Background

The filamentous ascomycete Fusarium graminearum is the main pathogen causing Fusarium head blight (FHB) in wheat (McMullen et al., 1997). The consequences of host plant infection lead to significant quantitative and economic losses (Goswami and Kistler, 2004), as the infection results in incompletely formed grains. During infection, Fusarium graminearum produces a variety of secondary metabolites, low-molecular-weight molecules that are generally not necessary for the growth or developmental processes of the fungus but result in contamination of the grain with harmful mycotoxins (Shwab and Keller, 2008). Contaminated grain thus becomes unusable as food for humans and animals.

One of these mycotoxins is butenolide, which was first isolated in 1967 and shown to be toxic to mitochondrial respiration in rat cardiac muscle tissue (Wang et al., 2009). Further studies revealed that mitochondria of muscle tissue are affected. This includes swelling, disruption of the double membrane, and disruption of mitochondrial respiration (Wang et al., 2007 and 2009; Pei et al., 2013). Whether plant mitochondria are also affected by butenolide has not yet been investigated in detail. However, the impairment of animal mitochondria by butenolide could be an indication of a possible effect of this metabolite on plant mitochondria. Therefore, understanding the function of these substances on mitochondria during infection is of great importance for the knowledge of fungus–plant interaction in general and should help to produce resistant plants in the future.

Because the measurement of mitochondrial respiration in cells from plant cultures may not fully reflect the real situation, we describe here a method for measuring mitochondrial respiration parameters in wheat paleae (paleae superior) using an XF24 Extracellular Flux Analyzer. To date, no measurements of mitochondrial respiratory parameters have been performed in wheat paleae. The XF24 analyzer is a respirometer based on a multi-well plate that measures oxygen consumption rate (OCR) by changes in the fluorescence of solid-state fluorophores (Gerencser et al., 2009). To obtain a mitochondrial respiration profile, mitochondrial agents are automatically injected through various ports during testing (Divakaruni et al., 2014). This procedure involves preparation of samples for the XF24 analyzer and the measurement of mitochondrial parameters by addition of specific mitochondrial inhibitors.

The analysis of mitochondrial respiration in these samples is an important addition to existing studies and also offers the possibility to test the effects of early infection of plants with harmful fungi on mitochondrial respiration parameters. This could contribute to the development of resistant plants as well as specifically acting plant protection products. In addition to questions on resistance to fungal attack, this protocol can be used to investigate questions relating to mitochondrial activity in wheat in more detail or to use mitochondrial parameters as a supplement to classical phenotyping.