Jasmonates during senescence

Abstract

Jasmonic acid and derivatives are oxylipin signaling compounds derived from linolenic acid. Jasmonates accumulate during natural and dark-induced senescence but the increase in these compounds is not essential for the initiation or progression of these senescence processes. Here we report that during natural and dark-induced senescence the increase in jasmonate levels does not trigger jasmonate signaling. Furthermore we provide evidence that jasmonate production might result from membrane turnover during dark-induced senescence.

Jasmonic acid (JA) and derivatives, collectively termed jasmonates, are enzymatically formed oxylipins that accumulate upon stress treatments such as pathogen infection and herbivore deterioration. These compounds have been shown to play an important role in the defense of necrotrophic pathogens and insects as well as ozone tolerance.¹ The effects of jasmonates on the transcriptional level have been studied in detail and several jasmonate-responsive genes are known. The typical jasmonate response encompasses the induction of the expression of jasmonate biosynthesis genes, which results in a positive feedback on jasmonate levels, defense genes such as the vegetative storage protein (VSP) genes and genes related to antioxidant metabolism.² Exogenous application of jasmonates has also been reported to induce senescence-like phenotypes such as yellowing of leaves and induction of senescence-associated genes. Levels of endogenous jasmonates increase during senescence.^3,4

Senescence-like phenomena can also be induced by treatments such as incubation in darkness and flotation on sorbitol. Recently it was shown using transgenic Arabidopsis LOX2RNAi lines, which do not accumulate JA and 12-oxophytodienoic acid (OPDA) during natural senescence or upon dark/sorbitol-induced senescence processes, that the rise in the endogenous jasmonate levels is not necessary for the initiation or progression of leaf yellowing during aging or upon dark-induced senescence-like processes.⁴ In contrast, yellowing and expression of senescence-associated genes upon sorbitol treatment was significantly slower in these lines indicating that oxylipins are involved in processes induced by sorbitol stress.

In the course of the characterization of gene expression in these lines during senescence processes we noticed that expression of JA-responsive genes such as VSP1, lipoxygenase 2 (LOX2) and allene oxide synthase (AOS) is clearly induced at 24 h after sorbitol flotation as expected based on the increased jasmonate levels. However, no increase in the case of VSP1 or even a clear downregulation in the case of LOX2 and AOS during aging or upon dark incubation was detectable (Fig. 1). Similar results for the expression of LOX2 and AOS during natural and dark-induced senescence have also been reported by van der Graaf et al.⁵ Also the accumulation of OPDA containing galactolipids has been described as a stress and jasmonate response.⁶ Similarly to the lack of jasmonate-responsive gene induction, no clear rise in the levels of arabidopsides is detectable during natural or dark-induced senescence processes.⁴ This is remarkable because especially in the case of dark incubation there is such a dramatic increase of JA levels (more than 200-fold). What might be the reason for this lack of JA response?

Figure 1.

Expression of JA-responsive genes during natural senescence and upon dark incubation and sorbitol stress determined by qRT-PCR. Plants were grown on soil at 22°C under a 9 h photoperiod (100 µmol photons m⁻²s⁻¹). For experiments in natural senescence, rosette leaves No. 9–15 were harvested at 6, 8 and 10 weeks after sowing. For dark treatment leaves No. 5–15 of 6-week-old plants were detached and incubated on wet tissue in petri-dishes for 3 and 7 days in the dark. For sorbitol experiments leaves from 7- to 8-week-old plants were detached and floated on 500 mM sorbitol solution for 24 and 48 h. the number of transcripts was normalized to actin 2/8 cDNA fragments. VSP1, vegetative storage protein 1; LOX2, lipoxygenase 2; AOS, allene oxide synthase. Data represent means of at least three independent replicates ± SD.

One possibility is that an additional, stress-specific factor is necessary to trigger the JA response. A similar concept of multiple signaling systems has been discussed in the context of systemic wound responses.⁷ Another possibility is that the reason for the lack of the JA response resides within one of the known signal transduction components of the JA signaling pathway itself. For instance the missing JA-response despite high JA levels might be due to low conversion of JA to JA-Ile. Alternatively, the JAZ proteins as negative regulators might not be degraded e.g., because the not-degradable splice variants of the JAZ proteins are expressed. Further research is needed to experimentally address this question. The fact that induction of JA biosynthesis genes is rather downregulated during aging and upon dark treatment is also noteworthy in the context of increased jasmonate production. This indicates that during aging and upon dark incubation the amount of RNA/protein of JA biosynthetic enzymes which is present in the tissue is sufficient to ensure an increased production of oxylipins.

It is well known that during senescence increased hydrolysis of membrane lipids takes place⁸ and most of the released fatty acids will be metabolized to acetyl-CoA. In Arabidopsis leaves around 60 and 77% of the fatty acids esterified in monogalactosyldiacylglycerols (MGDG) and digalactosyldiacylglycerols (DGDG), respectively, constitute linolenic acid, the precursor of jasmonate biosynthesis.⁹ Therefore, the breakdown of chloroplast lipids probably results in the release of a considerable amount of linolenic acid. The fact that the dramatic increase of jasmonates during dark incubation does not induce jasmonate signaling as described above suggests that the high jasmonate levels might be the result of enhanced release of linolenic acid from membranes which will then be metabolized to jasmonates. In this respect we analyzed levels of linolenic acid esterified in MGDG and DGDG as well as free linolenic acid in the LOX2RNAi lines, which are impaired in the lipoxy-genation of linolenic acid, the first step of jasmonate biosynthesis in comparison to the wild-type. Also the dde2 mutant was included because this mutant is also defective in the production of OPDA and JA but in contrast to the LOX2RNAi lines is able to catalyze the oxygenation of linolenic acid to 13-hydroperoxyoctadecatrienoic acid. This 13-LOX product can be metabolized by different pathways to a variety of oxylipins.¹⁰ In leaves of 6-week-old plants levels of esterified linolenic acid and free linolenic acid were similar in the wild type, the LOX2RNAi lines and dde2 (Table 1 and Fig. 2). Upon dark incubation the amount of linolenic acid esterified in MGDG/DGDG decreased similarly in the wild type, the LOX2RNAi lines and dde2. Levels of free linolenic acid did not show clear changes in the wild-type in response to dark treatment. In contrast, in both LOX2RNAi lines free linolenic acid accumulated to levels 4- to 5-fold of wild-type levels (Fig. 2). This is in agreement with the hypothesis that efficient metabolism of free linolenic acid to jasmonates takes place in the wild-type and might explain the accumulation of high jasmonate levels. In contrast to the LOX2RNAi lines the dde2 mutant did not show accumulation of free linolenic acid upon dark treatment and contained levels similar to the wild-type (Fig. 2). This can be explained by the fact that this mutant can produce other lipoxygenase products such as hydroxy fatty acids. In support of this explanation, higher levels of hydroxy fatty acids have been reported in this mutant upon wounding.¹¹

Table 1.

Levels of linolenic acid esterified in monogalactosyldiacylglycerols (MGDG) and digalactosyldiacylglycerols (DGDG) in leaves of wild-type, LOX2RNAi lines2 and 9 and dde before treatment and 7 days after dark incubation

Linolenic acid	WT		LOX2i-2		LOX2i-9		dde2
[nmol g−1 FW]	MGDG	DGDG	MGDG	DGDG	MGDG	DGDG	MGDG	DGDG
0 days	757 ± 59	466 ± 68	673 ± 10	515 ± 135	738 ± 93	462 ± 40	763 ± 133	609 ± 124
7 days	305 ± 44	187 ± 23	266 ± 48	183 ± 51	306 ± 52	272 ± 191	307 ± 38	349 ± 118
Difference	452	279	407	332	432	190	456	260

For quantification of MGDG/DGDG, samples were extracted and analysed by LC-MS/MS as described in Seltmann et al.⁴ Data represent means of four independent replicates ± sd.

Figure 2.

Accumulation of free linolenic acid in leaves of wild-type, LOX2RNAi lines 2 and 9 and dde2 before treatment and after 3 and 7 days of dark incubation. For quantification of linolenic acid, samples were extracted and analyzed by LC-MS/MS as described for JA/OPDA in Seltmann et al.⁴ elution was performed with a linear gradient (50% 1 mm ammonium acetate 50% acetonitril to 100% acetonitril within 5 min). Detection was performed in the selected ion recording mode without fragmentation with m/z of 277 for linolenic acid and 269 for the internal standard n-heptadecanoic acid (C17:0). Data represent means of four independent replicates ± SD.

Taken together, these data show that the production of high jasmonate levels does not necessarily result in activation of the jasmonate signaling pathway and that the accumulation of these oxylipins might be an effect of lipid metabolism in certain processes which can be suppressed without affecting the process itself.

Addendum to: Seltmann MA, Stingl NE, Lautenschlaeger JK, Krischke M, Mueller MI, Berger S. Differential impact of lipoxygenase 2 and jasmonates on natural and stress-induced senescence in Arabidopsis thaliana. Plant Physiol. 2010;152:1940–1950. doi: 10.1104/pp.110.153114.