Global changes, through their widespread influence on all biological processes, can significantly impact plant life cycles and functions (Norby and Luo, 2004). Elevated levels of CO₂ and ozone, increased temperatures, as well as higher drought frequency, all alter the content and composition of plant metabolites by disrupting the physiological stoichiometry in plants (Kuokkanen et al., 2001; Xu and Zhou, 2006; Prior et al., 2011; Xu et al., 2014, 2015), thus influencing plant survival and distribution. Furthermore, by influencing the chemical composition of senesced litter that fuels the metabolism of soil heterotrophs, the climate-induced physiological alterations in plants could also influence soil nutrient cycling and ecosystem productivity. For example, climate-induced changes in the composition of senescing leaves (Top and Filley, 2014; Suseela et al., 2015) could affect the subsequent detritivory (Currano et al., 2008; Couture et al., 2012) that regulates the cycling of soil carbon and mineral nutrients (Aerts, 1997; Liu et al., 2009; Suseela et al., 2013), which in turn could influence ecosystem productivity. These protracted influences of environmental stress on ecosystem performance could be mediated in part by climatic regulation of defense compounds that retain their biological-inhibitory properties even after tissue senescence.

Polyphenols, specifically tannins, are ecologically relevant, multifaceted carbon-based secondary metabolites that are common in most plant species (Kraus et al., 2003a). These compounds perform multiple protective functions and facilitate the interactions of plants with their biotic and abiotic environments during the active growth of tissues and after tissue senescence. In green tissues, as protective compounds, tannins play a critical role in plant—herbivore and plant-to-plant interactions (Salminen and Lempa, 2002; Kraus et al., 2003a; Barbehenn and Constabel, 2011), and also serve as a photo-protectant (Close and Mcarthur, 2002). Tannins in senescent leaves can account for up to 25% leaf dry weight (Kraus et al., 2003b) and thus can be a major form of carbon reaching the belowground ecosystems. Tannins in senescent tissues also retain their ability to complex with proteins (Hagerman et al., 1998) and inactivate soil enzymes (Triebwasser et al., 2012), which could hinder soil N mineralization (Hättenschwiler and Vitousek, 2000; Kraus et al., 2003a; Adamczyk et al., 2013; Tharayil et al., 2013). Thus, environmental conditions that exist during plant growth, by regulating the production of tannins in green tissues, can influence carbon and nitrogen cycling in soils.

Tannins can be broadly classified into condensed tannins (CTs) and hydrolysable tannins (HTs; Kraus et al., 2003a), with gymnosperms and monocots producing primarily CTs, and dicots able to produce either HTs or CTs alone or as mixtures (Bate-Smith, 1977; Haslam, 1988; Kraus et al., 2003a; Triebwasser et al., 2012). Condensed tannins, also known as proanthocyanidins, are polymers of flavan-3-ols such as catechin and gallocatechin (Xie and Dixon, 2005). Hydrolysable tannins are composed of gallic acid units linked via an ester linkage to a glucose functional group, and they can be further classified as gallotannins and ellagitannis by the respective presence or absence of C–C linked galloyl groups (Hartzfeld et al., 2002). In plants, tannin production is genetically (Scioneaux et al., 2011), as well as environmentally, controlled (Tharayil et al., 2011). Environmental conditions that affect the production of tannins include photoperiod, soil pH, moisture and nutrient availability, herbivory, and atmospheric CO₂ and O₃ (Herms and Mattson, 1992; Bussotti et al., 1998; Kraus et al., 2003a; Cohen and Kennedy, 2010; Jaakola and Hohtola, 2010; Lindroth, 2010; Malisch et al., 2016). Along with their total quantity, the biological reactivity of tannins is closely regulated by the chemistry of the polymer, viz. identity of monomers, substitution pattern of the phenolic ring, and the degree of polymerization (Kraus et al., 2003b). Although tannin quantities have been shown to respond to environmental changes, little is known about the influence of climate on the chemical composition of tannins. Further, less is known about the changes in the composition of tannins between green and senesced tissues, which are primarily influenced by the overall resorption metabolism during tissue senescence.

We investigated the influence of warming and altered precipitation on tannin production in Quercus rubra, an important species in many forested ecosystems of eastern North America (Prasad et al., 2007-ongoing). Specifically, we sought to determine the interactive effects of temperature and precipitation on (1) the content and molecular composition of tannins, (2) the association of tannins within different tissue fractions (operationally defined as extractable and non-extractable tannins) and (3) whether climatic effects on content and chemical composition of tannins differ between green and senescent leaves. We hypothesized that the climate stresses we imposed (drought, increased temperature) would induce the production of tannins that are quantitatively and compositionally different, and that the climate-induced changes in composition of tannins in green leaves would also influence the content of tannins that are retained in the senescent leaves.

Climatic stresses that disrupt cellular functioning in plants initiate readjustments in metabolic pathways, which in turn facilitate the acclimation of plants to their new environment. The resulting reprogramming of important metabolic pathways, including the phenylpropanoid pathway, citric acid cycle, glycolysis, and urea cycle, results in the upregulation of several metabolite classes such as amino acids, phenolic acids, sugars, organic acids, sugar alcohols, polyamines, and polyols (Bohnert et al., 1995; Peñuelas et al., 2013; Suseela et al., 2015). Drought and warmer temperatures have been shown to influence many different morphological (yield, growth) and physiological (rate of photosynthesis, stomatal conductance, leaf pigmentation, water potential, protein concentrations, etc.) responses in plants (Benjamin and Nielsen, 2006; Rennenberg et al., 2006; Praba et al., 2009; Anjum et al., 2011). However, the influence of environmental stressors on the content and composition of polymeric defense compounds across green and senesced plant tissues is less well-known.

In this study, climate-induced changes in leaf tannin composition influenced not only the quantity of tannins, but also the proportion of HTs to CTs, monomer composition and mean degree of polymerization of CTs, composition of HTs, the association of tannins within the cells (extractable and non-extractable), and the composition of the tannins remaining in the senescent leaves. In general, plants growing in favorable conditions (Ambient^*No Warm, Wet) produced less tannins per unit leaf mass than those growing in more stressful conditions (Dry, Ambient^*Warm). Increase in tissue content of tannins with increasing environmental stress has been frequently reported (Bussotti et al., 1998; Cohen and Kennedy, 2010). Reallocation of nutrients in plants based on the favorability of growth and nutrient conditions is expected under two major hypotheses: the carbon/nutrient balance and the growth/differentiation balance (Herms and Mattson, 1992). The increase in tannin production under Dry^*Warm conditions could be a defense strategy in plants to protect the resources that they have already acquired (Herms and Mattson, 1992; Wright et al., 2010; Massad et al., 2014). At the same time, in the Wet treatments, because growth conditions were presumably closer to optimal, the decrease in tannin production could be a result of the plant preferentially allocating C for growth instead of defense (Bryant et al., 1983; Tuomi, 1992). Also, a greater concentration of tannins binding with cellulose and lignin matrices (Zucker, 1983; Bussotti et al., 1998) could potentially create a physical impedance to cell expansion. A similar allocation pattern of photosynthates for growth, rather than for defense compounds, has been previously reported in other tree species (Donaldson et al., 2006), where the plant growth was negatively correlated with phenolics and CTs. In a previous study conducted at BACE, the tannin content in senesced leaves of Acer rubrum was greater in Dry than in Ambient treatments, while the senesced leaves from Wet and the Ambient treatments had similar tannin contents (Tharayil et al., 2011). This contrasting pattern could be attributed to a difference in the physiological responses of the two species to stress, and also to differences in precipitation patterns between the two growing seasons. In the 2009 growing season (April to October; Tharayil et al., 2011), 720 mm of rain fell, as opposed to 606 mm during the 2013 growing season. Further, only half as much rain fell in the last 4 months of the 2013 growing season (July to October) as fell during that period in 2009 (210 mm and 428 mm, respectively). The larger amount of precipitation received by A. rubrum during the 2009 growing season would have led to a similar physiological water status between Ambient and Wet treatments, which could have resulted in similar tannin content between these two precipitation treatments (Tharayil et al., 2011). In the present study, the scarcer and less evenly distributed precipitation of 2013 likely exposed Q. rubra in Ambient to a greater physiological water stress, which could explain the observed similarity in tannin content between Ambient and Dry treatments.

Along with the total quantity of tannins, climate influenced both the monomer composition and mean degree of polymerization of tannins, the two parameters that are seldom investigated despite their regulatory influence on the potential biological reactivity of tannins (Zucker, 1983; Kraus et al., 2003b; Tharayil et al., 2011; Triebwasser et al., 2012). Reactivity of CT is a function of the hydroxylation pattern of the B-ring, and tannins with trihydroxy B-rings (PD) are more reactive than those with dihydroxy B-ring (PC). Both Wet and Dry treatments, despite their contrasting influences on total content of CTs, reduced the relative proportion of PD units. Compared to PCs, the PD units are formed only during later parts of cell development (Stafford et al., 1989). Thus, the lower accumulation of the PDs in wet climates could indicate a metabolic modification in CT biosynthesis. Under the metabolic stress in Dry conditions the triphenolic vicinal hydroxyls predispose PDs to oxidation and polymerization reactions (Close and Mcarthur, 2002; Aron and Kennedy, 2008) resulting in a lower proportion of PDs. This could partly explain the lower proportion of PDs in Dry treatments despite a higher production of CTs. Compared to Ambient and Dry treatments, the proportion of PD units and the mean degree of polymerization were higher under Wet treatments. The biological reactivity of tannins is a function of their degree of polymerization, where tannins with greater chain length have a higher capacity to complex with and precipitate proteins. Thus, though the leaves that were formed under ambient growing conditions and Wet treatments had a lower tannin quantity, these tannins would have a greater capacity to defend herbivory due to their higher biological reactivity contributed by a greater polymerization and higher proportion of PD units. Environmental conditions have been shown to influence the degree of polymerization of tannins (Cohen and Kennedy, 2010). A lower polymerization of tannins exposed to non-optimal growing conditions has been reported before (Tharayil et al., 2011) in senesced leaves of Acer rubrum from the same site. In Vitis vinifera, environmental stressors, especially moisture deficit, have been reported to increase the mean degree of polymerization of tannins (Kennedy et al., 2002; Cohen and Kennedy, 2010). In Onobrychis viciifolia the influence of drought on the mean degree of polymerization of CTs was dependent on otogeny of the plant (Malisch et al., 2016). These results indicate that the influence of environment on molecular composition of tannins could be regulated by the physiological stress perceived by the plant (as opposed to the magnitude of the treatment applied), the identity of the species, as well as their growth stage.

Distributions of tannins within leaves are highly species-specific (Kraus et al., 2003a), and within a species the cellular localization of tannins, herein operationally defined as solvent extractable and non-extractable tannins, can change based on environmental conditions (Gagné et al., 2006). In green leaves, CTs are usually stored in the cell vacuoles (Stafford, 1988; Bussotti et al., 1998; Marles et al., 2003), and thus are sequestered away from potential interactions with the structural matrix and plant metabolic compounds. During leaf maturation the tannins penetrate the cell walls (Bussotti et al., 1998), which may result in an increase in the fiber-bound proportion of tannins. These complexes can protect the cell wall or organelle against microbial attack, and can also delay the decomposition after senescence of the tissue (Zucker, 1983). Irrespective of the warming treatment, the leaves from the Wet treatment had a greater proportion of total CT that was non-extractable during the sequential solvent extraction. While we cannot be certain of the exact complexation of the tannins, some of them may be in association with cellulose and pectin (Le Bourvellec et al., 2009; Padayachee et al., 2012a,b; Jakobek, 2015) in the cell walls and middle lamellae. A high affinity of PCs binding to pectin has been observed (Le Bourvellec et al., 2009; Watrelot et al., 2013; Jakobek, 2015), meaning that tannins in the Wet treatment with the highest percentage of PCs and greater chain-length could have a greater complexation capacity with cell wall components. Alternatively, the greater chain-length and higher proportion of PC units would have resulted in lower extractability of tannins from the leaf tissues, resulting in the observed lower content of tannins in Wet treatments. Also, the Wet treatments, despite a lower content of CTs, have a higher percentage of HTs, which have a higher enzyme inhibition capacity compared to CTs (Triebwasser et al., 2012). Thus, overall, the leaves formed under Wet treatments would have a similar herbivore deterrence contributed by tannins, despite a lower content of CTs in the tissue.

Climate influenced the proportion of HTs and CTs. Across most treatments, the dominant type of tannin in Q. rubra was CT, with lower content of HTs. A similar, lower proportion of HT in red oak species has been reported before (Barbehenn et al., 2005). Compared to the Ambient^*NoWarm control, all climate treatments had higher percentages of HTs, which may reflect the ease of maintenance, mobility, and greater responsiveness of HTs relative to CTs (Zucker, 1983; Shure et al., 1998). Because HTs are less associated with the structural matrix of the plants than CTs, they offer less resistance to normal cell growth and expansion, making them a preferable type of defense compound over CTs in environments that favor active plant growth (Zucker, 1983), as evidenced by the higher percentages of HTs in the Wet treatments. HTs are also thought to be metabolically cheaper; the metabolic cost for the formation of proanthocyanidin (monomers of CT) is 0.395 ATP equivalent g⁻¹, where as that of HTs is 0.27 equivalent g⁻¹ (Lewis and Yamamoto, 1989).

Both gallotannins and ellagitannins also exhibit antioxidant properties (Barbehenn et al., 2006), and thus may play a role in the inhibition of higher radical formation in leaves, especially under climatic stress. Ellagitannins can also be highly variable in structure (Zucker, 1983). This could have specific relevance inhibiting digestive enzymes in herbivore digestive tracts (Zucker, 1983), which would be an effective defense mechanism against herbivory for the plant. In most climates in this study, the green leaves exhibited higher content of ellagitannins, however, the Ambient^*No Warm control had the highest percentage of ellagitannins compared to all other treatments, suggesting that the alteration of climate in any way can significantly alter the proportion of certain types of tannins present, either by production rates or utilization of certain hydrolysable tannin structural components, such as the glucose molecule (Zucker, 1983).

During tissue senescence, following the disruption of membrane-bound vesicles, CTs can form insoluble complexes with proteins and cell wall carbohydrates (Kraus et al., 2003a; Marles et al., 2003), which could contribute to an increase in the percent of non-extractable CTs in the senesced tissue. Differences in the content of tannins in green leaves compared to senesced leaves observed in this study may be the result of different overall resorption strategies of the plants. Considering their protein complexation capacity, polymeric nature, and the C-C linkages, the CT would less amenable to enzyme mediated catalysis and subsequent resorption. Due to their lower complexity and the presence of more labile ester bonds, the gallotannins would be more amenable to resorption during tissue senescence than CTs. Compared to the green tissues, the greater mobilization of HTs during senescence is reflected in the lower proportion of HTs in the senesced tissues across the treatments. The CT content of the Wet treatments were similar in both green and senesced tissues, which could be primarily attributed to the greater degree of polymerization and a greater PC content of tannins produced in this treatment, which would have resulted in a greater association of these tannins with the cell wall.

Tannins can complex with carbohydrates and proteins and this complexation can persist after the leaf senesces, potentially delaying decomposition. With the increase in CTs and non-extractable CTs for some of the treatments, this might mean that those leaves would break down more slowly, lose less material to leaching, and possibly provide more sustained nutrient input into the soil. The higher tannin content of foliage observed under less favorable growing conditions could be partly countered by the changes in allocation patterns at the tree level, since trees exposed to climatic stress often produce less leaf biomass. However, the reduced carbon input through litterfall in such ecosystems, coupled with higher content of phenolic compounds in these tissues, might impose a greater constraint on nutrient cycling in these soils. Extractable phenolics and tannins are lost rapidly from the leaves, suggesting that leaching is a primary route for loss of tannins from leaves (Benner et al., 1988; Schofield et al., 1998; Hernes et al., 2001), and lower molecular weight tannins are lost more rapidly than higher molecular weight tannins (Schofield et al., 1998). Thus, the Wet treatments with their higher length CTs could better retain some of the tannin-related defense capabilities which could interfere with decomposition processes. However, the overall herbivory and decomposition processes are influenced not only by the content of anti-nutrients in the tissues, but also by their overall nutrient content (Suseela et al., 2013; Almuzini et al., 2017), which in turn is also regulated by environmental stressors.

Overall, our study elucidates multiple factors that regulate the production and seasonal change of tannins in Q. rubra. In agreement with the nutrient-balance hypothesis, the tannin concentrations in tissues formed under favorable climates were lower, but the tannins produced were more complex and potentially more protective due to higher chain lengths of CTs and greater proportions of HTs. When exposed to climatic stress, Q. rubra responded by producing a greater quantity of tannins that were of shorter chain length. The differential influence of the environment on the production dynamics of plant metabolites is important in the context of global changes and carbon and nitrogen cycle feedbacks. Although over longer time scales climate modifies ecosystem processes through shifts in species composition, over shorter time scales the physiological adaptations of plants to changing climate could influence soil C and nutrient cycling through changes in chemical composition of biomass. These changes in tannin content and composition may alter forest dynamics, not only by influencing decomposition and nutrient cycling dynamics, but also by regulating herbivore dynamics.

ST and NT contributed to the conceptualization of the project and design of the experiment, JD provided the experimental framework, ST collected and analyzed the data, CP and NT guided the analyses, ST drafted the article, all authors contributed to the critical revision of the article.