Research ArticleSIGNALING AND RESPONSE

Lipoxygenase6-Dependent Oxylipin Synthesis in Roots Is Required for Abiotic and Biotic Stress Resistance of Arabidopsis

Wiebke Grebner, Nadja E. Stingl, Ayla Oenel, Martin J. Mueller, Susanne Berger

Published April 2013. DOI: https://doi.org/10.1104/pp.113.214544

Abstract

Jasmonates are oxylipin signals that play important roles in the development of fertile flowers and in defense against pathogens and herbivores in leaves. The aim of this work was to understand the synthesis and function of jasmonates in roots. Grafting experiments with a jasmonate-deficient mutant demonstrated that roots produce jasmonates independently of leaves, despite low expression of biosynthetic enzymes. Levels of 12-oxo-phytodienoic acid, jasmonic acid, and its isoleucine derivative increased in roots upon osmotic and drought stress. Wounding resulted in a decrease of preformed 12-oxo-phytodienoic acid concomitant with an increase of jasmonic acid and jasmonoyl-isoleucine. 13-Lipoxygenases catalyze the first step of lipid oxidation leading to jasmonate production. Analysis of 13-lipoxygenase-deficient mutant lines showed that only one of the four 13-lipoxygenases, LOX6, is responsible and essential for stress-induced jasmonate accumulation in roots. In addition, LOX6 was required for production of basal 12-oxo-phytodienoic acid in leaves and roots. Loss-of-function mutants of LOX6 were more attractive to a detritivorous crustacean and more sensitive to drought, indicating that LOX6-derived oxylipins are important for the responses to abiotic and biotic factors.

Oxylipins are ubiquitous signaling molecules that are derived from polyunsaturated fatty acids by enzymatic and nonenzymatic processes. In plants, the biosynthesis and function of oxylipins of the jasmonate family in aboveground tissues has been investigated in detail. Jasmonates comprise 12-oxo-phytodienoic acid (OPDA), jasmonic acid (JA), and derivatives of JA. In leaves, jasmonates accumulate in response to abiotic factors such as wounding, drought, osmotic stress, darkness, and ozone and during interactions with organisms such as herbivores, pathogens, and mutualistic organisms (Wasternack, 2007). The relevance of jasmonates in wound response, ozone tolerance, and the defense against herbivores and necrotrophic pathogens in leaves has been well investigated using mutants in JA biosynthesis and signaling (Browse, 2009a). In addition, jasmonates play an important role in flower development, and Arabidopsis (Arabidopsis thaliana) mutants in the JA pathway are male sterile (Browse, 2009b). The first step in jasmonate biosynthesis is catalyzed by 13-lipoxygenases (LOXs). The resulting 13(S)-hydroperoxyoctadecatrienoic acid (13-HPOTE) is converted by allene oxide synthase (AOS) and allene oxide cyclase to OPDA (Wasternack, 2007). These enzymatic steps are located in plastids. OPDA is transported to peroxisomes and converted to JA. JA can be further metabolized to different derivatives that take place mainly in the cytosol. The conjugation of JA with Ile is an important step because jasmonoyl-Ile (JA-Ile) has been identified as a biologically active jasmonate (Staswick and Tiryaki, 2004). OPDA is also biologically active without conversion to JA derivatives. In contrast to all other jasmonates, the OPDA structure contains an electrophilic α,β-unsaturated carbonyl group that renders OPDA more reactive than JA. Therefore, OPDA is classified as a reactive electrophile species with unique signaling properties different from other jasmonates (Farmer and Davoine, 2007).

Of the six lipoxygenase genes present in Arabidopsis, four genes encode 13-LOX. For the respective enzymes LOX2, LOX3, LOX4, and LOX6, it was shown that linolenic acid is the preferred substrate and that 13-HPOTE is formed in vitro (Bannenberg et al., 2009). All four enzymes are proposed to be located in plastids. LOX2 is highly expressed in leaves; expression is up-regulated by jasmonates and stress treatments such as wounding and osmotic stress (Bell and Mullet, 1993; Seltmann et al., 2010a). LOX2 was shown to contribute the majority of jasmonate synthesis upon wounding and osmotic stress and during senescence in leaves (Bell et al., 1995; Glauser et al., 2009). LOX2 is also responsible for the accumulation of arabidopsides (Glauser et al., 2009), which are galactolipids containing esterified OPDA in plastids by direct oxidation of galactolipids (Zoeller et al., 2012). LOX3 and LOX4 are required for the development of fertile flowers (Caldelari et al., 2011). LOX6 shows overall low expression (Bannenberg et al., 2009). Recently, it was reported that LOX6 contributes to the fast accumulation of JA and JA-Ile in wounded leaves and is required for the fast increase of JA and JA-Ile in distal leaves after wounding (Chauvin et al., 2013).

In contrast to leaves and flowers, little is known on jasmonate biosynthesis and function in roots. Expression of the plastid-localized enzymes of jasmonate synthesis LOX2, AOS, and allene oxide cyclase2 is very low in roots (Zimmermann et al., 2004). By contrast, enzymes such as 9-LOX and α-dioxygenase1 are strongly expressed in roots. These enzymes are involved in the biosynthesis of oxylipins different from jasmonates, and 9-LOX products have been shown to regulate lateral root development because mutants in LOX1 and LOX5 produce more lateral roots (Vellosillo et al., 2007). However, jasmonate function in roots is still obscure. Here, we analyzed jasmonate accumulation in roots upon different stress treatments and show that mutants defective in LOX6 are impaired in stress-induced jasmonate synthesis and are more susceptible to drought and detritivore feeding.

RESULTS

Oxylipin Profiles in Roots and Shoots

The plastid localization and low expression in roots of several enzymes of the first steps of JA biosynthesis prompted us to investigate whether the oxylipin profile mirrors the expression of oxylipin biosynthetic enzymes. To enable gentle recovery of root material, plants were grown in a hydroponic system. In this system, plant parts below the hypocotyl were cultivated in darkness. Levels of hydroxyoctadecatrienoic acids (HOTEs) and jasmonates in leaves and roots were analyzed. OPDA levels were around 1 nmol g–1 dry weight both in roots and shoots (Fig. 1). 9-HOTE and 13-HOTE can be synthesized nonenzymatically as well as enzymatically via 9-LOXs and 13-LOXs, respectively. In agreement with published data (Grun et al., 2007; Triantaphylidès et al., 2008), 13-HOTE (0.28 nmol g–1 dry weight) constituted the highest amount of free HOTEs in leaves followed by 9-HOTE (0.16 nmol g–1 dry weight). The high amount of 13-HOTE is in accordance with the high expression of 13-LOXs in leaves. In roots, levels of 13-HOTE and 9-HOTE were similar (0.67 and 0.64 nmol g–1 dry weight, respectively) and higher compared with leaves. Amounts of OPDA (0.8 nmol g–1 dry weight) were in roots only slightly lower than in the shoot (1 nmol g–1 dry weight), indicating that roots contain 13-LOX products and especially jasmonates, despite the low expression of LOX2 and AOS. Basal levels of JA and JA-Ile were very low and at the detection limit in roots and leaves. In addition to free jasmonates, levels of arabidopsides were also analyzed. The presence of arabidopsides in leaves has been reported (Stelmach et al., 2001; Hisamatsu et al., 2005; Kourtchenko et al., 2007). In roots, levels of all arabidopsides were below the detection limit (Supplemental Fig. S1).

Figure 1.
Figure 1.

Oxylipin levels in roots and leaves. Roots and shoots of 6-week-old hydroponically grown plants were harvested, and free OPDA, JA, JA-Ile, and hydroxy fatty acids were determined. Data represent the mean of at least three biological replicates ± sd. The experiment has been repeated with similar results.

Synthesis of Jasmonates Is Induced upon Wounding in Roots

Leaves respond to wounding with an accumulation of arabidopsides, OPDA, JA, and JA-Ile (Buseman et al., 2006; Koo et al., 2009). Therefore, we tested if levels of these oxylipins also increase in roots. To avoid exchange of signals between shoots and roots, both parts of the plant were separated and wounded, and oxylipin levels in leaves and roots were determined after incubation of 30 min, 2 h, and 24 h. As expected from published data, in leaves, all oxylipins accumulated (Fig. 2; Supplemental Fig. S1). Levels of OPDA increased from 1 to 8 nmol g–1 dry weight and remained high. JA increased from the limit of detection to around 11 nmol g–1 dry weight at 0.5 and 2 h and dropped to 1 nmol g–1 dry weight at 24 h. JA-Ile showed the most transient kinetic with a maximum of 2.7 nmol g–1 dry weight at 30 min. In roots, levels of arabidopsides were also below or slightly above the detection limit after wounding (Supplemental Fig. S1). The course of JA and JA-Ile accumulation in roots was similar to leaves (Fig. 2). JA levels increased at 30 min to 5 nmol g–1 dry weight and started to decline, with 4 nmol g–1 dry weight at 2 h. JA-Ile accumulated to 1.7 nmol g–1 dry weight at 30 min and declined to 0.6 nmol g–1 dry weight at 2 h. Interestingly, OPDA levels decreased from 0.8 nmol g–1 dry weight to 0.3 nmol g–1 dry weight at 30 min and showed a further decrease at later time points. This might be due to conversion of the OPDA present in the root to JA and JA-Ile. This raises the question if the roots are able to produce OPDA on their own or if OPDA is synthesized in plastids in the shoot and transported into the root, where it is further metabolized to JA and JA-Ile upon stress stimuli.

Figure 2.
Figure 2.

Oxylipin accumulation in roots and leaves in response to wounding. Roots and shoots were separated, wounded, and harvested after 30 min, 2 h, and 24 h after wounding. Zero hours indicate untreated plant material. Data represent the mean of at least three biological replicates ± sd. The experiment has been repeated with similar results.

Roots Synthesize OPDA Independent from the Shoot

To test the possibility of OPDA import from the shoot into the root, grafting experiments with the mutant delayed dehiscence2 (dde2) were performed. This mutant is deficient in expression of AOS and therefore does not produce OPDA, JA, or JA-Ile. Both grafting combinations with the wild type and dde2 were performed, and for control experiments, wild-type shoot was grafted on wild-type root.

Grafting was done at seedling stage, and plants were grown for an additional 7 weeks before wounding experiments were performed. The 30-min time point was chosen for analysis because strongest changes were seen at this time point (Fig. 2). Basal and wound-induced oxylipin levels in the control grafts were similar to nongrafted wild-type plants (Fig. 3). In the first combination, dde2 shoot was grafted on ecotype Columbia (Col-0) rootstock. As expected, in the dde2 mutant leaves, OPDA, JA, and JA-Ile were barely detectable both under basal and wound-induced conditions, while the wild-type roots contained normal basal OPDA levels, indicating that this organ produces OPDA without the need for a shoot with JA-biosynthetic competence (Fig. 3). After wounding of roots, JA and JA-Ile increased similarly as in nongrafted plants. In the second combination, Col-0 shoot was grafted on dde2 rootstock. As expected, OPDA, JA, and JA-Ile levels strongly increased in leaves upon wound treatment (Fig. 3). All three oxylipins were at the detection limit in dde2 roots before and after wounding. This indicates that there is no significant transport of oxylipins from the shoot to the root.

Figure 3.
Figure 3.

Oxylipin accumulation in partially jasmonate-deficient plants. Levels of OPDA, JA, and JA-Ile in shoots and roots of dde2 shoot grafted on Col-0 rootstock (left) and of Col-0 shoot grafted on dde2 rootstock (middle) basal and 30 min after wounding. Control grafts were performed with Col-0 shoots on Col-0 roots (right). Black bars indicate nonwounded; gray bars indicate wounded. Data represent the mean of at least three biological replicates ± sd. The experiment has been repeated with similar results.

LOX6 Is Required for Maintaining Basal OPDA Levels in Shoots and Roots

To investigate the contribution of the different 13-LOXs on oxylipin production in roots, expression of the four 13-LOXs and oxylipin levels in the single mutants lox2 and lox6 as well as in the lox3/lox4 double mutant defective in the expression of the redundant LOX3 and LOX4 were determined in roots and leaves of nontreated plants and 30 min after wounding.

In wild-type leaves and roots, OPDA is the major jasmonate under basal conditions (Fig. 2). The lox2 and lox3/lox4 mutant lines displayed similar or even elevated OPDA levels (lox2, 1.3 and 1 nmol g–1 dry weight; lox3/lox4, 4 and 5.2 nmol g–1 dry weight in roots and leaves, respectively), while OPDA levels in the lox6 mutant were at the detection limit under basal conditions both in leaves and roots (Fig. 4B). A second independent lox6 mutant showed similar effects (Supplemental Fig. S2). Hence, LOX6 appears to be essential for maintaining basal OPDA levels in both organs. In roots, this is consistent with the expression profile because LOX6 exhibited the highest basal transcript level among the four LOXs (Fig. 4A). In leaves, this result was rather unexpected because LOX6 showed only low expression, while LOX2 is strongly expressed. In all LOX mutants, levels of JA and JA-Ile were as low as in the wild type.

Figure 4.
Figure 4.

Involvement of 13-LOXs in the wound response in roots and leaves. Roots and shoots were separated, wounded, and harvested after 30 min (gray bars). As control, untreated plant samples were analyzed (black bars). A, Transcript levels of LOX2, LOX3, LOX4, and LOX6 were determined by quantitative RT-PCR. B, Levels of OPDA, JA, and JA-Ile were determined in Col-0 and different LOX mutants. Data represent the mean of at least three biological replicates ± sd. Stars indicate significant differences of the level in the mutant compared with the corresponding wild-type sample (*P < 0.05; **P < 0.01). The experiment has been repeated with similar results.

LOX6 Is Essential for JA Signal Production in Response to Wounding in Roots

Because OPDA is a precursor of JA and JA-Ile, deficiency in basal OPDA accumulation in the lox6 mutant might affect stress-induced accumulation of JA and JA-Ile. In wild-type roots, as shown in Figure 2, OPDA levels drop after wounding, while JA and JA-Ile transiently accumulate. This suggests that preformed and de novo-formed OPDA is rapidly converted to JA and JA-Ile. In wounded roots of the lox6 mutant, levels of JA and JA-Ile were dramatically lower than in the wild type (Fig. 4B; Supplemental Fig. S2). This indicates that LOX6 is not only essential for basal OPDA but also for wound-induced accumulation of JA and JA-Ile in roots. Although LOX3 and LOX4 are more highly expressed in wounded roots (Fig. 4A), these LOXs appear not to be involved in root jasmonate production because wound-induced levels of all three jasmonates are not lower in lox3/lox4 roots compared with the wild type (Fig. 4B). Also, jasmonate amounts in lox2 roots were similar or slightly higher than in wild-type roots.

Unlike roots, leaves of the lox6 mutant exhibited a strong increase of OPDA, JA, and JA-Ile after wounding. However, the JA level was significantly lower in wounded lox6 leaves, suggesting that LOX6 is involved in, but not essential for, wound-induced accumulation of jasmonates in leaves. In the lox3/lox4 mutant, wound-induced levels of OPDA, JA, and JA-Ile in leaves were similar to the wild type (Fig. 4B) despite a strong up-regulation of both genes by wounding (Fig. 4A). JA accumulated to lower levels in leaves of the lox2 mutant upon wounding. The effect on OPDA levels was even more dramatic because OPDA did not increase in lox2 leaves upon wounding but showed a clear decrease (Fig. 4B). This is in agreement with LOX2 being a major contributor to wound-induced oxylipin production in leaves.

LOX6 Is Involved in Oxylipin Formation in Roots in Response to Osmotic and Drought Stress

Jasmonates also accumulate in leaves in response to stresses such as osmotic stress and drought (Creelman and Mullet, 1995; Seltmann et al., 2010b; de Ollas et al., 2013). Therefore, we investigated if LOX6 also contributes to oxylipin synthesis in response to these stress factors. Roots of intact plants grown in the hydroponic system were incubated in 500 mm sorbitol, and oxylipin accumulation was analyzed after 24 h.

In wild-type roots, OPDA levels increased about 3.6-fold after sorbitol treatment (Fig. 5). Also, JA and JA-Ile levels were clearly elevated. The increase in JA and JA-Ile in lox2 roots was similar to the wild type, while the rise in OPDA was lower. Upon osmotic stress, roots of lox3/lox4 showed wild-type levels of all jasmonates. In line with the results on wound stress, lox6 roots exhibited strong differences in oxylipin production compared with the wild type. lox6 roots did not accumulate OPDA, JA, or JA-Ile after sorbitol treatment. This indicates that LOX6 is also required for oxylipin accumulation in roots in response to osmotic stress.

Figure 5.
Figure 5.

Osmotic stress-induced oxylipin accumulation in roots and shoots of Col-0 and different LOX mutants. Levels of OPDA, JA, and JA-Ile were determined 24 h after replacing the medium with 500 mm sorbitol (gray bars). Medium of control plants was replaced with water. Data represent the mean of at least three biological replicates ± sd. Stars indicate significant differences of the level in the mutant compared with the corresponding wild-type sample (*P < 0.05; **P < 0.01). The experiment has been repeated with similar results.

In leaves of the wild type, OPDA levels rose about 3-fold (Fig. 5). JA and JA-Ile strongly accumulated, with JA especially showing a dramatic increase up to 52 nmol g–1 dry weight. No clear elevation of these oxylipins was detected in lox2 leaves, consistent with a major role of LOX2 in the response of leaves to osmotic stress. In leaves of lox3/lox4, all oxylipins rose, but to a smaller extend than in the wild type. No differences were obvious in the levels of OPDA and JA, while JA-Ile levels were even slightly higher in leaves of the lox6 mutant after osmotic stress treatment compared with the wild type.

To investigate the contribution of the LOXs to drought stress, the liquid medium was removed from plants grown in the hydroponic system, and roots were exposed to air. In leaves, JA-Ile levels were at the detection limit, and JA levels remained below 0.1 nmol g–1 dry weight after 48-h drought treatment (Fig. 6). Similarly, no strong increase in OPDA levels was detectable. By contrast, roots responded with an accumulation of OPDA and JA. Compared with osmotic stress, increases were moderate, reaching about 7 and 0.3 nmol g–1 dry weight for OPDA and JA, respectively. JA-Ile levels were at the detection limit but nevertheless tended to rise (Supplemental Fig. S3). Also, in lox2 and lox3/lox4 mutants, levels of JA and OPDA increased. By contrast, in lox6 plants, OPDA and JA were basal and, after drought treatment, below the detection limit. This suggests that LOX6 is also responsible for drought-induced accumulation of jasmonates in roots.

Figure 6.
Figure 6.

Drought-induced oxylipin accumulation in roots and shoots of Col-0 and different LOX mutants. Levels of OPDA and JA were determined in untreated plant samples (black bars) and 48 h after removing the liquid medium from the roots (gray bars). Data represent the mean of at least three biological replicates ± sd. Stars indicate significant differences of the level in the mutant compared with the corresponding wild-type sample (*P < 0.05; **P < 0.01). The experiment has been repeated with similar results.

Defect in LOX6 Expression Results in Increased Sensitivity to Drought

Drought stress eventually results in wilting and death of the plant. To assess whether the lack of LOX6 leads to altered tolerance to water limitation, plants were grown in soil, and at the age of 4 weeks, watering was stopped. Wilting symptoms were documented, plants were rewatered, and the survival rate was determined. The lox6 mutant showed earlier wilting symptoms compared with the wild type. Enhanced sensitivity of lox6 to water limitation was supported by a lower survival rate of plants after rewatering (Fig. 7, A and B). Only 14% of lox6 plants, compared with 78% of wild-type plants, survived the treatment. The altered drought tolerance might be due to lower levels of jasmonates or other 13-LOX products. To distinguish between these possibilities, dde2 plants were tested in addition to the lox6 mutant. Interestingly, dde2 plants did not show the wilting and survival phenotype of lox6 but were similar to wild-type plants (Fig. 7, A and B). This indicates that mechanisms other than lack of OPDA and derivatives of JA are responsible for enhanced drought sensitivity of lox6.

Figure 7.
Figure 7.

Wilting phenotype of Col-0, lox6, and dde2. Drought-stressed plants were rewatered, pictures were taken 2 d after rewatering (A), and the surviving plants were scored (B). Data represent the percentage of surviving plants relative to total plants and are the mean of at least three biological replicates ± sd. The experiment has been repeated with similar results. [See online article for color version of this figure.]

Defect in LOX6 Expression Renders Roots More Attractive to Detritivores

Jasmonates are important signals in defense responses against herbivores and detritivores. In particular, it was shown that freshly detached leaves of dde2 plants are consumed by woodlice (Porcellio scaber), which usually prefer dead tissue (Farmer and Dubugnon, 2009). Woodlice are soil-living organisms that are in frequent contact with plant roots. Therefore, this organism provides a suitable bioassay to test whether the lower levels of jasmonates in roots of lox6 result in fewer defenses against detritivores and render them more attractive as potential food. Leaves and roots of hydroponically grown plants were provided to rough woodlice that were starved for 2 d prior to the experiment. Pictures were taken at different time points. No clear differences were detectable in the consumption of leaves of lox6 and wild-type plants (Fig. 8B). However, in agreement with published data (Farmer and Dubugnon, 2009), dde2 leaves were consumed faster than wild-type leaves (Supplemental Fig. S4B), confirming that jasmonate deficiency of leaves results in enhanced attractiveness. Compared with leaves, roots were eaten considerably slower. Roots of lox6 plants were consumed more rapidly than roots of wild-type plants (Fig. 8A). After 8 d, 5% of the original root material was left in lox6 compared with 36% in the wild type, respectively. This indicates that, consistent with the result of wound and osmotic stress, LOX6 is important to restrain crustacean feeding in roots but not in leaves. Roots of dde2 plants were also preferred over wild-type roots (Supplemental Fig. S4A), suggesting that jasmonates are responsible for the root defense against woodlice.

Figure 8.
Figure 8.

Feeding of woodlice on roots of Col-0 and lox6. Roots (A) and leaves (B) of wild-type and lox6 plants were provided to woodlice, and pictures were taken at 0 h and after 4 d (leaves) and 8 d (roots). The area of roots (C) and leaves (D) was quantified at the beginning of the experiment and at the time points indicated. Data represent the area relative to the original area. The experiment has been repeated with similar results. [See online article for color version of this figure.]

DISCUSSION AND CONCLUSION

Differential Impact of 13-LOXs on Oxylipins Biosynthesis in Roots and Leaves

In this work, the accumulation of jasmonates in leaves and roots after different stress treatments was compared, and the LOX isoform involved in root jasmonate synthesis was identified. Roots accumulated JA/JA-Ile in response to wounding, osmotic stress, and, to a lesser extent, drought. OPDA levels increased upon osmotic stress and drought but decreased upon wounding. Roots synthesize jasmonates independently of the shoot despite the low expression of several JA-biosynthetic enzymes in Arabidopsis roots. This was concluded from wounding experiments with grafted dde2 mutant and wild-type plants. LOX6 was identified as the main enzyme involved in the production of jasmonates in roots constitutively and upon wound, osmotic, and drought stress. The effects were very clear, and none of the mutations in the other LOX genes showed a strong effect on jasmonate production in roots, although LOX3 and LOX4 are expressed in roots and show induction by wounding (Fig. 4). This indicates that LOX2, LOX3, and LOX4 cannot substitute a loss of function of LOX6 in roots, even though all four LOXs have been shown to catalyze the formation of 13-HPOTE from linolenate in vitro.

Wounding of leaves or osmotic stress applied to roots resulted in accumulation of OPDA, JA, and JA-Ile in leaves, which is in agreement with published data (Koo et al., 2009; Zoeller et al., 2012). In leaves, the strongest effects on jasmonate levels were observed in lox2 mutants in which wound- and sorbitol-induced jasmonate accumulation was significantly lower compared with the wild type. This correlates with the very high expression of LOX2 in leaves (Fig. 4; Zimmermann et al., 2004) and indicates that LOX2 is the main enzyme responsible for jasmonate accumulation in leaves after both stresses. LOX2 is involved in the accumulation of the bulk of free jasmonates after wounding and osmotic stress and during senescence, but not after pathogen attack (Bell et al., 1995; Glauser et al., 2009; Seltmann et al., 2010b; Zoeller et al., 2012). Lower JA accumulation, compared with wild-type leaves, was also detectable in lox6 mutant leaves after wounding and in lox3/lox4 mutant leaves after osmotic stress. This suggests that LOX3, LOX4, and LOX6 contribute to stress-induced JA biosynthesis in leaves and that the contribution depends on the specific stress condition. This is in agreement with a recent report that all 13-LOXs contribute to the increase of JA in wounded leaves at a very early time point (190 s; Chauvin et al., 2013). Unexpectedly, we found that synthesis of basal OPDA was completely prevented in the lox6 mutant. This indicates that LOX6 is important for OPDA formation in leaves, although basal expression of LOX6 is very low (Fig. 4; Zimmermann et al., 2004; Vellosillo et al., 2007). Because LOX2 is highly expressed constitutively in leaves but cannot ensure basal production of OPDA, oxylipin biosynthesis is regulated on other levels than transcription of biosynthetic genes.

How Is the Stress-Induced Synthesis of Jasmonates Regulated?

As discussed above, expression of 13-LOXs did not always correlate with accumulation of jasmonates. In addition, changes of JA were not necessarily concomitant with OPDA. For instance, JA and JA-Ile levels increased in lox6 leaves despite very low basal levels of OPDA, and vice versa, increase of JA was reduced in the lox2 mutant even though basal levels of the precursor OPDA were available. Different mechanisms might account for these phenomena. Firstly, enzymatic activity might be regulated posttranslationally. The requirement for regulation of the activity of enzymes that are already present is also obvious from the very fast accumulation of jasmonates upon wounding (Glauser et al., 2009). Regarding the regulation mechanism of LOX2, it was reported that the ion channel FATTY ACID OXYGENATION UPREGULATED2 (FOU2) regulates activity of LOX2 in response to wounding (Bonaventure et al., 2007; Beyhl et al., 2009). Secondly, different LOXs may generate separate pools of 13-HPOTE with different metabolic fates. 13-HPOTE might be used for partially competing metabolic routes eventually leading to the accumulation of arabidopsides, OPDA, JA, and its metabolites, or channeled into other oxylipin pathways such as the hydroperoxy lyase or peroxygenase pathways (Feussner and Wasternack, 2002). For example in roots, only LOX6 products are channeled into the jasmonate pathway, while LOX3 and LOX4 products might be metabolized to oxylipins other than jasmonates and serve other functions. Thirdly, the conversion of OPDA to JA might be regulated. In roots, OPDA levels decrease in response to wounding concomitant with an increase in JA. This suggests that OPDA constitutively present in roots is used for JA synthesis. This is in accordance with a lack of JA accumulation in roots of lox6 mutants, where the basal OPDA level is at the detection limit. However, the decrease in OPDA (less than 0.7 nmol g–1 dry weight at 30 min) is not sufficient to account for the increase in JA (more than 3 nmol g–1 dry weight; Figs. 2 and 4), suggesting that de novo synthesis of OPDA is necessary. Changes in oxylipin levels similar to the changes in wounded roots have been demonstrated in unwounded, distal leaves of wounded plants. Also in this system, accumulation of JA and JA-Ile in distal leaves correlated with a decrease in OPDA (Koo et al., 2009). This indicates that the regulated step of JA production in both scenarios is the conversion from OPDA to JA. In Arabidopsis, OPDA reductase3 (OPR3) is the first enzyme of this pathway catalyzing the reduction of OPDA. OPR3 activity has been discussed to be regulated posttranslationally by homodimerization (Breithaupt et al., 2006). In wounded roots as well as distal leaves of wounded plants, OPDA is present and OPR3 is expressed, which is consistent with a regulation of JA production by affecting OPR3 activity on a posttranslational level. In the case of osmotic and drought stress, the process of jasmonate production seems to be regulated by different mechanisms, because here OPDA levels increase to similar (osmotic) or even higher (drought) levels than JA. It has to be taken into account that in contrast to the early time point measured after wounding, oxylipin levels were detected after 24 h of sorbitol or 48 h after drought treatment, respectively. Therefore, timing, oxylipin profiles, and regulation of oxylipin synthesis of the responses to these different stresses are obviously different.

Function of Oxylipins in Roots

There are several indications for a role of oxylipins in root responses to biotic and abiotic factors. Accumulation of jasmonates has been observed in Medicago truncatula in the mutualistic interactions with soil-borne microorganisms. Using allene oxide cyclase-RNA interference approaches, it was demonstrated that jasmonate synthesis is important for the development of arbuscular mycorrhizal symbiosis but not for nodule formation (Isayenkov et al., 2005; Zdyb et al., 2011). In addition, it was shown that jasmonate synthesis is important for defense in Arabidopsis and maize (Zea mays) against Pythium species, which are soil-borne oomycetes. The fatty acid desaturase mutant fad3/fad7/fad8 and jasmonate resistant1 mutant (jar1) in Arabidopsis and an opr7/opr8 double mutant in maize are more susceptible to Pythium spp. (Staswick et al., 1998; Vijayan et al., 1998; Yan et al., 2012). The jar1 mutant also develops more disease symptoms after challenge with Phytophthora parasitica (Attard et al., 2010). Furthermore, altering the expression of LOX3 in maize renders plants more susceptible to root knot nematodes (Gao et al., 2008). Even though it was not explicitly shown that jasmonate production in the root is relevant, the fact that these organisms originated from soil and that in the experiments the microorganisms/nematodes were applied to roots suggests that root-derived jasmonates are important in these interactions.

The identification of LOX6 as the one LOX responsible for stress-induced oxylipin accumulation in roots enabled the investigation of the function of oxylipins specifically in roots using the lox6 mutant. The experiments show that oxylipins in roots are involved in detritivore defense and drought tolerance. Detritivorous crustaceans feed faster on roots of lox6 and dde2 mutant plants (Fig. 8; Supplemental Fig. S4) and on leaves of dde2 (Supplemental Fig. S4; Farmer and Dubugnon, 2009). The fact that lox6 and dde2 mutations have similar effects indicates that jasmonates are responsible for deterring crustacean feeding in the wild type. Restraining detritivore attack might be even more important in roots than in leaves because roots are more accessible to this soil organism. The molecular basis of lower attractiveness of wild-type roots compared with dde2 and lox6 roots is not clear. For leaves of different plant species, it was demonstrated that jasmonates are important for induction of secondary metabolism and expression of defense proteins (Howe et al., 1996; Reymond et al., 2000; for review, see Browse and Howe, 2008). Comparison of the metabolite profile of wild-type and lox6 roots will contribute to an understanding of the mechanisms involved.

In contrast to animal feeding, drought tolerance was impaired in lox6 but not in dde2. This suggests that oxylipins different from jasmonates are important for drought tolerance. The product of 13-LOX, 13-HPOTE can be converted to a variety of oxylipins, such as ketones, aldehydes, epoxides, hydroxides, and divinyl ethers (Feussner and Wasternack, 2002). Several of these compounds have been shown to be biologically active, for instance, in regulating root growth and gene expression (Vellosillo et al., 2007). It will be interesting to elucidate which, and how, products of LOX6 regulate drought tolerance. Possibilities are the formation of a long-distance signal in the root that signals water limitation to the leaves. The involvement of oxylipins in root-to-shoot communication has also been suggested in response to wounding (Hasegawa et al., 2011). Alternatively, LOX6-derived products might directly play a role in regulating stomatal opening. Oxylipins such as coronatine, JA, and its methyl ester are discussed to regulate stomatal closure (Suhita et al., 2004; Melotto et al., 2006). Interestingly, LOX6 has been described to be expressed in guard cells (Leonhardt et al., 2004), which points to a role in stoma function. It is a challenge for the future to identify the oxylipins and mechanisms responsible for the impact of LOX6 on drought tolerance.

MATERIALS AND METHODS

Plant Material

Arabidopsis (Arabidopsis thaliana) wild-type Col-0, dde2, and LOX mutant lines lox2, lox3/lox4, and lox6 were used. The lox2 (Glauser et al., 2009), lox3/lox4, and both lox6 mutants (Caldelari et al., 2011; Chauvin et al., 2013) were kindly provided by E.E. Farmer. The lox6 mutant analyzed throughout the manuscript corresponds to the line SALK_138907. Absence of LOX6 expression is shown in Supplemental Figure S5. The lox6 line used in Supplemental Figure S2 corresponds to SALK_083650. The identity of both lox6 mutants was confirmed by PCR analysis using primers suggested by the iSect tool (http://signal.salk.edu/tdnaprimers.2.html). The dde2-2 mutant (von Malek et al., 2002) was kindly provided by B. Keller. For the dde2 and lox3/lox4 mutant, the male-sterile phenotype was monitored.

For investigations, plants were grown in soil at 22°C under a 9-h photoperiod (100 µmol photons m–2 s–1) in a climate chamber or hydroponically in a plant cabinet.

Hydroponic Cultivation

The hydroponic system was set up according to Tocquin et al. (2003). Seeds were surface sterilized and sown on 0.5-mL microtubes filled with the standard nutrient solution described in Tocquin et al. (2003) with 1% (w/v) Phyto Agar (Duchefa) and cultivated in a sterile box that was placed in a plant cabinet for 14 d at 20°C under a 9-h photoperiod (80 µmol photons m–2 s–1). For further cultivation, 2 mm of the microtube bottom was removed, and the tube was placed in a sterile 50-mL Falcon tube containing sterile liquid standard nutrient solution. To avoid the exposure of roots to light, the Falcons were placed in a light-tight box (internal dimensions, 152 × 104 × 102 mm) with the microtube placed through a hole in the lid of the box. The boxes, each containing 11 plants, were cultivated for another 4 weeks at 20°C under a 9-h photoperiod (80 µmol photons m–2 s–1) in a plant cabinet. One week before harvest, the standard nutrient solution was refilled.

Grafting of Arabidopsis Plants

Arabidopsis Col-0 wild-type and dde2 plants were grafted using 7-d-old seedlings based on a micrografting technique described by Turnbull et al. (2002). Briefly, surface-sterilized seeds were sown in petri dishes on Murashige and Skoog medium containing 1.5% (w/v) Suc. Seeds were stratified for 2 d at 4°C in the dark, and then petri dishes with seeds were transferred to a growth cabinet with 24°C and constant light (40 µmol photons m–2 s–1) and placed in a vertical orientation. After 5 d, light intensity was reduced to 25 µmol photons m–2 s–1. For grafting, seedlings were placed under a binocular and cut at the hypocotyl with a razor blade. Shoots and roots were placed in intimate contact on new petri dishes with Murashige and Skoog medium containing 0.5% (w/v) Suc. After 7 d of growing on vertically oriented plates in a growth cabinet at 24°C under constant light (25 µmol photons m–2 s–1), all successful grafted plants were transferred to petri dishes with Murashige and Skoog medium containing no Suc and grown for another 14 d at 20°C under a 9-h photoperiod (80 µmol photons m–2 s–1) in a plant cabinet. All surviving plants were then transferred to the hydroponic system and cultivated until they were 8 weeks old.

Wounding and Sorbitol Treatment of Plants

For wounding experiments 6 to 8 weeks old, hydroponically cultivated plants were used. First, the plants were cut above and below the microtubes, in which they had been sown for germination, to obtain the separated shoot and root. The part of the hypocotyl was removed. The shoots were squeezed with a forceps once across every leaf blade, and the roots were squeezed five times along its full length. Wounded shoots were incubated in the light and roots in the dark until shock freezing in liquid nitrogen and stored at –80°C for further analysis. Each replicate consisted of shoots/roots of at least three to four plants.

For sorbitol treatment, the standard liquid media of 6-week-old hydroponically cultivated plants were replaced by 0.5 m sorbitol solution or water (control). After 24-h incubation, the roots were washed with water, and shoots and roots were harvested as described above, shock frozen in liquid nitrogen, and stored at –80°C for further analysis. Each replicate consisted of shoots/roots of at least three to four plants.

RNA Isolation and Quantitative Reverse Transcription (RT)-PCR

Total RNA was extracted from ground plant material using TriFast reagent (PEQLAB) according to the manufacturer’s protocol. RNA concentration was determined spectrophotometrically. Remaining DNA was removed using RNase-Free DNase I (Fermentas) according to the manufacturer’s protocol. First-strand complementary DNA and real-time PCR were performed as described previously (Szyroki et al., 2001) using SYBR-Green Capillary Mix (ThermoFisher Scientific) and a CFX 96 Real-Time System C1000 Thermal Cycler (Bio-Rad). Primers used (TIB MOLBIOL) were as follows: LOX2 (At3G45140): forward, 5′-GCCATTGAGTTGACTTGTCC-3′, reverse, 5′-CACTTAGTTGTCTATTTGCCGC-3′; LOX3 (At1g17420): forward, 5′-TCCCTGCCGATCTAA-3′, reverse, 5′-GTTTGGGACGTAGCCA-3′; LOX4 (At1g72520): forward, 5′-GCTTGCTTAGATACGACACT-3′, reverse, 5′-ATG TGGTCTTCCGTGAGAGC-3′; and LOX6 (At1g67560): forward, 5′-AAGACTGTTACTGCGGTTG-3′, reverse, 5′-GGCTGTGAATACGAGGTATC-3′.

The number of transcripts was normalized to AtSAND (At2g28390) complementary DNA fragments (Czechowski et al., 2005) amplified by AtSANDfwd (5′-AACTCTATGCAGCATT-3′) and AtSANDrev (5′-GGTGGTACTAGCACAA-3′) primers.

Analysis of JA, JA-Ile, and OPDA in Arabidopsis

For analysis of oxylipins, fresh (shock-frozen) plant material was ground by mortar and pestle and afterward freeze dried. For shoots and roots, 25 and 18 mg of freeze-dried material, respectively, were extracted with 950 µL of ethyl acetate/formic acid (99:1, v/v). Dihydrojasmonic acid (50 ng), JA-nor-Val (50 ng), and [18O2]OPDA (50 ng) were added as internal standards. Extraction samples were homogenized with a ball mill for 3 min at 20 Hz. After centrifugation, the supernatant was dried in a vacuum concentrator, and the extraction step was repeated with 1 mL ethyl acetate/formic acid (99:1, v/v). After evaporation of the ethyl acetate, samples were dissolved in 40 µL acetonitrile/water (50:50, v/v) for liquid chromatography (LC)-tandem mass spectrometry (MS/MS) analysis.

Ultra-high-performance LC/MS/MS analyses were performed on a Waters Quattro Premier XE triple-quadrupole mass spectrometer with an electrospray interface (ESI) coupled to a Waters Acquity ultra-high-performance liquid chromatograph (UPLC). Chromatographic separations were carried out using an Acquity UPLC BEH C18 column (2.1 × 50 mm, 1.7-µm particle size with a BEH C18 guard column equipped with a prefilter) with the following solvent system: solvent A = 0.1% (v/v) formic acid in water, solvent B = acetonitrile. A gradient elution was performed at a flow rate of 0.25 mL min–1 at 40°C: 97% solvent A, followed by 0% solvent A in 7 min.

The ESI source was operated in negative ionization mode with a capillary voltage of 3.0 kV at 120°C. Quantification was performed using multiple reaction monitoring with a scan time of 0.025 s per transition (Supplemental Table S1). Cone voltage and collision energy was set at 20 eV, and the desolvation temperature was 400°C. Nitrogen was used as the desolvation and cone gas, with a flow rate of 800 and 50 L h–1, respectively. Argon was used as the collision gas at a pressure of approximately 3.10 × 10–3 bar.

Analysis of Free Hydroxy Fatty Acids

Analysis of free hydroxy fatty acids was performed similarly as described in Zoeller et al. (2012). Briefly, fresh (shock-frozen) plant material was ground by mortar and pestle and afterward freeze dried. For shoots and roots, 25 and 18 mg of freeze-dried material, respectively, were extracted with 800 µL isopropanol containing 0.5 mg triphenylphosphane and 0.75 mg butylated hydroxytoluene. After 20-min reduction, 15-hydroxy-eicosadienoic acid (150 ng) was added as internal standard. Samples were sonicated for 5 min and centrifuged. The supernatant was recovered, and the residue was further extracted with 1.5 mL chloroform/isopropanol (1:2, v/v) and 1.5 mL chloroform/methanol (2:1, v/v). After each extraction, samples were centrifuged, and the supernatants were combined. The combined lipid extract was dried under a stream of nitrogen at 60°C and reconstituted in 40 µL 1 mm ammonium acetate in water/acetonitrile (1:2, v/v) for LC-MS/MS analysis.

Ultra-high-performance LC/MS/MS analyses were performed on a Waters Quattro Premier XE triple-quadrupole mass spectrometer with an ESI coupled to an Acquity UPLC. Chromatographic separations were carried out using an Acquity UPLC BEH C18 column (2.1 × 50 mm, 1.7-µm particle size with a BEH C18 guard column equipped with a prefilter) with the following solvent system: solvent A = 1 mm aqueous ammonium acetate, solvent B = acetonitrile. A gradient elution was performed at a flow rate of 0.25 mL min–1 at 40°C: 65% solvent A, followed by 30% solvent A in 6 min.

The ESI source was operated in negative ionization mode at a capillary voltage of 3.0 kV at 120°C. Quantification was performed using multiple reaction monitoring with a scan time of 0.025 s per transition (Supplemental Table S1). Cone voltage and collision energy was set at 20 eV, and desolvation temperature was 350°C. Nitrogen was used as the desolvation and cone gas, with a flow rate of 800 and 50 L h–1, respectively. Argon was used as the collision gas at a pressure of approximately 3.10 × 10–3 bar.

Detritivore Feeding

The detritivore rough woodlouse (Porcellio scaber) was collected and cultivated as described in Farmer and Dubugnon (2009). For the feeding assay, detached leaves and roots of 6-week-old hydroponically cultivated plants were used. Approximately 15 isopods were fed with the detached plant organs. For quantification, digital pictures were taken, including a 5-cm2-size marker. The area of leaves and roots was calculated with Photoshop 7 (Adobe).

Drought Resistance Experiments

To investigate drought tolerance, 4-week-old plants grown in 30 mL soil (Einheitserde P, Klasmann-Deilmann, Geeste; pot dimensions: 5-cm height, 7-cm diameter at the top, 4.5-cm diameter at the bottom) at 22°C under a 9-h photoperiod (100 µmol photons m–2 s–1) in a climate chamber were watered for 16 h. The remaining water was removed, and the plants were grown for 25 d without watering. When all plants showed wilting symptoms, plants were rewatered, and the survival rate was calculated after 2 d.

For oxylipin analysis, the liquid medium of 6-week-old hydroponically grown plants was removed, and the plants were further cultivated at 20°C under a 9-h photoperiod (80 µmol photons m–2 s–1) in a plant cabinet. After 48-h incubation, the shoots and roots were harvested, shock frozen in liquid nitrogen, and stored at –80°C.

Statistical Analysis

For statistical analysis of differences between the wild type and several mutants, one-way ANOVAs (Fisher’s least significant difference test) were performed with SPSS statistics software (IBM).

In addition to the analyses performed by quantitative PCR, information on gene regulation was obtained using the Web site www.genevestigator.ethz.ch (Zimmermann et al., 2004).

Supplemental Data

The following materials are available in the online version of this article.

Acknowledgments

We thank E.E. Farmer for his help in setting up the woodlice system and providing LOX mutants.

Footnotes

  • The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Susanne Berger (berger{at}biozentrum.uni-wuerzburg.de).

  • www.plantphysiol.org/cgi/doi/10.1104/pp.113.214544

  • ↵1 This work was supported by the Graduiertenkolleg 1342 and the Sonderforschungsbereich 567.

  • ↵[C] Some figures in this article are displayed in color online but in black and white in the print edition.

  • ↵[W] The online version of this article contains Web-only data.

Glossary

OPDA
12-oxo-phytodienoic acid
JA
jasmonic acid
LOX
lipoxygenase
13-HPOTE
13(S)-hydroperoxyoctadecatrienoic acid
JA-Ile
jasmonoyl-Ile
HOTE
hydroxyoctadecatrienoic acid
MS
mass spectrometry
UPLC
ultra-high-performance liquid chromatograph
LC
liquid chromatography
ESI
electrospray interface
Col-0
Columbia
RT
reverse transcription
  • Received January 17, 2013.
  • Accepted February 23, 2013.
  • Published February 26, 2013.

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Lipoxygenase6-Dependent Oxylipin Synthesis in Roots Is Required for Abiotic and Biotic Stress Resistance of Arabidopsis
Wiebke Grebner, Nadja E. Stingl, Ayla Oenel, Martin J. Mueller, Susanne Berger
Plant Physiology Apr 2013, 161 (4) 2159-2170; DOI: 10.1104/pp.113.214544
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