Identification of Oxylipins involved as signaling molecules in wheat during infestation by the Russian Wheat Aphid

Introduction

The main objective during the study is to identify a qualitative and quantitative method for determining the presence of oxygenated lipids (novel compounds) during the defense responses of wheat against the Russian Wheat Aphid. Furthermore, Oxylipins identified will be quantified and discussed. A method developed as part of Ph.D study by Sculze, 2005 titled “Oxylipins and their involvement in Plant responses to biotic and abiotoc stress”, was initially used as blueprint for extraction of these compounds. Never before has a specific method been used on wheat to extract and identify these Oxylipins. Some minor adaptations have been made to this method to suite the extraction of these compounds from wheat. However, this method proved to be very difficult in identifying lipids present in wheat. The so called pentafluorobenzyl (PFB) esters could not be qualitatively and quantitatively detected by using a GCMS equipped with only an EI+ ionization mode. When carboxyl groups of the Oxylipins are derivatized or estrified to PFB esters, they can only be detected by negative chemical ionization (NCI) or chemical ionization (CI). Therefore, a slightly modified method of Weber et al. (1997) was used as alternative as described in a review article by Mueller et al. (2006). This proved to be a better quantitative and qualitative method that can be used to detect Oxylipins in wheat.

These responses against infestation by RWA are crucial in fully understanding plant defense and in improving cultivars in order for them to be fully resistant to the RWA. These early reactions involve the enzyme Lipoxygenase (lipid oxygenating enzyme) which oxidizes lipids derived from the cell walls when aphids start to feed. During my visit to the University of Muenster in 2010, I conducted three experiments involving the role of Lipoxygenase in the resistance reactions of wheat and rice suspension cultures against chitosan as influenced by Ulvan (a polysaccharide extracted from Ulva sp. or common sea lettuce). My research was discussed on a weekly basis within our research group. A separate research report on these results is available on request and was submitted in 2010. This, however, will not be included in my thesis for there is no relevance.

Further studies regarding chitosan treated seeds are being discussed. A seed treated with chitosan before sowing them possibly induces defense responses. Oxylipins involved in the seeds needs further investigation to determine the role of these oxygenated lipids from seed germination to plant establishment.  

As for the identification of Oxylipins in established plants, RWA Biotype 1 was multiplied under controlled conditions. Hereafter, Wheat plants (Tugela cv. DN) containing a resistance gene was planted and infested with RWA Biotype 1 (± 20 aphids per plant) at the three leaf stage. Control plants were kept un-infested. The infested wheat plants were kept spatially apart for the duration of the trial. Wheat plants were harvested at 0 hours post infestation (h.p.i), 96 h.p.i. and immediately frozen in liquid nitrogen. Oxylipins were extracted using the modified method of Weber et al. (1997). Hereafter the Oxylipins were analyzed using a Perkin Elmer Clarus 500 GC equipped with a Clarus 560 S ion trap mass spectrometer in EI+ ionization mode. All chromatograms are available on request. Compounds were identified by their characteristic fragmentation pattern by using the NIST 2.0 library (2008). Various fatty acids were identified but not yet quantified. Differences in both unique and quantity of Oxylipins in infested and un-infested plants were identified (See chromatogram, Fig. 1).

The major fatty acids found to be present were as follows: (Methyl esters)

The Oxylipins (fatty acids) were identified. Oxylipins in the control un-infested plants at 0 h.p.i. and can be assumed to be constitutively present in the plant at very low levels. However, these Oxylipins were more prominent in plants infested with RWA biotype 1.

Hendri is passionate about his work as researcher and also an enthusiastic collector and breeder of exotic succulent plants. He enjoys cycling, running, to gym and to go on hiking trails.

BLGY1643 (Module coordinator)

BTNY2626 (Practical)

BTNY3724 (Practical)

BTNY3744 (Practical)