A method for profiling classes of plant hormones and their metabolites using liquid chromatography‐electrospray ionization tandem mass spectrometry: an analysis of hormone regulation of thermodormancy of lettuce (Lactuca sativa L.) seeds

Abstract

SummaryA highly selective and sensitive method for the simultaneous analysis of several plant hormones and their metabolites is described. The method combines high-performance liquid chromatography (HPLC) with positive and negative electrospray ionization-tandem mass spectrometry (ESI±MS/MS) to quantify a broad range of chemically and structurally diverse compounds. The addition of deuterium-labeled analogs for these compounds prior to sample extraction permits accurate quanti®cation by multiple reaction monitoring (MRM). Endogenous levels of abscisic acid (ABA), abscisic acid glucose ester (ABA-GE), 7H -hydroxyabscisic acid (7 H -OH-ABA), phaseic acid (PA), dihydrophaseic acid (DPA), indole-3-acetic acid (IAA), indole-3-aspartate (IAAsp), zeatin (Z), zeatin riboside (ZR), isopentenyladenine (2iP), isopentenyladenosine (IPA), and gibberellins (GA) 1 , GA 3 , GA 4 , and GA 7 were determined simultaneously in a single run. Detection limits ranged from 0.682 fmol for Z to 1.53 pmol for ABA. The method was applied to the analysis of plant hormones and hormonal metabolites associated with seed dormancy and germination in lettuce (Lactuca sativa L. cv. Grand Rapids), using extracts from only 50 to 100 mg DW of seed. Thermodormancy was induced by incubating seeds at 338C instead of 238C. Germinating seeds transiently accumulated high levels of ABA-GE. In contrast, thermodormant seeds transiently accumulated high levels of DPA after 7 days at 338C. GA 1 and GA 3 were detected during germination, and levels of GA 1 increased during early postgerminative growth. After several days of incubation, thermodormant seeds exhibited a striking transient accumulation of IAA, which did not occur in seeds germinating at 238C. We conclude that hormone metabolism in thermodormant seeds is surprisingly active and is signi®cantly different from that of germinating seeds.

High Temperature-Induced Abscisic Acid Biosynthesis and Its Role in the Inhibition of Gibberellin Action in Arabidopsis Seeds

“…8#-hydroxylation pathway, is accumulated in the seeds imbibed at supraoptimal temperature (Chiwocha et al, 2003). ABA content in thermoinhibited seeds showed only a slight increase after 12 h of imbibition (Fig.…”

Plant Cold Acclimation: The Role of Abscisic Acid

“…For example, ABA biosynthesis is reported for the maintenance of thermodormancy of lettuce seeds (Yoshioka and others 1998). Chiwocha and others (2003) detected a transient increase in ABA in thermodormant seeds, but thereafter the levels remained constant for 8 days. However, analysis of the ABA metabolites revealed continuous synthesis and turnover of ABA.…”

Vacuolar Transport of Abscisic Acid Glucosyl Ester Is Mediated by ATP-Binding Cassette and Proton-Antiport Mechanisms in Arabidopsis

“…S5 and S6). Since ABA-GE levels were reported to increase during seed maturation and germination (Chiwocha et al, 2003;Seiler et al, 2011), we hypothesized that AtABCC14 may be involved in ABA-GE transport. In a targeted approach, we tested the Arabidopsis ABCC transporters AtABCC1, AtABCC2, and AtABCC14 for their ability to transport ABA-GE using membrane vesicles isolated from yeast heterologously expressing these proteins.…”

“…In contrast to the oxidative pathway, the inactivation of ABA by Glc conjugation is reversible, and hydrolysis of ABA-GE catalyzed by b-glucosidases results in free ABA (Dietz et al, 2000;Lee et al, 2006;Xu et al, 2012). ABA-GE levels were shown to substantially increase during dehydration and specific seed developmental and germination stages (Boyer and Zeevaart, 1982;Hocher et al, 1991;Chiwocha et al, 2003). Furthermore, ABA-GE is present in the xylem sap, where it was shown to increase under drought, salt, and osmotic stress (Sauter et al, 2002).…”