Ethylene is considered a key regulator of plant developmental and physiological processes ranging from seed germination to senescence (Pierik et al., 2006). Additionally, it acts as a crucial signaling molecule in the abiotic stress tolerance mechanism because plants modulate ethylene to activate signaling pathways that protect them from the harmful effects of abiotic stress (Cao et al., 2007; Peng et al., 2014a; Shen et al., 2014; Zhang et al., 2016). However, a decisive conclusion on whether ethylene plays a positive role in plant response to abiotic stress could not be reached at this time. While many researchers reported a positive role of ethylene or its precursor 1-aminocyclopropane-1-carboxylate (ACC) in stress tolerance of various plant species, such as corn, Arabidopsis, tomato, grapevines (Lin et al., 2012; Yang et al., 2013; Freitas et al., 2017; Gharbi et al., 2017; Xu et al., 2019), other researchers claimed a negative role of ethylene in some plant growth (such as Cucurbita pepo, tomato, Arabidopsis, and tobacco) under abiotic stress (Albacete et al., 2009; Wi et al., 2010; Dong et al., 2011; Cebrián et al., 2021). The role of ethylene in the response regulation to abiotic stress depends on its level in plant tissue and plants’ sensitivity to it, as the optimal ethylene level for normal plant growth may vary at different stages and in different plant species (Khan et al., 2008; Tao et al., 2015).

Ethylene, derived from methionine, is converted into S-adenosyl-L-methionine (SAM) by SAM synthetase. SAM is then converted to 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthetase (ACS). Finally, ACC is converted to ethylene by ACC oxidase (ACO; Yang and Hoffman, 1984; Kende, 1993). As ACO is involved in the final step of the ethylene biosynthesis pathway, ACO gene encoding the ACO enzyme is an ideal candidate to study ethylene production in plants; its regulatory role in ethylene production has been reported in many horticultural crops (Houben and Van de Poel, 2019). For example, petunia has been broadly used as a model in plant genetics research (Vandenbussche et al., 2016). The ethylene production in petunia floral organs has been investigated and coincided with biosynthesis-related genes PhACO1, PhACO3, and PhACO4 expressions (Tang et al., 1993; Tang and Woodson, 1996; Huang et al., 2007). Recently, our research group also validated the association between ethylene production and gene expression in the flower of P. hybrida cv. “Mirage Rose” (Xu et al., 2020). Furthermore, when genes were edited using the CRISPR/Cas9 tool, there was a significant reduction in ethylene production in floral organs and prolongation of flower longevity (Xu et al., 2020, 2021), However, editing of the genes reduced ethylene production in seeds and negatively affected seed germination (Naing et al., 2021a).

As described above, ethylene can act as either a positive or negative regulator of abiotic stress tolerance, whereas the expression of the genes (ACS and ACO) involved in ethylene biosynthesis pawthway and of the genes ethylene resistance 2 (ETR2), ethylene response sensor 1 (ERS1), ethylene insensitive 2 (EIN2), and EIN3-like (EIL) involved in ethylene signaling pathway is critical in plant responses to abiotic stress (Dong et al., 2011; Peng et al., 2014a; Müller and Munnébosch, 2015; Yang et al., 2015; Gharbi et al., 2017; Riyazuddin et al., 2020). However, the expression of the genes associated with stress tolerance varied depending on the plant species; for example, ETR1 was upregulated in cotton under both short- and longtime salt treatments (Peng et al., 2014b), while its expression was suppressed in Arabidopsis under salt stress (Zhao and Schaller, 2004). Although the molecular mechanisms underlying the involvement of ethylene in abiotic stress tolerance have been widely studied in other plants, it remains unknown in petunia. It is interesting to elucidate how ethylene is involved in the abiotic stress tolerance mechanisms in petunia. Therefore, we used petunia mutants with edited ethylene biosynthesis genes (ACO1 or ACO3) and wild type (WT) to investigate their tolerance to salt and drought stresses, and we assessed the expressional changes of ethylene biosynthesis, receptor, and signaling genes between the before and after stress conditions.

Several plant species have revealed key physiological processes that control ion, water, and reactive oxygen species (ROS) homeostasis during abiotic stress (Munns and Tester, 2008; Miller et al., 2010; Tavakkoli et al., 2011). Overall, stomata density was reduced during abiotic stress to control transpiration rates, delay water loss, and mitigate the negative effects of osmotic stress and ion toxicity on shoot growth (Passioura and Munns, 2000; Abbruzzese et al., 2009; Hughes et al., 2017; Naing et al., 2017). Additionally, under abiotic stress, plants generated antioxidants, such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and proline enzymes to scavenge abiotic stress-induced ROS to protect them from oxidative stress (Müller and Munnébosch, 2015; Naing et al., 2018, 2021b). Moreover, abscisic acid (ABA) was observed to be involved in petunia drought stress resistance due to its rise in petunia leaves during drought stress (Kim et al., 2012). Similarly, the application of exogenous ABA improves drought tolerance in other plant species (Waterland et al., 2010; Du et al., 2013; Zeinali et al., 2014; Wei et al., 2015). Therefore, the changes in the expression of the genes involved in ABA synthesis and signaling pathways, including the antioxidant genes (SOD, CAT, and POD), proline-related gene (Osmotin), and the genes encoding 9-cis-epoxycarotenoid dioxygenase 1 (NCED1), abscisic aldehyde oxidase 31 (AAO31), and phospholipase Dα (PLDα), should be assessed before and after stress conditions.

In this study, we exposed wild-type (WT) petunia cv. “Mirage Rose” and two petunia mutant (phaco1 and phaco3) seedlings to salt and drought stresses and investigated their tolerance to the stresses to learn how ethylene is involved in the stress tolerance mechanism by assessing the morphological and physiological parameters associated with stress tolerance. Moreover, we discovered the molecular mechanisms underlying the involvement of ethylene in stress tolerance, by analyzing the genes involved in ethylene biosynthesis, ethylene signaling, antioxidants, and ABA biosynthesis.

The involvement of ethylene in the abiotic stress tolerance mechanism has been reported in many plant species, however, the role of its involvement in the mechanism remains unclear because it can act as a positive or negative regulator of the stress tolerance depending on its concentrations, plant species, and plant growth stages (Albacete et al., 2009; Wi et al., 2010; Dong et al., 2011; Freitas et al., 2017; Xu et al., 2019; Cebrián et al., 2021). In this study, we examined how ethylene affects salt and drought stress tolerance in Petunia hybrida cv. Mirage Rose using the ethylene biosynthesis genes (PhACO1 and PhACO3) mutants (phaco1 and phaco3) and WT plants.

There were no significant changes in plant growth and physiological parameters between the mutants (phaco1; 91-1 and 36-4, phaco3; 14-10 and 32-15) and WT or among the mutants before they were exposed to any stress. However, ethylene levels produced in the leaves of mutants were significantly lower than those of WT, despite no significant variation in ethylene production among the mutants. When they were exposed to salt and drought stresses, the differential response of the plants to the stresses was distinctly observed. Under salt stress, we observed a reduction of plant growth, RWC and SPAD values, and stomata density, and this could be due to the more Na⁺ content accumulation (four or five folds) in the stressed plants than before stress conditions. Because the high Na⁺ content will cause osmotic stress to the plants and might disrupt the water uptake and photosynthesis efficiency, resulting in decreased plant growth. Furthermore, the plants reduce stomata density and/or close stomata to delay water loss for sustained growth against stress. In this study, the elevation of K⁺ content was also observed in all plants under salt stress. It seemed that the plants triggered K⁺ to compete with Na⁺ in membrane transport and enzymatic functions to reduce the harmful effects of Na⁺ (Munns and Tester, 2008; Shabala, 2013; Deinlein et al., 2014; Sun et al., 2015). The importance of K⁺/Na⁺ content in salt stress tolerance has been reported in many plant species (Nadeem et al., 2006; Zhang et al., 2008; Karlidag et al., 2013; Han et al., 2014; Singh and Jha, 2016). However, the imbalance of K⁺/Na⁺ content was observed in the plants, whereas increased K⁺/Na⁺ content in WT plants over mutants would lessen the harmful effect of Na⁺ content in the former more than the latter. This was associated with the results of higher plant height, fresh weight, RWC, SPAD value, number of open stomata in the WT than the mutants. Abiotic stress often increases ROS generation in different organelles (Naing and Kim, 2021). Enzymatic antioxidants, such as SOD, CAT, and POD, are involved in the defense mechanism against the toxic effects of excess ROS; specifically, SOD removes O 2 − by catalyzing its dismutation, while CAT and POD remove H₂O₂ (Gill and Tuteja, 2010). Similar to these enzymes, proline is also involved in the defense mechanism and is effective in scavenging OH⁻ (Smirnoff and Cumbes, 1989). Meanwhile, Osmotin induces proline biosynthesis by activating Δ¹-pryrroline-5-carboxylate synthetase (P5GS), which catalyzes the rate-limiting step in proline biosynthesis (Delauney and Verma, 1993; Kavi Kishor et al., 1995). In this study, the suppression of SOD, CAT, POD, and Osmotin were observed in all plants compared to before stress conditions. The plants most likely consumed antioxidants and prolines to scavenge the salt-induced ROS, whereas the expression level of Osmotin was still significantly higher in the WT than the mutants, which might contribute to greater stress tolerance of the WT over the mutants. Previous studies using exogenous proline and transgenic plants with increased proline content revealed the importance of prolines in salt stress tolerance (Khedr et al., 2003; Simon-Sarkadi et al., 2006).

Similar to the salt stress, the reduction of the plant growth, RWC, stomata density, and SPAD values were also observed in the plants under drought stress. Additionally, all stomata were closed under drought stress. Because the plants were not watered for about 10 days, they cannot sufficiently uptake water and their RWC was significantly declined. This made the plants reduce the stomata density and close the stomata to maintain RWC and control the high transpiration rate. Moreover, a reduction in chlorophyll content or SPAD value is also one of the major responses to early drought stress. Such reduction in plant growth and disruption of the physiological process by abiotic stress has been observed in petunia (Arun et al., 2016; Ai et al., 2018; Naing et al., 2021b). In this study, the WT plant was discovered to tolerate drought stress more than the mutants. This was associated with the existence of greater plant growth and physiological parameters in WT over the mutants. Unlike the salt stress, we observed an upregulation of CAT, POD, and Osmotin genes in the mutants, but this was not observed in WT plants. It was likely that as the mutants were highly sensitive to drought stress, they strongly triggered antioxidant and proline-related genes to scavenge the drought-induced ROS for their survival. Whereas, WT plants that were less sensitive to the drought stress compared to the mutants might utilize existing antioxidants and prolines to protect them from the stress without further upregulating the genes. Because no significant changes in SOD expression were identified in the mutants or WT, it appeared that SOD is not involved in petunia drought tolerance. Abscisic acid (ABA) is the primary chemical signal for drought sensing. Its increase in concentration under drought stress regulates stomatal closure to prevent water loss (Zhang et al., 2004; Mishra et al., 2006). NCED and AAO are key regulatory genes of ABA biosynthesis (Seo et al., 2000; Thompson et al., 2000; Harb et al., 2010), and PLDα affects ABA signaling by mediating the effects of ABA on stomatal closing and opening. (Zhang et al., 2004; Mishra et al., 2006). Kim et al. (2012) discovered that petunia induced ABA under drought stress and improved tolerance to the stress. Similarly, other studies also reported an improvement of drought tolerance by applying exogenous ABA in other plant species (Waterland et al., 2010; Du et al., 2013; Zeinali et al., 2014; Wei et al., 2015). Harb et al. (2010) also reported that PLDα was upregulated as an early response in Arabidopsis leaves under moderate drought stress. In this study, we observed drought-induced upregulation of NCED1, AAO31, and PLDα in the mutants but not in the WT plants except PLDα. In a study by Kim et al. (2012), there were no significant changes in NCED1 and AAO31 expression in petunia under drought stress, despite the increase in leaf ABA concentration. In addition, they indicate that de novo ABA synthesis may not be a significant contributor to the increase in ABA concentrations in petunia leaves during drought. Therefore, although the upregulation of the NCED1 and AAO31 genes were not observed in the WT of this study, as observed by Kim et al. (2012), ABA concentration may increase in the WT leaves under drought stress. However, higher expression of the genes in the mutants than the WT could be explained that as the mutants were extremely sensitive to drought stress, they highly induced the ABA via upregulation of the genes to mediate the severity of the stress. In contrast to NCED1 and AAO31, PLDα was significantly upregulated in the WT, which was also similar to the finding of Kim et al. (2012), who suggested that PLDα expression promoted stomatal closing because phosphatidic acid generated through PLDα activity binds to ABI1/PP2C, a key component of ABA signaling, involved in stomatal regulation (Mishra et al., 2006). We also agreed with their suggestion because, in our study, higher PLDα expression in the mutants than in the WT was associated with more closing of the stomata in the mutants than in the WT.

Under the stresses, the induction of ethylene in WT was approximately two- or three-fold higher than the mutants. Furthermore, we observed the stress-induced ACS1 expression in all plants except WT under drought stress. However, the stresses did not significantly upregulate the altered ACO1 or ACO3 genes in their respective mutants, but they were all increased in the WT. Salt-induced ACS and ACO expression was observed in tobacco and cotton (Cao et al., 2007; Shen et al., 2014; Peng et al., 2014b; Xu et al., 2019), and the authors suggested that the promotion of ethylene production is necessary for plant adaptation to the salt stress condition. Similarly, Arraes et al. (2015) also reported drought-induced upregulation of ACS and ACO expression in soybean. Naing et al. (2021b) recently discovered drought-induced ACO1 expression and ethylene production in the petunia. In this study, greater stress tolerance of the WT over the mutants also indicated that ethylene was necessary for the modulation of stress response and adaptation in the petunia, and it seemed that editing of the PhACO1 or PhACO3 suppressed the ethylene production lower than the threshold level that is required for stress adaptation, thereby making the mutants more sensitive to the stresses, compared to WT. This finding was consistent with those of previous studies revealing the positive role of ethylene in abiotic stress tolerance in other plant species (Lin et al., 2012; Yang et al., 2013; Freitas et al., 2017; Gharbi et al., 2017; Xu et al., 2019). Jiang et al. (2013) reported that ethylene overproduction in the eto1 mutant results in salinity tolerance due to improved Na⁺/K⁺ homeostasis through an RBOHF-dependent regulation of Na⁺ accumulation. de Zélicourt et al. (2018) also discovered that Enterobacter sp. SA187 induces salt stress tolerance in Arabidopsis through the production of KMBA that activates the ethylene pathway, and Na⁺/K⁺ content observed in the plant inoculated with SA187 was lower than that of noninoculated plants. Therefore, in this study, the existence of a higher Na⁺/K⁺ or lower K⁺/Na⁺ content in the mutants over the WT could be due to the lower ethylene production in the mutants caused by editing of the ACO1 or ACO3. Moreover, previous studies also reported that ethylene can regulate stomatal closure (Shi et al., 2015) and activate ABA synthesis through the transcription of NCED1 (Rakitin et al., 2009; Sun et al., 2010), this would help the WT to more modulate the severity of the stresses than the mutants.

Ethylene signaling is necessary for plant responses and adaptation to abiotic stress, whereas the expressions of ETR2, ERS1, EIN2, and EIL, which are involved in the ethylene signaling pathway, are critical for plant responses to abiotic stress (Cao et al., 2007; Lei et al., 2011; Peng et al., 2014a,b; Arraes et al., 2015; Ge et al., 2015; de Zélicourt et al., 2018). Peng et al. (2014a) reported that salt-induced EIL1 confers salinity tolerance by inhibiting ROS accumulation in Arabidopsis, and loss-of-function mutant ein3-1 and the double mutant ein3eil1 were extremely sensitive to salinity, In addition, in Arabidopsis, loss-of-function of EIN2 became more sensitive to salinity, and overexpression of the C-terminus of EIN2 in ein2-5 suppressed the salinity sensitivity (Cao et al., 2007; Lei et al., 2011; Peng et al., 2014a). Moreover, we observed salt-induced ETR2 upregulation in cotton (Peng et al., 2014b). In guard cells of Arabidopsis leaves, ETR1 and ERS1 mediate both ethylene and H₂O₂ signaling, highlighting ethylene-mediated regulation of H₂O₂ concentrations during salinity stress (Ge et al., 2015). Similarly, de Zélicourt et al. (2018) also claimed that blocking the ethylene receptors by AgNO3 decreased the plants’ tolerance to salt stress. In this study, we observed transcriptional changes of the receptor and signaling genes in the WT and mutants between the before stress and after stress conditions, indicating the necessity of ethylene signaling in petunia stress adaptation. However, as observed in the aforementioned studies, salt-induced significant elevation of ethylene signaling genes (EIL1 and EIN2) and receptor gene (ERS1 and ETR2) were observed in WT, but not in the mutants except ERS1 in some mutants. This supports greater WT tolerance to the salt stress over the mutant. Moreover, other studies also hypothesized the involvement of ethylene signaling in abiotic stress tolerance (Splivallo et al., 2009; Garnica-Vergara et al., 2016; Verbon and Liberman, 2016). Under drought stress, in contrast to salt stress, the genes were upregulated in the mutants that were highly sensitive to the drought, but not in the WT that was moderately sensitive to the drought. It showed that the expression patterns of the ethylene signaling genes were opposing between the salt and drought stresses, indicating that regulation of ethylene signaling in response to abiotic stress tolerance varied depending on the types of abiotic stress. Arraes et al. (2015) reported that ETR expression was significantly higher in the leaves of drought-sensitive soybean cultivars than drought-tolerant soybean cultivars when they were exposed to the drought stress for 150 min. However, it is still unknown the reason why the transcriptional changes of the genes were not observed in the WT between the before stress and after stress conditions, even though they were moderately sensitive to the drought. Other hormonal signaling pathways, including the ABA signaling pathway, may be involved in drought stress adaptation, whether ethylene-dependent or -independent because the ABA signaling gene PLDα that regulates stomatal closure was significantly upregulated in the WT. In addition, we observed ethylene-induced ABA synthesis in grapevine (Rakitin et al., 2009; Sun et al., 2010). To the best of our best knowledge, there has been no report revealing the involvement of ethylene signaling in petunia under abiotic stress. Therefore, further research would be needed to study deep insight as to how ethylene signaling is involved in petunia stress adaptation. Overall, these results indicated how ethylene is involved in the abiotic stress tolerance mechanism of petunia and alerted the researchers to consider this in the future when editing the ethylene biosynthesis genes using CRISPR/Cas9 for improvement of flower longevity and quality. The occurrence of the slight variation in plant growth, physiological parameters, and gene expression among the mutants could be attributed to the differences in edited genes and/or their deletion patterns (genotypes).

We discovered that editing PhACO1 or PhACO3 in P. hybrida cv. “Mirage Rose” reduced ethylene production in the leaves of the phaco1 and phaco3 mutants and reduced tolerance to salt and drought stress, compared to the WT. This was proven by analyzing the plant growth and physiological parameters, indicating superior plant growth and physiological parameters in the WT to the mutants. Furthermore, we revealed the molecular mechanism by which ethylene is involved in stress tolerance by analyzing the expression levels of antioxidant and proline-related genes, ABA synthesis and signaling genes, and ethylene receptor and signaling genes in the mutants and WT plants for both before and after stress conditions. We observed that the transcriptional regulation of genes in response to the stresses varied between the mutants and WT plants. These results indicate the necessity of ethylene for abiotic stress adaptation in petunia to some extent and provide a better physiological and molecular understanding of the role of ethylene in the abiotic stress response in petunia. Additionally, the finding alerts that when editing the ethylene biosynthesis genes for the prolongation of postharvest fruit, vegetable, and flower quality, careful consideration should be taken to avoid the negative effects of ethylene reduction toward reduced tolerance to the abiotic stresses.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

AN designed the study, wrote, and revised the manuscript. AN and JC conducted the experiments. HK did gene expression. JX and MC assisted the experiment. CK supervised the project. All authors read and approved the final manuscript.

This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01485801)” Rural Development Administration, Republic of Korea.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2022.844449/full#supplementary-material