Introduction
The increasing severity of extreme heat events (i.e . heatwaves) has threatened the survival of many species worldwide. These frequent heatwaves have contributed to increased mortality across various forest types (Allen et al. 2010; McDowell & Allen 2015; McDowellet al. 2016; Adams et al. 2017; Ogaya, Liu, Barbeta & Peñuelas 2020) and to major reductions in crop yield (Siebers et al. 2015; Zampieri, Ceglar, Dentener & Toreti 2017; Ingvordsenet al. 2018). It has long been recognized that high temperatures and drought that occur during heatwaves, are the two major environmental factors that will challenge future plant productivity (Mittler 2006; Brodribb, Powers, Cochard & Choat 2020). Yet our understanding of how plants respond metabolically to the combination of high temperatures and drought in a climate with increasing CO2 concentrations is far from complete.
Genomic, metabolomic, and proteomic studies on molecular responses to the combination of high temperatures and drought stress have largely been focused on model plant species (e.g. Vile et al. 2012, Killi et al. 2017, Zintaet al. 2018) and herbaceous crops (Jagadish et al. 2011; Obata et al. 2015; Perdomo, Conesa, Medrano & Ribas-carbó 2015; Templer et al. 2017; Li et al. 2019; Zhou et al.2019; Alhaithloul, Soliman, Ameta, El-Esawi & Elkelish 2020). These studies have shown that responses to multiple stressors tend to be unique and cannot be inferred from the response to either stress experienced independently (Rizhsky et al. 2002, 2004a, Prasad et al. 2011, Vile et al. 2012, Suzuki et al. 2014, Zhao et al. 2016, Lawas et al.2018, Sewelam et al. 2020). For example, when Arabidopsis plants were exposed to heat+drought, the molecular responses were dominated by drought-specific transcriptomic changes (Rizhsky et al. 2004a), but in wheat and sorghum, molecular responses were dominated by heat-specific transcriptomic changes (Aprile et al. 2013; Johnsonet al. 2014). To date, little progress has been made characterizing molecular responses to the combination of heat and drought in woody species. In recent studies on eucalyptus and citrus trees, increases in antioxidants, amino acids, citrate, and cinnamate were found in response to heat+drought that were not found in response to either independent stress (Zandalinas, Balfagón, Arbona & Gómez-Cadenas 2017; Correia et al. 2018). On the other hand, Berini et al. (2018) showed that the levels of other metabolites (catechin and two unspecified resin acids) in paper birch and balsam fir were similar among plants treated with heat, drought, and heat+drought. These few examples suggest the degree to which woody plants respond to the combination of heat and drought is metabolically complex and species-specific.
Surprisingly, many recent studies examining plant responses to future climate-change type scenarios such as high temperatures and drought, tend to overlook changes in atmospheric CO2concentration and fail to incorporate it into the experimental design. Rising atmospheric CO2 and other greenhouse gases are driving global climate change (IPCC 2014), but few investigations have included elevated CO2 (eCO2) as an additional environmental factor when examining responses to multiple abiotic stressors (Li, Tiiva, Rinnan, Riitta, Julkunen-Tiitto, Anders & Rinnan 2020). Because there is limited data on the molecular responses of woody plants to the combination of high temperatures, drought and eCO2, and because the few existing studies suggest plant responses to multiple stressors tend to be unique compared to responses to either independent stress, our first objective is to quantify a subset of metabolic compounds in two woody species exposed to repeated heatwaves, drought, and heat+drought stress under eCO2to determine if their responses to the combined stress are unique or shared with either individual stress.
One of the major causes of cellular-level stress during unfavorable abiotic conditions is the overproduction of reactive oxygen species (ROS). ROS can be beneficial as signaling molecules under normal growing conditions, but under severe stress their quantities become toxic (Ślesak, Libik, Karpinska, Karpinski & Miszalski 2007; Foyer & Noctor 2009; Singh et al. 2016). During plant stress, some compounds that scavenge ROS activity can accumulate at high intracellular concentrations without hindering critical cellular metabolism (e.g . polyamines). Polyamines are biochemical indicators of environmental stress and their accumulation has been shown to improve tolerance to several types of stress. However, their functions under stress are still not fully understood. Polyamines can stabilize macromolecules, scavenge radicals, promote the production of antioxidant systems, and serve as compatible solutes and signaling molecules (review by Minocha et al. 2014). At the same time polyamines catabolism does produce ROS (Gupta, Sengupta, Chakraborty & Gupta 2016). To date, little is known about how polyamine metabolism is affected by multiple co-occurring stressors, especially the combination of heat and drought stress (Cvikrová, Gemperlová, Martincová & Vanková 2013).
Oxidation of large complex cellular components such as DNA, RNA, proteins, lipids, and smaller molecules such as free amino acids (AAs) with ROS alter cellular activity (Sharma, Jha, Dubey & Pessarakli 2012; Ahmad et al. 2017). When proteins or free AAs interact with ROS, the AA side chains are modified, causing both structural and functional changes to the compound (Stadtman & Levine 2003). Some of these oxidation reactions are reversible, such as those reactions with methionine and cysteine and may have an antioxidant function (Stadtman & Levine 2003; Kim 2020). Free AAs are also important for cell signaling and regulating plant responses to multiple stressors. For instance, during water stress some AAs serve as osmolytes to maintain turgor pressure prolonging cellular metabolism (Rai 2002; Sharma & Dietz 2006; Sharma et al. 2019). Given the central role AAs have in cellular protection and in nitrogen (N) and carbon (C) metabolism, and due to the lack of clarity on how AAs and their downstream products (e.g . polyamines and soluble proteins) are affected by multiple stressors, here we examine the impact multi-stress exposure has on AAs and their downstream products.
Carbon metabolism can be disrupted by high temperature or drought exposure, but the consequences can be most severe when both stressors co-occur (Rizhsky et al. 2002; Birami et al. 2018). These stressors can also have contrasting effects on C allocation. In beech saplings, Blessing, Werner, Siegwolf & Buchmann (2015) found that heat stress increased C allocation to roots Ruehr et al. (2009) found it decreased under drought. Because heat and drought stress can disrupt C uptake, mobilization, and utilization, the depletion of essential nonstructural carbohydrates (NSC; including starch and soluble sugars) is likely when both stressors co-occur (Birami et al. 2018). However, recent studies on the impacts of drought and high temperature stress on NSC dynamics have yielded mixed results. Some studies examining drought stress observed a depletion in NSC (e.g.Mitchell et al. 2013; Sevanto, Mcdowell, Dickman, Pangle & Pockman 2014; Maguire & Kobe 2015) while others have found an increase or no change (e.g. Anderegg et al. 2012; Gruber, Pirkebner, Florian & Oberhuber 2012). There is evidence that the accumulation of NSC, particularly soluble sugars, during drought may be linked to drought tolerance in some species (Piper 2011; O’Brien, Burslem, Caduff, Tay & Hector 2015). High temperature stress has also generated mixed responses in NSC content where declines or no changes have been observed (Wilson 1975; Rowland-Bamford, Baker, Allen Jr. & Bowes 1996; Zha, Ryyppö, Wang & Kellomäki 2001; Adams et al.2013). Although total NSC often decline in response to high temperatures, the accumulation of some soluble sugars may be an important trait related to heat tolerance (Niinemets 2010), especially the accumulation of sucrose and glucose (Liu & Huang 2000). Because the accumulation of soluble sugars may improve drought and heat tolerance, and because eCO2 has also been shown to have a positive effect on soluble sugar accumulation (Vu, Newman, Allen, Gallo-Meagher & Zhang 2002), here we examine changes in soluble sugar content in plants exposed to these three environmental factors.
The main goal of the study is to examine several primary metabolites involved in N and C metabolism in paper birch (Betula papyrifera ) and white spruce (Picea glauca ) subjected to repeated summer heatwaves, drought, and eCO2. Paper birch and white spruce are functionally dissimilar species that occupy the same geographic region in the boreal and hemi-boreal zones. Paper birch is a broadleaf angiosperm with a rapid growth rate compared to white spruce, a slow growing conifer. Conifers invest heavily in C-rich protective resins that are used against herbivore and pathogen attack which for many angiosperms including birch, are only produced in low concentrations (Trapp & Croteau 2001). These two species are also on different ends of the leaf economic spectrum. White spruce is on the conservative end with a long leaf life span, low specific leaf area, and an overall more expensive energy investment for leaf construction as compared to paper birch that has a short leaf lifespan, high specific leaf area, and cheaper leaf construction costs. The structural and functional differences between white spruce and paper birch are critical for predicting how C and N metabolism may be impacted by multiple abiotic stressors. For instance, it has been suggested that resource conservative species, such as spruce, are more resistant to C loss during stress than less conservative species such as birch (Saura-Mas & Lloret 2007). Because conifers and angiosperms allocate C resources and energy differently to growth and secondary metabolism (e.g. leaf construction and defense compounds), the way in which C and N metabolism is altered by abiotic stress is expected to differ between them as well. These species have already demonstrated that recurrent heatwave stress affects their gas exchange, growth, and the ability to photosynthetically acclimate differently (Gagne, Smith, McCulloh, in press). Therefore, it may be predicted that shifts in metabolite pools will also occur in a species-specific manner.
In the current study conducted under eCO2 conditions, we sought to answer the following questions: Are changes in N and C metabolism in response to heat+drought stress unique or shared with either individual stress? Will shifts in N and C metabolite pools be greater in birch compared to spruce under combined stresses? Will the changes observed after one year of heat+drought stress be carried over into the following season? We hypothesize that: 1) plant responses to combined heat+drought stress will be unique from either independent stress, 2) that N and C metabolite pools in birch will show a greater response to heat+drought stress than spruce, and 3) changes observed in N and C metabolism after one year of stress will be carried over into the following growing season.