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

Abstract

To create a metabolic sink in the jasmonic acid (JA) pathway, we generated transgenic Nicotiana attenuata lines ectopically expressing Arabidopsis (Arabidopsis thaliana) jasmonic acid O-methyltransferase (35S-jmt) and additionally silenced in other lines the N. attenuata methyl jasmonate esterase (35S-jmt/ir-mje) to reduce the deesterification of methyl jasmonate (MeJA). Basal jasmonate levels did not differ between transgenic and wild-type plants; however, after wounding and elicitation with Manduca sexta oral secretions, the bursts of JA, jasmonoyl-isoleucine (JA-Ile), and their metabolites that are normally observed in the lamina, midvein, and petiole of elicited wild-type 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 that 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 deaminase and trypsin proteinase inhibitor, two JA-inducible defense genes, was strongly decreased in both transgenic lines without influencing the expression of JA biosynthesis genes that were uncoupled from the wounding and elicitation with M. sexta oral secretions-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 spatiotemporal characteristics of the jasmonate metabolic network.

Figure 1.

N. attenuata plants ectopically expressing AtJMT (35S-jmt) or additionally silenced for the expression of NaMJE (35S-jmt/ir-mje) are indistinguishable from wild type during rosette stage growth but produce flowers with altered morphology. A, N. attenuata plants ectopically expressing AtJMT (35S-jmt-1) and additionally silenced for NaMJE (35S-jmt/ir-mje-1) do not differ from wild-type plants during rosette stage (approximately 30-d-old). B, Flowers of 35S-jmt and 35S-jmt/ir-mje plants have short styles (white arrows indicate stigma position) and closed corollas compared to wild-type plants. C, Relative transcript abundance (mean ± sd, n = 5) of AtJMT and NaMJE in leaf laminas of wild-type, 35S-jmt, and 35S-jmt/ir-mje-1 plants 1 h after mechanical wounding and application of M. sexta oral secretions to the wounds (W + OS). No AtJMT expression was detected (n.d.) in wild-type tissues after W + OS elicitation. NaMJE relative transcript levels in 35S-jmt/ir-mje-1 leaf laminas after W + OS elicitation were reduced to approximately 5% of wild-type levels but were unchanged in 35S-jmt-1.

Figure 2.

N. attenuata 35S-jmt and 35S-jmt/ir-mje plants have altered JA-methylation and MeJA-demethylation activities. Leaf discs (0.4 cm²) were infiltrated with a control solution (Mock) and 0.5 μg of JA (A), unlabeled MeJA (B), or 0.25 μg synthetic MeJA labeled with ¹³C ([1, 2, 13-¹³C]MeJA; C). JA methylation (A), MeJA demethylation (B), and the remethylation of the subsequently released JA (C) were analyzed by quantifying 0.5, 10, and 45 min increases (mean ± sd, n = 5) in MeJA (JA → MeJA), JA (MeJA → JA), and [1, 2-¹³C]MeJA levels after infiltration in the elicited leaves. After JA infiltration, 35S-jmt-1 and 35S-jmt/ir-mje-1 leaves showed larger MeJA accumulations than wild type that came at the expense of other JA metabolites (Supplemental Fig. S2). MeJA deesterification was strongly impaired in 35S-jmt/ir-mje-1 and only slightly reduced in 35S-jmt-1 plants. Left section of C: high-resolution time-of-flight MS measurement of (calculated m/z = 228.158) [1, 2, 13-¹³C]MeJA-infiltrated leaf areas—zoom in of the spectral range m/z 227 to 229—revealed the production of a m/z signal at 227.155 characteristic for ¹³C₂-MeJA ([1, 2-¹³C]MeJA, calculated m/z = 227.155) formed by remethylation of the deesterified ¹³C₃-MeJA in 35S-jmt-1 and 35S-jmt/ir-mje-1, but not in wild-type leaf samples. ¹³C atoms are circled. Right section of C: [1, 2-¹³C]MeJA levels. Asterisks represent significant differences between wild-type and transgenic lines (unpaired t test; * P < 0.05, ** < 0.001, *** < 0.0001). n.d., Not detected.

Figure 3.

AtJMT-catalyzed MeJA formation depletes W + OS-induced JA levels. Mean (±sd, n = 5) constitutive and induced JA (A) and MeJA (B) levels (nmol g⁻¹ FW⁻¹) after mechanical wounding and application of M. sexta oral secretions to the puncture wounds (OS elicitation) in laminas of wild-type, 35S-jmt (35S-jmt-1, 35S-jmt-2), and 35S-jmt/ir-mje (35S-jmt/ir-mje-1, 35S-jmt/ir-mje-2) plants. Prior to elicitation, JA and MeJA levels in leaf laminas of 35S-jmt and 35S-jmt/ir-mje lines did not differ from those in wild type. The JA burst observed in wild-type leaf laminas after W + OS elicitation was largely absent in 35S-jmt and 35S-jmt/ir-mje plants but was mirrored, due to AtJMT activity, by a large burst of MeJA. Asterisks represent significant differences between wild-type and transgenic lines (unpaired t test; * P < 0.05, ** < 0.001, *** < 0.0001).

Figure 4.

Ectopic expression of AtJMT in 35S-jmt and 35S-jmt/ir-mje has profound, but differential, effects on spatiotemporal accumulation patterns of jasmonates in lamina, midribs, and petioles. Mean (±sd, n = 5) of jasmonate levels—OPDA, JA, MeJA, JA-Ile, and OH-JA (the sum of 12- and 11-OH-JA)—in wild-type, 35S-jmt-1, and 35S-jmt/ir-mje-1 leaves after OS elicitation (W + OS). Leaves were harvested 0 to 5 h after elicitation, dissected into leaf lamina, midvein, and petiole (see schematics above line graphs), and analyzed separately. Different jasmonates attained their maximum levels in different tissues postelicitation. MeJA hyperaccumulation occurred at the expense of JA-Ile and JA accumulation but only retarded the accumulation of OH-JA. AtJMT ectopic expression also had different consequences on the accumulation of JA-Ile and other JA-amino acid conjugates (Supplemental Fig. S4). Asterisks (35S-jmt-1) and plus signs (35S-jmt/ir-mje-1) represent significant differences between wild-type and transgenic lines (unpaired t test; */+ P < 0.05, **/++ < 0.001, ***/+++ < 0.0001).

Figure 5.

Alterations in W + OS elicited jasmonate levels in systemic unelicited leaves of 35S-jmt-1 and 35S-jmt/ir-mje-1 are most pronounced in petioles and differ from those observed in elicited leaves. Mean (±sd, n = 5) levels jasmonates—OPDA, JA, MeJA, JA-Ile, and OH-JA (the sum of 12- and 11-OH-JA)—in wild-type, 35S-jmt-1, and 35S-jmt/ir-mje-1 leaves after OS elicitation (W + OS). Untreated leaves growing on the same plants as in Figure 4 with a minimal angular distance above the treated leaf, and hence orthostichous to the treated leaf, were considered systemic leaves. Leaves were harvested 0 to 5 h after elicitation, dissected into leaf lamina, midvein, and petiole (see schematics above line graphs), and analyzed separately. All jasmonates attained their maximum levels in petioles. Accumulation of JA, OPDA, and OH-JA was largely reduced in systemic petioles, although MeJA formation was less pronounced than in elicited tissues (Fig. 4). Jasmonate levels detected in systemic leaves were, except for OPDA and depending on the tissue type, 10- to 60-times lower than in treated leaves. Asterisks (35S-jmt-1) and plus signs (35S-jmt/ir-mje-1) represent significant differences between wild-type and transgenic lines (unpaired t test; */+ P < 0.05, **/++ < 0.001, ***/+++ < 0.0001).

Figure 6.

Alterations in jasmonate pools and relative composition are tissue dependent and most pronounced in petioles. A, Hierarchical clustering analysis performed, using the Pearson correlation as metric, on tissue-specific jasmonate signatures using vectors defined by the different levels reached at the different sampling times by a given jasmonate in a given tissue. Jasmonate profiles in systemic petioles relate the most to the signatures elicited after OS elicitation (W + OS) of treated leaves. The structure of locally elicited jasmonate signatures in wild type is not retained in 35S-jmt-1 and 35S-jmt/ir-mje-1 leaves as revealed by the clustering. B, Mean (±sd, n = 5) summed levels of the five most abundant jasmonates (OPDA, JA, MeJA, JA-Ile, and OH-JA) detected at 0 to 5 h after W + OS elicitation in local (top section) and systemic (bottom section) leaf tissues (lamina, midvein, and petiole) of wild-type, 35S-jmt-1, and 35S-jmt/ir-mje-1. Major quantitative changes in jasmonate pools were detected in midveins and petioles (Supplemental Fig. S6). Asterisks represent significant differences between tissues of wild-type and transgenic plants (unpaired t test; * P < 0.05, ** < 0.001, *** < 0.0001).

Figure 7.

Transcript levels of JA biosynthesis and two direct defense genes are differentially affected by AtJMT ectopic expression. Mean (±sd, n = 5) transcript abundance (relative to NaACTIN) of JA biosynthesis genes in leaf lamina—AOS (NaAOS), lipoxygenase3 (NaLOX3), OPDA reductase (NaOPR3)—does not differ between wild-type and 35S-jmt-1 and 35S-jmt/ir-mje-1 plants before and after OS elicitation (W + OS). In contrast, constitutive and W + OS-induced transcript abundance of Thr deaminase (NaTD) and trypsin proteinase inhibitor (NaTPI), two direct defense genes regulated by the JA signaling pathway, were decreased in 35S-jmt-1 and 35S-jmt/ir-mje-1 compared to those in wild-type plants. OS-induced leaf laminas were harvested when peak expression levels have been reported for the selected genes. Asterisks represent significant differences between wild-type and transgenic lines (NaAOS, NaTD, NaLOX3, NaOPR3 unpaired t test, * P < 0.05; NaTPI Welch’s test, * P < 0.05). n.d., Not detected.

Figure 8.

Model of OS-induced JA metabolism and signaling in leaf lamina of 35S-jmt and 35S-jmt/ir-mje plants. AtJMT ectopic expression in concert with NaMJE silencing creates a powerful metabolic sink in the JA pathway that outcompetes herbivory-induced JA and JA-Ile bursts, and in turn, compromises NaCOI1-mediated activation of NaTD and NaTPI expression but not that of JA biosynthesis genes. Font size and arrows’ thickness are proportional to the intensity of metabolite fluxes and activation of gene expression in leaf laminas tissues after simulated M. sexta herbivory.