Terpenes are one of the major group of volatile organic compounds (VOCs) released from plants (Holopainen and Blande, 2013), of which isoprene, monoterpenes and diterpenes are formed via methylerythritol-4-phosphate pathway (MEP) in plastids, and sesquiterpenes are formed via mevalonate pathway (MVA) in cytoplasm (Zuo, 2019). The formation and emission of terpenes are regulated by environmental conditions, such as temperature, CO₂, water, light, insect feeding (Zuo et al., 2019; Zheng et al., 2020; Tian et al., 2021). For the emitters, terpenes contribute to floral scents, fruit aromas and crop quality, and play important roles in attracting pollinators and seed dispersers (Mostafa et al., 2022). In ecosystems, terpenes not only serve important functions in ecological relationships between emitters and other plants or insects (Llusia et al., 2013; Vivaldo et al., 2017), but also play essential roles in protecting emitters against various stresses (Holopainen, 2011; Zuo, 2019).

Climate warming is one of global challenges not only for humans but also for plants, as high temperature seriously impacts plants by inducing reactive oxygen species (ROS) accumulation and damaging photosystems (Mathur et al., 2014; Wang et al., 2014). It is widely reported that high temperature can promote terpene emission from plants (Jardine et al., 2017; Guidolotti et al., 2019). Meanwhile, the terpene emission is beneficial to plant thermotolerance (Holopainen and Blande, 2013; Zuo et al., 2017; Tian et al., 2020). Isoprene-emitting Arabidopsis thaliana transformed with isoprene synthase gene (ISPS) enhanced the tolerance to high temperature at 40°C and even 60°C (Loivamäki et al., 2007; Sasaki et al., 2007; Zuo et al., 2019). After knocking down the ISPS, grey poplar (Populus pruinosa Schrenk) decreased photosystem II (PSII) efficiency and CO₂ assimilation rate in exposure to high temperature (Behnke et al., 2007). When isoprene emission from Vismia guianensis was inhibited by fosmidomycin (Fos) blocking MEP pathway, the plant reduced PSII efficiency under high temperature (Rodrigues et al., 2020). In fumigation with monoterpenes, Quercus ilex (Loreto et al., 1998; Peñuelas and Llusià, 2002) and Q. suber (Delfine et al., 2000) improved thermotolerance by declining leaf damage and maintaining photosynthetic abilities. In addition, monoterpenes and isoprene can also enhance plant tolerance to O₃, as blocking their synthesis by Fos resulted in ROS accumulation and photosynthesis reduction under O₃ stress (Loreto and Velikova, 2001; Loreto et al., 2004).

For the thermotolerance mechanisms of isoprene and monoterpenes, they has been mainly hypothesized that these small molecules can stabilize chloroplast membranes by intercalating into membranes against leakiness, and directly quench ROS as antioxidants (Sharkey et al., 2008; Velikova et al., 2011; Zuo et al., 2017). However, isoprene cannot dissolve into cellular membranes in great quantity, and is not involved in the formation of thylakoid membrane acyl lipids (Harvey et al., 2015). Meanwhile, there is a lack of an in vivo evidence about isoprene and monoterpenes with very low concentration quenching ROS in plant cells. Thus, the two hypotheses about the thermotolerance mechanisms are suspected by more and more people.

Recently, a new role about isoprene and monoterpenes regulating gene expression has been reported. In myrcene or ocimene fumigation, A. thaliana up-regulated the expression of the genes as transcription factors or involving in stress or defense responses (Godard et al., 2008). Riedlmeier et al. (2017) and Wenig et al. (2019) found that α-pinene and β-pinene fumigation can raise expression of the genes related with salicylic acid-mediated innate immune responses. In exposure to isoprene, A. thaliana changed expression of the genes that coded for proteins functionally associated with flavonoid and phenylpropanoid biosynthesis, cell wall synthesis, photosynthetic light reaction, stress responses, etc. (Harvey and Sharkey, 2016). When A. thaliana and tobacco (Nicotiana tabacum) were transferred into ISPS to become isoprene emitters, altered expression was found in the genes related with signaling networks and growth regulators, and up-regulated expression was found in the genes related with stress tolerance (Zuo et al., 2019). These findings demonstrate that monoterpenes and isoprene should play important signaling roles in plant tolerating stresses (Lantz et al., 2019; Zuo et al., 2019; Tian et al., 2020).

Cinnamomum camphora (L.) J. Presl releases a wide spectrum of terpenes, and is used as Chinese herbal medicine, a flavor and fragrance agent, and excellent evergreen tree species for landscaping, etc. (Shi et al., 2016; Li et al., 2018; Yakefu et al., 2018). This species has 4 main chemotypes, including eucalyptol (EuL), camphor (CmR), linalool (LnL) and borneol (BeL) chemotypes (Luo et al., 2021). During one year, the 4 chemotypes of C. camphora improved monoterpene emission in hot months by raising expression of the genes associated with monoterpene synthesis (Tian et al., 2021). C. camphora is widely cultivated in southern China, an area frequently harmed by high temperature (Ma et al., 2019). In outdoor experiments, ROS accumulation and photosynthetic ability decline were found in the 4 chemotypes of adult C. camphora with inhibiting monoterpene synthesis under high temperature weather (Xu et al., 2022). Similar results were also found in indoor seedlings of EuL under high temperature, suggesting that monoterpene emission is beneficial to the plant tolerating high temperature (Zuo et al., 2017). When monoterpene synthesis in adult CmR was blocked by Fos, the fumigation with terpinene and β-pinene enhanced the plant thermotolerance by altering expression of 73 genes, demonstrating that the 2 monoterpenes may act as signals to enhance the plant thermotolerance (Tian et al., 2020).

Eucalyptol, camphor, linalool and borneol are the uppermost monoterpenes of C. camphora. Their emission amount more than 50% of total monoterpene emission amount in the corresponding chemotype, and seriously declined by more than 91% when Fos blocked the monoterpene synthesis (Tian et al., 2021). However, it is still unknown the roles of these uppermost monoterpenes in C. camphora tolerating high temperature. Therefore, in the present study, the ROS metabolism and photosynthetic abilities were investigated in the 4 main chemotypes of C. camphora that were fumigated with the corresponding uppermost monoterpenes under high temperature after Fos blocking monoterpene synthesis. The related gene expression was analyzed in EuL and CmR. Meanwhile, the thermotolerance differences among the uppermost monoterpenes were analyzed. The present findings not only uncover the thermotolerance mechanisms of the uppermost monoterpenes in C. camphora, but also provide a solid evidence for the new insight that monoterpenes and isoprene serve signaling functions in plants tolerating high temperature.

Fos is an effective inhibitor for isoprene and monoterpene synthesis by blocking MEP pathway (Tian et al., 2021). In exposure to O₃, ROS accumulation was detected in P. australis leaves with blocking isoprene synthesis by Fos and in Q. ilex leaves with blocking monoterpene synthesis (Loreto and Velikova, 2001; Loreto et al., 2004). Meanwhile, P. australis that was blocked isoprene synthesis accumulated ROS under high temperature stress (Velikova and Loreto, 2005). Under high temperature, EuL seedlings and adult CmR plants remarkably increased ROS and TBARS content after their monoterpene synthesis was blocked (Zuo et al., 2017; Tian et al., 2020). In this study, the similar increase was also found in the 4 chemotypes of C. camphora in the treatment with Fos+38°C. Moreover, Fos+38°C+monoterpene (eucalyptol, camphor, linalool, and borneol) treatments significantly declined the ROS and TBARS content, demonstrating that the 4 uppermost monoterpenes served important functions in regulating ROS levels ( Figures 1 , 2 ). Although monoterpenes can quench ROS and free radicals in vitro (Wojtunik et al., 2014), their scavenging abilities against ROS in vivo are suspected for their internal low concentration and lacking a direct scavenging evidence.

ROS accumulation can cause oxidative stress, which is harmful to the cells. There are a large and integrated non-enzymatic and enzymatic antioxidants in plants to regulate ROS levels and reduce oxidative stress. For non-enzymatic antioxidants, Car, VE, AsA, GSH and flavonoids exhibit strong antioxidant abilities (Koopman et al., 2010; Ma et al., 2019; Shen et al., 2022). In the 4 chemotypes, Car content significantly decreased in 38°C treatment and further decreased in Fos+38°C treatment ( Table 1 ), which should result from the down-regulation of the genes related with Car biosynthesis, such as ZDS, VDE and ZEP in EuL, as well as ispG, idi, GPS, crtB and ZEP in CmR. Compared with Fos+38°C treatment, Car content increased in Fos+38°C+monoterpene treatments for the up-regulation of the related genes. Meanwhile, the genes in VE, AsA, GSH and flavonoids biosynthesis and in GSH metabolic transformation were up-regulated and down-regulated, respectively, in Fos+38°C+monoterpene (eucalyptol and camphor) treatments compared with Fos+38°C treatment ( Figures 5 , 6 ; Supplementary Tables 2, 5 ), which were also beneficial to non-enzymatic antioxidant formation. These results were consistent with previous studies in CmR fumigated with terpinene and β-pinene (Tian et al., 2020), suggesting that monoterpenes may regulate expression of the genes associated with non-enzymatic antioxidant formation, and then adjust ROS levels.

Enzymatic antioxidants can scavenge ROS through enzymatic reaction, and the enhancement of their activities is induced by ROS accumulation. In the treatment with Fos associating with high temperature, Eul seedlings and adult CmR plants increased the activities of antioxidant enzymes in response to the ROS accumulation (Zuo et al., 2017; Tian et al., 2020). In this study, the activities of SOD and POD also increased in the 4 chemotypes of C. camphora in the treatment with Fos+38°C. Moreover, Fos+38°C+monoterpene treatments declined their activities in contrast to Fos+38°C treatment ( Figure 3 ). The corresponding alterations were also found in expression of the genes SOD2 (encoding SOD) and CAT (encoding catalase) in EuL treated with Fos+38°C+E5 ( Figure 5A , Supplementary Table 2 ), and the genes SOD1 and CAT in CmR treated with Fos+38°C+C5 ( Figure 6A , Supplementary Table 5 ). AsA-GSH cycle includes several enzymes to serve antioxidant function. APX, gpx and GSR encode AsA peroxidase, GSH peroxidase and GSH reductase, respectively (Caverzan et al., 2012; Passaia et al., 2014; Couto et al., 2016). High temperature and acid rain stresses increased their activities in C. camphora to quench ROS (Ma et al., 2019). In this study, the expression of the 3 genes in EuL and 1 gene APX in CmR increased in 38°C treatment, further increased in Fos+38°C treatment, and then decreased in Fos+38°C+E5 and Fos+38°C+C5 treatments, which may be caused by the variations of the ROS levels ( Figures 5 , 6 ; Supplementary Tables 2, 5 ). It can be speculated that monoterpenes reduce ROS levels by promoting non-enzymatic antioxidant formation, and ROS accumulation resulted from blocking monoterpene formation under high temperature stress raises antioxidant enzyme activities by inducing related gene expression.

In previous studies, Chl and Car content significantly reduced in adult CmR plants in Fos+38°C treatment, but increased in Fos+38°C+β-pinene and Fos+38°C+terpinene treatments, due to the alterations of related gene expression (Tian et al., 2020). Similarly, the down-regulation of the genes in Chl and Car biosynthesis in EuL and CmR may lead to the decrease of Chl and Car content, respectively, in the treatments with 38°C and Fos+38°C. In contrast to Fos+38°C treatment, Fos+38°C+E5 and Fos+38°C+C5 treatments increased the photosynthetic pigment content by raising expression of the related genes ( Figures 5 , 6 ; Supplementary Tables 3, 6 ).

In the 4 chemotypes, the Chl fluorescence intensity (O to P) decreased in 38°C treatment, and then reduced to the minimum level in the treatment with Fos+38°C ( Figure 4 ), which were similar with previous findings in EuL seedlings and adult CmR plants (Zuo et al., 2017; Tian et al., 2020). The decline of O to J was interpreted as the decline of PQ pool (Strasser et al., 1995), as HST that coded for homogentisate solanesyltransferase in PQ biosynthesis was down-regulated in EuL ( Figure 5C ; Supplementary Table 4 ). For J to P, its decrease was caused by the blockage of electron transport at PSII donor side (Gao et al., 2016). The treatments with 38°C and Fos+38°C inhibited expression of the genes associated with PSII complex and PSII oxygen-evolving enhancer proteins in EuL and CmR, which may restrain PSII assembly and water photolysis and lead to the decline of electron supply at PSII donor side. Meanwhile, the down-regulation was also found in the genes associated with the assembly of Cytb₆-f, PSI and ATP synthase, as well as encoding plastocyanin and ferredoxin-NADP⁺ reductase, which may block electron transport, NADP⁺ reduction and ATP formation, and lower assimilatory power (ATP and NADPH) generation. However, these cases reversed to better statuses in Fos+38°C+E5 and Fos+38°C+C5 treatments ( Figures 5 , 6 ; Supplementary Tables 4, 7 ).

Compared with 28°C, the fumigation with 1, 3 and 5 µM eucalyptol at 28°C not changed the φPo and φD_O in EuL, indicating that monoterpene fumigation no affect the photosynthetic abilities in C. camphora under normal temperature ( Supplementary Figure 1 ). In contrast to 38°C, a decline was found in φPo in the 4 chemotypes of C. camphora treated with Fos+38°C ( Table 2 ). This was similar with the reduction of ΔF/Fm′ in Q. ilex with blocking monoterpene synthesis under O₃ stress (Loreto et al., 2004). For φDo, Fos+38°C treatment aggravated its increase. These demonstrated that an aggravated suppression has happened in the quantum yield and electron transport in Fos+38°C treatment, with massive light energy absorbed by photosynthetic pigments consuming as heat (Zhao et al., 2016). The variations of the 2 Chl fluorescence transient parameters were consistent with that in EuL seedlings and adult CmR plants in Fos treatment associating with high temperature stress (Zuo et al., 2017; Tian et al., 2020). Compared with Fos+38°C treatment, Fos+38°C+monoterpene treatments increased φPo in the 4 chemotypes, but decreased φDo ( Table 2 ), indicating that the uppermost monoterpene fumigation was beneficial to maintaining PSII efficiency. This was similar with Q. ilex maintaining higher ΔF/Fm′ in fumigation with sabinene, α-pinene and cis-β-ocimene (Loreto et al., 1998), and adult CmR plants maintaining higher PSII efficiency in fumigation with terpinene and β-pinene (Tian et al., 2020).

Under high temperature, blocking monoterpene synthesis reduced photosynthetic rate in Q. ilex (Loreto et al., 1998), Q. suber (Delfine et al., 2000) and EuL seedlings (Zuo et al., 2017) in indoor experiments. Compared with high temperature, blocking monoterpene synthesis under high temperature reduced the stomatal conductance in EuL seedlings, but raised the intercellular CO₂ concentration, indicating that the photosynthetic rate reduction with blocking monoterpene synthesis under high temperature is non-stoma limitation. Under high temperature weather, the reduction was also detected in the photosynthetic abilities in the 4 chemotypes of outdoor adult C. camphora with blocking monoterpene synthesis (Xu et al., 2022). This reduction should not only result from the decline of the photosynthetic pigment content, PSII efficiency and assimilatory power, but also result from the decline of CO₂ assimilation abilities, due to the down-regulation of related genes in EuL and CmR ( Figures 5 , 6 ; Supplementary Tables 4, 7 ). In contrast to Fos+38°C treatment, Fos+38°C+E5 and Fos+38°C+C5 treatments up-regulated expression of the genes associated with CO₂ assimilation, suggesting that the uppermost monoterpenes were beneficial to maintaining photosynthesis in C. camphora under high temperature.

In fumigation with monoterpenes (α-pinene, β-pinene, myrcene, and ocimene), A. thaliana up-regulated expression of the genes that were involved in defense (Godard et al., 2008) and innate immune responses (Riedlmeier et al., 2017; Wenig et al., 2019). Both endogenous (Zuo et al., 2019) and exogenous (Harvey and Sharkey, 2016) isoprene exhibited inducing effects on the gene expression in several biological pathways and stress responses. In previous study, terpinene and β-pinene served important functions in adult CmR tolerating high temperature by adjusting expression of the genes associated with ROS metabolism and photosynthetic abilities (Tian et al., 2020). In this study, the uppermost monoterpenes also exhibited similar roles in improving thermotolerance in C. camphora, indicating that they also serve signaling functions.

In previous studies, isoprene improved plant photosynthetic abilities by stabilizing thylakoid membranes (Pollastri et al., 2019), which has been hypothesized by intercalating into membranes against leakiness (Sharkey et al., 2008; Velikova et al., 2011). However, isoprene cannot dissolve into cellular membranes in great quantity, and is not involved in the formation of thylakoid membrane acyl lipids (Harvey et al., 2015). When CmR was treated with Fos+38°C+C5, a reduction was found in the cell membrane damage. The levels of membrane lipid molecules showed variation tendencies to the control at 28°C, and the expression of the genes related with these membrane lipid metabolism was also altered accordingly (submitted). This demonstrates that monoterpenes and isoprene might stabilize thylakoid membranes by regulating related gene expression, which is beneficial to maintaining plant photosynthetic abilities under high temperature.

In the previous study, the fumigation with β-pinene and terpinene at 10 µM (Fos+38°C+β-pinene and Fos+38°C+terpinene) recovered the ROS metabolism and photosynthetic pigment levels in CmR to the levels at 28°C (Tian et al., 2020), while the recovery effects were carried out in fumigation with the 4 uppermost monoterpenes at 5 µM, suggesting that the uppermost monoterpenes had stronger thermotolerance abilities. In contrast to Fos+38°C treatment, the fumigation with the 4 uppermost monoterpenes at 5 µM caused different variation ratio in the ROS metabolism, photosynthetic pigment levels and photosynthetic abilities in the corresponding chemotype. Among of them, camphor fumigation (Fos+38°C+C5) showed high variation ratio in most of indexes, while eucalyptol fumigation (Fos+38°C+E5) showed low variation ratio, indicating that camphor might have strong abilities in reducing ROS levels and maintaining photosynthesis under high temperature, and eucalyptol might have weak abilities ( Table 3 ). These differences should be caused by the recovery effects of related gene expression, with high in Fos+38°C+C5 treatment but low in Fos+38°C+E5 treatment, which was similar with different monoterpene (α-pinene, β-pinene, myrcene, and ocimene) fumigation inducing different defense responses in gene expression in A. thaliana (Godard et al., 2008; Riedlmeier et al., 2017; Wenig et al., 2019). This indicated that monoterpenes might have different signaling effects in improving C. camphora thermotolerance.

The data presented in the study are deposited in the NCBI SRA database, accession number PRJNA909422.

ZZ and CX conceived the main idea of the study. CX, BW, and QL performed the experiments and analyzed the data. YM, TZ, YW, and YC took part in the experiments. ZZ wrote and modified the paper. All authors contributed to the article and approved the submitted version.