Mangrove forest distributes in the intertidal coastal areas of the tropics and subtropics, which possesses four high characteristics, namely, high productivity, high return rate, high decomposition rate, and high resistance as one of the unique marine ecosystems (Wang, 2019), and plays a pivotal role in the global climate change due to its special ecological characteristics in the world (Wang and Gu, 2021). Global climate change has a strong impact on the stability of mangrove ecosystems (Alongi, 2015). Since 1950, it has been observed in a number of climate changes and extreme weather events associated with low or high temperatures (Pachauri et al., 2014). Temperature is one of the key abiotic factors, which can affect the growth and development of mangrove ecosystems (Hatfield and Prueger, 2015).

Climate warming leads to early phenol-logical development, making plants more susceptible to the subsequent low-temperature damage in the spring (Gu et al., 2008). Chilling stress (0–12°C) of plants results from low temperature (Allen and Ort, 2001). It has been observed that chilling stress can affect the leaf morphology of plants. Most studies that focused on maize, rice, and other plants confirmed that cold temperature exposure reduced leaf expansion and growth, resulting in browning, wilting, and chlorosis (Boese and Huner, 1990; Yoshida et al., 1996). Low temperature can result in sudden leaf loss, branch and stem reductions, and even mortality of various plants including mangrove plants (Peng et al., 2013, 2015; Fei et al., 2015; Lovelock et al., 2016).

Because of the great reactive oxygen species (ROS) produced by chilling-exposed plant tissues, from the perspective of plant physiology, oxidative stress is one of the earliest challenges facing the plant (Finkel and Holbrook, 2000; Mittler, 2002).

The imbalance between ROS production and removal could induce oxidative stress (Finkel and Holbrook, 2000). Chilling as the abiotic stress would break the balance of ROS in plants such as maize and winter wheat (Okuda et al., 1991; Prasad et al., 1994). Accumulation of a certain quantity of ROS would activate the antioxidase system to alleviate the damage from overoxidation (Mittler, 2002; Foyer and Noctor, 2005). For example, the activity of ascorbate peroxidase (APX) increases the hydrogen peroxide (H₂O₂) in spinach chloroplasts, and ROS also enhances the enzyme activity [superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT)] in mangrove plants (Peng et al., 2015). The accumulation of low-molecular-weight osmotic adjustment substances (e.g., proline, betaine, soluble sugars) is considered to enhance cold tolerance and had been confirmed in citrus and two coffee species (Kushad and Yelenosky, 1987; Jouve et al., 1993). Chilling exposure induces the movements of the membrane (Örvar et al., 2000) and the change in malondialdehyde (MDA) contents (Campos et al., 2003). Low temperature affects the balance between metabolic reactions, the fluidity of cell membranes, and the activity of antioxidant enzymes; it also leads to cell oxidation, photosynthesis impairment, DNA strand disruption, and protein instability (Ruelland et al., 2009). Mangrove plants are sensitive to low temperatures and can hardly survive the long-term chilling stress, but they still show different tolerance to low temperatures (Lovelock et al., 2016).

In this study, the response mechanisms of five mangrove species (Rhizophora Stylosa, Bruguiera Gymnorrhiza, Aegiceras Corniculatum, Avicennia Marina, and Kandelia obovata) exposed to a long-term chilling treatment are elaborated, the ability of cold tolerance is analyzed, and the understanding of ecophysiological effects on mangrove plants is mentioned under global climate change.

Plants are subjected to temperature fluctuations and have evolved mechanisms to minimize the negative effects at temperature extremes (Chinnusamy et al., 2003). Generally, different species of plants exposed to low temperatures enable to induce different metabolic patterns (Farooq et al., 2009). This study suggested that chilling stress affected leaf morphology, antioxidant enzymes, lipid peroxidation, hydrogen peroxide, and proline content, and the tested mangrove plants responded in different ways.

This study was carried out for the morphological recording of the five mangrove plants under low-temperature stress. Leaves are sensitive to low temperature, and the morphological changes would be more obvious when the low-temperature stress is enhanced (Boese and Huner, 1990). According to the morphological characteristics of the plants under chilling stress, the cold tolerance of different mangrove species was revealed. In this work, the leaf morphology of K. obovata was the best and A. corniculatum was the second, while the damage to B. gymnorrhiza and A. marina was more serious, and it was irreversible damage. Previous studies also compared the leaf morphology of three mangrove species (K. obovata, A. corniculatum, and A. marina), but the difference in leaf morphology was not obvious in short-term chilling exposure (Peng et al., 2015). Nevertheless, the impaired symptom caused by low temperature was different, the leaves of A. marina were browned, and the leaves of B. gymnorrhiza were curled and wilted. During the 20-day chilling exposure, five mangrove plants were damaged to a certain extent.

In addition to leaf morphology, physiological and biochemical indicators of five mangrove species were studied. It was found that cold exposure affected H₂O₂, MDA, proline, SOD, POD, and CAT activities. Chilling has been proven to induce the accumulation of ROS and impair the normal functioning of the plants at the cellular level (Apel and Hirt, 2004; Ruelland et al., 2009). The H₂O₂ is a ROS and leads to oxidative injuries due to chilling stress (Prasad et al., 1994). The high H₂O₂ accumulation in the leaves indicates that cells are beginning to be damaged by chilling stress and is also an antioxidant signal (Penfield, 2008). In the experiment, an increase in H₂O₂ was shown in all mangrove plants, but more evident in A. marina. Although K. obovata had also high H₂O₂ contents, it showed slight damage due to its same high antioxidant enzyme activity.

Furthermore, the chilling application causes greater membrane injury as assessed by lipid peroxidation due to ROS production (Apel and Hirt, 2004). MDA is the product of lipid peroxidation and is used to evaluate the stability of the cell membrane (Bonnes-Taourel et al., 1992). It is also considered a cold sensitivity marker (Campos et al., 2003). H₂O₂ accumulation and consequently lipid peroxidation occurred. The obtained results indicated that lower values of the H₂O₂ and MDA contents in A. corniculatum reflected higher antioxidative capability in comparison to A. marina and R. stylosa. It seemed likely that the double cumulative pressure of H₂O₂ and MDA induced more severe chilling injury in A. marina. The response in cold-tolerant plants was more pronounced than in cold-sensitive plants, consistent with the results reported in previous studies. These studies also reflected strong membrane damage in cold-sensitive plants (Campos et al., 2003). The contents of proline in A. marina and R. stylosa were higher than in A. corniculatum and K. obovata. Drastic MDA changes on the 15th day in B. gymnorrhiza were probably a sign that the injury has exceeded the reasonable tolerance.

In contrast, ROS is thought to act as a signal and to be regulated by antioxidants that could scavenge the reactive oxygen to alleviate the injury (Mittler, 2002). The accumulation of H₂O₂ increased earlier than the change in enzyme activity, which may also explain the signal function of ROS. Previous studies have reported that the first defensive line for ROS is constituted by the SOD, removing O₂^– radical and producing H₂O₂ within cells (Alscher et al., 2002). The elimination of H₂O₂ is inseparable from the participation of POD and CAT. In mangrove plants subjected to chilling stress, SOD, POD, and CAT activities were stimulated. In this study, the continuously increased POD activity cooperated with CAT to scavenge the H₂O₂. Comparing the POD and CAT activities, CAT was the most effective enzyme in cell protection against H₂O₂ exposed to chilling stress. However, other enzymes may cooperate or replace CAT activity in longer chilling stress. It may be deduced that higher CAT activity was the effective way against chilling stress in R. stylosa. Although the enzyme activity of A. marina was high in the early stage, the decline time was earlier than other species. This may be the reason why A. marina had better cold tolerance in the early stage, and the damage was serious in the later stage. B. gymnorrhiza was sensitive and accumulated active oxygen to low-temperature stress, and its antioxidant enzyme activity was negative, thus, the excessive active oxygen could not be cleared in time. K. obovata had a higher antioxidative capability, reflecting a higher tolerance to cold stress, which was associated with the high activities of POD and CAT when chilling stress was applied.

In addition to antioxidant enzymes, it has been noted that proline accumulation is higher in plants exposed to abiotic stresses (Jouve et al., 1993). The accumulation of proline is effective to scavenge hydroxyl radicals, protect and stabilize ROS scavenging enzymes and has regulatory functions as a signal molecule (Szabados and Savoure, 2010). In previous studies, the accumulation of proline could influence chilling tolerance in A. corniculatum (Peng et al., 2015). In this study, B. gymnorrhiza intended to accumulate more proline, which might be the main method to regulate oxidative injury. Higher proline content and CAT activity seemed to play a key role in the detoxification of oxidative stress in K. obovata.

In the five mangrove species, the significant correlation between antioxidant enzymes (SOD, POD, and CAT) and H₂O₂ also revealed its inherent correlation mechanism encountered due to chilling stress. POD and CAT are important enzymes for removing H₂O₂ and alleviating the damage caused by ROS such as H₂O₂ to cells. Proline also plays an important role in regulating the redox state of cells. The level of cold tolerance is a complex process of multifactor coordination.

The five mangrove species differed in their morphological, physiological, and biochemical characteristics under chilling treatment. Lower H₂O₂ and MDA contents were the chilling tolerance performance of A. corniculatum. The results indicate that the chilling tolerance mechanisms of five mangrove plants are the increasing activity of antioxidant enzymes and the content of proline. Moreover, proline may play a more important role in alleviating the effect of chilling stress in B. gymnorrhiza and K. obovata. The stronger active oxygen scavenging mechanism explained the ability of R. stylosa, A. marina, and K. obovata to resist low-temperature stress, and meanwhile, it shows that the level of active oxygen and the activity of antioxidant enzymes are important physiological parameters for the evaluation of chilling tolerance. There is a synergistic competition mechanism in the antioxidant system, which is more conducive to enhancing the ability of mangrove plants to withstand low temperatures. Compared with other mangrove species, K. obovata has better performance exposed to chilling stress and is an alternative plant for planting in the higher latitude region or preparing for the impact of chilling stress for climate change.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

S-MW wrote the manuscript and undertook the experiments. Y-SW designed, conceptualized, and supervised the manuscript. B-YS modified the manuscript and undertook the experiments. Y-YZ, L-FC, X-YM, and X-ML undertook the experiments. 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.

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.