Bosch, M.; Wright, L. P.; Gershenzon, J.; Wasternack, C.; Hause, B.; Schaller, A.; Stintzi, A. Jasmonic acid and its precursor 12-oxophytodienoic acid control different aspects of constitutive and induced herbivore defenses in tomato Plant Physiology 166, 396-410, (2014) DOI: 10.1104/pp.114.237388
The jasmonate family of growth regulators includes the isoleucine conjugate of jasmonic acid (JA-Ile) and its biosynthetic precursor 12-oxophytodienoic acid (OPDA) as signaling molecules. In order to assess the relative contribution of JA/JA-Ile and OPDA to insect resistance in tomato, we silenced the expression of OPDA reductase (OPR3) by RNA interference. Consistent with a block in the biosynthetic pathway downstream of OPDA, OPR3-RNAi plants contained wild-type levels of OPDA but failed to accumulate JA or JA-Ile after wounding. JA/JA-Ile deficiency in OPR3-RNAi plants resulted in reduced trichome formation and impaired monoterpene and sesquiterpene production. The loss of these JA/JA-Ile-dependent defense traits rendered them more attractive to the specialist herbivore Manduca sexta with respect to feeding and oviposition. Oviposition preference resulted from reduced levels of repellant mono- and sesquiterpenes. Feeding preference, on the other hand, was caused by increased production of cis-3-hexenal acting as a feeding stimulant for M. sexta larvae in OPR3-RNAi plants. Despite impaired constitutive defenses and increased palatability of OPR3-RNAi leaves, larval development was indistinguishable on OPR3-RNAi and wild-type plants, and much delayed as compared to development on the JA/JA-Ile insensitive (jai1) mutant. Apparently, signaling through JAI1, the tomato ortholog of COI1 in Arabidopsis, is required for defense while the conversion of OPDA to JA/JA-Ile is not. Comparing the signaling activities of OPDA and JA/JA-Ile, we found that OPDA can substitute for JA/JA-Ile in the local induction of defense gene expression, but the production of JA/JA-Ile is required for a systemic response.
Ziegler, J.; Abel S. Analysis of amino acids by HPLC/electrospray negative ion tandem mass spectrometry using 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) derivatization Amino Acids 46, 2799-2808, (2014) DOI: 10.1007/s00726-014-1837-5
A new method for the determination of amino acids is presented. It combines established methods for the derivatization of primary and secondary amino groups with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) with the subsequent amino acid specific detection of the derivatives by LC–ESI–MS/MS using multiple reaction monitoring (MRM). The derivatization proceeds within 5 min, and the resulting amino acid derivatives can be rapidly purified from matrix by solid-phase extraction (SPE) on HR-X resin and separated by reversed-phase HPLC. The Fmoc derivatives yield several amino acid specific fragment ions which opened the possibility to select amino acid specific MRM transitions. The method was applied to all 20 proteinogenic amino acids, and the quantification was performedusing l-norvaline as standard. A limit of detection as low as 1 fmol/μl with a linear range of up to 125 pmol/μl could be obtained. Intraday and interday precisions were lower than10 % relative standard deviations for most of the amino acids. Quantification usingl-norvaline as internal standard gave very similar results compared to the quantificationusing deuterated amino acid as internal standards. Using this protocol, it was possible to record the amino acid profiles of only a single root from Arabidopsis thaliana seedlings and to compare it with the amino acid profiles of 20 dissected root meristems (200 μm).