Impact of heat stress on photosynthetic characteristics of C. nankingense
Photosynthesis is a critical plant physiological process that is responsible for plant growth and survival, and accordingly it is of vital importance of plant photosynthesis responds to stress. Photosynthesis is one of the most sensitive physiological processes to heat stress (Allakhverdiev et al. 2008; Ashraf & Harris 2013; Hüve et al. 2019; Okereke et al. 2022). Several studies have demonstrated that heat stress reduces leaf photosynthetic activity due to inhibition of multiple rate-limiting processes and damage to key components of photosynthetic machinery (Ashraf & Harris 2013; Salvucci & Crafts-Brandner 2004; Sharkey 2005; Schrader et al. 2007; Hüve et al. 2011; Zhu et al. 2018). Typically, moderate heat stress results in readily reversible changes in photosynthetic activity, primarily as the result of reductions in stomatal conductance to water vapor (Gs) (Hüve et al., 2019; Okereke et al., 2022), and severe heat stress leads to non-stomatal slowly reversible or non-reversible inhibition of photosynthesis due to damage of the photosynthetic machinery (Salvucci and Crafts-Brandner 2004; Sharkey 2005; Schrader et al. 2007; Hüve et al. 2011; Zhu et al. 2018). Photosynthetic electron transport, in particular, PSII has been considered to be a highly heat-sensitive component of photosynthetic apparatus (Gombos et al. 1994; Schrader et al. 2004; Hüve et al. 2011).
In our study, net assimilation rate, An, was not affected by plant exposure to 35°C, although there was evidence of reduced rate of increase of Gs after leaf enclosure to leaf chamber and increase in light level (Fig. 1a, b). Such delayed responses of photosynthetic characteristics have been observed after heat exposure (Hüve et al. 2019), and are expected to reduce leaf carbon gain in fluctuating environments. Leaf exposure to 45°C treatment resulted in a major reduction of An without concomitant reductions in Gs, indicating non-stomatal reduction of foliage photosynthetic activity (Fig. 1a, b) as was also confirmed by increased intercellular CO2 concentration (Fig. 1c). This result is in agreement with the reports showing that severe heat stress can reduce the cyclic transport of electrons and thylakoid permeability (Schrader et al. 2004; Sharkey 2005). Our results are in accordance with previous reports suggesting that severe heat stress leads to non-stomatal inhibition of photosynthesis, and milder heat stress to stomatal limitations (Zou et al. 2017; Turan et al. 2019).
Heat stress-dependent stimulation of leaf volatile organic compound (VOC) emissions and concentrations in C. nankingense
Under optimal conditions for photosynthesis, volatile organic compounds (VOC) emitted by plants account for a few percentage of terrestrial fixation, but this percentage can greatly increase during stress conditions that inhibit net primary productivity and stimulate emissions of some class of volatiles (Niinemets 2010; Jardine et al. 2020). In several species, the increase in temperature has been demonstrated to strongly increase the emission of various terpenes (Staudt and Bertin 1998; Grote et al. 2013; Loreto & Schnitzler 2010; Maja et al. 2016). To understand the impact of temperature on terpene emissions, it is important to consider that only some species are strong constitutive terpene emitters, and terpenes might be emitted right after their synthesis (de novo emissions) or from specialized storage (storage emissions) (Grote et al. 2013; Copolovici & Niinemets 2016). Moderately high temperatures can enhance constitutively synthesized de novo terpene emissions primarily by reversibly enhancing the activity of terpene synthases and substrate availability (Niinemets et al. 2002; Grote et al. 2013). In addition, heat stress can lead to induction of expression of terpene synthases and start of terpene release in non-constitutive emitters (Copolovici & Niinemets 2016). In addition, in storage emitters, severe stress can enhance terpene emissions by breaking the trichomes or increasing the permeability of glandular trichome outer surfaces (Guenther et al. 1993; Jansen et al. 2009; Grote et al. 2013). In addition, larger molecular size volatiles and semi-volatiles might be released from leaf surface waxes, especially when temperature increases (Himanen et al. 2015; Joensuu et al. 2016). Both induction of emissions as well as enhancement of storage emissions have been observed in heat-shocked Solanum lycopersicum and Nicotiana tabacum (Copolovici et al. 2012; Pazouki et al. 2016; Turan et al. 2019).
Chrysanthemum nankingense leaves have small amount of glandular and non-glandular trichomes, and thus volatile emission might come from both de novo synthesis and specialized storage (Guo et al. 2020). In our study, no terpene emissions were observed in control plants at 25 ℃, and the release of mono- and sesquiterpenes was initiated only after 3 h 45 ℃ treatment (Fig. 2). Given that the dominant monoterpene induced by heat stress, camphor, was not found in leaf extracts (Fig. 4), we conclude that the release of monoterpenes reflects de novo induction of monoterpene synthesis in heat-stressed leaves. Given that sesquiterpenes were also observed in leaf extracts (Fig. 4), the heat stress-induced emissions of the sesquiterpene (E)-β-caryophyllene could be related to the breakage of glandular trichomes or increases permeability of storage cells. However, leaf extracts demonstrated presence of 10 other sesquiterpenes, but only (E)-β-caryophyllene was observed in leaf emissions (Fig. 2). This suggests that the emitted (E)-β-caryophyllene might also come from de novo synthesis as has been observed in response to different stresses (Hansen & Seufert 2003; Jiang et al. 2017; Kanagendran et al. 2018).
Analysis of leaf extracts further demonstrated that total sesquiterpene concentration, and the concentrations of most individual sesquiterpenes detected increased after plant exposure to 45 ℃ (Fig. 4). There is limited information of heat stress impact on terpene contents, and the results are controversial. In conifers Pinus sylvestris and Picea abies, foliage terpene concentrations were enhanced by growth at higher temperature (Sallas et al. 2003), but in the annual herb Artemisia annua terpenoid content was not affected by growth temperature (Daussy & Staudt 2020). In the conifer Pseudotsuga menziesii, growth under high temperature reduced foliage terpene concentrations in one provenance and did not affect the concentrations in another provenance (Duan et al. 2019). In the perennial herb Valeriana jatamansi grown in a Free Air Temperature Increase Experiment, the concentration of the sesquiterpene globulol was increased by high temperature, but not the concentration of other compounds (Kuandal et al. 2018). Clearly more work is needed to gain an insight into the impact of heat stress of foliage terpenoid contents.
Heat stress increases the attractiveness of C. nankingense leaves to Spodoptera litura (tobacco cutworm, TCW)
Our study showed that the volatile terpenoids emitted from C. nankingense after 3 h exposure 45 ℃ increased plant attractiveness for TCW. Enhanced attractiveness of the plants to TCW could be mediated by the induced emissions of (E)-β-caryophyllene. However, (E)-β-caryophyllene is typically associated with attraction of pest and herbivore enemies rather than attraction of herbivores themselves. For example, (E)-β-caryophyllene emitted from maize (Zea mays) roots strongly attracted an entomopathogenic nematode of the pest Diabrotica virgifera virgifera (Rasmann 2005; Köllner et al. 2008). In addition, Z. mays leaves infested by TCW emitted (E)-β-caryophyllene that strongly attracted a larval parasitoid Cotesia marginiventris (Köllner et al. 2008). On the other hand, it has been also observed that (E)-β-caryophyllene could attract the pest D. virgifera virgifera larvae to locate the maize plant (Robert et al. 2012). Moreover, (E)-β-caryophyllene could function as a host location signal for the rice (Oryza sativa) pest Sogatella furcifera (Wang et al. 2015), and it also attracted the whitefly (Bemisia tabaci) to the leaves of rosemary (Rosmarinus officinalis) (Sadeh et al. 2017). Our results are consistent with the observations that (E)-β-caryophyllene could serve as host location signal for TCW.
Inhibition of TCW feeding by heat-dependent increases of C. nankingense leaf sesquiterpene concentrations
The comparison of feeding area between control plants and 45 ℃-treated plants suggested that elevated concentrations of sesquiterpenes could inhibit the infestation of TCW (Fig. 5). In particular, total sesquiterpene concentration and concentrations of γ-elemene, β-copaene, isogermacrene D, γ-muurolene, germacrene D, (E)-β-caryophyllene, (E)-β-farnesene, and germacrene D-4-ol were negatively correlated with the total leaf area consumed and the percentage of leaf area removed by TCW. This implies that acclimation to heat stress resulted in higher resistance to TCW larval infestation. Anti-feedant activity and toxicity of sesquiterpenes to related herbivore Spodoptera littoralis larvae has been observed in multiple studies, including 38 sesquiterpenes extracted from leaves of different Celastraceae species (Gonalez et al. 1997), two eremophilane sesquiterpenes isolated from the forb Senecio adenotrichus (Ruiz-Vasquez et al. 2017), and terpenoids from the forb Origanum vulgaris (Agliassa & Maffei 2018). It has been further observed that among eight tropical woody species, the essential oil extracted from leaves of Piper pseudolanceifolium and Ocimum campechianum most strongly repelled the food grain pest Tribolium castaneum; in these species, germacrene D and (E)-β-caryophyllene were the dominant components of the essential oil (Caballero-Gallardo et al. 2014). In our study, the concentrations of these two sesquiterpenes increased after heat stress in C. nankingense leaves, and this was associated with reduced TCW feeding area, underscoring the high repellence of germacrene D and (E)-β-caryophyllene.