Species-specific metabolic response to combined heat+drought stress
The differences observed in metabolic response of young white spruce and paper birch trees to the combination of heatwave and drought stress versus the response to either independent stress is indicative of the problem of generalizing plant responses across species to multiple stressors acting simultaneously in the growth environment. White spruce showed many unique metabolic responses to the combined stress, while paper birch displayed few. The HD birch tended to share responses with the D plants (e.g. total leaf N, Spd, Spm, and several AAs) or the response was between that of the D andH plants (e.g. soluble sugars). These findings partially support our first hypothesis that plant responses to the combination of heatwave and drought stress will be unique from either independent stress. Furthermore, the unique responses in spruce did not remain constant over time, but instead were mainly limited to the first year of heatwave exposure. A potential explanation for this is that “ecological stress memory” is responsible for the adjustments between the first and second year of treatment (Walter, Beierkuhnlein, Jentsch & Kreyling 2013). The idea behind ecological stress memory is that individual plants will respond to a stress event differently if they have previously been exposed to that stress.
The spruce exhibited many distinctive metabolic responses to the combined stress, especially in the first season (e.g. AAs, PAs, TSP, chlorophyll a +b ) whereas the birch did not. These findings do not support our second or third hypotheses that N and C metabolite pools in birch will show a greater response to heat+drought stress than the spruce, and that the changes observed in N and C metabolism after one year of stress will be carried over into the following growing season. In the first season of prolonged HDstress, relatively more metabolic adjustment occurred through the accumulation of several AAs and TSP whereas in the second season ofHD stress, adjustment occurred through changes in PAs and fructose. The major adjustments to individual AAs from the HDtreatment in spruce show how carbon and nitrogen assimilation can become completely disrupted under multiple stressors. For example, Ser, Gly, and Gln are involved in the recycling of carbon from photorespiration (Blackwell, Murray & Lea 1990). Photorespiration is known to increase during both drought and high temperatures (Jordan & Ogren 1984; Wingleret al. 1999) and it is very likely that the activity of this pathway was elevated in the HD spruce, as indicated by the 5-fold increase in Ser and 2-fold increase in Gln. Unfortunately, during the analysis Gly did not separate from Arg and Thr, therefore changes in Gly content in 2016 could not be assessed. Furthermore, the elevated levels of Phe and Trp suggest a greater proportion of carbon may have been allocated to protective compounds, e.g. phenolics. Such response was observed in Eucalyptus exposed to heat+drought triggering a novel accumulation of cinnamate, a substrate central to phenylpropanoid biosynthesis derived from Phe (Correia et al. 2018). Proline is a well-known indicator of osmotic stress and both Pro and GABA have been shown to provide protection from a number of environmental stress factors (Bouché & Fromm 2004; Liang, Zhang, Natarajan & Becker 2013b). The substantial increases in Pro and GABA indicate the combination of heat and drought may have drastically increased osmotic stress in these plants that was not experienced under either independent stress. Two decades ago, it was suggested that until recent climate warming, drought may have been the only factor limiting growth of white spruce (Barber, Juday & Finney 2000). If elevated temperatures had not been a selective pressure in white spruce’s recent life history (i.e. no transgenerational epigenetic inheritance), these plants may have responded by overcompensating in AA accumulation when experiencing both heat and drought stress for the first time. But by the second season, the plants had already experienced the combined stress and therefore did not have the same over-compensatory response due to ecological stress memory. The first year may have primed their stress-response systems for a more prepared response the following year (Hilker & Schmülling 2019). This may also explain why no carry-over effect was observed at the start of the second season. Our findings highlight the importance of not inferring plant responses to multiple stressors based on the responses to either independent stress for these data show that the addition of high temperatures during a prolonged drought can have major consequences on primary metabolism in spruce that are not experienced when either stress is applied independently.
The only unique responses in the HD birch plants from either of the individual stressor was the accumulation of Trp and Phe in late August 2016. Phenylalanine and Trp are aromatic AAs that are precursors to a wide variety of secondary metabolites, the phenylpropanoids derived from Phe, and auxin, phytoalexins, glucosinolates, and alkaloids derived from Trp (Radwanski & Last 1995; Tzin & Galili 2010). All of these compounds have significant roles in protection against abiotic and biotic stress and their production is stimulated by stress (Dixon & Paiva 1995). The novel accumulation of Phe and Trp in the HDplants suggests there were likely accumulations of other phenolic compounds involved in the catabolism of Phe and Trp. It was surprising that the HD birch did not accumulate sucrose in either year in response to the combined stress. These plants also did not accumulate Pro in either year which is why sucrose may may accumulated since it often replaces Pro as the dominant osmolyte under the combination of high temperatures and drought (Rizhsky et al. 2004b). Also, to our surprise, the combined stress did not uniquely alter soluble inorganic ion concentration or other soluble sugar concentrations (aside from xylose+arabinose in late 2017).