathogen (Galiana et al., 1997). A 30-kDa protein sharing similarity with a tomato RNase has been isolated from leaves of Engelmannia pinnatifida and shown to have broad-spectrum antifungal activity (Huynh et al., 1996). An extracellular RNase activity in rust-infected wheat leaves has also been reported (Barna et al., 1989). Although several wound-responsive RNases have been identified, the signaling pathways responsible for RNase induction are rarely addressed.

RNases LE, NE, and NW belong to the widespread RNase T2 family of ribonucleases. Members of this family of secretory RNases have been identified in all organisms so far examined, encompassing viral, bacterial, mammalian, and plant systems (Irie, 1997). Despite the widespread presence of this family, little is known regarding the functions of the enzymes. The exception is the S-RNase family, known to be involved in gametophytic self-incompatibility in several plant families (Golz et al., 1995). In most cases, changes in gene expression patterns or activity levels provide the most useful information for predicting functions of the enzymes. For instance, several members of the RNase T2 family exhibit increased activity or expression of the corresponding gene during senescence or under phosphate deprivation conditions (Bariola and Green, 1997). Such regulatory patterns have led to the hypothesis that the enzymes participate in a phosphate remobilization pathway, either by scavenging phosphate from extracellular sources or by degrading intracellular RNA (Goldstein et al., 1989). Recently, a novel function has been proposed for the RNase T2 family, in which extracellular RNases are hypothesized to be involved in regulating cell membrane permeability (MacIntosh et al., 2001). Such an idea could explain the cytotoxicity of viral T2 RNases (Hulst et al., 1994) as well as other unexplained effects of secreted RNases.

The Arabidopsis thaliana genome contains at least five genes, RNS1 to RNS5, with high similarity to the RNase T2 family (Taylor and Green, 1991; G. C. MacIntosh et al., unpublished), and RNase activity has been demonstrated for the products of three of the RNS genes (Bariola et al., 1994). Like other plant T2 RNases, the RNS genes are differentially regulated by senescence and phosphate starvation (Bariola et al., 1994; Taylor et al., 1993). In order to investigate the effects of wounding on the expression of this family of Arabidopsis RNases, we examined the RNase profile of wounded plants. We found not only T2 enzymes, but also other activities increased in damaged tissues in response to mechanical wounding, including several with both RNase and DNase activity. Within the RNase T2 family, RNS1 activity increases, and the RNS1 transcript accumulates in both wounded and systemic unwounded leaves of wounded plants. Unexpectedly, we found that the regulation of these activities by wounding is independent of both jasmonic acid and oligosaccharides, indicating that a novel, unidentified wound signaling pathway may be operating in Arabidopsis. Additionally, the systemic induction of RNS1 suggests that the enzyme may have a defensive function.

Results

Wounding induces several RNase activities in Arabidopsis

In order to examine the effect of wounding on RNase expression in Arabidopsis, 2-week-old seedlings, or stems or leaves of 4-week-old plants were wounded. Samples were harvested at subsequent timepoints, and protein extracts were analyzed on RNase activity gels (Figure 1). Significant alterations in RNase activity are seen in the activity gels, including not only one well-characterized member of the RNase T2 family, RNS1, but also several other activities not likely to be related to this family. In Figure 1(a), equivalent timepoints were taken from leaves of wounded and unwounded plants to control for any possible circadian regulation of RNase activities. Several activities can be seen even in unwounded leaves, the most prominent of which is a strong band of activity at 25 kDa; however, no RNases appear to be regulated in a circadian manner (Figure 1a, Control lanes). Similar controls were taken for stems and seedlings, and there, too, no circadian regulation is evident (data not shown). In contrast, in leaves, stems, and seedlings (Figure 1a–c, respectively) multiple activities are induced by wounding, including an activity that migrates at approximately 35 kDa (Figure 1a–c, top arrow), two at 33 kDa (middle arrow; separation of the two 33-kDa bands can be seen in Figure 2a,b), and a fourth of approximately 23 kDa (bottom arrow, labeled RNS1).

Figure 1.

Multiple RNase activities are induced by wounding.

Activity gels showing the increase in RNase activity in Arabidopsis at various timepoints after wounding. Bands seen on the gel are areas in which RNA cast in the gel has been degraded. Each lane contains 100 μg of protein from wounded leaves (a), stems (b), or seedlings (c).Time, in hours, from wounding to harvesting is indicated above each lane of the gels. Arrows to the right of the gels indicate increased activities. Control lanes contain protein from unwounded leaves. MW, molecular weight marker (Gibco-BRL). Sizes of the MW markers in kDa are shown to the left in (a). hpw, hours post wounding.

Figure 2.

Bifunctional nuclease activities are induced by wounding.

(a) Protein samples (100 μg for RNase gel; 75 μg for DNase gel) from wounded stems were run on activity gels containing either RNA (upper panel) or DNA (lower panel) as a substrate. Both gels contain 9% acrylamide in order to separate the two activities at 33 kDa. Arrows to the left of the gels indicate wound-regulated bifunctional activities. hpw, hours post wounding.

(b) Stem protein samples (100 μg) from both Columbia (Col) and RLD (RLD) ecotypes were run on RNase activity gels. Unwounded samples are shown on the left, and stem samples taken 3 h after wounding are on the right. The lower band (lower arrow) is not found in unwounded Columbia stems but is present after wounding. Arrows indicate the upper and lower bands of the 33-kDa nuclease doublet.

Past studies have determined that a well-characterized RNase T2 enzyme, RNS1, is induced by phosphate starvation in seedlings and migrates at 23 kDa on RNase activity gels (Bariola et al., 1994, 1999). In the wounded samples, a 23-kDa activity is also induced (Figure 1a–c; bottom arrow, labeled RNS1). This activity can be seen within 3 h and remains elevated for at least 48 h after wounding. Northern analysis reveals that the RNS1 transcript is induced in wounded leaves (see below). Additionally, an insertional knock out mutant of RNS1 fails to induce the 23-kDa band after wounding (N. D. LeBrasseur and P. J. Green, unpublished). Taken together, Northern analysis and mutant results confirm that the 23-kDa activity induced by wounding is indeed RNS1. Thus, as in other plant systems, RNase T2 enzymes are also induced by wounding in Arabidopsis. However, our results also demonstrate that RNase activities not related to T2 enzymes respond to wounding in Arabidopsis.

A pair of 33-kDa activities induced in all three wounding experiments has a timecourse of induction similar to that of RNS1. In leaves, the doublet of activity at 33 kDa is induced within 6 h (Figure 1a, middle arrow). In stem tissue and in seedlings, the upper band of the doublet is evident even in unwounded samples. Still, both bands are enhanced after wounding, reaching an apparent maximum by 24 h (Figure 1b,c), and high levels of these activities can be found as late as 48 h post wounding (hpw).

Interestingly, unlike the sustained induction of the 23- and 33-kDa RNases, a fourth activity, at 35 kDa, is induced transiently. In leaves, this activity appears within 6 h of wounding (Figure 1a). In stems and in seedlings, the 35-kDa activity appears more rapidly, with induction evident within 3 h (Figure 1b,c). In all three cases, the activity is no longer visible by 24 hpw. Unlike RNS1, these wound-inducible activities exhibit characteristics that distinguish them from the RNase T2 family, as discussed below.

Multiple bifunctional nucleases are increased by wounding

Previous studies using the gel assay system shown in Figure 1 demonstrated that a doublet of bifunctional nucleases capable of degrading both RNA and DNA migrate at 33 kDa; these nucleases are normally expressed at low levels but are upregulated in a recently isolated mutant of Arabidopsis (M. L. Abler et al., unpublished). To investigate whether the 33-kDa RNase activities increased by wounding are bifunctional nucleases, protein extracts of wounded stem tissue were run on 9% acrylamide RNase and DNase gels. While other activity gels in this study contained 11% acrylamide, the lower acrylamide percentage used in these gels results in better separation of the two activities that migrate at approximately 33 kDa, as can be seen by comparing Figures 1(b) and 2(a). Additionally, the increase in the intensity of the bands seen in the RNase activity gel at 33 kDa is mirrored in the DNase gel (Figure 2a), confirming that this doublet has bifunctional nuclease activity.

In addition to the 33-kDa doublet, another activity that increases upon wounding appears to have bifunctional activity. The transient 35-kDa RNase activity seen in Figure 1 also degrades DNA (Figure 2a). Although the DNase activity gels are more sensitive than the RNase gels, resulting in broader bands, and although several RNA-specific degrading activities do not appear on the DNase gel, the patterns of the bifunctional bands are identical in the two gels. Therefore, wounding causes an induction not only of T2 RNases in Arabidopsis, but also of several nuclease activities, including a previously unidentified nuclease at 35 kDa.

Past studies of RNase expression profiles in Arabidopsis have shown that cultivar variation is present. Specifically, the lower band of the 33-kDa doublet can normally be seen at low levels in the cultivar RLD but is absent in Columbia (M. L. Abler et al., unpublished). However, the lower band can be induced in Columbia upon wounding (Figure 2b). Thus, wounding indicates that although this activity is differentially regulated under non-wounded conditions in Columbia and RLD, it is not altogether absent from Columbia.

RNS1 is induced by wounding locally and in non-damaged, systemic leaves of wounded plants

As seen in Figure 1, an RNase activity that migrates at approximately 23 kDa is induced during wounding. During phosphate starvation in Arabidopsis seedlings, a 23-kDa activity is also induced (Bariola et al., 1994). The increase in this activity is due to an increase in the protein levels of RNS1 (Bariola et al., 1999) and corresponds to an increase in transcript accumulation (Bariola et al., 1994). Additionally, as mentioned, wounded leaves of an RNS1 T-DNA insertion mutant lack the 23-kDa activity (N. D. LeBrasseur and P. J. Green, unpublished). Therefore, we were interested in examining whether the RNS1 transcript also accumulates in response to wounding. Rosette leaves of adult plants were wounded and then harvested at subsequent timepoints. RNA blot analysis demonstrates that the RNS1 transcript accumulates within 3 h and is still elevated as late as 48 hpw (Figure 3a). Western blot analysis confirms that the protein accumulates with a timing similar to the activity increase (data not shown).

Figure 3.

RNS1 activity and transcript increase transiently in wounded and in systemic, non-wounded leaves.

(a) Northern blot of wounded leaves. Arabidopsis leaves were wounded and harvested at subsequent timepoints. RNA was extracted, and 10 μg was electrophoresed, blotted, and probed with RNS1. Blots were then stripped and reprobed with EF-1α to control for loading. hpw, hours post wounding.

(b) Systemic induction of the RNS1 transcript shown by RNA gel blot analysis. Unwounded rosette leaves adjacent to the wounded leaves were harvested at the timepoints indicated above the lanes. The gel contained 10 μg total RNA per lane. RNA from seedlings harvested 9 h after wounding was included for comparison (W). The blot was stripped and reprobed with EF-1α as a loading control. hpw, hours post wounding.

(c) RNase activity gel showing systemic induction of RNS1 activity. Each lane contains 100 μg protein. The arrow indicates the RNS1 band. No other induced activities were detected in systemic leaves. Protein samples from wounded leaves are included for comparison of RNS1 activity levels.

We also investigated whether the Arabidopsis response to wounding includes systemic induction of RNase or nuclease activities in non-wounded tissue. Unwounded rosette leaves adjacent to wounded leaves were harvested, and RNA was examined by Northern blot. The RNS1 transcript is induced in unwounded leaves. The increase is transient and rapidly returns to uninduced levels between 6 and 8 hpw (Figure 3b), unlike the more sustained increase of RNS1 in wounded leaves (Figure 3a). Wounded seedlings (9 hpw; Figure 3b, lane W) were included in the blot for a comparison of transcript levels induced locally and systemically. Additionally, protein extracts were examined by RNase activity gel assay to determine whether RNS1 or nuclease activities are induced in unwounded leaves of wounded plants. In the unwounded leaves, the 23-kDa RNS1 band is induced (Figure 3c, left), indicating that RNases may play a role in the systemic wounding response in Arabidopsis. This modest increase relative to that in wounded leaves was observed in multiple experiments between approximately 6 and 12 hpw. Local, wounded samples are included in the gel for comparison (Figure 3c, right). No other bands aside from RNS1 are induced systemically (not shown). Note that the activity gel in Figure 3(c) was incubated at pH 6.0, instead of pH 7.0, to enhance RNS1 activity, since no other activities were seen to be induced systemically at pH 7.0 (see Experimental procedures). As a result, the relative activity of RNS1 is higher compared with the constitutive 25 kDa band. It is interesting that the systemic activity increase does not appear to reflect fully the amount of transcript induced (compare 3 and 6 h in Figure 3b with 6 and 12 h in Figure 3c). The discrepancy may be due to a possible post-transcriptional mechanism of regulation of RNS1 in the systemic tissue.

The induction of RNS1 and the nucleases is independent of jasmonic acid

Wounding responses in Arabidopsis are controlled by jasmonic acid (JA)-dependent and -independent signaling pathways (Titarenko et al., 1997). The presence of distinct wounding pathways in Arabidopsis has led to the proposal that the function of multiple signals is to control local and systemic gene expression differentially. Specifically, it has been suggested that the JA-dependent pathway controls induction of systemic responses, while an antagonistic oligosaccharide-dependent pathway directs gene activation locally at the site of damage (Rojo et al., 1999). Under this assumption, RNS1 would be expected to be regulated by the JA-dependent pathway, as its transcript is induced systemically. In contrast, the bifunctional nuclease activities, which are not systemically wound-responsive, would be regulated independent of JA. The JA-insensitive mutant coi1 (Feys et al., 1994) has been widely used to demonstrate dependence of the wound-induction of specific transcripts on JA (Reymond et al., 2000; Titarenko et al., 1997). We investigated whether the wound induction of RNS1 or the nuclease activities are regulated by the JA-dependent pathway by wounding leaves of coi1 plants. As expected, like the wild type, wounded coi1 plants display increased levels of the 35-and 33-kDa activities (Figure 4a). The transient 35-kDa nuclease can be seen in both wild type and mutant at 6 hpw, and the nuclease bands at 33 kDa are induced at 6 and 24 hpw in both wild type and coi1. However, wounded coi1 leaves unexpectedly also had increased RNS1 activity. In fact, in contrast to systemic responses previously characterized in Arabidopsis, both local and systemic wound-induction of RNS1 is independent of JA. RNS1 activity is induced in coi1 local wounded tissue (Figure 4a, upper panel) and in systemic leaves (Figure 4a, lower panel). RNA blot analysis demonstrates that the RNS1 transcript also accumulates in the coi1 mutant upon wounding (Figure 4b). Local 3-h and systemic 6-h timepoints are shown (Figure 4b, L and S, respectively). Systemic RNS1 levels at 6- and 24-h timepoints were also similar in coi1 and WT plants (data not shown).

Figure 4.

RNS1 and nuclease induction by wounding is independent of jasmonic acid.

(a) Top panel; activity gel showing induction of all four wound-responsive activities in both the wild type (WT) and the JA-insensitive coi1 mutant (coi1). Arrows to the right indicate the induced activities, including the nuclease at 35 kDa (top arrow), the doublet at 33 kDa (middle arrow), and RNS1 (bottom arrow). Bottom panel; systemic induction of RNS1 in the coi1 mutant. hpw, hours post wounding.

(b) Northern blot analysis of wounded wild type (WT) and coi1 (coi1) plants. The blot contains RNA (10 μg) from unwounded leaves (U), local 6-h wounded leaves (L), and systemic leaves 3 h after wounding (S). The blot was probed with RNS1, stripped, and reprobed with EF-1α as a loading control.

(c) Activity gels containing 100 μg of protein from leaves of JA-treated (JA) or untreated (control) plants. Plants were treated with methyl jasmonate or buffer alone, and leaves were harvested at 0, 3, 6, and 24-h timepoints. W, protein from leaves 24 h after wounding. Arrows to the right of the gel indicate the running positions of wound-inducible activities.

(d) Northern analysis of RNA (20 μg) from leaves of JA-treated (JA) or untreated (control) plants. Plants were treated and timepoints taken as in (c). W, RNA from leaves 6 h after wounding. Blots were probed with RNS1, then sequentially stripped and reprobed with AOS to control for JA-treatment and with EF-1α as a loading control.

To confirm that JA does not affect the induction of RNS1 or the nucleases, young plants were treated with methyl jasmonate (MeJA) and examined for RNase activity (Figure 4c). Efficacy of the MeJA treatment is shown by the accumulation of the allene oxide synthase transcript (AOS), a component of the jasmonate pathway known to be induced by JA (Laudert and Weiler, 1998; Figure 4d). However, MeJA treatment does not cause RNS1 accumulation after 3, 6, or 24 h of treatment (Figure 4d). Likewise, the 35- and 33-kDa nucleases, as well as RNS1, are not induced by MeJA at the activity level (Figure 4c). Thus, we have demonstrated that the wound-inducible RNase and nuclease activities are not controlled by the JA-dependent signaling pathway. Recent results from an independent microarray experiment also demonstrated that the local expression of RNS1 is induced in a JA-independent manner (Reymond et al., 2000), supporting our observations.

RNS1 is not induced by the oligosaccharide pathway

It has been suggested that the JA-independent wound response in Arabidopsis is controlled by oligosaccharide elicitors, and transcripts whose accumulation after wounding is not dependent on JA can be induced by oligogalacturonic acids (OGAs), proteinase-inhibitor inducing factor (PIIF) fractions from tomato leaves, and chitosan treatments (Rojo et al., 1999), all of which are rich in oligosaccharides. To test if one of these known inducers of certain JA-independent wound-inducible genes would also induce RNS1 transcript accumulation, an OGA-rich fraction known as ‘TFA-PIIF’ (generously provided by Dr E. E. Farmer; purified according to Bishop et al., 1984) was used to treat detached rosette leaves. Leaves were floated in an MS medium either with or without the addition of TFA-PIIF and harvested from both conditions at subsequent timepoints. RNS1 transcript levels were analyzed by RNA gel blot analysis (Figure 5a).

Figure 5.

OGA-rich fractions do not induce RNase activities.

Leaves were detached from 4-week-old wild type plants and floated in buffer with (+ PIIF) or without (– PIIF) the addition of TFA-PIIF. Samples were harvested at timepoints indicated above the lanes (in hours).

(a) Northern blot containing 10 μg RNA per lane. W, RNA from leaves 6 h after wounding. The blot was probed with RNS1, then sequentially stripped and reprobed with CK to control for PIIF-treatment and with eIF-4A as a loading control.

(b) Activity gel containing 100 μg protein per lane. Arrows to the right of the gel indicate the normal running positions of the wound-inducible activities.

As seen in Figure 5(a), the slight wounding involved in detaching leaves causes a higher than normal basal level of transcript in the samples after 3 h. However, in contrast to most known Arabidopsis JA-independent wound-induced transcripts, the presence of TFA-PIIF in the medium does not induce RNS1 compared with the control (Figure 5a). Blots were also probed with choline kinase (CK), an OGA-induced transcript (Rojo et al., 1999), to demonstrate efficacy of the OGA treatment. The wounding involved in the detachment technique induces CK expression at the 90-min timepoint, regardless of the presence or absence of OGAs and consistent with the timing of CK wound-induction seen previously (Rojo et al., 1999). However, as expected, the TFA-PIIF-treated samples have increased levels of CK compared with the control samples at both 3- and 6-h timepoints (Figure 5a). Thus, while CK levels increase due to the treatment, RNS1 levels are not affected by the PIIF fraction. Interestingly, the wound-inducible nuclease activities, as well as RNS1 activity, are also not induced by the treatment, as seen by RNase activity gel assay (Figure 5b). Throughout the treatment timecourse, the only activity detected is a major band at 25 kDa, which is not regulated by wounding. This activity is also not affected by the TFA-PIIFs, and no other activities are induced, including the wound-responsive 35- and 33-kDa nucleases and RNS1.

Thus, although the treatment was effective, oligosaccharides do not control the induction of the RNase and nuclease activities by wounding. These results were unexpected, considering that all the activities were also independent of JA. Therefore, we hypothesize that in addition to oligosaccharides and JA, an additional signal exists that directs expression of nuclease and RNase activities in wounded tissue.

Discussion

In this paper, we have demonstrated that several Arabidopsis RNase and nuclease activities are co-ordinately regulated by wounding. Unlike other wound-inducible proteins previously studied in Arabidopsis, these activities are not controlled by jasmonic acid signaling or by oligosaccharide elicitors. The systemic induction of RNS1 makes this ribonuclease especially intriguing. The systemic induction of RNS1 not only supports the existence of a novel pathway for the regulation of systemic wound responses, but also is suggestive of a defensive role for this RNase.

The increases in all the activities occur in a co-ordinate manner and provide us with a unique perspective into Arabidopsis wound signaling mechanisms. Our understanding of the wound response in Arabidopsis is currently highlighted by the presence of two distinct, antagonistic pathways: JA-dependent and -independent. The JA-independent pathway controls local induction of transcript accumulation and has been shown to be regulated by OGA elicitors probably released from injured plant cell walls (Rojo et al., 1999). The three nuclease activities and RNS1 are strongly induced locally by wounding (Figures 1 and 2). However, they are not induced by treatments with OGA-rich fractions (Figure 5). In fact, in several repetitions of the OGA treatments, the slight wound-induction of RNS1 caused by detaching the leaves appeared to be inhibited by the presence of the OGAs (for example, compare + PIIF and -PIIF at 6 h in Figure 5a). Given that the RNase and nuclease activities are not induced by the PIIF treatments, we conclude that another signal besides OGAs is likely operating to direct the local induction of these activities upon wounding.

The local response of RNS1 and the nucleases to wounding was also not controlled by the JA-dependent signaling pathway, as shown by the strong wound-induction of these activities in the coi1 mutant (Figure 4a). Further, RNS1 transcript accumulated to high levels in systemic coi1 tissues. JA is an important signaling molecule in gene induction in unwounded systemic leaves in Arabidopsis (Titarenko et al., 1997). However, the systemic induction of RNS1 did not depend on JA (Figure 4a,b). To our knowledge, RNS1 is the first gene in Arabidopsis shown to be induced systemically by wounding in a JA-independent manner and therefore indicates the existence of an alternate long-distance signaling pathway.

It has been proposed that dehydration itself might be the causal induction factor of some JA-independent wound-inducible genes, including RNS1, which was shown by microarray analysis to be induced by dehydration (Reymond et al., 2000). However, the dehydration experiments performed involved detaching the rosette leaves from the roots, which itself induces RNS1 (Figure 5). Our preliminary results indicate that dehydration itself does not induce RNS1 expression but may potentiate the wound-induction (N. D. LeBrasseur and P. J. Green, unpublished results). The induction of certain wound-inducible genes in potato and tomato are affected by ABA levels (Peña-Cortés et al., 1989), so ABA could be considered a possible signal for the wound-induction of RNS1. However, RNS1 is still induced by wounding in the abi1, abi2, and aba1–1 mutants (N. D. LeBrasseur and P. J. Green, unpublished results), indicating that ABA perception and signal transduction are not required for the wound induction.

Given its involvement in the wound response in Solanaceous plant species and in the regulation of various programmed cell death processes (Jones, 2001), ethylene is another candidate for the signal involved in the induction of RNS1 both locally and systemically. However, the wound-induction of RNS1 also occurs in the ein2 mutant, suggesting that ethylene perception is not required (Reymond et al., 2000). Salicylic acid (SA) is required for many plant-pathogen defense responses (Alvarez, 2000), and cross-talk between SA, JA, and ethylene pathways is emerging as an important regulatory method for activating multiple resistance mechanisms (Pieterse and Van Loon, 1999). However, SA does not appear to be involved in the regulation of RNS1 by wounding, as the transcript is still induced in SA-deficient, NahG-expressing plants (E. E. Farmer, pers. comm.). Additionally, analysis of AFGC microarray data in the Stanford Microarray Database available on the web (http://genome-www4.stanford.edu/MicroArray/SMD/Wisman and Ohlrogge, 2000) indicates that RNS1 is not induced by treatment with the SA-analog, BTH, or by various pathogens that induce systemic acquired resistance, which is known to cause the accumulation of SA within the plant (Alvarez, 2000).

Other possible signals for the induction of RNS1 include reactive oxygen species (ROS). ROSs are commonly produced in plants in response to both pathogen and herbivore attacks (Grant and Loake, 2000; Orozco-Cárdenas and Ryan, 1999). In tomato, H2O2 acts as a second messenger for the induction of defense–related genes induced systemically at later timepoints than the earlier, signaling-related genes (Orozco-Cárdenas et al., 2001). The timing of RNase and nuclease responses coincides with these later responses that are dependent on H2O2 signaling. However, microarray analysis of NO- and H2O2-treated Arabidopsis cultured cells shows no induction of RNS1 after 3 h of treatment (AFGC microarray expts 9371 and 7523), although peroxide effects in plants may differ from those in cell culture. The involvement of ROSs, as well as other possibilities, for example, electrical signals, in RNase induction during the Arabidopsis wound response will prove an important avenue of study for defining alternate pathways regulating defense responses.

The systemic increase in RNS1 appears to be regulated at multiple levels. Studies with reporter constructs controlled by the RNS1 promoter region indicate that the local increase in RNS1 transcript caused by wounding is likely due to transcriptional regulation (N. D. LeBrasseur and P. J. Green, unpublished results). However, while the transcript increases to significant levels systemically, the activity increase is not as great (compare Figures 3b, 3 and 6h, with Figures 3c, 6 and 8h). It is possible that a post-transcriptional mechanism may also exist that regulates the amount of RNS1 protein translated or the activity of the translated protein in systemic tissue.

In addition to its interesting regulatory properties, the systemic induction of RNS1 implies that the RNase may have an important function during the wound response. It is striking that the RNS1 transcript was the most highly induced in local wounded tissue of all transcripts examined in two independent microarray experiments: one examined 150 genes enriched for those implicated in defense responses (Reymond et al. 2000), and the second examined 600 genes, approximately half of which were hypothesized to be involved in RNA metabolism and RNA turnover (M. A. Pérez-Amador and P. J. Green, unpublished). The high level of transcript accumulation of RNS1 may be indicative of an important role for the protein product of this gene. Additionally, the transcript and the activity are induced in non-damaged systemic tissue (Figure 3b,c), and the transcript was also found to be elevated in systemic leaves 2 h after wounding in the microarray analysis described above (M. A. Pérez-Amador and P. J. Green, unpublished). Since recycling of nutrients and degradation of bulk cellular nucleic acid, two of the proposed functions of secretory RNases, should not be necessary in unwounded tissues, it is possible that RNS1 has a defensive function. Normally, RNS1 is expressed solely in flowers (Bariola et al., 1994). The presence of RNases in the pistil may contribute to protection of the structure from pathogens (Bariola et al., 1994). In fact, it has been demonstrated that application of an extracellular RNase to tobacco leaves inhibits growth of both TMV and a fungal pathogen (Galiana et al., 1997). Likewise, local and systemic induction of RNS1 could contribute to protection of the plant against invasion of pathogens following wounding. Although it is not known how RNases could achieve such defensive roles, wound signal molecules are known to produce membrane-associated changes within the plant cells, including membrane depolarization (Thain et al., 1995). Recently, it has been proposed that T2 RNases can affect membrane stability or permeability (MacIntosh et al., 2001), suggesting a possible link. The role of RNS1 in defense mechanisms will be readily testable in a recently isolated RNS1 T-DNA insertional mutant and in plants that overexpress RNS1 activity (N. D. LeBrasseur and P. J. Green, unpublished).

In contrast to the systemically induced RNS1, the bifunctional activities were induced only in local, damaged tissue. These activities may also have defensive roles in the local tissue, or they may be functioning in other aspects necessary during the wound response, such as recycling of nucleotides from damaged cells or rebuilding of damaged vascular tissue. Cells damaged by wounding would contain phosphate and other molecules that could be recycled for use in active, growing parts of the plant. Similar functions have been hypothesized for T2 RNases that are induced by phosphate-starvation and senescence in several species, including tomato and tobacco (Howard et al., 1998). Activities at 33 kDa, as well as RNS1, are induced in phosphate-starved seedlings (see Figure 10 in Bariola et al., 1994), and bifunctional nucleases are induced by senescence in Arabidopsis (Pérez-Amador et al., 2000). Possibly, RNS1 and the nucleases are induced during wounding as part of this wide-spread recycling mechanism.

It is possible that the systemic induction of RNS1 could be explained as a sort of ‘priming’ of the plant for nucleotide recycling in as-of-yet undamaged tissue. However, the nucleases, as well as RNS2 and RNS3, which encode two additional RNases shown to be induced during conditions requiring nutrient recycling, such as phosphate starvation and senescence (Bariola et al., 1994; Taylor et al., 1993), are not induced locally or systemically by wounding, at least indicating that other hydrolytic enzymes thought to be involved in phosphate recycling and remobilization are not generally activated by wounding. In fact, RNS1 is the only one of the five Arabidopsis genes related to the RNase T2 family that is induced in either local or systemic tissue by wounding (data not shown). Such specificity indicates that the enzyme may have an important role in the wound response.

Although local induction of hydrolytic activities by wounding has been found in several other plant species (Galiana et al., 1997; Lers et al., 1998), little is known regarding the signal pathways involved in their regulation. We have now identified a wide array of activities that are regulated by wounding in Arabidopsis, including RNase and nuclease activities that show both sustained and transient patterns of induction. Given its unique responses to wounding, independent of all known wound regulators, RNS1 will be a valuable marker for identifying novel signals that operate in the Arabidopsis wound response. More importantly, to our knowledge, RNS1 is the first RNase shown to be induced systemically by wounding. This result points us towards new ideas regarding the function of the widespread T2 family of RNases. Specifically, in addition to having a role in phosphate recycling as has been previously hypothesized, RNS1 may have a more direct defense-related function, possibly involved in protection of the plant from further attack after a wound-stimulus has been detected.

Experimental procedures

Plant materials and treatments

Unless otherwise stated, the Columbia ecotype of Arabidopsis thaliana was used throughout this study. Soil-grown plants were grown in chambers under 16 h of light in 50% relative humidity at 20°C. For seedling experiments, seeds were surface-sterilized and germinated on Arabidopsis growth medium as described (Taylor et al., 1993). The coi1 seeds were kindly provided by Dr J. G. Turner (University of East Anglia, Norwich, UK). Mutant coi1 plants were selected by germinating on MS medium supplemented with 50 μM methyl jasmonate as described (Feys et al., 1994). The plants were then transferred to soil and grown for an additional 4 weeks before wounding treatments were performed.

Stems or leaves of 4- to 6-week-old plants or leaves of 14-day-old seedlings were wounded using ridged flat-tipped tweezers and harvested at subsequent timepoints. For non-wounded, systemic material, leaves on either side of the wounded leaf were harvested. All samples were frozen in liquid N2 immediately after harvesting and stored at −80°C until used for RNA or protein extractions. Wounding experiments were performed a minimum of three times. Representative blots or gels are shown.

Jasmonic acid treatments were conducted on 4-week-old plants that were placed in enclosed boxes. Methyl Jasmonate (MeJA; Bedoukian Research, Inc., Danbury, CT) was diluted 1 : 25 in ethanol to a final concentration of 190 mm. A cotton-tipped applicator was soaked with 50 μL of the MeJA solution and placed in the container with the plants. An applicator soaked with ethanol alone was placed in a separate container with control plants. Four boxes were used in each of two experiments, for 2- and 24-h timepoints of both MeJA-treated and control plants. Two additional experiments were conducted for 3-, 6-, and 24-h timepoints, for a total of four replications. Representative blots and gels are shown in Figure 4(c),(d).

For OGA-treatments, rosette leaves of 4-week-old plants were removed by slicing the petiole with a razor blade. Approximately 20 leaves were floated on 40 ml of MS medium (Life Technologies, Rockville, MD, USA) supplemented with 0.5% (w/v) sucrose in each of two 250-ml flasks. One flask also contained 250 μg/ml of the OGA-rich TFA-PIIF fraction (provided by Dr E. Farmer, Université de Lausanne, Switzerland). Flasks were shaken on a rotary platform at approximately 50 R.P.M. Six or seven leaves were removed from the flasks at 1.5-, 3-, and 6-h timepoints and immediately frozen in liquid nitrogen. OGA treatments were performed twice using this method, and the Northern blot and activity gel in Figure 5 are representative of this method. Additionally, two replicates of treatments of seedlings grown in liquid culture, according to Rojo et al. (1999), were performed using a PIIF fraction kindly provided by Dr G. Howe (Michigan State University, MI, USA). Similar results were obtained in all four repetitions; however, the greatest induction of the positive control, choline kinase, was seen using the TFA-PIIF treatments of detached leaves.

RNA extraction and Northern hybridization

Total RNA from Arabidopsis samples was extracted as previously described (Newman et al., 1993). RNA (10 μg per lane) was separated by electrophoresis in 3% (w/v) formaldehyde/1.2% (w/v) agarose gels and blotted to Nytran Plus nylon membrane (Schleicher and Schuell, Keene, NH, USA). The RNA blots were hybridized as described in Taylor and Green (1991) using a 32P-labeled RNS1 probe. To control for loading, the same RNA blots were stripped in distilled water at 90–95°C for at least 20 min and then hybridized with a 32P-labeled probe for the Arabidopsis translation elongation factor EF-1α (EST accession number R29806) or pea translation initiation factor eIF-4A (Taylor et al., 1993). A choline kinase probe, kindly provided by Dr J. Sánchez-Serrano (Universidad Autónoma de Madrid, Spain), was used as a positive control for OGA treatments (Rojo et al., 1999). A probe for allene oxide synthase (Laudert et al., 1996) was used as a positive control for MeJA treatments. Quantitation of hybridization was performed using Phosphorimager (Molecular Dynamics, Sunnyvale, CA. USA) analysis to confirm increased CK and AOS levels in response to PIIF- and JA-treatments, respectively, as well as increased RNS1 levels in local wounded and systemic leaves (data not shown).

Protein extraction and detection of RNase and DNase activities

Total protein was extracted as described (MacIntosh et al., 1996). Homogenates were clarified by centrifugation, and soluble protein was quantified by the method of Bradford (1977). 100 μg total protein was loaded in each lane.

RNase and DNase activities were assayed using activity gels as described (Yen and Green, 1991), with minor modifications. After the isopropanol wash, before incubation, gels were washed in 100 mm Tris–HCl containing 2 μM ZnCl2 for 20 min in order to restore Zn2+ required for certain RNase and DNase activities. Gels were washed and incubated at pH 7.0, except for the RNase gels of systemic activity in Figures 3(c) and 4(a) (lower panel), which were incubated at pH 6.0 to enhance resolution of RNS1 activity, which is most active between pH 5.0 and 6.0 (N. D. LeBrasseur and P. J. Green, unpublished). Similar results were seen at pH 7.0 (data not shown). Separating gels contained 11.3% (w/v) acrylamide, except the gels in Figure 2, which contained 9% acrylamide for increased separation of the doublet at 33 kDa.

Acknowledgements

The authors would like to thank Dr E. E. Farmer (Université de Lausanne, Switzerland) for helpful discussions and sharing of unpublished results, TFA-PIIF fractions, and critical reading of the manuscript. We also thank Dr S. Y. He for critical reading of the manuscript, Dr J. Sánchez-Serrano (Universidad Autónoma de Madrid, Spain) for the CK clone, Dr J. G. Turner (University of East Anglia, Norwich, UK) for coi1 seed, and L. R. Danhof for technical assistance. This work was supported by National Science Foundation Grants MCB0096394 and IBN9408052 and Department of Energy Grant DE-FG02–91ER20021 to P.J.G.

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