Background

Ethylene is a gaseous plant hormone that plays an important role in the regulation of numerous physiological and developmental plant processes, such as seed germination, root and shoot development, cellular growth regulation, carbon assimilation, senescence of leaves and flowers, organ abscission, and fruit ripening (Abeles et al., 1992; Pierik et al., 2006; Olsen, 2010; Wang et al., 2013; Dubois et al., 2018). Ethylene also acts as a signaling molecule in responses to biotic and abiotic environmental stresses, such as temperature extremes, droughts, floods, shading, radiation, nutrient deficiency, and mechanical and chemical damage (Yang and Hoffman, 1984; Iqbal et al., 2013; Dubois et al., 2018). The amount of ethylene production depends on the plant species, developmental stage of the plant, and organ type (Cristescu et al., 2013). Apart from endogenous or stress-induced ethylene production, production in plant organs can also be induced by exogenous application of the plant growth regulator ethephon (2-chloroethylphosphonic acid). Ethephon has been widely used in agriculture for different applications, including fruit ripening and coloration, organ abscission to aid harvesting, bloom delay, and suppression of vegetative growth (Nickell, 1994).

Accurate quantification of ethylene evolution from ethephon as well as endogenous ethylene production following different treatments is necessary to correlate production with respective plant responses and elucidate the functions and role of ethylene. According to Cristescu et al. (2013), there are three main methods to detect ethylene in plants: gas chromatography detection, electrochemical detection, and optical detection. These can be used for periodic or continuous measurement depending on the type of plant material and experimental design. Gas chromatography (GC) is most widely used for separation and analysis of ethylene, due to its small sample requirement, high selectivity, and fast analysis. Although more sensitive detection techniques have been developed, such as a laser-based sensing technique (Cristescu et al., 2013; Gwanpua et al., 2018), GC detection is still the most applicable method for tree crops, since non-destructive and/or continuous sampling is not possible. This applies particularly to studies that focus on trees in orchard systems and that are subject to climatic effects.

In fruit tree crops, the quantification of ethylene has mainly been used to assess the effect of endogenous ethylene production in developing fruit, e.g., on fruit coloration (Yin et al., 2001; Chervin et al., 2005; Wang et al., 2007), or in harvested fruit, e.g., on fruit quality and shelf life after harvest (Tseng et al., 2000; Gwanpua et al., 2018). However, few studies investigated ethylene production in leaves, buds, flowers, or other vegetative material from tree crops. Examples are GC detection of ethylene in leaves of citrus (Tudela and Primo-Millo, 1992), in shoot portions of apple (Sanyal and Bangerth, 1998), and in floral buds and leaves of mango (Bindu et al., 2017), peach (Liu et al., 2021) and litchi (Cronje et al., 2022) trees. However, most of these studies only provided limited information on the sampling and analysis techniques used to successfully reproduce the techniques for application in other crops with similar plant organs. For this purpose, we developed a protocol to easily determine ethylene evolution in leaves and apical buds of litchi as part of a recent study, which used ethephon to induce bud dormancy and delay panicle emergence for more consistent floral initiation in litchi (Cronje et al., 2022). To determine the mode-of-action of ethylene inside the leaf and bud tissue, as well as the downstream processes, such as relative expression of ethylene-, dormancy-, and flowering-related genes, ethylene concentration needed to be quantified reliably and accurately in these plant organs to correlate the results with those obtained from corresponding biochemical and molecular analyzes (Cronje et al., 2022). The sampling technique and incubation period was adapted specifically for litchi to account for low overall ethylene production and wound ethylene release after detaching of the respective plant organs. The quantification protocol was derived from a protocol for the quantification of ethylene evolution in tomato leaves developed by Kim et al. (2016) and used a porous layer open tubular (PLOT) TG-Bond Q+ column (Thermo Scientific) for direct separation of ethylene. While several related studies used packed alumina (Al₂O₃) columns that are recognized for the separation of hydrocarbons from C1 to C5, the use of the mentioned PLOT column is considered advantageous since it is specifically developed for selective separation of acetylene (C₂H₂), ethylene (C₂H₄) and ethane (C₂H₆) to baseline. Additionally, the use of a capillary column in conjunction with a flame ionization detector has proven to be highly sensitive with a linear calibration range of three orders of magnitude, i.e., 0.08 nL to 80 nL of injected C₂H₄. Although the protocol was specifically developed for leaves and buds of litchi, it can be equally applied to plant material from other fruit tree crops by making appropriate changes to container sizes.