Metabolic responses to individual heat and drought stress
High temperatures alone had a minimal impact on spruce in both years of treatment. Polyamines (and to a lesser extent potassium) were the only compounds affected by the heatwaves which occurred only at the end of the second year. The decline in Put and Spd observed with Hand/or HD at the end of second growing seasons may have been due to their oxidation and consumption into Spm. Elevated levels of Spm were also observed in the H birch at this time signifying its involvement in cellular stress response. Spermine plays a major role in protecting plants from heat stress through its involvement in elevating transcript levels of heat shock-related genes (Sagor, Berberich, Takahashi, Niitsu & Kusano 2013). Alternatively, the decline of Put and Spd may also point to a fatigued defensive system at the end of the two-year experiment (Sgobba, Paradiso, Dipierro, de Gara & de Pinto 2015). Polyamine accumulation increases tolerance to a variety of stressors (reviewed by Rhee, Kim & Lee (2007); Shi & Chan 2014), but due to the production of H2O2 via PA catabolism, their long-term accumulations may have toxic effects (Bhattacharjee 2005; Mohapatra, Minocha, Long & Minocha 2010; Minochaet al. 2014). Additionally, the H spruce showed a moderate increase in K at the end of second growing season that was not observed in the water limited D or HD plants which may suggest theH plants had higher rates of evaporative cooling which also increased water and nutrient uptake (Pregitzer & King 2005).
In general, the lack of response in birch to the heatwave only treatment suggests that the birch may have been better equipped to tolerate high temperatures than drought. Even so, we expected to detect some metabolic adjustment in response to air temperatures near 40C. The few metabolic adjustments observed were contrasting and only occurred at the end of the 2016 season where the H birch produced nearly twice as much Spd and 30% less chlorophyll a +b as the C plants. Elevated levels of Spd suggests these plants may have had greater heat tolerance (Shao, Wang & Yu-Fen 2015), while lower chlorophyll a +b indicates the plants were experiencing oxidative stress (Havaux & Tardy 1999). During the heatwave events, these plants likely had increased rates of daytime respiration (Liang, Xia, Liu & Wan 2013a) which would have led to the consumption of large quantities of carbohydrates and other compounds such as proteins (Araújo, Tohge, Ishizaki, Leaver & Fernie 2011). Alternatively, the lack of apparent response may have been due to the complexity of plant responses to high temperature, and we may not have measured the compounds that were most impacted. We did not, for example, measure heat shock proteins and dehydrins (Hanin et al. 2011; Jacob, Hirt & Bendahmane 2017; Aspinwall et al. 2019).
Unexpectedly, the drought treatment had a minimal impact on metabolism across the two seasons in both species. Drought stress is known to induce the production of various osmoprotectants including compatible solutes that help maintain osmotic balance and protect cells during dehydration. Proline is a well-studied compatible solute and has been shown to have many protective functions during plant stress (Kemble & Macpherson 1954; Demiral & Türkan 2005; Kaushal, Gupta & Bhandhari 2011; Per et al. 2017). Glycine and GABA also act as important compatible solutes that alleviate osmotic stress (Di Martino, Delfine, Pizzuto, Loreto & Fuggi 2003; Renault et al. 2010). Unlike theHD spruce, the -treated spruce showed a lack of Pro, GABA, and Gly accumulation suggesting other osmolytes, such as betaines or polyols, may be responsible for maintaining cell turgidity and restoring osmotic homeostasis during drought. This lack of Pro accumulation is a somewhat uncommon response among species under drought (Hossain & Hoque 2014; Zandalinas et al. 2017). Although neither the D or HD birch showed evidence of Pro, Gly, or GABA accumulation, Spd, a compound with anti-senescent properties (Nambeesanet al. 2010), was reduced in the water-limited birch (Dand HD ) at the end of the first season. Spermine was not elevated at this time which indicates Spd may have been oxidized to form 1,3-diaminopropane as opposed to Spm, a response indicative of increased oxidative stress (Cvikrová et al. 2013).
During drought conditions soluble sugars such as hexoses (e.g. glucose, fructose), can also accumulate in conjunction with Pro and polyamines to act as osmoprotectants (Sengupta, Chakraborty, Saha, Gupta & Gupta 2016; Templer et al. 2017). Although not significantly different from C, the D birch did have elevated levels of foliar fructose and glucose+galactose at the end of the first season compared to the H birch, and the D spruce had elevated levels of fructose at the end of the second season compared to the H spruce suggesting that these sugars may be facilitating osmotic homeostasis under water stress. Still, the mechanisms utilized to cope with prolonged drought stress in these species is still not clear. We can speculate that the dominant osmolytes may be various soluble sugars under water stress (including those not quantified here, e.g.sorbitol, (Lo Bianco, Rieger & Sung 2000), but due to the prolonged nature of the drought treatment (> 3 months) which continuously suppressed photosynthesis (Gagne, Smith, McCulloh, in press), carbohydrate reserves may have been depleted by the end of August. It is also likely that photosynthates were translocated from the leaves to the stem and roots for post-drought recovery (O’Brien et al. 2015; Tomasella, Petrussa, Petruzzellis, Nardini & Casolo 2020).
Overall, we found that white spruce exhibited many unique molecular responses to the combined stress, while paper birch displayed few and tended to share responses with the D plants. The differences in metabolic response of young white spruce and paper birch trees to the combination of heatwave and drought stress under eCO2versus the response to either independent stress shows how generalizing plant responses to multiple stressors is problematic. Our data highlights the species-specific nature of metabolic adjustment to multiple stressors which should be considered when making predictions about forest response to future climate scenarios. The data also suggest that paper birch may lack the ability to metabolically adjust to extreme heat events in the future, which may limit their future distribution within boreal forests.