Ectopic expression of AtJMT in Nicotiana attenuata . Creating a metabolic sink has tissue-specific consequences for the jasmonate metabolic network and silences downstream gene expression

To create a metabolic sink in the jasmonic acid (JA) pathway, we generated transgenic Nicotiana attenuata lines ectopically expressing Arabidopsis thaliana JA O-methyltransferase ( AtJMT , 35S -jmt ) and additionally silenced in other lines the N. attenuata methyl jasmonate (MeJA) esterase (35S -jmt /ir- mje ) to reduce the de-esterification of MeJA. Basal jasmonate levels did not differ between transgenic and wild type (WT) plants; however, after wounding and elicitation with Manduca sexta oral secretions (W+OS), the bursts of JA, jasmonoyl-isoleucine (JA-Ile) and their metabolites that are normally observed in the lamina, midvein and petiole of elicited WT leaves were largely absent in both transformants but replaced by a burst of endogenous MeJA that accounted for almost half of the total elicited jasmonate pools. In these plants, MeJA became a metabolic sink which affected the jasmonate metabolic network and its spread to systemic leaves, with major effects on 12-oxo-phytodieonic acid, JA and hydroxy-JA in petioles and on JA-Ile in laminas. Alterations in the size of jasmonate pools were most obvious in systemic tissues, especially petioles. Expression of threonine desaminase and trypsin proteinase inhibitor , two JA-inducible defense genes, was strongly decreased in both transgenic lines without influencing the expression of JA biosynthesis genes which were uncoupled from the W+OS-elicited JA-Ile gradient in elicited leaves. Taken together, this study provides support for a central role of the vasculature in the propagation of jasmonates and new insights into the versatile spatio-temporal characteristics of the jasmonate metabolic network.


INTRODUCTION
Plant cells, both those proximal to a wound site as well as those in more distal locations respond to tissue damage with large-scale changes in their transcription and metabolism, changes which help to promote survival of the entire plant (Reymond et al., 2000;Yan et al., 2007;Koo and Howe, 2009). Oxylipins, derived from fatty acid oxidation, play a central role in these responses: some function as direct defenses (Weber et al., 1999), others elicit the expression of defense related genes (Browse, 2005;Gfeller et al., 2010), or act as synomones in tritrophic interactions (Allmann and Baldwin, 2010). Jasmonic acid (JA) and its different derivatives collectively referred to as jasmonates, are the oxylipins whose biosynthesis and functions have been best characterized. They regulate responses to biotic and abiotic stresses (Baldwin et al., 1994;Devoto and Turner, 2005;Browse and Howe, 2008), but they also control different developmental processes throughout the life cycle of higher plants, including seed dormancy, flower morphogenesis and fruit formation (Creelman and Mullet, 1995;Hause et al., 2000;Stintzi and Browse, 2000;Li et al., 2004).
Initial steps in jasmonate biosynthesis occur in plastids and are associated with the quick biochemical activation of specific lipases and oxidizing enzymes upon tissue damage.
Trienoic fatty acids released from lipid membranes are first dioxygenated by 13lipoxygenase enzymes to form the oxylipin 13-HPOT. The combined catalytic action of allene oxide synthase (AOS) and allene oxide cyclase (AOC) converts 13-HPOT to cis-OPDA (12-oxo-phytodienoic-acid). The next catalytic steps require the export of OPDA from plastids and its import into the peroxisome where it is reduced by OPDA reductase enzymes (OPR). Three subsequent cycles of β -oxidation finally yield JA, which is transported to the cytoplasm and serves as a precursor for the synthesis of a broad range of JA derivatives (Gfeller et al., 2010). Our understanding of JA biosynthesis is largely biased towards the transcriptional regulation of JA biosynthesis genes (Howe and Schilmiller, 2002;Paschold et al., 2008) and several transcription factors modulating the accumulation of JA (Chung et al., 2008;Skibbe et al., 2008). However, fatty acid precursor availability is another essential determinant of the flux and metabolism of the JA pathway. Moreover the nature of the rate-limiting steps may also be context dependent. For instance, over expression of AOS in Arabidopsis and Nicotiana tabacum or AOC in tomato did not increase basal JA levels, but amplified the JA burst that is elicited in response to mechanical wounding (Laudert et al., 2000;Stenzel et al., 2003;Miersch et al., 2004). JA levels in leaves of many plant species increases within a few minutes after mechanical damage, which is too rapid to result entirely from the transcriptional regulation of JA biosynthesis genes. In

Generation of transgenic plants
In Arabidopsis thaliana (At), the homologue of Brassica campestris and Brassica rapa NTR1 (Nectarin1) genes has been shown to encode for a jasmonic acid O-methyltransferase (AtJMT, At1g19640) (Seo et al., 2001). The AtJMT gene-product methylates JA, but not OPDA, and its constitutive expression resulted in strongly increased basal endogenous MeJA levels (Seo et al., 2001). To re-route JA metabolism and evaluate the consequences for the regulation of the complete octadecanoid pathway, we generated stable transgenic lines All transgenic lines were indistinguishable from WT plants during rosette stage growth under controlled growth conditions (Fig. 1A). As is commonly reported for many mutants and transgenic lines altered in JA metabolism or signaling (Wasternack, 2007), flowers of 35S-jmt and 35S-jmt/ir-mje morphologically differed from those produced by WT plants. For all transgenic lines selected, style elongation was reduced to approximately half of that in WT flowers and the opening of the corolla limbs was impaired (Fig. 1B). Transcript measurements with gene-specific primers confirmed high relative expression levels of AtJMT in 35S-jmt-1 and 35S-jmt/ir-mje-1 but not in untransformed WT plants (Fig. 1C). After wounding and application of Manduca sexta oral secretions (W+OS), the expression of NaMJE was comparably high in 35S-jmt-1 and WT leaves and reduced by 95% in 35S-jmt/irmje-1 leaves.

35S-jmt and 35S-jmt/ir-mje plants have altered JA methylation and MeJA demethylation activities
We next verified that transgenic lines had deregulated JA methylation and MeJA demethylation activities. To assess AtJMT activity in vivo, we infiltrated 0.5 µg JA into WT, 35S-jmt-1 and 35S-jmt/ir-mje-1 leaf tissues and quantified increases in MeJA levels over a 45 min time period ( Fig. 2A) The impairment of MeJA de-methylation in 35S-jmt/ir-mje-1 was verified by measuring, as described above, the turnover of infiltrated MeJA (Fig. 2B). For simplicity, we do not describe here accumulation patterns of JA metabolites produced after MeJA deesterification ( Fig. S2B). While MeJA infiltration into WT and 35S-jmt-1 leaf laminas resulted in a rapid (0.5 min after infiltration) increases of free JA levels, the release of JA was, at the same time, reduced by ~ 80 % in MeJA-infiltrated leaf areas of 35S-jmt/ir-mje-1 ( MeJA-infiltrated 35S-jmt leaves were lower by up to 30 % compared to WT and the MeJA-to-JA conversion efficiencies did not differ among the three sampling times. These observations suggested that de-esterified MeJA was quickly re-methylated in 35S-jmt-1 leaves. To evaluate the importance of this reaction in both 35S-jmt-1 and 35S-jmt/ir-mje-1, we performed the same experiment as above using 0.25 µg of synthetic MeJA labeled with 13 C on the two first carbons and on the methylester group ([1, 2, 13-13 C]MeJA ). Similar differences were detected between 35S-jmt/ir-mje-1, 35S-jmt-1 and WT plants during [1,2,  about as low as before elicitation (0.03 nmol g -1 FW -1 ), they reached 2.9 nmol g -1 FW -1 in 35S-jmt and 7.9 nmol g -1 FW -1 in 35S-jmt/ir-mje after W+OS elicitation. This shift of the JA flux towards methylation did not translate into increased MeJA emissions -less than 0.01 % of endogenous maximum MeJA levels could be recovered from in the headspace of 35S-jmt and 35S-jmt/ir-mje leaves (Fig. S3).

Re-routing JA metabolism in 35S-jmt and 35S-jmt/ir-mje has profound, but differential, effects on jasmonate spatio-temporal accumulation patterns
We examined large-scale alterations in the jasmonate profiles caused by the depletion of the W+OS-induced JA pools (Fig. 4) (Table   SI).
Formation of OPDA, the JA precursor, increased in a consistent biphasic manner in the leaf laminas of all 3 genotypes after a single W+OS elicitation (Fig. 4). The second phase in OPDA accumulation occurred in all three genotypes after the waning of the JA burst.
Importantly, OPDA levels attained similar levels in WT, 35S-jmt and 35S-jmt/ir-mje-1 plants in response to elicitation, indicating that the manipulation of JA metabolism in these two transformants did not modify upstream components of the JA pathway. As observed above, tissues. As expected, the topology of this network was strongly distorted in the two transgenic lines (Fig. S5). Most importantly, the topology of the network of the two transgenic lines almost perfectly overlapped, suggesting that silencing of NaMJE provided likely little additional effect to the AtJMT-mediated alterations.

AtJMT ectopic expression affects jasmonate pools in a tissue-dependent manner
To determine whether the observed changes in jasmonate levels and the distortions of the jasmonate co-expression network resulted only from the diversion of JA metabolism towards MeJA or also involved deregulation of the jasmonate biosynthetic capacities, we calculated the sum of the 5 main abundant jasmonates detected in the individual tissues for each time point (Fig. 6B). As expected, jasmonate pools of elicited leaf laminas of 35S-jmt-1 and 35S-jmt/ir-mje-1 plants clearly differed in their relative composition from that of WT counterparts, but the sum of jasmonate levels in transgenic plants attained comparable maximum levels after 1h as in WT and vanished more slowly. Important differences in the relative composition and size of the jasmonate pools of local vascular tissues of transgenic lines were also observed when comparing with those of WT plants. But more important, for this comparison, maximum jasmonate pools elicited in midveins and petioles of the two transgenic lines were significantly decreased compared to those of WT -respectively by 44 % ( P < 0.001) and 60 % (P < 0.001) in 35S-jmt-1 and by 60 % ( P < 0.001) and 54 % (P = 0.014) in 35S-jmt/ir-mje-1.
As expected from the individual kinetics of Fig. 4 and 5, the size of the jasmonate pools elicited in systemic lamina tissues was considerably smaller than in local tissues (~ 0.15 nmol g -1 FW -1 vs more than 8.5 nmol g -1 FW -1 1 h post elicitation) and did not significantly differ between the two transgenic lines and WT. Compared to WT tissues, relative reductions in jasmonate pools of transgenic plants were more pronounced in systemic than in local vascular tissues. These decreases were most severe in petiole samples of systemic leaves -85 % reduction in 35S-jmt-1 ( P = 0.03) and 91 % reduction in 35S-jmt/ ir-mje-1 ( P = 0.03) after 2 h -and from a quantitative standpoint mainly attributable to decreases in OPDA and JA formation ( Fig. 5) which did not directly result from AtJMT-catalyzed MeJA production. This result was best visualized when the relative differences in total jasmonate production in transgenic lines vs WT were plotted for the different tissue types from the local to the distal lamina ( Fig. S6). We conclude from the above results that, at least at the elicitation sites, AtJMT ectopic expression caused a redirection of the JA flux towards MeJA without deregulating the entire JA biosynthetic machinery. To evaluate this conclusion, we quantified transcript levels of central JA biosynthesis genes in W+OS elicited leaf laminas. We also measured the expression of two direct defense and JA-inducible genes, NaTD (Kang et al., 2006) and

AtJMT ectopic expression differentially affects JA biosynthesis and JA-dependent
NaTPI (Horn et al., 2005), as references for the output of jasmonate signaling in the two transgenic lines. For both gene categories, transcriptional analyses were performed at times at which maximum transcript levels have been observed in previous studies (Halitschke and Baldwin, 2003;Halitschke et al., 2004). Maximum levels NaLOX3, NaAOS and NaOPR3 transcripts were similar in all three genotypes (Fig. 7). In contrast, the relative abundance of the two highly JA-induced defense transcripts of NaTD and NaTPI significantly differed before and after W+OS elicitation ( Fig. 7), suggesting an uncoupling in the transcriptional regulation of JA biosynthesis and the expression of these two direct defense genes in the transgenic lines.

DISCUSSION
This study demonstrates that ectopically expressing AtJMT in N. attenuata is sufficient to create a strong metabolic sink in the endogenous JA pathway which out-competes endogenous catalytic reactions controlling the availability, bioactivity and catabolism of free JA (Fig. 8). Individual accumulation patterns, especially those of JA-Ile, total jasmonate pools and networks were altered with different degrees of intensity and in a tissue-dependent manner in transgenic lines. This strong redirection of JA metabolism impaired the accumulation of defense-related transcripts but did not affect transcript levels of JA biosynthesis genes in transformed plants.

Creating a metabolic sink in the JA pathway
Methylation is one of the catalytic reactions used by plants to adjust their pools of active hormones to environmental conditions. Conversely, manipulating the scale of this reaction and characterizing its consequences at the metabolic and organismic level has been shown to be a valuable approach to uncover new signaling outputs and to revisit the The expression ´metabolic sink´ has previously been used when diverting or inactivating the metabolic and/or signaling outputs of biosynthetic pathways (Yao et al., 1995;Li and van Eck, 2007). Decreases in herbivory-induced jasmonate levels reported here that result from the depletion of JA levels by AtJMT, were as large or larger than those of RNAibased approaches previously used in our group to silence NaLOX3 (Halitschke and Baldwin, 2003) or NaJAR4 and NaJAR6 (Wang et al., 2008) expression. This metabolic sink did not deregulate jasmonate biosynthetic capacities, since no effects were seen in the transcript abundance of JA biosynthesis genes and the accumulation of upstream elements of the JA pathway, such as OPDA. However, we cannot fully rule out that changes had occurred in the activity of enzymes, since in N. attenuata, critical steps within the JA pathway are not Compartmentalization of the enzymes controlling JA synthesis and metabolism within the different cellular organelles may be responsible for these differences. JMT is predicted, since the amino acid sequence lacks an apparent organ-specific transit signal peptide, to act within the cytosol and therefore to strongly compete with the formation of JA-Ile by JAR enzymes which have been shown to be localized in the cytosol (Hsieh et al., 2000). Additionally, the almost complete depletion of the JA-Ile pool may in part have resulted from the reduced NaTD expression which mediates Ile formation (Kang et al., 2006). The contribution of NaTD down-regulation to the depletion of JA-Ile pools may also explain why less pronounced alterations were seen for other JA-amino acid conjugates than for JA-Ile (Fig. S4). An explanation for not having observed developmental alterations resembling those of N. likely be that the metabolic sink created in the two transgenic backgrounds affected only inducible levels of expression of this gene and not its developmental regulation.

Tissue-specific spread of jasmonate bursts and the role of petioles
Different jasmonates reach their maximum levels in different tissues (Hause et al., 2000;Glauser et al., 2008) and importantly JA-Ile, the bioactive jasmonate, accumulates preferentially at wound sites and was the only jasmonate with a clear "burst" character in distal laminas, as previously shown (Koo et al., 2009). As previously reported in Arabidopsis

JA biosynthesis and direct defense transcript levels are uncoupled in transgenic lines
JA biosynthesis, at least at the elicitation sites, was not repressed by the sink created within JA metabolism. This hypothesis was verified at the transcriptional level by the absence of differences, before and after induction, for central genes of the JA biosynthesis pathway (Fig. 7). In other plants species, over-expression of AOS or AOC has also been shown to not NaLOX3 and JA-Ile forming genes (NaJAR4 and NaJAR6) that NaAOS, NaAOC and NaOPR3 expression was not directly linked to JA-Ile synthesis. In contrast, the abundance of NaTD and NaTPI transcripts, which have been consistently shown by microarray analysis to be among the most highly JA and JA-Ile inducible genes in N. attenuata, was strongly impaired in both transformants and this effect was also observed in systemic lamina tissues of 35S-jmt-1 plants (Fig. S8). Such uncoupling between the expression of JA biosynthesis genes from that of "late"-induced defense-related genes has been demonstrated and extensively studied at the temporal and spatial levels in tomato (Howe et al., 2000;Ryan, 2000;Strassner et al., 2002). Moreover, tomato mutants differentially affected in these two classes of genes, as were our transgenic lines, have been reported. For instance, in the def-1 mutant which is deficient in wound-induced JA accumulation and in turn impaired in PI expression, AOS expression, as well as that of LOX-D involved in JA biosynthesis, is not affected (Howe et al., 2000). Homozygosity of the resulting T 2 plants was determined by screening for the resistance to the antibiotic hygromycin and the number of insertions was determined as described in (Gase et al., 2010), by southern blot hybridization of genomic DNA using a PCR fragment of the hptII gene as a probe (Fig. S1).

Plant treatments
For all experiments, plant treatments were randomly assigned among rosette stage plants and the first fully elongated leaf (+1 position) was treated. Manduca sexta feeding was simulated by wounding the leaf lamina with a fabric pattern wheel on both sides of the midrib and immediately applying 20 µl of M. sexta OS (diluted 1:10 in water) to the fresh wounds (W+OS); this procedure which is referred to as OS-elicitation, provides a convenient means of accurately standardizing herbivore elicitation of N. attenuata plants and allowing for detailed kinetic analyses of the elicitation process. M. sexta oral secretions (OS) were collected from 3 rd -4 th -instar larvae reared on N. attenuata WT leaves as described in Roda et al. (2004). Eggs of the tobacco hornworm M. sexta were obtained from North Carolina State University (Raleigh, NC, USA).
For jasmonate profiling, leaves of similar size and at the same developmental stage were harvested after W+OS treatment and, before being flash-frozen, rapidly dissected with a scalpel into three distinct tissue types: petioles (the vascular tissue connecting leaf laminas to the plant's shoot), midveins (the leaf's major vein acting as a vascular manifold) and the lamina (expanding right and left of the midvein). Petioles were flash-frozen immediately after being detached (less than 10 seconds). The midvein and the right and left sections of the leaf lamina were dissected and flash-frozen within ~ 10 seconds. The treated leaf was analyzed for local responses. The untreated leaf growing on the same plant with a minimal angular distance above the treated leaf and therefore orthostichous to the treated leaf was considered the systemic leaf.
Briefly, 15 µL of the resulting extracts were analyzed for jasmonates using a Varian 1200L liquid chromatography-MS/MS/MS system (Varian, Palo Alto, CA, USA) working with an electrospray ionization source (ESI). Negative or positive ionization mode was used depending on the jasmonate structure (Table SI) OPDA (1.28) and OH-JA (1.06) calculated versus the JA IS (Table SI) were obtained by measurement of dilution series of pure OPDA, OH-JA and JA IS dissolved into a leaf matrix.
Elemental formulas of [1, 2-13 C]MeJA and 12-COOH-JA-Ile were verified by ultrahigh pressure liquid chromatography time-of-flight MS using the method and calculation settings described in Gaquerel et al. (2010).

Collection of petiole exudates
The petiole exudates (PEX) collection protocol was modified from previously published methods (King and Zeevaart, 1974;Maldonado et al., 2002;Park et al., 2007). WT, 35S-jmt-1 and 35S-jmt-2 rosette stage plants of similar size were used for petiole exudate collection. For one replicate, three leaves of similar developmental stage were gently detached with a scalpel at the very base of their petioles and then subsequently re-cut in 1 mM EDTA solution (pH 7.5) to prevent callose deposition and closure of phloem vessels. Leaf laminas were mechanically wounded with a fabric pattern wheel and M. sexta oral secretions were applied to the wound sites (W+OS). Petioles of three induced leaves of one genotype were then immersed into a fresh 1.5 mL 1 mM EDTA solution (pH 7.5). After 2.5 h the collection solution was renewed and PEX collected for another 2.5 h combined with the first fraction.
PEX were freeze dried and dissolved in 70% methanol prior to the analysis for jasmonates.

in vivo JMT and MJE enzyme activity assays
To examine AtJMT and NaMJE activities, we infiltrated with a syringe JA, MeJA or [1, 2, 13-13 C]MeJA (labeled with 13 C on the three first carbons counting from the methylester group) dissolved in 1% DMSO in distilled water into leaves of rosette stage plants.
Concentrations of the solutions were for JA of 12.5 µg/mL, for MeJA of 12.5 µg/mL and for 13 C 3 -MeJA of 6.25 µg/mL. The amounts of infiltrated substrate per leaf discs were of 0.5 µg for JA and MeJA and of 0.25 µg for 13 C 3 -MeJA. To evaluate the effect of the infiltration on endogenous jasmonate production, similar volumes of 1% DMSO solution (Mock infiltration) were infiltrated with a syringe into leaves of equal size. After 0.5, 10 and 45 min, a leaf disc of 0.4 cm 2 was removed from the infiltrated leaf area with a cork borer and immediately flashfrozen in liquid nitrogen until analyses for jasmonates.

Quantitative real-time PCR analysis
Total RNA from five biological replicates per line was extracted as described in Linke at al. (2002). RNA extracts were treated with DNAse using the DNA-free™ Kit from Ambion (Applied Biosystems/Ambion, Austin). cDNA was synthesized from 500 ng RNA using SuperScript II Reverse Transcriptase (Invitrogen, Germany) and a poly-T primer. Quantitative real-time PCR (qRT-PCR, Stratagene 500 Mx3005P, Waldbronn, Germany) was conducted with 30 ng cDNA using the core reagent kit (Eurogentec, http://www.eurogentec.be) and pairs of gene specific primers (Table SII). qPCR products were either detected by gene-specific double fluorescent dye-labeled TaqMan® probes or after reaction with SYBR Green (qPCR Core Kit for SYBR Green I; Eurogentec, Köln, Germany). Relative gene expression was calculated using a 200-fold dilution series of cDNAs synthesized from RNA samples of the same experiment and normalized, according to Pfaffl et al. (2002), by the expression value of the N. attenuata ACTIN gene.

Statistical analysis
Most statistics were performed with StatView (Abacus Concepts Inc.,
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