Independently silencing two JAR family members impairs levels of trypsin proteinase inhibitors but not nicotine

Abstract

Jasmonic acid (JA)-amino acid conjugates are important JA metabolites that activate JA responses. However, our understanding of their involvement in herbivore defenses is limited. We identified a new Arabidopsis jasmonate resistant 1 (JAR1) homologue in Nicotiana attenuata (N. attenuata) and named it jasmonate resistant 6 (JAR6). JAR6 clustered closely with Arabidopsis JAR1 and the recently reported jasmonate resistant 4 (JAR4), another JAR1 homologue in N. attenuata, in a phylogenic analysis. The strong elicitation of JAR6 transcripts by wounding and treatment with Manduca sexta (M. sexta) oral secretions (OS), which mimics herbivore attack, suggests it plays a role in herbivore defense. Independently silencing JAR4 or JAR6 by transforming N. attenuata with inverted repeat JAR4 or JAR6 constructs significantly reduced levels of not only JA-Ile plus JA-Leu but also JA-Val in OS-elicited leaves, suggesting JAR4 and JAR6 are functionally redundant and their amino acid substrates are not highly specific to individual amino acids. A new JA conjugate, JA-Gln, whose levels are much higher than those of the other JA conjugates in WT plants, was not affected in JAR4- or JAR6-silenced lines, implying that another JA-conjugating enzyme exists in N. attenuata. Neither JA-ACC, the second most abundant JA conjugate in Arabidopsis seedlings, nor JA-Met or JA-Trp, was detectable in N. attenuata. Levels of trypsin proteinase inhibitors (TPIs) in JAR4- and JAR6-silenced plants were significantly reduced, but nicotine levels were normal. We conclude that both JAR4 and JAR6 conjugate JA to Ile, Val, and Leu, and that both positively regulate TPI activity.

Arabidopsis Pollen Fertility Requires the Transcription Factors CITF1 and SPL7 That Regulate Copper Delivery to Anthers and Jasmonic Acid Synthesis

“…Collected tissues were flash-frozen in liquid N 2 prior to subsequent manipulations. Given that the internal JA concentration in tissues of plants grown under control conditions is low, we scaled up the JA extraction procedure described (Wang et al, 2007) to enable us to quantify and compare JA concentration in different mutant lines. Briefly, 400 to 500 mg of frozen tissues was ground to a fine powder using a mortar and a pestle.…”

“…Given that some samples from the spl7 single and many samples from the citf1 spl7 double mutant did not contain detectible JA, we replaced the missing values with a value of the detection limit for the instrument, 1 ng/g fresh weight. JA concentrations in leaf tissues of different plant lines using the extraction procedure described by Wang et al (2007) are shown in Supplemental Figure 7A.…”

A Comparison of Two Nicotiana attenuata Accessions Reveals Large Differences in Signaling Induced by Oral Secretions of the Specialist Herbivore Manduca sexta

“…At 1 h after either treatment, UT showed about 70% higher levels of JA-Ile than did AZ. JAR4 and JAR6 have been identified as the enzymes that conjugate JA and Ile to form JA-Ile (Wang et al, 2007); we measured the transcript levels of both genes (Fig. 5B, middle and bottom).…”

Empoasca leafhoppers attack wild tobacco plants in a jasmonate-dependent manner and identify jasmonate mutants in natural populations

“…The reduced OPDA (OPC-8:0) undergoes three cycles of β-oxidation involving acyl-CoA transferase (ACX) (33) and finally forming (3R, 7S)-JA. JA can be modified, e.g., by jasmonyl-O-methyl transferase (JMT) to form methyl-jasmonic acid (MeJA) (34) or by JASMONATE RESISTANT (JAR) that conjugates isoleucine to form JA-Ile (35,36). JA-Ile activates the SCF COI1 -JAZ complex (37), which transcriptionally activates genes involved in the biosynthesis of defense molecules, among other responses (38,39).…”