Coevolution and photoprotection as complementary hypotheses for autumn leaf reddening: a nutrient‐centered perspective

Abstract

There has been recent discussion in the Forum of New Phytologist on the topic of adaptive functions of red, anthocyanin pigments in senescing leaves of temperate deciduous trees species in autumn. In this Letter, we (1) respond to some points raised previously in the Forum, (2) present the argument that soil nutrient characteristics should also be considered among the suite of environmental factors potentially driving evolution of autumn leaf color, and (3) provide evidence that nutrient deficiencies in soils of eastern North America (ENA) could also potentially account for the greater abundance of red-leafed species compared with Europe, especially when considered in combination with temperature and irradiance trends described by others (Zohner & Renner, 2017; Zohner et al., 2017; Renner & Zohner, 2019). This nutrient-centered perspective dovetails with both photoprotection and signaling hypotheses, as nutrient deficiencies both increase the need for photoprotection as a result of reduced photosynthetic capacity (Evans & Seemann, 1989; Martin et al., 2002; Hikosaka, 2004) and render the plant a lower-quality food source for herbivores (Ball et al., 2000; reviewed in Awmack & Leather, 2002). Cited as evidence against the photoprotection hypothesis, Pena-Novas & Archetti (2020a) reiterated Manetas's (2006) question of why plants would evolve anthocyanins as light-attenuating pigments if they ‘absorb mainly green light, which is relatively harmless’. Agati et al. (2021) addressed the blue and red light-absorbing properties of anthocyanins in their Forum letter (also see Landi et al., 2021); here we defend the importance of green light to photosynthesis, and the value of a green light-attenuating pigment for photoprotection. First, while it is true that chlorophyll in solution absorbs green light less efficiently than red or blue wavelengths, whole-leaf absorptance of green wavelengths is not zero but, rather, closer to 70–75% (e.g. Hughes & Smith, 2007; Liu & van Iersel, 2021). This is because plants possess accessory pigments including carotenoids, which expand the photosynthetic action spectrum into blue-green wavelengths; also, absorption of blue and red wavelengths by the uppermost mesophyll cells renders green light the primary driver of photosynthesis in the lower mesophyll (Sun et al., 1998; Liu & van Iersel, 2021). A similar process to the latter occurs at larger structural scales in the forest, as the overstory preferentially absorbs red and blue light, leaving the understory enriched in green light (Smith et al., 2017). There is also evidence that plants can grow exclusively on green light (Johkan et al., 2012), that green light is used more efficiently than red and blue light at high photosynthetic photon flux densities (PPFDs) (Landi et al., 2020; Liu & van Iersel, 2021), that photosynthesis increases with increasing intensity of green light (Johkan et al., 2012), and that green light can induce photoinhibition of photosynthesis (Hughes et al., 2005; Zhang et al., 2010; Cooney et al., 2015). More pertinent to this discussion, however, is the question of whether attenuation of green photons by anthocyanins translates into photoprotection. This issue has been directly addressed by studies comparing changes in maximum quantum yield of photosystem II (or Fv/Fm), a popular metric for quantifying photo-oxidative stress, in tissues with or without anthocyanin, under green vs red light (which anthocyanins do vs do not absorb efficiently, respectively). Consistent with a photoprotective function of anthocyanins, these studies show more dramatic declines in Fv/Fm and/or total chlorophyll fluorescence in green than red tissues under green light, but similar declines under red light (Hughes et al., 2005, 2008; Zhang et al., 2010). However, as Pena-Novas & Archetti (2020a) emphasized, although studies such as these have demonstrated a photoprotective effect of anthocyanins, others have not. Accordingly, in the past decade, researchers have begun to systematically re-examine these seemingly contradictory cases, and have generally ruled in favor of the photoprotection hypothesis. For example, Logan et al. (2015) repeated Burger and Edwards’ (1996) experiment using red- and green-leafed cultivars of Coleus. While Burger and Edwards reported no difference in quantum yield of O2 evolution following exposure to high light stress, a more detailed fluorescence and biochemical analysis by Logan et al. (2015) revealed that green-leafed varieties sustained similar photosynthetic efficiency by upregulating nonphotochemical quenching (NPQ, e.g. the xanthophyll cycle), an alternative form of photoprotection; under red light (which anthocyanins do not absorb efficiently), both varieties utilized NPQ to the same degree. This finding is consistent with those of other studies which concluded that anthocyanins serve as an alternative photoprotective mechanism to increased NPQ (Hughes et al., 2012; Moy et al., 2015; Ramírez-Valiente et al., 2015). Importantly, Logan et al. (2015) also demonstrated that this difference in NPQ does not necessarily translate into reductions in dark-adapted Fv/Fm or ΦPSII (quantum yield efficiency of photosystem II (PSII) in the light-adapted state). These were the primary metrics used by several studies cited by Pena-Novas & Archetti (2020a) as evidence against the photoprotection hypothesis, including Lee et al. (2003), Manetas et al. (2003) and Karageorgou & Manetas (2006). We note, however, that the findings of these latter two studies actually supported the photoprotection hypothesis, as both showed significantly lower Fv/Fm in green than in red leaves under white light (although this result was de-emphasized by Karageorgou & Manetas, 2006). Gould et al. (2018) also directly addressed conflicting reports of photoprotection by anthocyanins by teasing apart the effects of leaf age, temperature, duration and intensity of light source on Fv/Fm and xanthophyll cycle pigments in wild-type (WT), anthocyanin-deficient ttg1-1 and anthocyanin-rich pap1-D mutants of Arabidopsis. Their results showed that photoprotective effects of anthocyanins were observed only when PPFDs were both high-intensity (above saturating values) and sustained (> 2 h); similar results were reported for anthocyanic vs acyanic tissues of Colocasia esculenta, which exhibited no difference in Fv′/Fm′ or NPQ until after 2 h of sunlight exposure (Hughes et al., 2014). These results help to explain those of Kyparissis et al. (2007), who in fact noted that, ‘Our [red and green] test plants displayed progressive reductions in PSII effective yield and gs, and increases in NPQ; however, their photoprotective capacity was not exceeded, as indicated by the absence of both a plateau in NPQ development and photoinhibitory damage, probably because the imposed stress was insufficient to drive the photoprotective potential of our test plants to their limits.’ Similarly, in Esteban et al. (2008), leaves were exposed to relatively low-intensity PPFDs (300 μmol m−2 s−1) and for a short duration (30 min). The remaining study cited by Pena-Novas & Archetti (2020a) as contradicting the photoprotection hypothesis for leaf reddening (Hormaetxe et al., 2005) examined photoprotection by red carotenoids sequestered in chromoplasts of winter-red evergreens, and is therefore not relevant to the discussion of autumn leaf reddening (which involves vacuolar anthocyanins and chloroplasts/gerontoplasts). Another point made by Karageorgou & Manetas (2006) and evoked by Pena-Novas & Archetti (2020a) regarded the seemingly ill-positioned location of anthocyanins in the vacuole, relative to the source of photo-oxidative reactive oxygen species (ROS), presumably the chloroplast. It is our view that the position of anthocyanins in the vacuole is optimal for its function as a sunscreen, as the vacuole is the largest organelle in the plant cell, comprising up to 90% cytoplasmic volume (Tan et al., 2019). One large umbrella is more effective than many tiny umbrellas (i.e. chloroplasts), especially given that chloroplasts often migrate to vertical cell walls under high light stress (Davis & Hangarter, 2012), leaving underlying cells vulnerable when photoprotection is needed most. This photoprotective strategy is analogous to structural shading reported in the desert plant Retama raetam, which uses outer-canopy stems to shield photosynthetically active lower canopy stems during the dry season (Mittler et al., 2001). There is also ample evidence that anthocyanins can neutralize ROS from the vacuole. While some ROS (e.g. superoxide) cannot cross the vacuolar tonoplast in their original form (Takahashi & Asada, 1983), these molecules are short-lived, and are either rapidly protonated into OH˙ or converted by superoxide dismutase to H2O2, which can freely penetrate the tonoplast and enter the vacuole (Takahashi & Asada, 1983; Yamasaki et al., 1997). Accordingly, the vacuole also contains high concentrations of peroxidases (Zipor & Oren-Shamir, 2013; Zipor et al., 2015). Anthocyaninless tissues and mutants lacking anthocyanins have also been shown to accumulate more H2O2 and O2˙ compared with red tissues or WT plants (Gould et al., 2002; Kytridis & Manetas, 2006; Zhang et al., 2012), indicating that anthocyanins reduce ROS accumulation (Zhang et al., 2012). Manetas himself reported (in another 2006 paper) that ‘vacuolar anthocyanins may be an effective in vivo target for oxy-radicals, provided that the oxy-radical source and the anthocyanic detoxifying sink are in close vicinity’ (i.e. in mesophyll rather than epidermal cells; Kytridis & Manetas, 2006). Renner & Zohner (2020) criticized Pena-Novas & Archetti (2020a) for including ‘green’ leafed species in their analysis, claiming that ‘any test of whether species from North America are more likely to turn red than non-American species during leaf senescence, must include only species that exhibit autumn leaf senescence, i.e. deciduous species’. Species defined as ‘green’ by Archetti (2009) included both evergreen species and deciduous species, which the authors observed to senesce green. Our suggestion is to continue to include evergreens, but to score autumn color in deciduous species based on their color during autumn in the tree’s native range. Archetti (2009) scored autumn color based on personal observation in one geographic region, Gloucestershire, in the UK. Although the authors indicated that field guides were consulted in cases of conflicting observations, many deciduous species were nevertheless scored as ‘green’, despite exhibiting colorful autumn hues in their native locations (e.g. Betula nigra, Cornus canadensis and Quercus falcata, among many others). This may be a result of the relatively mild climate of the UK, or from abundant fertilization, which is known to delay the onset of leaf senescence (Fu et al., 2019). Fertilizing late in the season has even been reported to result in retention of leaves until killed by frost (Sakai & Larcher, 1987). Regardless, we feel strongly that the deciduous species marked ‘green’ by Archetti (2009) should be carefully re-evaluated to ensure the color scoring reflects that of their native locations, especially before they are applied to any additional analyses. We do, however, agree with Pena-Novas & Archetti (2020a) that evergreen species should be included in the overall study of leaf color change. However, we suggest that, in addition to scoring these species as ‘evergreen’, they could also be scored based on leaf color during senescence, whenever it occurs. Evergreen leaves often senesce with vibrant yellow and red hues (Fig. 1), although this tends to occur in spring and summer rather than the autumn (e.g. Poudyal et al., 2012), and receives far less study. There is also evidence that the color of young leaves of deciduous species in spring mirrors that of sensing leaves in autumn (Lev-Yadun et al., 2012), suggesting that selective pressures driving red leaf color are present during other seasons as well. A second reason we argue that evergreens should be included in the discussion of autumn leaf color is the possibility that plants with evergreen, red-deciduous and yellow-deciduous leaves could represent strategies along a continuous resource spectrum (Wright et al., 2004; Onoda et al., 2017). We discuss this possibility further in the next section. Lastly, while on the topic of scoring leaf color, Renner & Zohner (2019) stated: ‘Following Archetti (2009), orange was grouped with yellow because both colours are caused by xanthophylls.’ However, our own (unpublished) observations of autumn-orange leaf cross sections (as well as those of D. W. Lee, pers. comm.) are consistent with findings of Junker & Ensminger (2016), and suggest that orange color is imparted by low levels of anthocyanins, rather than increased carotenoids. Although not clearly communicated in their methods, Archetti (2009) also scored orange leaves as red (M. Archetti, pers. comm.). For these reasons, we suggest that future studies group orange leaves with red. The only abiotic environmental variables tested so far as potential selective pressures driving evolution of autumn leaf reddening have been related to climate – namely, high irradiance during autumn, cold temperatures and precipitation (Zohner & Renner, 2017; Zohner et al., 2017; Renner & Zohner, 2019; Pena-Novas & Archetti, 2020b). Yet, soil variables are strong, and in some cases stronger, predictors of leaf traits than climatic variables (Maire et al., 2015). The relative availability of plant soil nutrients varies by orders of magnitude over biogeographical gradients (Vitousek, 2004; Huston, 2012) and can be affected by several factors, including climate, topography, organic matter content, pH, soil particle size and parent material. Low soil fertility has long been known to favor evolution of evergreen over deciduous leaves (Monk, 1966; Chapin, 1980; Aerts, 1995). This trend has been supported in a broad range of biomes, including the temperate deciduous forest (citations in Givnish, 2002). We believe that an important clue to understanding why some deciduous species evolved to synthesize anthocyanins in autumn leaves lies in the fact that anthocyanin production in leaves is known to correlate with many of the same variables associated with evergreen leaves, for example, low leaf nitrogen (N), low maximum photosynthesis, and high leaf mass per unit area (LMA) (Bongue-Bartelsman & Phillips, 1995; Hodges & Nozzolillo, 1996; Kumar & Shanna, 1999; Kytridis et al., 2008; Peng et al., 2008; Nikiforou et al., 2011; Larbat et al., 2012; Carpenter et al., 2014; Meng et al., 2020), including during autumn (Lee et al., 2003; Schaberg et al., 2003; Anderson & Ryser, 2015). Studies in Arabidopsis have also demonstrated that anthocyanin production is part of the N limitation response, as is increased lignin production (which would contribute to increased LMA) (Peng et al., 2008). Increased LMA under N deficiency corresponds with greater leaf thickness and toughness, which are achieved via reallocation of leaf N to structural (rather than photosynthetic) proteins, cell wall thickening, denser cell packing, increased mesophyll layers, greater sclerophylly and increased allocation to major veins (Takashima et al., 2004; Wright et al., 2004; John et al., 2017). These characteristics maximize leaf longevity by leaves more of environmental stress & and for herbivores to and et al., 2003; et al., leaves also often et al., 1998; Karageorgou et al., and are lower-quality food for herbivores (Ball et al., 2000; Awmack & Leather, 2002). a low leaf N also corresponds to reduced photosynthetic capacity per unit leaf mass to in photosynthetic (Evans & Seemann, 1989; Martin et al., 2002; Hikosaka, 2004) and reallocation of N to structural (Takashima et al., low leaf N is associated with reduced nutrient leaves and reduced photosynthetic anthocyanins could be in photoprotection and signaling low leaf reddening reflects an to that have soils (i.e. but not to an evergreen This is consistent with observations that senescing leaves of species to be and are even green until killed by frost Archetti, photosynthesis and leaf longevity represent the major of the leaf which account for an of in traits related to and N (Wright et al., 2004; Onoda et al., 2017). We therefore feel that these trends in leaf characteristics could represent important and further study. The greater and of red-deciduous species in eastern North America than in by et al., Renner & Zohner, have far been to two primary (Lev-Yadun & and the of more dramatic temperature in autumn compared with and (Zohner et al., irradiance & Zohner, and seasons (Zohner & Renner, which these authors argue a photoprotective hypothesis for autumn leaf In Pena-Novas & Archetti showed that the native of red-deciduous species in North America are by greater precipitation on than green-leafed species, and that yellow-deciduous species to occur in on than green-leafed species as however, their analysis could not any factors that could account for of red- vs yellow-deciduous We suggest that factors (e.g. soil nutrient content, or even structural could help to further observed a test of the hypothesis that low soil fertility drive evolution of red autumn we compared the following for temperate deciduous of and pH, organic effective total N and Consistent with our hypothesis, soils of deciduous in were significantly less in metrics examined compared with Europe, with exhibiting a range of total soil N in Europe, for example, was the for and (Fig. Although the that has over has no these from they would be under they are nevertheless to one with the original that are more red-leafed species in than in These can also be for For example, Renner & Zohner (2020) the hypothesis could explain the greater of autumn leaf reddening in than in our suggest that reduced nutrient availability should render trees lower-quality to in more Similarly, the yellow-deciduous trees of and noted by & may not anthocyanins in autumn as a result of high soil N (Fig. despite photoinhibitory during autumn. it is that seasons in than in reported by Zohner & Renner could be related to soil as previously nutrient stress leaf senescence & Ryser, 2015; et al., 2019). photoprotection by anthocyanins functions to nutrient during autumn, should red-deciduous species necessarily exhibit nutrient efficiency relative to It is our that, if anthocyanins function as an alternative photoprotective strategy to increased NPQ (as in Hughes et al., 2012; Moy et al., 2015; Ramírez-Valiente et al., similar (rather than nutrient in red- vs yellow-deciduous species should serve as for a photoprotective hypothesis, especially if nutrient in anthocyanin-deficient mutants (as shown by et al., A question is whether (or species to nutrient should exhibit greater efficiency than those with more optimal nutrient While some authors have to a leaf nutrient and efficiency (e.g. Aerts, et al. showed a nutrient and efficiency for and but only after for leaf mass during if autumn leaf reddening has evolved in to low leaf red-deciduous species exhibit more nutrient than yellow species, but not directly because of anthocyanins per of the studies comparing nutrient of red- and yellow-deciduous species for leaf mass during senescence et al., 2003; et al., 2014; Pena-Novas & Archetti, We therefore that future studies include leaf mass factors in their efficiency et al., for these studies also to occur in soil nutrients are presumably for both red- and yellow-deciduous this could potentially by Pena-Novas & Archetti question is not why autumn colours but why they evolved only in some of the signaling hypothesis argue that species with red autumn leaves evolved in by autumn trees is while argue that red-leafed species are for in temperate with especially cold and we suggest lower-quality if other photoprotective strategies that plants may to photo-oxidative stress anthocyanins (e.g. selective pressures drive a species to anthocyanins over these other as by Pena-Novas and Archetti studies directly this we a based on the studies First, et al. (2003) showed that acyanic mutants of red-deciduous species significantly less N than wild-type suggesting that anthocyanins are part of an photoprotective strategy which NPQ cannot for anthocyanins have been More Moy et al. (2015) in photoprotection and photosynthetic during leaf senescence in a red-deciduous and yellow-deciduous Their results showed that the red-deciduous chlorophyll and associated with and (e.g. in the season a compared with the yellow-deciduous and also exhibited of photoprotective xanthophyll cycle pigments and in the before senescence, consistent with a greater on anthocyanins for photoprotection than It is that the of photosynthetic the (i.e. the of render this mechanism of less effective than vacuolar Consistent with this Schaberg et al. (2003) showed that with low leaf N in and autumn color, compared with with nutrient in soils a senescence strategy by of leaf including those used in photosynthesis, vacuolar anthocyanins more for photoprotection than A larger study comparing the of chlorophyll and photoprotective strategies for red- vs yellow-deciduous species is needed to test this hypothesis One of the hypothesis is that it for both and of autumn leaf A study of factors and intensity of red in by Schaberg et al. (2003) showed that the predictors of and more autumn leaf reddening were low leaf N and high during the season high in autumn, It is known that N deficiency accumulation in leaves during the season as a result of in associated with reduced & and that is into in the autumn et al., The effects of high and low N on anthocyanin production in the of light have been demonstrated in plant (e.g. et al., 2007; et al., 2010; et al., et al., 2020), and to result from of factors related to anthocyanin (e.g. and which result in greater anthocyanin than et al., we that low leaf N and high may serve as the for autumn leaf and that the function is both signaling and photoprotection. We would to Landi and for their on this and and the the and and the

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