A rapid and robust method for simultaneously measuring changes in the phytohormones ABA, JA and SA in plants following biotic and abiotic stress

Abstract

We describe an efficient method for the rapid quantitative determination of the abundance of three acidic plant hormones from a single crude extract directly by LC/MS/MS. The method exploits the sensitivity of MS and uses multiple reaction monitoring and isotopically labelled samples to quantify the phytohormones abscisic acid, jasmonic acid and salicylic acid in Arabidopsis leaf tissue.

Figure 1

The phytohormone extraction method is reproducible. (a) The abundance of the phytohormones ABA, JA, SA and the SA glycoside expressed as a ratio of the added internal standard. Standard error for the seven replicates is less than 10% of the mean value. Glycosylated derivatives can also be determined by ratioing relative to the unglycosylated internal standard. (b) Determination of the absolute value of phytohormones present in the stressed tissue demonstrates that this method can accurately capture the dynamic range of phytohormones with small amounts of starting material (10 mg).

Figure 2

Comparison of absolute yields from freeze dried (FD) or fresh frozen Arabidopsis leaf material. Yields of both JA and SA were consistently higher using fresh frozen material, probably due to a combination of both analyte insolubilisation or volatilisation during the freeze drying process.

Figure 3

Linearity of detection of phytohormones. To ensure the method is capable of capturing the range of differences in phytohormones expected during stress associated experiments, five diluted deuterated standards were compared to unlabelled "stressed controls" in ratios indicated. a-c demonstrates that for ABA, JA and SA respectively, there is a statistically significant linear increase in abundance of the expected ion over a 20 fold range.

Figure 4

Phytohormone response metrics. To determine whether 10 mg was sufficient sample to elicit a linear response in LC/MS signal, phytohormones were determined in 5, 10 and 15 mg amounts of freeze dried material. ABA, JA and SA and SA-glyc (a-d respectively) all show a linear response with increasing amounts of sample (R² > 0.997).

Figure 5

The extraction method is capable of capturing the dynamic response of phytohormones to inducing stresses. JA, ABA and SA levels were determined following stresses designed to elevate specific levels of each hormone (a-c). (a) Following wounding by mechanical damage JA levels increase 8 fold within 5 minutes and increase over the following 2 h. (b) Two hours desiccation of detached leaves (at 60% RH) is sufficient to increase foliar ABA levels 8 fold. (c) Challenge with the virulent bacterial pathogen, DC3000 or the DC3000 hrp mutant elicits increases in SA levels 21 hpi in wild-type but not the SA biosynthetic mutant, sid2. (d) Challenge with virulent DC3000 induces ABA in Arabidopsis leaves within 6 hours post inoculation (hpi).