Temperatures above the normal optimum are sensed as temperature stress by all living organisms. Plant response to high-temperature stress (HTS) during early regeneration stages determines recruitment success and the diversity and abundance of a given species with cascading effects on community composition and ecosystem function (Walck et al., 2011). Among suitable microsite conditions appropriate for seed germination, the temperature is one of the most critical drivers that alter seed viability and shape seed germination success (Walck et al., 2011). Temperature changes can affect seed dormancy and persistence in soil, preventing, delaying, or enhancing the processes and mechanisms of seed germination, seedling development, and seedling establishment (Walck et al., 2011; Chen et al., 2021). However, due to abrupt variation in climate, which is driven by anthropogenic activities, suitable temperature conditions for successful seed development and germination have been significantly altered in natural landscapes (Liu et al., 2018; Uriarte et al., 2018; Wan et al., 2018). Most often temperature stress goes hand in hand with severe drought further impeding the regeneration process due to low levels or lack of water.

Severe climate variations have led to extreme weather resulting in unprecedented phenomena such as El Niño and La Niña. Sudden changes in climate trigger drastic temperature alterations accompanied by heatwave conditions, intense rainfall, or shifts in seasonality that lead to prolonged drought and extreme flooding (Jentsch and Beierkuhnlein, 2008). While extreme weather events may be transitory, they have dramatic ecological consequences that could significantly impact ecosystem function and forest regeneration compared to just increases in average temperatures (Franklin et al., 2016). However, we have very limited knowledge on how early plant regeneration stages respond to extreme weather events, especially for forest tree species (Hou et al., 2014; Concilio et al., 2015; Luna et al., 2019). Understanding how these events may affect plant regeneration processes and future forest composition will allow us to propose suitable strategies for forest management. However, these events are very difficult to simulate under field conditions to conduct controlled experimental studies and chamber experiments provide a suitable alternative, especially for the exploration of how such extreme conditions can affect the early regeneration process.

Current scientific knowledge on how plant regeneration is affected by HTS is mostly based on studies conducted on the model plant Arabidopsis thaliana and a few agricultural species (Wahid et al., 2007; Toh et al., 2008; Tan et al., 2013; Quint et al., 2016; Begcy et al., 2018; Gao et al., 2020). Stress tolerance can profoundly shape seed germination, seedling growth, and development, metabolism, and physiology (Han et al., 2019). Seed germination requires suitable temperatures for breaking the dormancy and initiating biochemical mechanisms, such as protein and plant hormones mobilization and activation (Miransari and Smith, 2014; Gao et al., 2017; Vishwakarma et al., 2017; Agostinetto et al., 2018). When seeds face HTS, higher probabilities of seed mortality, slow germination rates, and germination failure will occur (Weitbrecht et al., 2011; Tan et al., 2013; Tangney et al., 2019) and the resulting seedlings will be less resilient due to low stored resources and more vulnerable to future stressors due to impaired development of shoot and root growth (Uriarte et al., 2018).

Under conditions of environmental stress, plants are able to adapt and regulate their growth through the production of specific phytohormones, which influences division and differentiation of cells and plant regeneration, thus providing stress resilience (Peleg and Blumwald, 2011; Fahad et al., 2014; Quint et al., 2016). The relationship between stresses and plant hormones has shown that diverse plant species have specific responses at different regeneration stages under HTS. Plants can regulate the level of hormones, and therefore metabolism, during regeneration through stress signaling related to specific gene expression (Wahid et al., 2007; Verma et al., 2016; Zhu, 2016). Sensing and stress signaling are two of the most important responses that plants have evolved in response to elevated temperatures that minimize damage and ensure the protection of cellular homeostasis. Both plant hormones and reactive oxygen species also contribute to temperature stress signaling, which disturbs cellular homeostasis that can lead to severe retardation in growth and development, and finally impact plant survival (Kotak et al., 2007).

Phytohormones are good candidates to assess HTS resilience in plants as they interact during plant growth and play a critical role in providing strategies for plant adaptation to environmental stress. Phytohormones such as abscisic acid (ABA), indole-3-acetic acid (IAA), cytokinins, gibberellins (GAs), ethylene, brassinosteroids (BRs), jasmonic acid (JA), and salicylic acid (SA) regulate pivotal functions in different physiological and biochemical plant processes (Suzuki et al., 2014). The phytohormone ABA, a sesquiterpene, which controls several developments and growth processes of plants such as leaf abscission, and inhibition of fruit ripening (Vishwakarma et al., 2017) is considered as the “stress hormone” that responds to a diversity of biotic and abiotic stressors including heavy metal, drought, salinity, temperature, and radiation stress (Zhang, 2014). It can specifically affect plant regeneration through extending seed dormancy (Suzuki et al., 2014) and inhibiting seed germination by delaying the radicle expansion and weakening of endosperm, as well as enhancing expression of transcription factors (Graeber et al., 2010). The response to environmental stresses is also driven by IAA (Zhao et al., 2020), an essential hormone for the process of somatic embryogenesis, which plays a key role in regulating cell cycling, formation of vascular tissues and pollen, governing seedling growth, and embryo, leaf, and root development (Suzuki et al., 2014). IAA and brassinosteroids hormones have the ability to stimulate ethylene production, and along with ABA can induce seed germination by rupturing testa and endosperm, while ABA alone has the opposite effects on seed germination (Suzuki et al., 2014). Phenolic phytohormones, such as salicylic acid (SA), mediate plant responses to abiotic stresses such as drought, chilling, heavy metal toxicity, heat, and osmotic stress (Rivas-San Vicente and Plasencia, 2011) as well as against biotic stresses such as pathogens and pest attacks (Bari and Jones, 2009). The role of SA in seed germination has been controversial. There are conflicting reports suggesting that it can either inhibit germination (Rajjou et al., 2006; Xie et al., 2007) or increase seed vigor under different abiotic stress conditions (Rajjou et al., 2006; Alonso-Ramírez et al., 2009), which has been linked to the concentration of SA applied exogenously in experimentation (Rivas-San Vicente and Plasencia, 2011). This phenolic compound has a wide range of distribution in plants, as well as various levels of expression among species and plays a key role during photosynthesis, transpiration, and ion uptake and transport (Alonso-Ramírez et al., 2009). However, the contribution of phytohormones to early plant regeneration processes is mainly studied in mature plants and specifically in the model plant, A. thaliana (Miransari and Smith, 2014; Lymperopoulos et al., 2018).

Species with a wide variety of ecological attributes such as the members of the genus Ficus, commonly known as figs, provide a good model system to understand the mechanisms that underlie the environmental response and endogenous phytohormones expression during the early regeneration process (Hao et al., 2010, 2011a, 2013; Jin et al., 2015; Castillo-Díaz et al., 2021; Chen et al., 2021). Ficus comprises more than 750 species in diverse life forms such as shrubs, vines, lianas, hemiepiphytes, and non-hemiepiphytes (Berg, 1989; Harrison et al., 2003; Harrison and Shanahan, 2006). Hemiepiphytes, which make up the half of Ficus species characteristically start their life as epiphytic plants in the forest canopy and send their areal roots to the ground to become terrestrial plants as adults (Harrison et al., 2003), while non-hemiepiphytic species begin life on the forest floor and become mature adults and continue their terrestrial growth form. These contrasting growth forms that accompany different eco-physiological mechanisms are a consequence of environmental adaptations resulting in strong evolutionary changes, making this genus an ideal model system for comparative plant eco-physiological studies (Harrison, 2005; Harrison and Shanahan, 2006; Hao et al., 2013; Castillo-Díaz et al., 2021; Chen et al., 2021). Many figs are keystone species that have a relatively low abundance compared to their influence on the ecosystem (Paine, 1969; Mills and Doak, 1993; Power et al., 1996). Thus, their loss could cause a disproportionately large effect on communities as they fulfill important functions in biological and ecological interactions (Mills and Doak, 1993; Harrison, 2003; Seekar et al., 2010; Peabotuwage et al., 2019). Despite their importance, we have limited knowledge on the responses of plant hormones to temperature stress during the early regeneration of Ficus.

In this study, we assessed the effects of temperature stress on the early regeneration process (seed germination, seedling emergence, and seedling survival) and three endogenous phytohormones, namely indole-3-acetic acid (IAA), abscisic acid (ABA), and salicylic acid (SA), of eight Ficus species after subjecting them to temperature stress during seed imbibition to seedling development. Using both hemiepiphytic (Hs) and non-hemiepiphytic congeneric species (NHs), we tested the following hypotheses: (1) hemiepiphytic species will have greater resilience to temperature stress and a higher probability of successful germination, seedling emergence, and survival compared to non-hemiepiphytes. Previous studies have shown the evolutionary strategies developed by hemiepiphytic species to face and overcome a variety of environments resulting in greater germination and seedling survival (Hao et al., 2011a; Chen et al., 2021). (2) The concentration of ABA would be higher during seed germination under greater temperature stress in both groups but the change would be greater for hemiepiphytic species. (3) The phytohormone IAA concentration would be higher during germination and decrease with the seedling emergence, with greater secretion when plants are subjected to temperature stress, especially in hemiepiphytic species. (4) We also expect that SA concentrations will be markedly higher in hemiepiphytic species under greater temperature stress during seed germination and seedling emergence.

The assessment of the effect of temperature stress during seed imbibition, seed germination, seedling emergence on the early regeneration stages (seed germination, seedling emergence, and seedling survival) of the eight Ficus species showed that Ficus microcarpa had the highest survival under all temperature stress treatments (Supplementary Figure 2). Seedling survival was highest for hemiepiphytic species (47.35 ± 0.27%) when the temperature stress treatment was experienced during seedling emergence and seedling survival was lowest for the same treatment for non-hemiepiphytic species (26.18 ± 0.16%).

During natural regeneration transient stress conditions can impose physiological barriers that can drastically inhibit growth limiting seed germination, seedling emergence, and, finally, survival. Temperature is one of the most important abiotic factors that can influence the successful transition from one early regeneration stage to another (Baskin and Baskin, 2014a). The interaction of temperature with other inherent biotic conditions, such as phytohormones and plant growth form, can further modulate the regeneration success and recruitment into the next life-history stage (Chen et al., 2021). How the early regeneration stages of contrasting growth forms respond to temperature stress is an important fundamental investigation in plant sciences. In this study, we provide detailed evidence of how temperature stress affects the early regeneration process and phytohormones in eight Ficus species belonging to two divergent growth forms. Our results show that in both hemiepiphytic and non-hemiepiphytic species, seeds are more sensitive to temperature stress when stress is experienced early in the regeneration process, with stress experienced during imbibition affecting emergence and survival and stress experienced during germination affecting emergence. There was no effect from the temperature stress if it was received after seedling emergence. Thus, our detailed empirical study shows that environmental variability affects the transition from on early regeneration stages to the next (Clark et al., 1998).

Temperature variations significantly impact plant regeneration, altering forest structure and composition (Christian, 2001; D’Antonio et al., 2001; Seabloom et al., 2003; Hou et al., 2014). The importance of how temperature stress affects plant regeneration has only become even greater as temperatures warm due to climate change making temperature a major abiotic stress factor for plant growth (Grant et al., 2017; Liu et al., 2018; Uriarte et al., 2018; Wan et al., 2018). The stress experienced during regeneration can have cascading effects altering plant species composition, richness, and community composition, significantly altering or damaging the functions of the original ecosystem (Yan et al., 2015). In our study, when temperature stress was applied during seed imbibition and seed germination, we found positive effects on seedling emergence for non-hemiepiphytic species but seedling survival probabilities were higher for hemiepiphytic species. These contrasting advantages can be a priority effect favoring both growth forms with faster seedling development and recruitment (Alexander et al., 2015; Catelotti et al., 2020).

Prior studies have identified unique evolutionary characteristics and clear differences in the functional traits between the hemiepiphytic and non-hemiepiphytic species in the genus Ficus (Hao et al., 2013; Castillo-Díaz et al., 2021; Chen et al., 2021). We found that temperature stress during seed imbibition and seed germination had no significant effect on latter regeneration stages such as seed germination, seed emergence, and seedling survival of hemiepiphytes. These results indicate that seeds and seedlings of hemiepiphytic species had better stress tolerance during the early regeneration stages with more seedlings surviving the stress condition compared to non-hemiepiphytes when exposed to temperature stress, consistent with previous studies (Hao et al., 2013; Chen et al., 2021). Thus, this result partially supports our first hypothesis, since hemiepiphytic germination and emergence were not significantly different between the two groups. The resistance mechanism of abiotic stress in hemiepiphytes resulting in greater seedling survival may help them become more competitive when faced with temperature stress leading to an increase in the colonization and re-assembly of the hemiepiphytic Ficus species under drier and warmer canopy conditions. This scenario might be equally positive for seedling emergence of non-hemiepiphytic species when faced with temperature stress. The ability to conserve water from early regeneration (Chen et al., 2021) to sapling and tree size classes (Hao et al., 2011a,b, 2013) has provided a greater advantage for hemiepiphytic species, which is crucial for survival in drought conditions that can be important under canopy conditions. In germination chamber experiments conducted by Chen et al. (2021), on 15 Ficus species, hemiepiphytic species were more resilient to drought conditions and germination peaks at 25/15°C, day and night temperature but seedling survival decreased when the day and night temperature were increased to 35/25°C. This is consistent with what we observed in our temperatures stress treatment which was provided as a HTS event lasting 8 h. Our study provides further evidence that temperature stress imposes greater limitations on the early regeneration stages of these species and the response is significantly modulated by phytohormones.

Several environmental factors, particularly variations in temperature, are considered significant drivers of the early regeneration process of Ficus species. Temperature changes significantly influence the germination of Ficus benjamina L. var. nuda seeds, and the optimum germination temperature for this species was between 20 and 35°C or 30°C/20°C (Fu et al., 2008). Ficus species present a variety of germination responses under the effects of temperature as well as red:far-red light because of their different habitat preferences (Chen et al., 2013). The extremely high temperature at 51–53°C has resulted in irreversible leaf damage on potted seedlings of Ficus insipida Willd, a neotropical pioneer tree species (Krause et al., 2010); even though they could survive and develop new leaves under heat stress, temperatures higher than 51–53°C led to significant damage to the canopy due to lower heat acclimation capacity. Our study, one of the few conducted to explore the effect of temperature stress on specific stages of the early regeneration process, shows how the temperature stress, received for a short duration simulating a heatwave and the timing of this stress event, could affect the early regeneration stages influencing their downstream demographic processes through modulating seedling survival.

Growth form is an important factor that underpins the ecosystem structure and diversity (Rowe and Speck, 2005). The impact of temperature stress on the successful transition through each early regeneration stage can explain Ficus rarity as well as co-existence (Harrison, 2006; Chen et al., 2021). We did not see strong effects on germination and emergence of both growth forms when seeds were subjected to temperature stress in the first 24 h period during which imbibition takes place. However, seedling survival was affected indicating that there is a carryover effect for the latter regeneration stages from the temperature stress exposure during seed imbibition. Seed imbibition controls seed dormancy and seed germination (Preston et al., 2009) and it is an important driver of water uptake, including passive water uptake by the dry seeds, negligible water uptake, and seed germination and seedling growth (Finch-Savage and Leubner-Metzger, 2006). Here we show that under temperature stress, seed imbibition was very resilient, and thus, a highly significant factor determining the stability of metabolic activities that result in seed germination. The importance of the relative contribution from each early regeneration stage can be used to identify the most important stage that contributes to an individual fitness (Eriksson and Ehrlén, 2008). In both experimental and field studies, it has been shown that recruitment limitation became progressively higher as the Ficus species move forward from one early regeneration stage to the next, and environmental filters were most influential during the seedling emergence stage which creates a niche bottleneck when transitioning from an emerging seedling to become an established seedling (Castillo-Díaz et al., 2021; Chen et al., 2021). Our study shows that events occurring very early in regeneration have cascading effects on the latter regeneration stages.

In our study, the endogenous phytohormones, ABA, IAA, and SA, showed the growth form-specific responses to the temperature stress in different regeneration stages. In contrast to our second hypothesis, ABA concentration was greater for imbibition compared to germination or emergence. However, many studies would not differentiate between two stages considering seed imbibition the first step toward germination. Hence, it can be considered that the germination stage showed the greatest increase in ABA concentration. Among the phytohormones in our study, ABA and IAA were the major phytohormones to mediate temperature stress and SA was less sensitive. Consistent with our third hypothesis, the ABA levels declined significantly in non-hemiepiphytes following exposure to temperature stress during germination and it increased by threefold in hemiepiphytes and onefold in non-hemiepiphytes when temperature stress was experienced on emerging seedlings. Our results are in contrast to what was observed in imbibed A. thaliana seeds, where ABA levels were elevated at high temperatures (Toh et al., 2008). With IAA the two growth forms presented contrasting results for temperature stress experienced during germination, with increases observed for hemiepiphytes and decreased concentrations observed for non-hemiepiphytes. When stress was experienced by emerging seedlings both groups of species showed increased concentrations for both ABA and IAA. Physiological studies with excised stem segments have implicated that IAA regulates cell elongation (Cleland, 2010). However, supporting evidence on how IAA impacts the early regeneration process from germination to seedling emergence is sparse at best and focuses on the model plant A. thaliana. At high temperatures (29°C) these seedlings exhibit dramatic hypocotyl elongation compared with seedlings grown at 20°C (Gray et al., 1998). We also detected a corresponding increase in the level of IAA in Ficus seedlings grown at high temperatures, suggesting that temperature regulates auxin synthesis or catabolism to mediate this growth response.

Some of our results supported our fourth hypothesis but the expression of this phytohormone between treatments and growth forms was complex. The SA response of the non-hemiepiphytic Ficus species to temperature stress showed that they were more sensitive to temperature stress resulting in significantly lower SA values during seed imbibition but no significant effect was seen when stressed during germination. This pattern was reversed with increased SA concentrations when heat stress was received during emergence. This contrasts with the results observed in A. thaliana where seed germination and the seedling establishment was accompanied by an increase in SA (Alonso-Ramírez et al., 2009) but it is consistent with the inhibitory effect of SA observed in the A. thaliana and barley and maze germination (Guan and Scandalios, 1995; Rajjou et al., 2006; Xie et al., 2007). In plants, many hormones may interact to provide a complex response. For example, cross-talk between ABA and SA signaling can result in increased synthesis of ABA-regulated proteins, such as late embryogenesis abundant proteins, dehydrins, and heat shock proteins, which provide resilience to HTS (Rajjou et al., 2006). Our results where the regeneration stage had specific phytohormonal responses are consistent with the idea that the phytohormone effect on growth depends on the plant species and developmental stage (Rivas-San Vicente and Plasencia, 2011).

Other studies have shown that phytohormone can influence the temperature stress response in Ficus plants as a regulator of plant growth and development, and mediate the damage caused by stress. For example, brassinosteroids can alleviate the injury in Ficus concinna seedlings caused by high temperature by increasing antioxidant defense and sustaining glyoxalase systems (Jin et al., 2015). Our results are consistent with previous studies on the different bio-physiological mechanisms in the two growth forms (Hao et al., 2010, 2013; Chen et al., 2021). In Ficus species, the optimal maximum temperature for germination rises gradually during the summer in subtropical environments and optimum temperatures are present year-round in the tropics, which is consistent with the flowering and fruit setting observed in subtropical to tropical environments where the majority of the Ficus species are found. While germination is enhanced as temperature increases, seedling emergence and survival is repressed by temperatures higher than the optimal conditions (Castillo-Díaz et al., 2021; Chen et al., 2021). The distribution of these species within the tropical to the subtropical range is limited in higher elevations where autumn and nighttime temperatures fall below the upper limit for germination conditions (Castillo-Díaz et al., 2021; Chen et al., 2021). Therefore, the seed sensitivity to temperature is an ecologically important role in the detection of and responding to the appropriate timing for germination under natural conditions (Yoshioka et al., 1998; Baskin and Baskin, 2014b). Our study shows that the temperature stress experienced as transient extreme events may be modulated by phytohormone expression providing greater resilience to temperature stress in hemiepiphytic species, which had greater survival under all HTS treatments.

Plant regeneration is more sensitive than vegetative growth to many environmental stresses. Although HTS is becoming an increasingly more prevalent and common stressor due to recent global warming much of its effects are studied in crop species. Our study highlights the importance of understanding how HTS affects the early regeneration of ecologically important flora such as the keystone species in the Ficus genus. In our study both hemiepiphytic and non-hemiepiphytic the seeds of Ficus species were more sensitive to temperature stress when stress is experienced early in the regeneration process but with no effect when received after emergence. Further, seedling survival favored hemiepiphytes regardless under HTS received during imbibition, germination as well as seedling emergence which was modulated by phytohormones in a species- and regeneration stage-specific manner. The different eco-physiological mechanisms between the hemiepiphytic and non-hemiepiphytic species in Ficus make this group a good comparative model for understanding the patterns of subtropical and tropical tree species response to climate change. We conclude that the sensitivity to HTS is an ecologically important functional trait for detecting and responding to the appropriate timing for seed germination and seedling development. In the future, the Ficus study still needs to focus more on their bio-physiological mechanism, plant and seed anatomy, and molecular research to elucidate the relationships among plant and seed structure, physiological mechanism, genes expression, and abiotic stress.

The dataset presented in this study is available in the online Figshare Digital Repository, which can be accessed in the following link: https://doi.org/10.6084/m9.figshare.16851007.v1.

CF: conceptualization, methodology, investigation, formal analysis, and writing of the original draft. HC: conceptualization, methodology, formal analysis, and writing with review and editing. DC-D, BW, and K-FC: methodology and writing with review and editing. UG: conceptualization, methodology, formal analysis, supervision, funding acquisition, and writing with review and editing. All authors contributed to the article and approved the submitted version.

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.

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