Condensed tannins: arbiters of abiotic stress tolerance?

Gourlay and Constabel (2019) remind us that in temperate forests, condensed tannins (CTs) affect not only trophic interactions such as herbivory and leaf decomposition, but also processes intrinsic to leaf growth and development. Condensed tannins, also known as proanthocyanidins (PAs), are non-structural, polymeric end products of the flavonoid pathway. The flavonoid biosynthetic pathway is well studied, but there are still major gaps in our understanding of how CTs are formed and compartmentalized (Zhao et al. 2010, Brillouet et al. 2013, Wang et al. 2018). In short, the central phenylpropanoid pathway gives rise to various hydroxycinnamates and flavonoids, including flavan-3-ols, which polymerize to form the CT oligomers (Figure 1). It has long been recognized that with their capacity to absorb UV radiation and scavenge damaging radicals, flavonoids contributed to the survival and early evolution of land plants (Stafford 1991, Agati and Tattini 2010, Albert et al. 2018). In recent decades, interest has expanded into the importance of CTs as defensive antifeedants and antimicrobials with additional ecophysiological roles pertinent to nutrient cycling (Madritch and Lindroth 2015). The report of Gourlay and Constabel (2019) now steers the conversation back toward the intersection of CTs with leaf health and functional resilience (Figure 1). Suggested impacts of condensed tannin (CT) manipulation. Sunlight as well as fluctuations in Calvin cycle capacity due to nutrient and water availability can lead to reactive oxygen species (ROS) excesses in the chloroplast (green) and photosystem II (PSII) damage, or photoinhibition. Reactive oxygen species such as H2O2 can also exit in the chloroplast or be generated in the cytosol. Condensed tannins and monomeric flavonols inhibit ROS accumulation both preventively by absorbing UV radiation, chelating reactive metals that participate in radical generation, and reactively by scavenging peroxyl radicals. By extension, CT abundance and compartmentalization should affect ROS-retrograde signaling pathways in the cytosol. Retrograde signaling can trigger programmed cell death (PCD) and leaf senescence, itself a radical generating process that affects leaf area duration. Finally, CT accrual interfaces with that of phenolic glycosides (PGs) via uncharacterized mechanisms to condition trophic interactions important for tree fitness, perennial growth and habitat productivity. The authors present a novel finding that leaf CTs mediate oxidative stress tolerance in vivo with physiologically relevant outcomes. This is noteworthy in part because flavonoids comprise only one facet of a plant arsenal based also on ascorbic acid and glutathione as well as various pigments and retrograde signaling for redox control and the neutralization of harmful radicals (Kleine and Leister 2016, Smirnoff 2018). The work of Gourlay and Constabel significantly extends in vitro work using non-plant systems where the ability of flavan-3-ols and other flavonoid monomers to oxidize free peroxyl radicals, prevent their chain propagation and inhibit damaging effects to cellular components has already been demonstrated (Jovanovic et al. 1996, Arora et al. 1998). The physiological relevance of such studies has been challenged in the past due in part to kinetic constraints which predict that the flavonoid concentrations required for effective scavenging may be greater than would be achievable in vivo (Forman et al. 2014). This point was suggested earlier by Stafford (1991) in tempering the idea that UV-absorbing flavonoids also functioned as radical scavengers in primitive plants. In addition, direct interactions between dietary flavonoids, glutathione and catalase have been reported (Sudheesh et al. 1999, Myhrstad et al. 2002, Krych and Gebicka 2013, Majumder et al. 2017). Given the plethora of interacting mechanisms for redox control, the new findings support an interesting notion that increased CT abundance can result in a physiologically beneficial gain in radical scavenging in leaves. It was perhaps fortuitous that overexpressing CT-regulating MYB134 or MYB115 (hereafter MYB 134/115) had only a minor effect on the levels of other key flavonoids, especially flavonols like quercetin (Mellway et al. 2009, James et al. 2017). Quercetin glycosides have broad subcellular distribution and strong radical scavenging abilities (Husain et al. 1987, Jovanovic et al. 1996, Agati and Tattini 2010). In this respect, Gourlay and Constabel appear to have threaded a needle. While it seems intuitive that reducing flavonol levels might lead to damage, there is also evidence that a constitutive increase in their cytosolic abundance can be damaging. Flavonols can act as pro-oxidants (Krych and Gebicka 2013), and in the presence of H2O2, levels well below those thought to be required for effective intracellular scavenging have been shown to damage DNA (Sakihama et al. 2002, Labieniec et al. 2003, Azam et al. 2004). Current animal system models suggest that cytosolic flavonoids provided in the diet operate at very low concentrations, less by radical scavenging and more as receptor ligands for the prevention of oxidative imbalances via the nuclear factor erythroid 2-related (NRF2) signaling pathway (Forman et al. 2014, Loboda et al. 2016). Plants lack orthologs for the NRF2 signaling pathway, perhaps not surprisingly since flavonols are light-regulated UV filters and probably too abundant to serve as signaling molecules. Flavonol levels in the cytosol are partly controlled by mitochondrial and chloroplast sensing, as well as by various transporters and vesicular trafficking (Giraud et al. 2008, Morishita et al. 2009, De Clercq et al. 2013, Zhao 2015). Subcellular compartmentalization, though as yet poorly understood, therefore deserves further consideration for the enhanced constitutive scavenging observed in the high-CT transgenic poplars. One distinguishing feature of plant compared with animal cells is of course the large central vacuole where flavonoids can concentrate. Not only does the vacuole act as a flavonoid reservoir, but polymerization of CTs is thought to increase in the vacuole (Figure 1), albeit by mechanisms that remain unknown (Jiang et al. 2015, Wang et al. 2018). Additionally, at least on the basis of in vitro studies, the enzymatic polymerization of catechin monomers increases their antioxidative capacity on a per carbon basis (Kurisawa et al. 2003). Given these contexts, demonstration of the ameliorative effects on photoinhibition by Gourlay and Constabel provides new impetus for exploring the partnership between compartmentalization and flavonoid pathway fluxes in abiotic stress tolerance. One of several questions raised by the current findings concerns the mechanism of photosystem II (PSII) protection. Preemptive UV filtering through the vacuole and enhanced radical scavenging in the cytosol both remain as mechanisms (Figure 1). However, the effect of MYB134/115-regulated CT increases on their subcellular distribution and oligomerization prior to vacuolar import is unclear. The so-called ‘tannosome’ transport model is based on evidence that suggests a chloroplastic origin for CTs followed by their delivery to the vacuole in tannosome vesicles derived from chloroplasts (Brillouet et al. 2013). Other models support the biosynthesis of CTs and their oligomerization at the endoplasmic reticulum (Liu et al. 2016). Ultimately, greater certitude about the topology and capacity of CT trafficking will lend clarity as to how increased CT accrual leads to decreased PSII damage. In addition to the matter of compartmentalization, future studies will need to determine whether CT localization within the leaf is a factor in its remedial effects. Most UV filtering occurs in the leaf epidermis (Stapleton and Walbot 1994), but in quaking aspen (Populus tremuloides Michx.) at least, CTs are normally less abundant in the upper epidermis than in other cell layers (Kao et al. 2002), including the palisade mesophyll where most photosynthetic reactive oxygen species (ROS) occur. Altered localization of CT accrual including the possibility of ectopic accrual in the upper epidermis needs to be considered as a possible contributor to effects such as those reported by Gourlay and Constabel. Room for varied cell-specific and subcellular localization of CTs suggests potential for dynamic interactions and tradeoffs involving CTs, abiotic stress tolerance and defense (Figure 1). One defining feature among the Salicaceae (poplars and willows) is wide genetic diversity when it comes to foliar CT and salicinoid phenolic glycoside (PG) levels (Orians et al. 2000, Harding et al. 2005). Such variation probably serves an important purpose since CTs and PGs function differently in chemical defense of aspens (Hemming and Lindroth 1995). In fact, a clear allocation tradeoff between CTs and PGs with trophic implications has been reported for the MYB134 poplar lines (Mellway et al. 2009, Kosonen et al. 2012). Recent work has shown a positive correlation between CT concentration and poplar resistance to rust (Melampsora larici-populina) (Ullah et al. 2018). Little is known about the genetic mechanisms that underlie the partitioning of metabolic carbon between CTs and PGs in these species. However, low-nitrogen-induced CT enhancements costly to PG accrual, and to the availability of primary metabolites for growth (Harding et al. 2005, 2014), can now be viewed in the added context of shifts in the demand for antioxidants in response to adverse conditions. Finally, leaf senescence is an inherently oxidative process during which nutrients derived from the turnover of cellular contents are resorbed by the stem and roots (Zimmermann and Zentgraf 2005) (Figure 1). Whether the filtering and antioxidant properties of CTs conditionally modulate programmed turnover of cellular contents during senescence has not been evaluated. Condensed tannin polymers are more persistent than flavonoid monomers or small CT oligomers as leaves senesce (Kandil et al. 2004). However, measurement of CT levels in senescing leaves has not been without challenge since their extractability may decrease due to extensive polymerization and binding prior to leaf abscission (Lindroth et al. 2002). One possible outcome of elevated CT level is the possible delaying effect that it could have on the initiation of seasonal or premature leaf senescence. As recently reviewed by Petrov et al. (2015), leaf senescence is triggered in part by retrograde signaling pathways in response to specific ROS signals from the chloroplast and mitochondria (Figure 1). Delayed onset of seasonal senescence would therefore conceivably lead to reduced resorption of leaf resources by the plant before killing conditions terminate the process. How scenarios like these might play out in nature will depend on the regulation of CT biosynthesis as well as on the processes that control CT trafficking and the locations of CT accrual in the cell. Much also remains to be learned about CT accrual in the contexts of changing temperatures, precipitation and atmospheric CO2 levels, work that has begun (Kosonen et al. 2012) and presumably will continue with the current findings of Gourlay and Constabel in mind. None declared.