*Communication* **Effects of Short-Term Exposure to Low Temperatures on Proline, Pigments, and Phytochemicals Level in Kale (***Brassica oleracea* **var.** *acephala***)**

**Valentina Ljubej 1, Erna Karalija 2, Branka Salopek-Sondi <sup>1</sup> and Dunja Šamec 1,3,\***


**Citation:** Ljubej, V.; Karalija, E.; Salopek-Sondi, B.; Šamec, D. Effects of Short-Term Exposure to Low Temperatures on Proline, Pigments, and Phytochemicals Level in Kale (*Brassica oleracea* var. *acephala*). *Horticulturae* **2021**, *7*, 341. https:// doi.org/10.3390/horticulturae7100341

Academic Editors: Agnieszka Hanaka, Jolanta Jaroszuk-Sciseł, ´ Małgorzata Majewska and Alessandra Francini

Received: 6 September 2021 Accepted: 22 September 2021 Published: 24 September 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Abstract:** Kale (*Brassica oleracea* var. *acephala*) is known as a vegetable with good tolerance of environmental stress and numerous beneficial properties for human health, which are attributed to different phytochemicals. In the present study, investigation of how low temperatures affect proline, pigments and specialized metabolites content was performed using 8-weeks old kale plants subjected to chilling (at 8 ◦C, for 24 h) followed by short freezing (at −8 ◦C, for 1 h after previous acclimation at 8 ◦C, for 23 h). Plants growing at 21 ◦C served as a control. In both groups of plants (exposed to low temperatures and exposed to short freezing) a significant increase in proline content (14% and 49%, respectively) was recorded. Low temperatures (8 ◦C) induced an increase of pigments (total chlorophylls 7%) and phytochemicals (phenolic acids 3%; flavonoids 5%; carotenoids 15%; glucosinolates 21%) content, while exposure to freezing showed a different trend dependent upon observed parameter. After freezing, the content of chlorophylls, carotenoids, and total phenolic acids retained similar levels as in control plants and amounted to 14.65 <sup>±</sup> 0.36 mg dw g<sup>−</sup>1, 2.58 <sup>±</sup> 0.05 mg dw g−<sup>1</sup> and 13.75 <sup>±</sup> 0.07 mg dw CEA g<sup>−</sup>1, respectively. At the freezing temperature, total polyphenol content increased 13% and total flavonoids and glucosinolates content decreased 21% and 54%, respectively. Our results suggest that acclimatization (23 h at 8 ◦C) of kale plants can be beneficial for the accumulation of pigments and phytochemicals, while freezing temperatures affect differently specialized metabolite synthesis. The study suggests that growing temperature during kale cultivation must be considered as an important parameter for producers that are orientated towards production of crops with an increasing content of health-related compounds.

**Keywords:** *Brassica oleracea* var. *acephala*; short-term cold stress; phytochemicals; pigments

#### **1. Introduction**

Climate changes that generally cause global warming can cause sudden, low temperature episodes. These types of events can significantly reduce crop production and quality. According to the review article by Ritonga and Chen [1], low temperature is one of the most harmful environmental stresses that higher plants face, along with drought stress. Low temperature stress significantly limits the geographical distribution of plants, but with climate changes and climate disturbances, crops can experience periods of extremely low temperatures uncommon for the region [2]. Crops that experience low-temperature stress suffer impaired growth, which affects and delays essential processes in their development such as flowering, development and fruiting. That may cause loss in crop yields and quality, and in extreme cases, the plant freezes, wilts and dies [2]. Plants differ in their tolerance to chilling (0–15 ◦C) and freezing (<0 ◦C) temperatures, and resistance is speciesor cultivar-dependent. Chilling tolerant plants often grow in temperate climatic regions, and they can increase their freezing tolerance by being exposed to chilling, non-freezing temperatures, a process known as cold acclimation [3]. Plant tolerance to low temperature may involve mechanisms related to carbohydrates, specialized metabolites and energy metabolism, transcription, signal transduction, protein transport and degradation [3–5].

The family Brassicaceae, often called Cruciferae or mustard family, represents a monophyletic group distributed worldwide, except for Antarctica, comprising of approximately 341 genera and 3977 species. The number of genera and species changes frequently in parallel with development of new methods that dissect the genetic diversity of the family in a greater detail [6]. One of the important members is the genus *Brassica*, which includes significant crop plants such as rapeseed and some commonly used vegetables with a long history of agricultural use around the world [7]. One of the key species is *Brassica oleracea* that includes vegetables with significant morphological diversity of plant organs and good adaptation to adverse environmental conditions [8]. According to the morphology and other characteristics *B. oleracea* includes several cultivar groups (Acephala, Botrytis, Capitata group, etc.). Among these groups, plants from Acephala group include leafy, nonheading cabbage varieties with common names of kale and collards. Kale (*B.oleracea* var. *acephala*) is known for its good tolerance of environmental stress such as salt, drought and frost hardiness and resistance [8–14]. Kale is easy and cheap for cultivation and production significantly increased from 3994 to 6256 harvested acres in US, in the time from 2007 to 2012, respectively [15]. In recent years, kale has been marked as a superfood [8] due to its beneficial effects on human health, which are related to the presence of specialized metabolites from the group of polyphenols, carotenoids and glucosinolates, but also important minerals, vitamins and dietary fibres [8,11,13,14]. Kale and collards have higher content of Ca, folate, riboflavin, vitamin C, K and A content than other cruciferous vegetables while their phytochemical content is comparable with other *Brassica* vegetables [8]. Kale possesses biological activities such as antioxidant and anticarcinogens activity, and protective properties in cases of cardiovascular and gastrointestinal system diseases (reviewed by Šamec et al. [8]). Kale tolerates both high summer temperatures and low winter temperatures, even freezing temperatures for a short period of time. Improved understanding of mechanisms related to kale low temperature tolerance can introduce improved cold resistance varieties, better equipped to deal with unusually low temperatures that might arise during the growth season. Low temperatures during kale cultivation have been reported to affect polyphenolic compounds [16,17], soluble sugars and glucosinolates [18], but most of these studies were conducted in the field where it is difficult to control other environmental parameters.

Tolerance of low temperatures in kale is associated with the content of specialized metabolites that can serve not only as a protective mechanism against environmental stressors for the plant, but also as a source of benefit compounds for human health. We hypothesize that phytochemicals and pigments may play a significant role in kale stress adaptation capability and that short term low temperature exposure affects specialized metabolite content. In the present study, we investigated cold induced changes in specialized metabolite content: polyphenols, glucosinolates and carotenoids, under short term chilling temperatures (8 ◦C and −8 ◦C) compared with the control (21 ◦C). In addition, we determined the effect of short cold stress on basic growth physiological parameters (chlorophyll *a*, chlorophyll *b*, total chlorophylls) and stress markers (proline content).

#### **2. Materials and Methods**

#### *2.1. Plant Growing and Stress Experiments*

Seeds were purchased from the family farm "Srdan Frani´ ¯ c", Vrgorac, Croatia. Seeds were sterilized and germinated as we have previously reported [13]. Approximately 20 seeds per plate were transferred to Petri dishes containing 1% agar (*w/v*). Plates were first stored in dark at 4 ◦C for 48 h, then transferred to the growth chamber and maintained at 21 ◦C for three days with a 16/8 h photoperiod. Seedlings were then placed in pots (diameter 55–73 mm; height 105 mm) containing Stender A240 substrate (Stender GmbH,

Schermbeck, Germany) and maintained in a growth chamber at 16/8 h photoperiod (light/dark) at 21 ◦C. All plants were watered regularly with the same amount of water and transplanted after 4 weeks into larger pots (diameter 130–145 mm; height 110 mm) with fresh substrate to ensure sufficient plant nutrients throughout the experimental period. After 8 weeks of growth, 30 healthy representative plants were selected for cold stress experiments.

The experimental design is shown in Figure 1. Plants from all three groups (control, chilling and freezing group) were harvested at exactly the same time, quickly frozen in liquid nitrogen, and stored at −80 ◦C followed by freeze-drying (Lyovac GT 2; Steris Deutschland GmbH, Köln, Germany) for further extraction and analysis.

**Figure 1.** Schematic diagram of the short-term cold experiments.

#### *2.2. Determination of Proline Content*

For proline determination, we extracted 30 mg of freeze-dried tissue with 70% ethanol, as previously reported [10]. For the reaction, a mixture of 100 μL of extract and 1 mL of the reaction mixture (containing 1% ninhydrin, 60% acetic acid, and 20% ethanol) was heated at 95 ◦C for 20 min, followed by cooling on ice. Absorbance was measured at 520 nm against control using a UV–VIS spectrophotometer (BioSpec-1601, Shimadzu, Kyoto, Japan). Quantification of proline was done according to calibration curve of proline (0–1.6 mM) and expressed as μmol g−<sup>1</sup> dw<sup>−</sup>1.

#### *2.3. Determination of Chlorophylls and Carotenoids Content*

To determine concentration of chlorophyll *a*, chlorophyll *b*, total chlorophylls, and carotenoids, 10 mg of freeze-dried tissue was extracted using 80% acetone until tissue discoloration. Samples were centrifuged and supernatants were collected for quantification by spectrophotometric absorbance reading at 663.2 nm for chlorophyll *a*, 646.8 nm for chlorophyll *b*, and 470 nm for carotenoids [19] and expressed as mg g−<sup>1</sup> dw<sup>−</sup>1.

#### *2.4. Determination of Polyphenolic Compounds*

For the determination of the content of polyphenolic compounds, extracts were prepared by mixing 60 mg of dried material with 2 mL of 80% methanol and the extraction was performed as previously reported [14].

For the determination of total polyphenols, total phenolic acids and total flavonoids, we used well-established methods suitable for small-scale volumes and for Brassicacea plants [20]. For the determination of total phenols content, Folin–Ciocâlteu method was used and the calibration curve was constructed using gallic acid as the standard. Results are expressed as milligrams of gallic acid equivalents per gram of dry weight (mg GAE g<sup>−</sup>1dw−1). For the determination of phenolic acids, a mixture of sodium nitrite and sodium molybdate was used and caffeic acid was used to construct the calibration curve. Results are expressed as caffeic acid equivalents per gram of dry weight (mg CAE g−1dw−1). Flavonoids were determined using Al2O3 method, catechin was used as the standard to construct the calibration curve, and results are expresses as catechin equivalents per gram of dry weight (mg CE g−<sup>1</sup> dw<sup>−</sup>1).

#### *2.5. Determination of Total Glucosinolates*

For the determination of total glucosinolates, an established method based on the reaction with sodium tetrachloropalladate II (Na2PdCl4) [21] was adapted as previously reported [14]. Sinigrin standard was used to construct the calibration curve and the results are expressed as sinigrin equivalents per gram of dry weight (mg g−<sup>1</sup> dw<sup>−</sup>1).

#### *2.6. Statistical Analysis*

All analyses were performed in at least three replicates and results are expressed as mean ± standard deviation (SD). All statistical analyses were performed using the free software PAST [22]. One-way ANOVA and post hoc multiple mean comparison (Tukey's HSD test) were performed and differences between measurements were considered significant at *p* < 0.05.

#### **3. Results**

#### *3.1. Effect of Cold Stress on Proline Content in Kale*

Increase in proline content was noticed after exposure to cold stress was strongly related to the drop of the temperature (Figure 2). After 24 h at chilling temperatures (8 ◦C), proline content was 14% higher than in the control plants, with additional increase to 49% in kale exposed to −8 ◦C for 1 h after 23 h of acclimation. The rise of proline, as one of cold stress markers, is a clear indication that kale plants perceive the administered temperatures as cold stress.

**Figure 2.** The proline content in kale plants exposed to cold stress. Value marked with different letters are significantly different at *p* < 0.05.

#### *3.2. Effect of Cold Stress on Content of Chlorophylls and Carotenoids in Kale*

Concentration of chlorophylls in chilled plants showed a significant increase in content of chlorophyll a, b and total chlorophylls (Figure 3), indicating that cold stressed plants increased the rate of synthesis of chlorophylls. In case of previous acclimatization of plants to chilling temperatures followed by exposure for freezing temperatures, the plant's photosynthetic apparatus was less affected and the content of photosynthetic pigments remained similar to the levels recorded for stress free control plants.

**Figure 3.** The content of chlorophyl a (**a**), chlorophyl b (**b**) and total chlorophylls (**c**) in kale exposed to low temperatures. Value marked with different letters are significantly different at *p* < 0.05.

The content of carotenoids in kale under low temperatures is shown in Figure 4. In the control, the content of carotenoids was 2.71 ± 0.06 mg g−<sup>1</sup> and their amount followed the same trend as that of chlorophylls—under 8 ◦C increase, but their content in plants subjected to freezing temperature was comparable with those in the control plants.

**Figure 4.** The content of carotenoids in kale exposed to low temperatures. Value marked with different letters are significantly different at *p* < 0.05.

The ratio of chlorophyll a and chlorophyll b, as well as the ratio of total chlorophylls and total carotenoids could provide useful information about adaptation of plants to stress conditions [19]. Our values for those parameters are shown at Table 1. The ratio of chlorophyll a/chlorophyll b remained stable for under chilling temperatures while significant increase was recorded for plants exposed to freezing temperature. The ratio of total chlorophylls/total carotenoids remained stable under cold stress.

**Table 1.** The ratio of chlorophyll a/chlorophyll b and total chlorophylls/total carotenoids in kale exposed to low temperatures. Value marked with different letters are significantly different at *p* < 0.05.


#### *3.3. Effect of Cold Stress on Content of Polyphenolic Compounds in Kale*

Significant changes in polyphenolic compounds was recorded in plants exposed to freezing temperatures and results are shown at Figure 5. For total phenolic acids, the trend was different and only plants at chilling temperature showed significantly higher content than in the control. For flavonoids, a slight increase was observed at chilling temperature, while total flavonoid content decreased at freezing temperature compared with the control and plants at chilling temperature, suggesting that polyphenolic compounds are main metabolites synthesized during acclimation and counteracting cold stress induced by freezing temperatures.

**Figure 5.** The content of total polyphenols (**a**), total phenolic acids (**b**) and total flavonoids (**c**) in kale exposed to low temperatures. Value marked with different letters are significantly different at *p* < 0.05.

#### *3.4. Content of Glucosinolates under Low Temperature*

The level of glucosinolates was majorly affected by cold stress as is shown at Figure 6. Exposure to chilling temperatures induced an increase in glucosinolates synthesis while a significant drop in glucosinolates content in plants exposed to freezing temperatures was recorded.

**Figure 6.** The content of total glucosinolates in kale exposed to low temperatures. Value marked with different letters are significantly different at *p* < 0.05.

#### **4. Discussion**

Proline is an amino acid that plays an important role in plant metabolism and development. Its accumulation is associated with plant stress response where it acts as an osmolyte, metal chelator, antioxidant defence molecule and signalling molecule (reviewed by Hayat et al. [23]. In the presented study, proline also acted as a protective molecule against cold stress with recorded increase in content correlating to the temperature drop (Figure 2). Similar results were reported for frost-resistant mutants of cauliflower (*Brassica oleracea* var. *botrytis*) by Hady et al. [24] and Fuller et al. [25] Proline accumulation is reported to be associated with cold stress tolerance in many other crops such as beans [26], sorghum [27], and rice as well [28]. Stressful environment cause changes in a variety of physiological, biochemical, and molecular processes in plants, which is evident from the proline levels mentioned above, but it can also affect photosynthesis, the most fundamental and complicated physiological process in all green plants [29]. Photosynthesis is an essential process for plants, and it is very sensitive to changes in environmental conditions [30]. Chlorophylls play important role in photosynthesis in harvesting and converting light energy in the antenna systems and charge separation and electron transport in the reaction centres [31]. Therefore, chlorophyll content is an important parameter to evaluate photosynthetic capacity. The level of chlorophyll *a*, *b*, and total chlorophylls and carotenoids was decreased after exposure to chilling temperature, while staying stable under freezing temperature in similar concentration as in control plants. Freezing temperatures normally cause a decrease in the ratio of total chlorophyll to carotenoids [12], but the acclimatization period in chilling period was much longer, so decrease in chlorophylls can also be a response to long exposure to chilling rather than freezing itself.

The ratio of total chlorophylls and carotenoids is an indicator of the plant greenness [19] and in our experiment chilling temperatures did not change significantly the ratio. The ratio of chlorophyll *a*/chlorophyll *b* may be an indicator of the functional pigment endowment and light adaptation of the photosynthetic apparatus [19]. Under stress conditions in plants, both chlorophyll *a* and chlorophyll *b* tend to decrease while, the chlorophyll *a*/*b* ratio tends to increase due to greater reduction in chlorophyll *b* compared to chlorophyll *a* [29]. Accordingly, our results indicate notable stress under freezing temperatures.

Kale is also considered a good source of carotenoids [32]. Carotenoids are essential pigments in photosynthetic organs along with chlorophylls where they act as photoprotectors, antioxidants, colour attractants, and precursors of plant hormones in non-photosynthetic organs of plants [33]. In addition, they play important roles in humans such as precursors of vitamin A, photoprotectants, antioxidants, enhancers of immunity, and oth-

ers [33]. Under our experimental conditions, in control plants, total carotenoid content was 2.71 ± 0.06 <sup>μ</sup>g g−1, increased to 3.10 ± 0.13 <sup>μ</sup>g g−<sup>1</sup> under 8 ◦C and decreased to 2.58 ± 0.05 <sup>μ</sup>g g−<sup>1</sup> under −<sup>8</sup> ◦C. Similarly, Hwang et al. [34] reported a gradual increase in total carotenoid content of kale during cold acclimatizationon for 3 days. They noted an increase in zeaxanthin content under cold stress. Mageney et al. [32] also reported high zeaxanthin contents in kale under traditional harvest conditions consistent with low temperatures. Consistent with reported papers, our results show that cold acclimatization may be beneficial for carotenoid content accumulation. According to our previously published work comparing the carotenoid content in kale with that of white cabbage and Chinese cabbage, kale tends to accumulate a higher amount of carotenoids, which may be related to its better tolerance to low temperatures [10,11].

Polyphenol compounds are considered an important player in the response of plants to abiotic stress, including low temperature stress [35]. The content of total polyphenols, phenolic acids and flavonoids in our experiments is shown in Figure 6. Polyphenol content differed between chilling and freezing where decrease and significant increase was recorded, respectively. Jurkow et al. [36] also reported increased levels of polyphenols in kale after moderate (−5 ◦C) and severe (−15 ◦C) frost compared to the pre-freezing (>0 ◦C) period. Similarly, Lee and Oh [16] reported increased levels of polyphenolic compounds in 3 weeks old kale exposed to 4 ◦C for 3 days. They also reported an increase in the content of phenolic acids, caffeic acid and ferulic acid, and flavonoid kaempferol after low temperature treatment at 4 ◦C. The same authors in another study [17] observed no differences in total flavonoid content in kale grown at 10 ◦C compared to the control. Our results for total flavonoids content are consistent with these findings; we found no significant changes in total flavonoids and phenolic acids at 8 ◦C, but a decrease in flavonoids at freezing (−8 ◦C) temperature was observed. The duration of exposure to low temperature and prior acclimatization may have an influence on the level of phenolic compounds under low temperature stress [18], and probably this is the reason for different trends published in different papers. In addition, individual phenolic compounds, or their conformation (e.g., conjugates, etc.) may change at low temperatures, which cannot always be observed by measuring total phenolic compounds content [37]. Low temperatures induce changes in cellular ultrastructure and localization of phenolic compounds, as has been reported for rapeseed (*B. napus* var. *oleifera*) leaves [38] and this may also affect polyphenolic compounds extraction and their detection.

Glucosinolates are well-known specialized metabolites characteristic for Brassicacea plants. Their contents in plants exposed to low temperatures compared to the control in our experiments are shown in Figure 6, where we observed an interesting trend. Under chilling temperature their content increased, while under additional freezing temperature their content decreased significantly. This trend is different from the one we reported previously, where the content of glucosinolates increases under chilling and freezing temperatures [12]. This different trend may be due to different experimental conditions and age of plants as reported in our previous work [12], where we exposed plants to freezing temperature for 1 h after 1 week of acclimation. Differences in glucosinolates trend reported in different papers are probably due to different experimental conditions, e.g., temperatures, duration of acclimation, etc., which may influence accumulation of different phytochemicals or be related to the kale variety.

#### **5. Conclusions**

In a present study, 8 weeks old kale plants were exposed to short-term low temperature stress (chilling and freezing) to study changes in proline, phytochemicals and pigments content under low temperature. Increases in proline content indicate that stress protective mechanisms were activated in plants at low temperatures and confirmed proline as a reliable stress marker for low temperature stress. Compared to the control, plants exposed to 8 ◦C (chilling temperatures) for 24 h accumulated significantly higher content of chlorophyll *a*, *b*, total chlorophylls, and carotenoid as well as content of total polyphenols, flavonoids and glucosinolates. This may indicate that short term (24 h) chilling temperature is beneficial for the accumulation of phytochemicals in kale. However, our results also suggest that freezing temperatures (−8 ◦C) may cause significant stress and decrease in pigments and phytochemical levels. Changes in measured phytochemicals indicate that they play significant role in chilling and freezing tolerance in used kale variant, but future more advanced metabolomics and transcriptomic studies will explain the mechanisms in more detail.

**Author Contributions:** Conceptualization, D.Š.; methodology, D.Š.; formal analysis, D.Š.; V.L.; investigation, V.L., D.Š., B.S.-S.; writing—original draft preparation, D.Š.; writing—review and editing, D.Š.; B.S.-S., B.S.-S., E.K.; visualization, D.Š.; supervision, D.Š.; project administration, D.Š.; B.S.-S.; funding acquisition, D.Š.; B.S.-S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Unity through Knowledge Fund, grant number 12/17, and by the Operational Programme Competitiveness and Cohesion 2014–2020 and the Croatian European regional fund under a specific scheme to strengthen applied research in proposing actions for climate change adaptation (Project No. KK.05.1.1.02.0005).

**Institutional Review Board Statement:** Not Applicable.

**Informed Consent Statement:** Not Applicable.

**Data Availability Statement:** Additional data are available upon request.

**Acknowledgments:** We thank Srdan Frani´ ¯ c for providing the seeds used in the experiment.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Effect of Cold Stress on Growth, Physiological Characteristics, and Calvin-Cycle-Related Gene Expression of Grafted Watermelon Seedlings of Different Gourd Rootstocks**

**Kaixing Lu 1, Jiutong Sun 1, Qiuping Li 1, Xueqin Li 2,\* and Songheng Jin 2,\***


**Abstract:** Recently, grafting has been used to improve abiotic stress resistance in crops. Here, using watermelon 'Zaojia 8424' (*Citrullus lanatus*) as scions, three different gourds (*Lagenaria siceraria*, 0526, 2505, and 1226) as rootstocks, and non-grafted plants as controls (different plants were abbreviated as 0526, 2505, 1226, and 8424), the effect of cold stress on various physiological and molecular parameters was investigated. The results demonstrate that the improved cold tolerance of gourd-grafted watermelon was associated with higher chlorophyll and proline content, and lower malondialdehyde (MDA) content, compared to 8424 under cold stress. Furthermore, grafted watermelons accumulated fewer reactive oxygen species (ROS), accompanied by enhanced antioxidant activity and a higher expression of enzymes related to the Calvin cycle. In conclusion, watermelons with 2505 and 0526 rootstocks were more resilient compared to 1226 and 8424. These results confirm that using tolerant rootstocks may be an efficient adaptation strategy for improving abiotic stress tolerance in watermelon.

**Keywords:** watermelon; rootstock; cold stress; antioxidant enzymes; gene expression

#### **1. Introduction**

Cold stress is one of the key environmental factors that severely affect plant growth and development, especially for watermelon (*Citrullus lanatus*). Watermelon is an important global fruit due to its economic importance and nutritional qualities, it contains a great quantity of antioxidants and may mitigate oxidative damage in tissues. Originating from Africa and tropical regions in Asia, watermelon is sensitive to low temperatures. The best growing conditions for watermelon are between 21 and 29 ◦C. When the temperature drops to 10 ◦C, watermelon stops growing and it even dies at temperatures below 1 ◦C [1]. Extreme weather frequently occurs in China, especially during the seedling stage, such as winter, late autumn, and early spring, in which watermelon usually suffers cold stress. Thus, it is hard to grow the fruit and obtain high yields. Therefore, the study of how to improve cold resistance in watermelon has become a vital part of watermelon breeding projects. One way to improve cold tolerance is to graft plants onto rootstocks with higher cold tolerance [2–7].

Grafting originated in Japan in the late 1920s and was initially used to improve resistance to soil pathogens [8]. Since then, the use of grafting has spread around the world. In Korea and Japan, approximately 95% and 92% of the land area, respectively, is cultivated with grafted watermelon [9]. Recently, grafting was proposed as a promising approach to improve tolerance to abiotic stress and can represent an efficient technique for reducing or eliminating losses in production. Over the years, many studies have demonstrated the ability of grafting to enhance salt tolerance in different plants, such as melon (*Cucumis melo* L.) [10], pumpkin (*Cucurbita moschata* D.) [4], cucumber (*Cucumis sativus* L.) [11], grapevines

**Citation:** Lu, K.; Sun, J.; Li, Q.; Li, X.; Jin, S. Effect of Cold Stress on Growth, Physiological Characteristics, and Calvin-Cycle-Related Gene Expression of Grafted Watermelon Seedlings of Different Gourd Rootstocks. *Horticulturae* **2021**, *7*, 391. https://doi.org/10.3390/ horticulturae7100391

Academic Editors: Agnieszka Hanaka, Jolanta Jaroszuk-Sciseł and ´ Małgorzata Majewska

Received: 23 August 2021 Accepted: 30 September 2021 Published: 11 October 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

(*Vitis vinifera* L.) [12], and watermelon [13]. Other studies have demonstrated that grafting induces resistance to high temperatures [14] and water stress [15], improves the yield and quality of watermelon under low potassium supply [16], and enhances nutrient uptake [17], among other things. However, to date, there are few studies concerning the results of grafting with appropriate rootstocks for cold tolerance in watermelon.

Accumulating evidence has demonstrated that certain stresses lead to oxidative stress by overproducing reactive oxygen species (ROS), such as superoxide anions (O2 −) and hydrogen peroxide (H2O2). The improved performance of grafted plants, especially under abiotic stress, is usually associated with the higher accumulation of osmolytes, such as proline, as well as higher antioxidant enzyme activity, such as that of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), which protect the cellular systems from the cytotoxic damage by ROS [18,19]. Moreover, some studies have shown that grafted plants have enhanced tolerance to stress via the induced the expression of some key genes, especially those related to photosynthesis. Li et al. [20] identified novel photosynthetic proteins in grafted cucumber seedlings. Similarly, Yang et al. [21] found that the amelioration of photosynthetic capacity in grafted watermelon seedlings under salt stress might be due to enzymes of the Calvin cycle. They further proved that rootstock grafting watermelon seedlings enhanced the gene expression of enzymes related to ribulose-1,5-bisphosphate (RuBP) regeneration under salt stress [22]. Xu et al. [7], who analyzed the transcriptomic results of grafted watermelon under cold stress, found 702 genes that were differentially expressed, among which 180 genes associated with photosynthesis were downregulated. These studies demonstrated that rootstocks could regulate gene expression patterns, especially those related to photosynthesis, in scions under stress.

Recently, different authors have demonstrated that the effects of grafting on plant growth and stress tolerance depend on different rootstocks [12,13,23,24]. Therefore, selecting suitable rootstocks with higher compatibility and resistance is a promising strategy. However, studies on rootstock screening to improve watermelon's cold tolerance are lacking and urgently required. Due to their vigorous root systems, bottle gourd (*L. siceraria* Standl) and pumpkin have been used as rootstocks by watermelon growers to improve fruit quality and sensory parameters [4]. Recently, some local bottle gourd genotypes that may have potential for use as a rootstock against stress tolerance were selected and bred by the Ningbo Academy of Agricultural Sciences, but no studies have been performed on them. Information about the molecular responses of grafted watermelon to cold stress is also limited. Therefore, this investigation aimed to understand the mechanisms by which grafted watermelon can develop improved cold resistance. The 'ZaoJia 8424' watermelon was chosen for grafting onto three gourd rootstocks to evaluate ROS and osmolyte accumulation, membrane stability, antioxidant defense system, and the expression level of photosynthesis-related genes. The performed study will help to explain cold-tolerant mechanisms in grafted horticultural crops, which may be useful when exploring potential rootstocks to improve watermelon cold tolerance.

#### **2. Materials and Methods**

#### *2.1. Plant Material and Treatments*

The watermelon (*Citrullus lanatus* (Thunb.) Matsum. and Nakai) cv. ZaoJia 8424 was used as a scion, and two cold-tolerant gourds (*L. siceraria* Standl. cv. LS0525, and LS0526) and a cold-sensitive gourd (*L. siceraria* Standl. cv. LS1226) were selected as rootstocks. The rootstock-grafted watermelon plants have been abbreviated as 2505, 0526, and 1226, respectively. The nongrafted watermelon plants (abbreviated as 8424) were used as controls. The watermelon and gourds used here were selected and bred by the Ningbo Academy of Agricultural Sciences of China.

The seeds of the gourds (rootstocks) and watermelons (scion and nongrafted plants) were sown on May 5 and 8 in a sand/soil/peat (1:1:1 by volume) mixture. The scion plants were grafted onto the rootstock six days after the watermelon scion seeds were sown using the method of Lee [25]. To improve graft formation, transparent plastic film

was used to cover the seedlings. All grafted seedlings were kept in the shade for 72 h. To ensure the stability of relative humidity, the plastic film was opened every day and completely removed 7 days after grafting. Then, all different seedlings were grown under the following conditions in the greenhouse: approximately 28/23 ± 1 ◦C (D/N) with 60% relative humidity, a 12 h photoperiod, and a photosynthetic photon flux density (PPFD) of <sup>300</sup> <sup>μ</sup>mol·m−<sup>2</sup> · <sup>s</sup>−. After the third true leaves had fully unfolded, 12 plants of uniform size from each group were transplanted into different pots (one seedling per pot) and cultivated in an illuminating incubator under the controlled environment described above. After another two days of acclimation, the plants were randomly distributed to two identical illuminating incubators; one was a control group, and the other was a cold stress group. The control group remained in the same conditions, while the experimental group was subjected to cultivation for two days with a temperature of 20/15 ◦C (D/N) and then for five days under 10/6 ◦C (D/N). Samples were collected at the same time, i.e., after one week from both control and cold-stressed plants (2-day acclimation period and 5 days under cold exposure). The third leaves from the tops of different groups were harvested and used for H2O2 and O2 − detection, each replicated three times. The remaining leaves were also sampled immediately and frozen in liquid N2 before being kept at −80 ◦C. Leaves from the same position were used to measure the same parameter.

#### *2.2. Chlorophyll Content and Plant Growth Measurements*

Chlorophyll content was measured spectrophotometrically according to Porra et al. [26]. The height and stem diameter of seedlings from each treatment were measured using a ruler and vernier caliper, respectively. The dry weights were determined after drying at 105 ◦C for 10 min and then at 70 ◦C for 72 h. The total number of leaves was counted on each plant.

#### *2.3. Measurements of ROS, MDA, and Proline Content*

Histochemical staining of O2 − was performed using nitroblue tetrazolium (NBT) [27]. Leaf stalks were immediately combined with 0.1% NBT (*w*/*v*) in 25 mmol L−<sup>1</sup> K-HEPES buffer (pH7.8) and kept at 25 ◦C in the dark for 4 h. After the leaves were boiled in 95% ethanol, they were photographed. The different staining colors indicated the different degrees of lipid peroxidation.

Detection of H2O2 in the leaves and measurements of O2 − generation, H2O2, and MDA content were all carried out according to our previous methods [28]. Proline content was analyzed according to the method of Bates et al. [29].

#### *2.4. Antioxidant Enzyme Analysis*

The extraction of antioxidant enzymes and the determination of those enzyme activities (SOD, CAT, and POD) were all carried out according to our previous methods [18], while the activity of GPX was measured following Huang et al. [30]. Electrophoretic separation of POD and CAT bands was performed as described by Lu et al. [18] and Woodbury et al. [31].

#### *2.5. Gene Expression Analysis*

Leaves of different watermelons were used to extract total RNA using the TRI Reagent (Takara Bio Inc. Dalian, China) according to the manufacturer's protocol. Reverse transcription reactions were performed using a PrimeScript RT Reagent Kit with genomic DNA Eraser (Takara Bio Inc. Dalian, China). Suitable primers were designed based on the National Center for Biotechnology Information (NCBI) and the watermelon genome sequence in the cucurbit genomics database. All primers used in this study are provided in Table 1. Quantitative gene expression analysis was performed using a Light Cycler 480 II, Roche Real Time PCR System (Roche Diagnostics Ltd., Basel, Switzerland). qPCR was carried out in a 20 μL reaction mixture containing 1 μL diluted first-strand cDNA, 125 nM of each primer, and 10 μL Lightcycler 480 SYBR Green I Master (Roche Diagnostics GmbH, Mannhein, Germany). The 2−ΔΔCt method was used to determine the relative change in gene expression [32], and melt curve analysis (from 55 to 94 ◦C) was used to determine the specificity of PCR amplification. All measurements were performed in triplicate, and the data are presented as the means and their standard errors.

**Table 1.** Gene-specific primers designed for qRT-PCR.


Note: *rbc*L: Rubisco large subunit (RBCL) gene; *TPI*: oftriose-3-phosphate isomerase gene; *FBPA*: fructose-1,6 bisphosphate aldolase gene; *FBPase*: fructose-1,6-bisphosphatase gene; *SBPase*: sedoheptulose-1,7-bisphosphatase gene; *PRK*: ribulose-5-phosphate kinase gene.

#### *2.6. Statistical Methods*

All data were statistically analyzed with SPSS 13.0 software (SPSS Chicago, IL, USA). Two-way analyses of variance (ANOVA) were used to evaluate the effects of rootstock and cold treatment. Tukey's honestly significant difference (HSD, *p* ≤ 0.05) post hoc test was performed to test the existence of statistical differences between different rootstocks under normal and cold-stressed conditions.

#### **3. Results**

#### *3.1. Growth Parameters*

Under control conditions, all the grafted seedlings exhibited higher growth rates than non-grafted 8424, among which 2505 and 0526 exhibited the best performance trend, followed by 1226 (Table 2). Cold stress caused remarkable changes, and the growth of 8424 was significantly inhibited, whereas the growth inhibition of grafted plants was clearly alleviated, and the level significantly varied depending on rootstock genotypes, among which 2505 and 0526 were better than 1226. All growth parameters under cold stress decreased significantly compared with the control, with 2505 and 0526 having a smaller decrease than 1226. These results indicate that grafting increased the tolerance of watermelon seedlings to cold stress, and the degree of resistance depended on the rootstock. These growth parameters were significantly influenced by both different rootstocks and cold stress, and a significant interaction of rootstock and cold was observed (Table 2).



Values are means of three replicates ± standard error (SE). Small case superscript letters in the same column show statistically significant differences among different rootstocks for the same parameter under normal and cold stress at *p* ≤ 0.05 according to Tukey's tests. *F*S: rootstock effect, *F*C: cold effect, *F*S×C: rootstock × cold interaction effect. \*, \*\*, and \*\*\*: significant at *p* ≤ 0.05, 0.01, and 0.001, respectively.

#### *3.2. Chlorophyll Content*

Photosynthetic pigments play a significant role in plant photosynthesis. Under control conditions, the chlorophyll content of grafted watermelon was significantly higher than that of non-grafted 8424 (Table 3). Under cold stress conditions, chlorophyll content decreased significantly, and the reduction was greater in 8424 (37%) and 1226 (34%) than in 2505 (26%) and 0526 (25%) compared with normal conditions (Table 3). No significant changes were found in the ratios of Chl *a*/*b* under normal conditions, while the ratio of Chl *a*/*b* in grafted seedlings was significantly higher than that in non-grafted 8424 under cold stress.

**Table 3.** Variability of total chlorophyll content and ratio of Chl *a/b* between different graft combinations after cold stress.


Values are means of three replicates ± standard error (SE). Small case superscript letters in the same column show statistically significant differences among different rootstocks for the same parameter under normal and cold stress at *p* ≤ 0.05 according to Tukey's tests. *F*S: rootstock effect, *F*C: cold effect, *F*S×C: rootstock × cold interaction effect. \*, \*\*, and \*\*\*: significant at *p* ≤ 0.05, 0.01, and 0.001, respectively.

#### *3.3. Oxidative Stress Evaluation*

The accumulation of H2O2 in leaves was reflected by necrotic lesions, which were easily detected by chlorophyll bleaching. Obviously, under control conditions (Figure 1A), the accumulation of H2O2 in grafted 1226 and non-grafted 8424 was higher than in grafted 2505 and 0526. Necrotic lesions were mainly located on the leaf base and main veins in 2505 and 0526 leaves, while in 1226 and 8424 leaves, even the small veins had brown spots. Furthermore, significant brown spot accumulation was observed in 1226 and 8424 leaves under cold stress conditions, while this phenomenon was slight in 2505 and 0526 leaves (Figure 1A). The measurement of H2O2 content (Figure 1C) matched the effects of H2O2 accumulation in leaves. Under control conditions, no significant difference was found in H2O2 content between grafted and non-grafted watermelons, whereas cold stress noticeably increased the H2O2 content in 8424 and 1226, which, compared with the controls, increased by 56.3% and 21.1%, respectively.

Figure 1B shows the results of O2 − generation and accumulation in the inoculated leaves by nitroblue tetrazolium staining. The trend in O2 − concentration was similar to that of H2O2. Under control conditions, more dark blue staining was detected in 8424, followed by 1226, whereas very little dark blue staining was found in 2505 and 0526. Cold stress improved the accumulation of O2 −, which was reflected in greater NBT staining spots, especially in 8424, where the blue color was sinificantly deepened. The influence of cold stress on the O2 − production rates of different seedlings was also measured (Figure 1D). Under control conditions, no significant difference was observed between grafted seedlings in terms of O2 − production rate, whereas in 8424, the O2 − production rate was significantly higher. Furthermore, cold stress induced a sharp increase in the O2 − production rate in 8424 (34.0%), while a lower increase was observed in 2505, 0526, and 1226, the rates

for which were increased by 26.8%, 24.3%, and 14.9%, respectively. The interaction of rootstocks × cold stress significantly affected the H2O2 content and O2 − production rate.

**Figure 1.** Effects of cold stress on the accumulation of H2O2 (**A**) and O2 − (**B**), as well as the content of H2O2 (**C**) and O2 − producing rate (**D**) in leaves of different watermelon seedlings. The numbers 0526, 2505, and 1226 represent watermelon grafted onto three different gourds, while 8424 means non-grafted watermelon. Scale bars correspond to 2 cm. Data are the mean ± SE. Bars with different letters indicate a significant difference (*p* < 0.05).

#### *3.4. Lipid Peroxidation and Proline Accumulation*

Lipid peroxidation, reflected by MDA content, usually accompanies ROS accumulation. Under control conditions, the MDA content was significantly higher in 8424 than in grafted seedlings (Figure 2A). A sharp increase was observed when seedlings were exposed to cold stress. The MDA content of 8424 underwent the greatest increase under cold stress (50.0%), and 0526 and 1226 showed intermediate increases (39.5% and 34.1%, respectively). A minimal increase was displayed by 2505 (18.6%). Under control conditions, no significant differences in the level of proline content were observed between grafted and non-grafted seedlings (Figure 2B). Although cold stress promoted leaf proline content, a significantly greater increase was observed in grafted seedlings than in non-grafted 8424. When compared with the corresponding controls, proline accumulation in 2505 and 1226 was increased by 79.2% and 66.9%, respectively, while in 0526 and 8424, the increase was only 57.4% and 49.1%, respectively. Moreover, the interaction of rootstocks × cold stress significantly affected the MDA and proline contents.

#### *3.5. Antioxidant Enzyme Activity*

The effect of cold stress on the activities of SOD, CAT, POD, and GPX in leaves of different watermelons is shown in Figure 3A–D. SOD is the first enzyme in the enzymatic antioxidative pathway. Under control conditions, SOD enzymatic activity was significantly higher in grafted 2505 and 0526 than in grafted 1226 and non-grafted 8424 (Figure 3A). Cold stress decreased the SOD activity of both grafted and self-rooted watermelons compared with the control, while the reduction was less notable in 2505 and 0526 (25% and 28%, respectively) than in 1226 and 8424 (31% and 32%, respectively). In contrast, cold stress increased the CAT activity of both grafted and self-rooted seedlings (Figure 3B). However, the values in grafted watermelons were significantly higher than those in non-grafted 8424

under both control and cold stress conditions. Conversely, the activity of POD was severely inhibited in both grafted and non-grafted seedlings when they were exposed to cold stress (Figure 3C). A lesser decrease was observed in plants grafted onto 0526 (17.7%), 1226 (23.4%), and 2505 (24.5%) than in non-grafted 8424 (30.3%). The GPX activity was reduced by cold stress in non-grafted 8424 (25.2%) (Figure 3D), while in grafted seedlings, cold stress led to a considerable increase in GPX activity (5.5%, 16.8%, and 21.3% for 0526, 2505, and 1226, respectively). Moreover, the activities of those enzymes were all significantly affected by the interaction of rootstocks × cold.

**Figure 2.** Effects of low temperature on MDA (**A**) and proline (**B**) content in leaves of different grafted seedlings. Data are the mean ± SE. Bars with different letters indicate a significant difference (*p* < 0.05).

**Figure 3.** Effect of low temperature on activities of SOD, CAT, POD, and GPX (**A**–**D**) in leaves of different grafted seedlings. Data are the mean ± SE. Bars with different letters indicate a significant difference (*p* < 0.05).

To evaluate the enzyme activity as stimulated by cold stress, polyacrylamide gel electrophoresis analysis of enzyme isoform patterns was performed. No significant differences were found in POD and GPX isoforms (data not shown), but there was a significant differ-

ence in SOD and CAT isoforms (Figures 4 and 5). At least five isoforms were detected in the leaves of different watermelon seedlings under both control and cold stress conditions, while CAT 6 was only found under cold stress conditions. The cold-induced increase in CAT enzyme activity coincided with the increased expression of CAT isoforms. Under both control and stress conditions, the expression of CAT isoforms in grafted watermelons was much higher than that in non-grafted 8424 (Figure 4). Conversely, the expression of SOD isoforms was downregulated by cold stress (Figure 5). Under control conditions, at least nine isoforms were detected in different seedlings, with SOD1–3 and 6–8 being the major ones, while under cold stress conditions, the expression of these isoforms was greatly diminished. Similar to CAT isoforms, the expression of SOD isoforms in grafted watermelons was also much stronger than in non-grafted 8424, among which 0526 displayed the highest expression, followed by 2505 and 1226. The differences observed in the SOD isoform patterns match the changes in total SOD activity described above.

**Figure 4.** CAT isozymes in leaves of different grafted seedlings (different numbers represent different bands).

**Figure 5.** SOD isozymes in leaves of different grafted seedlings (different numbers represent different bands).

#### *3.6. Gene Expression*

Quantitative expression analyses were used to evaluate the association of the selected genes with cold resistance, as well as their differential expression under stress conditions in different watermelon seedlings, and the results are shown in Figure 6. Grafting increased

the expressions of *Rbc*L, *TPI*, *FBPA*, *SBPase*, and *PRK*, whereas the expression of *FBPase* was only slightly upregulated in 0526, while it decreased in 2505 and 1226. The expression of *SBPase* was significantly upregulated by cold stress in both grafted and non-grafted seedlings, while the expressions of *TPI*, *FBPA*, and *SBPase* were only enhanced by cold stress in grafted watermelons and decreased in non-grafted 8424. The expressions of *Rbc*L and *PRK* were reduced in all four seedlings by cold stress. The expression of these six genes in grafted watermelons was higher than those in non-grafted 8424 under cold stress. Moreover, the interaction of rootstocks × cold significantly affected the expressions of *Rbc*L, *TPI*, *FBPA*, and *SBPase.*

**Figure 6.** qRT-PCR analysis of the studied genes. Total RNA was extracted from scion leaves, converted to cDNA, and subjected to comparative real-time RT-PCR quantification. Relative transcript levels from qRT-PCR are the mean ± SE of three replicates. Bars with different letters indicate a significant difference (*p* < 0.05).

#### **4. Discussion**

Grafting is a widely used technique in plants for improving tolerance to abiotic stress [10–13]. The results of the present study showed that grafted plant growth was significantly better than that of non-grafted 8424, especially under cold stress (Table 1). Furthermore, plants grafted on 2505 and 0526 rootstocks showed better tolerance to cold stress than the plants grafted onto 1226. The results demonstrate that grafting improved watermelon tolerance to cold stress, but the inhibition level varied depending on rootstock genotypes. Similarly, Ramón et al. [17] reported that sweet pepper (*Capsicum annuum* L.), when grafted with appropriate rootstocks, could overcome the negative effects of heat stress conditions. Yan et al. [13] also stated that the use of 'Kaijia No.1' rather than 'Jingxin No.2' or 'Quanneng Tiejia' improved watermelon salt tolerance. In the present study, the superior performances of 2505 and 0526 under cold stress conditions were attributed to the use of an appropriate rootstock, which can absorb more water and nutrients than 8424 and 1226. Our results indicate that rootstock genotypes might play a crucial role in resistance to abiotic stress.

The chlorophyll content reflects the physiological status of plants and is closely related to photosynthetic potential and primary production. The present study shows that the chlorophyll content increased significantly in grafted plants under normal conditions (Table 2), while no significant difference was found in the ratio of *Chl* a/b. Under cold stress conditions, a reduction in chlorophyll content was observed in 8424 and grafted watermelons, whereas these values were higher in the latter, indicating that the grafted watermelon alleviated the photosynthetic inhibition induced by cold stress. Similarly, Yan et al. [13] showed that the chlorophyll content increased in grafted watermelons under salt stress conditions, and Tao et al. [33] also indicated that cucumber plants grafted onto bitter melon (*Momordica charantia* L.) had higher chlorophyll contents than self-grafted plants. The reduced chlorophyll content in stressed plants could be attributed to decreased chlorophyll synthesis, faster chlorophyll degradation, or both.

Environmental stress leads to the accumulation of ROS, which are highly toxic to plants, causing necrotic symptoms [34]. The ROS concentration is a good indicator of oxidative stress. Our results show the greater accumulation of H2O2 and O2 − in nongrafted 8424 (Figure 1), whereas in gourd-grafted plants, this accumulation was relatively lower, indicating that gourd grafting alleviated the inhibition of oxidative stress. This difference was even clearer under cold stress conditions. Cold stress induced more severe oxidative stress in non-grafted 8424 plants than in grafted plants, as manifested by their high ROS accumulation. Among the three rootstocks, 2505 and 0526 were considered more cold-resistant than 1226, which suggests that the different degrees of oxidative stress induced by cold stress were closely related to the rootstock genotype. Similar observations for cold tolerance in grafted grapevines (*Vitis vinifera* L.) [35] and salt tolerance in grafted watermelons [13] have been reported, whereby plants using tolerant rootstocks displayed lower ROS accumulation than those using sensitive rootstocks.

An increase in ROS accumulation under abiotic stress parallels increased lipid peroxidation. MDA, as the final product of peroxidation, is responsible for cell membrane damage [36]. Our results show that, under control conditions, the degree of lipid peroxidation in 8424 and 1226 was significantly higher than that in 0526 and 2505, while upon exposure to cold stress, the MDA content in 8424 increased more pronouncedly than that in grafted watermelons (Figure 2A), indicating that the damage caused by cold injury in 8424 was more serious than that in grafted watermelons. Among the tested rootstocks, 2505 showed the lowest increase. The results confirm that rootstock grafting enhanced the cold tolerance of watermelon, but this was genotype dependent. These findings validate the results of previous investigations, wherin chilling injury increased MDA accumulation [19].

As a compatible solute, proline plays an important role in protecting enzymes from denaturation in order to stabilize the machinery of protein synthesis [37]. Proline accumulation during stress conditions was also thought to be correlated with tolerance. Our results also showed that cold stress promoted the accumulation of proline (Figure 2B), especially

in grafted seedlings, indicating that grafting improvs the cold tolerance of watermelon by promoting proline accumulation. These results are also similar to those of Krasensky and Jonak [38], who found that proline was accumulated under abiotic stress conditions in many plant species.

To alleviate oxidative damage under stress conditions, plants have developed a series of effective detoxification mechanisms, among which antioxidant enzymes play an important role [15,35]. In the present research, the activities of these antioxidant enzymes (SOD, CAT, POD, and GPX) were higher in grafted seedlings than in the non-grafted 8424 (Figure 3), which indicates that grafted seedlings counteracted oxidative stress by elevation the antioxidant enzyme activity so as to scavenge ROS and protected the membrane from damage. Compared to normal conditions, the CAT and GPX activities increased when plants were exposed to low temperatures, while the POD and SOD activities decreased. Among the three different rootstocks, 2505 and 0526 showed higher increments and lower reductions. This suggests that CAT and GPX might play a crucial role in antioxidant defense. The cold-induced increase in CAT enzyme activity was likely due to the enhanced expression of CAT isoforms (Figure 4), while the decrease in SOD enzyme activity was also correlated with the decreased expression of SOD isoforms (Figure 5). Similar results were also obtained in other studies, which reported that CAT played an important role in alleviating the oxidative injuries induced by low temperatures [39,40]. Other studies found that SOD activity [39] and POD activity [41] increased under cold stress conditions. Recently, Yan et al. [13] reported that SOD activity, as well as CAT, POD, and APX activity, were increased in watermelon grafted onto P2, which suggests this grafted seedling had a higher H2O2 scavenging capacity than those using other rootstocks. These contrasting reports indicate that different plants might employ different pathways to cope with oxidative stress. Here, CAT and GPX might play a more important role in impeding the accumulation of ROS under cold stress.

Abiotic stress is known to influence gene expression. As the last rate-limiting step in carbon fixation, the carboxylated activity of Rubisco is closely related to the Rubisco large subunit (RBCL) and RuBP [42]. In this study, the transcript levels of the Rubisco large subunit (*Rbc*L) and enzymes involved in RuBP regeneration were analyzed (Figure 6). As shown in Figure 6, the higher transcript level of *Rbc*L in grafted plants, especially under stress conditions, implies the existence of a correlation between cold tolerance and *Rbc*L expression, while the latter ameliorates the photosynthetic capacity. As the main genes encoding enzymes required for RuBP regeneration, triose-3-phosphate isomerase (TPI) and fructose-1,6-bisphosphate aldolase (FBPA) catalyze two triose-3-phosphates to fructose-1,6 bisphosphate (FBP). Fructose-1,6-bisphosphatase (FBPase) catalyzes the hydrolysis of FBP to fructose-6-bisphosphatase (Fru6P), while sedoheptulose-1,7-bisphosphatase (SBPase) catalyzes the conversion of sedoheptulose-1,7-bisphosphate (SBP) to sedoheptulose 7 phosphate (Sed7P). Ribulose phosphate epimerase (RuPE) and ribulose-5-phosphate kinase (PRK) are the last rate-limiting enzymes in RuBP synthesis. Except for *RuPE*, which was almost unchanged between different gourd-grafted and non-grafted plants (data not shown), and *FBPase*, in which there was no significant difference between plants under control conditions, the other genes of gourd-grafted plants were expressed much more highly than those of non-grafted plants, especially under cold stress. These results imply that gourd-grafted watermelons could sense cold-induced changes early, and reponse with quick regulations at the transcript level so as to guarantee greater RuBP regeneration, which would activate the carboxylated activity of Rubisco and result in higher carboxylation efficiency.

#### **5. Conclusions**

The selection of stress-tolerant rootstocks might be a promising approach to alleviate the detrimental effects of abiotic stress on watermelon productivity. In the present study, the results show that the cold-induced inhibition of growth was significantly ameliorated in gourd-grafted watermelons, as manifested by physiological indices, such as much better

growth parameters; much higher chlorophyll and proline contents; lower levels of ROS and lipid peroxidation; higher antioxidant enzyme activities, especially CAT and GPX; and higher expression levels of enzymes related to the Calvin cycle. Overall, as evidenced by the presented data, watermelon grafted with 2505 and 0526 rootstocks showed better resilience than those grafted with 1226 and 8424.

**Author Contributions:** K.L. performed the experiments. K.L. and J.S. wrote the manuscript. Q.L. participated in the low temperature stress treatment and the determination of physiological characteristics. X.L. participated in organization and discussion regarding the manuscript. X.L. and S.J. designed the research. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Key Research and Development Program of China, grant number 2019YFE0118900, the Zhejiang Provincial Natural Science Foundation of China, grant number LY18C06000, and Ningbo Natural Science Foundation, grant number 2017A610293.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All datasets generated for this study are included in the article.

**Acknowledgments:** We thank the National Key Research and Development Program of China (2019YFE0118900), the Zhejiang Provincial Natural Science Foundation of China (LY18C06000) and Ningbo Natural Science Foundation (2017A610293) for supporting this work.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Article* **Exogenous EBR Ameliorates Endogenous Hormone Contents in Tomato Species under Low-Temperature Stress**

**Parviz Heidari 1,\*, Mahdi Entazari 1, Amin Ebrahimi 1, Mostafa Ahmadizadeh 2, Alessandro Vannozzi 3, Fabio Palumbo <sup>3</sup> and Gianni Barcaccia <sup>3</sup>**


**Abstract:** Low-temperature stress is a type of abiotic stress that limits plant growth and production in both subtropical and tropical climate conditions. In the current study, the effects of 24-epi-brassinolide (EBR) as analogs of brassinosteroids (BRs) were investigated, in terms of hormone content, antioxidant enzyme activity, and transcription of several cold-responsive genes, under low-temperature stress (9 ◦C) in two different tomato species (cold-sensitive and cold-tolerant species). Results indicated that the treatment with exogenous EBR increases the content of gibberellic acid (GA3) and indole-3-acetic acid (IAA), whose accumulation is reduced by low temperatures in cold-sensitive species. Furthermore, the combination or contribution of BR and abscisic acid (ABA) as a synergetic interaction was recognized between BR and ABA in response to low temperatures. The content of malondialdehyde (MDA) and proline was significantly increased in both species, in response to low-temperature stress; however, EBR treatment did not affect the MDA and proline content. Moreover, in the present study, the effect of EBR application was different in the tomato species under low-temperature stress, which increased the catalase (CAT) activity in the cold-tolerant species and increased the glutathione peroxidase (GPX) activity in the cold-sensitive species. Furthermore, expression levels of cold-responsive genes were influenced by low-temperature stress and EBR treatment. Overall, our findings revealed that a low temperature causes oxidative stress while EBR treatment may decrease the reactive oxygen species (ROS) damage into increasing antioxidant enzymes, and improve the growth rate of the tomato by affecting auxin and gibberellin content. This study provides insight into the mechanism by which BRs regulate stress-dependent processes in tomatoes, and provides a theoretical basis for promoting cold resistance of the tomato.

**Keywords:** cold stress; cold-responsive genes; anti-oxidants; proline; malondialdehyde; hormone profiling

#### **1. Introduction**

Low-temperature stress in plants, categorized as freezing stress or chilling stress, is one of the main environmental stresses that adversely affects plant production across the world, especially in subtropical and tropical climate conditions. This environmental extreme is escalating due to global climate change and is, therefore, threatening sustainable crop production [1,2]. Cold stress impacts the photosynthetic system, impairing the cycle of carbon reduction, the thylakoid electron transport, and the stomatal control of CO2, providing enhanced accumulation of sugars, lipids peroxidation, and water balance disturbance [3–6]. Moreover, a low temperature negatively impacted plants, especially in regards to macromolecules activity, altering the fluidity of the membrane, and reducing osmotic

**Citation:** Heidari, P.; Entazari, M.; Ebrahimi, A.; Ahmadizadeh, M.; Vannozzi, A.; Palumbo, F.; Barcaccia, G. Exogenous EBR Ameliorates Endogenous Hormone Contents in Tomato Species under Low-Temperature Stress. *Horticulturae* **2021**, *7*, 84. https:// doi.org/10.3390/horticulturae7040084

Academic Editors: Agnieszka Hanaka, Jolanta Jaroszuk-Sciseł and ´ Małgorzata Majewska

Received: 9 March 2021 Accepted: 12 April 2021 Published: 17 April 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

potential of the cell [7]. Regarding the metabolic processes and pathways—cold stress affects antioxidant enzyme activities, membrane fatty acid compositions, and adjusting of the redox state and gene expression [8].

Once plants are exposed to stresses, such as cold stress, different kinds of reactive oxygen species (ROS) are generated, which can undertake a series of oxidation–reduction reactions. Plants defend themselves by enzymatic and non-enzymatic antioxidants [9]. An alternative defense strategy could be supplementing hormones, which could enhance antioxidant and detoxification ability in order to cope and tolerate stressful conditions [10,11]. Previous studies identified numerous hormones and signaling molecules associated with plant responses to particular stress. For instance: ethylene engaged in red light-induced anthocyanin biosynthesis in cabbage [12], the antioxidant system, and ABA in brassinosteroidinduced water stress tolerance of grapevines [13]. Coordination of signaling molecules and hormones positively influences the plant's responses to stress and ultimately its preservation in unfavorable conditions [14–16]. Moreover, brassinosteroids (BRs) as steroidal hormones are involved in an array of physiological and developmental processes via their active engagement in processes, such as antioxidant metabolism [4,17,18], photosynthesis [4,19], nitrogen metabolism [20,21], plant–water relations [22], and osmolyte accumulation [23] in various conditions [1,24–26]. Treatment with 24-epi-brassinolide (EBR) regulates the ascorbate–glutathione (AsA–GSH) component cycle in low-temperature stress on a temporal basis, leading to increased low-temperature tolerance in grapevines at the seedling stage [7]. Transcriptome analysis revealed that treatment with EBR in cold conditions raises the transcript levels of genes related to photosynthesis and chlorophyll biosynthesis, including those encoding for photosystem II (PSII) oxygen-evolving enhancer protein, photosystem I (PSI) subunit, light-harvesting chlorophyll protein complexes I and II, and ferredoxin [27].

Furthermore, BRs illustrated engagement in the regulation of ROS metabolism through the expression of many antioxidant genes that enhance the activity of antioxidant enzymes, such as catalase (CAT), superoxide dismutase (SOD), and peroxidase (POX) [17]. Under low-temperature conditions, plants, by active BR signaling and accumulation of the activate Brassinazole resistant 1 (BZR1) (a BR signaling positive regulator protein), elevate the respiratory burst oxidase homolog 1 *(RBOH1)* transcript levels and the apoplastic H2O2 production [28]. The *RBOH1* encodes NADPH oxidase that is involved in ROS in the apoplast, mainly for signaling purposes [25] Moreover, crosstalk between the alternative oxidase (AOX) pathway and BR plays a pivotal role in ameliorating plant tolerance to cold stress, and it has been shown that BR-induced AOX synthesis protects photosystems by bounding ROS synthesis exposed to low-temperature stress [29]. In young grapevine seedlings, foliar application of 24-epi-brassinolide adjusted proteins, free proline contents, and soluble sugars activates the antioxidant machinery to increase chilling stress tolerance [30].

Tomato is a popular garden fruit worldwide because of its edible fruits, rich in antioxidants, and capable of fighting against ROS. Overexpression of DWARF or exogenous EBR application enhances low-temperature tolerance by diminishing oxidative damage in tomato plants [31]. It is worth noting that ROS may also act as a signal in mediating BR-adjusted responses in low-temperature tolerance [32]. A previous study showed that, to protect the plants from oxidative damage, glutaredoxin (GRX), 2-cysteine peroxiredoxin (2-Cys Prx), and RBOH1 participate in a signaling cascade to mediate BR-induced low-temperature tolerance in tomatoes [31]. It was shown that BRs can interact with auxin, salicylic acid, cytokinin, abscisic acid, jasmonic acid, gibberellin, and ethylene, in controlling several morpho-physiological processes in plants [33]. The objective of the current study was to evaluate the effects of 24-epi-brassinolide (EBR) treatment on hormone content, antioxidant activity, the content of malondialdehyde (MDA) and proline, and gene expression of cold-responsive genes on the tomato species under low-temperature stress. This study provides insight into the mechanisms by which BRs regulate stressdependent processes in tomatoes and provides a theoretical basis for promoting cold resistance in tomato.

#### **2. Materials and Methods**

#### *2.1. Plant Materials and Growth Condition*

In this study, seeds of the two contrasting tomato species, cold-sensitive (*Solanum lycopersicum* cv. 'Moneymaker') and cold-tolerant (*S. habrochaites*, Accession 'LA1777') [34], were selected to investigate the effects of BR on tomato seedlings under lowtemperature stress. Firstly, seeds were sterilized using 2% sodium hypochlorite solution for 12 min and then washed with double distilled water and dried. The three sterilized seeds of each species were sown in plates containing 50% vermicompost and 50% perlite. In this study, 30 plates were used for each tomato species. The plates were maintained in a growth chamber at 23 ± 2 ◦C with the 16 h light/8 h dark cycling. After 40 days, tomato plates of each species were divided two groups; half of them were sprayed with 5 mg/L of 24-epi-brassinolide (EBR) and repeated after 6 h. After 3 h from the last spraying, each treatment was divided into two groups; the first group was transferred to a growth chamber at 9 ± 1 ◦C and the second group was maintained at 23 ± 1 ◦C. After three days, the leaves of each sample were cut and stored in liquid nitrogen and transferred to −80 ◦C for the next analysis. In the present study, control plants were cultivated at 23 ◦C and without spraying EBR.

#### *2.2. Hormone Profiling*

To analyze the free forms of the hormones, including abscisic acid (ABA), indole-3 acetic acid (IAA), and gibberellin (GA3), the young leaves (2.0 g) of each treatment were well-powdered using liquid nitrogen and then samples were crushed by cold methanol. The extract was achieved using 30 mL of 80% cold aqueous methanol in darkness at 4 ◦C. To determine the hormone content, 10 μL of the extract was injected. The concentration of each hormone was determined using HPLC (Unicam, Cambridge, UK) with a C18 reverse-phase column (4.6 × 250 mm Diamonsic C18, 5 μm, PerkinElmer, Ohio, USA) and column temperature was 35 ◦C, gradient elution, mobile phase in methanol, and 1 mL/min flow rate at a wavelength of 254 nm. The peak area of the standard was considered to determine the sample concentration. Moreover, the standards of ABA, IAA, and GA3 were received from Sigma–Aldrich (Steinheim, Germany). The content of IAA and GA3 was measured based on the method defined by Tang et al. [35]. The content of ABA was determined according to the method characterized by Li et al. [36].

#### *2.3. Lipid Peroxidation Assay and Proline Content*

The malondialdehyde (MDA) content has been identified as a marker of lipid peroxidation rate associated with oxidative stress. In the current study, 200 mg of fresh leaves were homogenized using 1% TCA (*w*/*v*). The MDA content was measured according to the method defined by Campos et al. [37]. Moreover, 0.5 g of leaves were homogenized by 10 mL of 3% sulfosalicylic acid to determine the proline content in each sample. In the current study, the free proline content was analyzed using a method described by Zhang and Huang [38].

#### *2.4. Enzyme Activity*

The tomato leaves (300 mg) were ground to a powder in liquid nitrogen and mixed in 3 mL of 0.1 M extraction phosphate buffer (pH 7.5) and the mixed sample was shortly vortexed. The homogenized samples were centrifuged at 13,000 rpm for 15 min at 4 ◦C. The supernatant of each sample was transferred to determine the enzyme activities. The glutathione peroxidase (GPX; EC 1.11.1.9) activity was distinguished using a method described by Mittova et al. [39], and catalase (CAT; EC 1.11.1.6) activity was measured as described by Aebi [40].

#### *2.5. RNA Extraction and Real Time PCR*

The leaves of tomato seedlings were well powdered in liquid nitrogen and the total RNA of each sample was extracted using RNX TM-Plus (SinaClon, Tehran, Iran), based on

the manufacturer's protocols. The extracted RNA samples were then treated by RNase-free DNase I (Thermo Fisher Scientific, Wilmington, MA, USA). The Nano Photometer (Implen N50, Munich, Germany) and a 1% (*W/V*) agarose gel were used to check the quality and quantity of extracted RNA samples. The cDNA was synthesized using 1 μg total RNA and M-MULV reverse transcriptase (Thermo Fisher Scientific, Wilmington, MA, USA) based on the instructions of manufacture. The real-time PCR reactions were run using RealQ Plus 2x Master Mix Green High ROXTM (Ampliqon, Odense, Denmark) on an ABI StepOne system. In this study, four genes belonging to the apetala2/ethylene responsive factor (AP2/ERF) gene family, involved in response to low-temperature stress [41] and the inducer of CBF expression 1 (ICE1), as a key transcription factor gene involved in cold stress tolerance [42], were selected to study the expression patterns by real-time PCR. The *elongation factor 1α* (*EF-1α*; *Solyc06g005060*) gene, as an internal control gene, was used to calculate the relative expression of target genes. The specific real-time PCR primers of genes were designed and evaluated by the online Primer3 Plus tool (Table 1). Finally, the relative expression levels of selected genes were calculated using the 2−ΔΔ*C*<sup>t</sup> method [43].

**Table 1.** List of used primers in real-time PCR reactions.


#### *2.6. Statistical Analyses*

All experiments were run in triplicate with three technical replicates, and the effect of the low temperature and EBR treatments on analyzed variables within each species was analyzed by one-way ANOVA and Tukey test using Minitab software (version 17). The final graphs were created using Prism 6 software (GraphPad Software Inc., San Diego, CA, USA) based on the average of each treatment and the standard deviation (SD).

#### **3. Results**

#### *3.1. Effects of EBR on Endogenous Hormones in Tomato Leaves Exposed to a Low Temperature*

After three days of exposure to low-temperature stress, the content of both GA3 and IAA hormones significantly decreased in cold-sensitive species, but not in cold-tolerant species, although a slight decrease of IAA content under stress was still observed (Figure 1). Exogenous EBR treatment could significantly increase the content of GA3 and IAA in coldsensitive species in comparison to the control under low-temperature stress. Moreover, the ABA content in both tomato species significantly increased in response to low-temperature stress. A sharp increase in the ABA content was observed in cold-sensitive species that received EBR treatment compared with the control under the low-temperature stress. The treatment with EBR had different effects in cold-sensitive and cold-tolerant species. In fact, although the trend of accumulation in response to a low temperature was similar to that observed in an untreated plant (-EBR), in the cold sensitive species, the treatment with EBR did not significantly affect the GA3, ABA, and IAA content in unstressed conditions, with respect to untreated plants (-EBR). However, it led to a higher accumulation of hormones under stress with respect to untreated plants. The opposite was true for cold-tolerant

species: in all cases (IAA, ABA, GA3), treatment with EBR led to a higher accumulation of hormones in unstressed plants with respect to untreated ones, whereas it did not affect the hormone concentration in stressed plants.

**Figure 1.** Profile of hormone content (in nanomoles per gram fresh weight (nmol/grfw)) of tomato leaves in response to temperature change and EBR application. Different letters above a bar show significant difference according to the Tukey's range test at *p* < 0.05.

The ABA/GA3 and ABA/IAA ratio increased in both species after three days of exposure to low-temperature stress (Figure 2). Interestingly, the ABA/GA3 and ABA/IAA ratio in cold-tolerant species were higher than the cold-sensitive species. However, the results of the current study revealed that EBR could not affect the ABA/GA3 and ABA/IAA ratio.

**Figure 2.** The ration of ABA/IAA and ABA/GA3 under low-temperature stress and EBR application.

#### *3.2. MDA and Proline Are Increased By a Low Temperature*

The content of MDA and proline significantly increased in both species, especially in the cold-tolerant species in response to low-temperature stress (Figure 3). Interestingly, EBR treatment could not affect the content of MDA and proline when compared with the control under normal temperature and low-temperature stress. However, in general, the EBR treatment slightly reduced the content of MDA and proline.

**Figure 3.** The profile of MDA and proline content under low-temperature stress and EBR treatment. Different letters above a bar show significant difference according to the Tukey's range test at *p* < 0.05.

#### *3.3. Effects of EBR on Activity of Antioxidant Enzymes*

According to the results of antioxidant activity, EBR treatment could affect the GPX activity in both tomato species (Figure 4). In normal conditions (23 ◦C), the EBR treatment increased the GPX activity compared to the untreated plants in both species, whereas in low-temperature stress, the treatment significantly enhanced GPX activity only in the cold-sensitive species. EBR treatment showed different effects in the tomato species under low-temperature stress (Figure 4). The cold-sensitive species, in plants not treated with EBR, showed an increase in CAT activity under cold stress, whereas the treatment with EBR seemed to impair the CAT response to cold. Interestingly, EBR treatment significantly increased the CAT activity in the cold-tolerant species.

#### *3.4. Effect of EBR on Expression Pattern of ERF Genes*

The expression pattern of four members of the ERF multigenic family, together with the MYC-like bHLH transcriptional activator ICE1, was investigated. The expression levels of ERF genes, as well as the ICE1 gene, were significantly affected by low temperatures compared to normal temperatures (Figure 5). Most studied genes were significantly upregulated in both species in response to low-temperature stress while the expression pattern of ERF13 was sharply downregulated in the cold-tolerant species. Furthermore, EBR treatment increased the expression levels of the ERF2, ERF13, ERF.B13, and ICE1 genes in the cold-sensitive genotype comparing to the control, while they were not affected by EBR treatment in the cold-tolerant species. Our results revealed that studied genes are involved in response to low-temperature stress and BR may associate with cold tolerance.

**Figure 5.** Expression patterns of studied genes under a low temperature (CS) and EBR treatment application; \* and \*\* above a bar shows a significant difference between the applied treatments and normal temperature treatments as control (C) at *p*-value < 0.05 and *p*-value < 0.01, respectively (according to Student's *t*-test).

#### **4. Discussion**

#### *4.1. EBR Improves Cold Tolerance by Affecting ABA Content*

Various interactions between plant hormones induce a heterogeneous network of plant responses that make it challenging to predict plant performance in response to adverse conditions [44,45]. Moreover, BR can regulate stress responses by cross-talking with other phytohormones [33,46]. In this study, the synergetic interaction was observed between BR and ABA in response to low-temperature stress, where endogenous ABA content significantly increased in the cold-sensitive species under low-temperature stress and EBR treatment. However, the ABA/GA3 and ABA/IAA ratios were not influenced by EBR application. Abscisic acid (ABA) is known as a stress hormone that is influenced by stress and raises plant durability during abiotic stresses, such as drought and cold stress [47]. Moreover, ABA can decrease the damage of dehydration by closing the stomatal pore and maintaining the cellular water [48,49]. However, several antagonistic effects have been observed between signaling components of BRs and ABA under different stress conditions [47,50]. One well-known case of crosstalk occurs at the GSK3-like kinase BIN2 (BRASSINOSTEROID-INSENSITIVE 2), which inhibits the signaling components of the BR pathway, but can be activated by ABA [51]. Moreover, it was stated that ABA negatively controls the BR signaling pathway via phosphorylation of BES1 (bri1-EMS-SUPPRESSOR 1) as a BR signaling positive regulator [52]. Furthermore, Divi et al. found that BR effects are masked by ABA in *Arabidopsis* responses to heat stress, and only in the ABA-deficient *aba1-1* mutant, BR application could make the positive effect [53]. On the other hand, Bajguz stated that BR can enhance the ABA content in *Chlorella vulgaris* under stress conditions [54]. Overall, it seems that the interaction between ABA and BR plays important role in increasing stress tolerance through controlling the synthesizing antioxidants, photosynthesis, and expression of stress response genes [55]. Our results indicated that application of BR is involved in low-temperature stress tolerance by directly/indirectly affecting the ABA content of the tomato species.

#### *4.2. EBR Application Affects the Auxin and GA Content under Low-Temperature Stress*

The synergetic interactions are stated between BR and auxin in regulating the cellular processes related to growth, such as cell proliferation and cell expansion [53,56,57]. Furthermore, it was defined that BR and GA are involved in several common cellular

processes and BR can regulate cell elongation from GA metabolism [58]. In the current study, the content of IAA and GA was significantly decreased in cold-sensitive species under low-temperature stress. The decrease in the content of GA and IAA through cold stress limits the plant growth and lets it withstand adverse environmental conditions, such as cold, salt, and osmotic stress [59,60]. Moreover, previous studies stated that the IAA content is decreased in response to abiotic stresses, including salinity and cold stress [61,62]. Cold stress can inhibit the activity of acropetal auxin transport by controlling the PIN2 as an auxin efflux carrier [62]. It seems that the transport of auxin from root to shoot is reduced in tomato seedlings under cold stress. Furthermore, the ABA/GA3 and ABA/IAA ratio were increased under low-temperature stress that revealed an antagonist interaction between ABA with auxin and GA in response to low-temperature stress.

The content of GA3 and IAA was significantly increased by EBR application in the coldsensitive species, compared with the control, under low-temperature stress. Various studies on the role of plant hormones in response to adverse conditions have been performed, but the exact interaction between BR with auxin and GA has not yet been determined, based on molecular information. The expression of many target genes that are involved in growth processes and stress response are commonly controlled by both BR and auxin [57,63,64]. Furthermore, BR and auxin may be involved in the induction of the phosphoinositide and calcium–calmodulin signaling as a second messenger in cellular signal transduction [57]. In the present study, the IAA content increased under low-temperature stress. It seems that BR might affect the polar transporter of auxin [65], and under low-temperature stress, the auxin transfer may increase from root to shoot. Moreover, BR can induce the expression of genes involved in GA biosynthesis, such as *GA3ox-2* [58]. Furthermore, BR can interact with DELLAs, as the GA suppressors, from BZR1, a BR signaling positive regulator [66–68]. In general, it seems that the application of EBR may affect the GA biosynthesis and increase the GA content in the tomato seedlings under low-temperature stress. Overall, the use of EBR treatment as a stimulant may induce some cellular signaling pathways associated with stress tolerance and reduce the adverse effects of stress on growth by increasing the content of growth-regulating hormones, such as GA and auxin.

#### *4.3. MDA and Proline Are Not Affected by EBR Treatment*

Abiotic stresses, such as low-temperature stress, hurt the cell membrane through enforced lipid peroxidation and membrane oxidation [11,69]. Antioxidant enzymes activity and the proline content were enhanced by the 28-homobrassinolide treatment in the *Brassica juncea* under cadmium stress. Moreover, the content of proline in roots was higher than in the leaves [70]. The content of MDA under salinity stress in rice seedlings was reduced by EBL treatment [71]. In this study, we discovered that MDA content significantly enhanced in the cold-sensitive species in low-temperature conditions, showing that the plasma membrane was affected and lipid peroxidation increased. In the same line, the increased activities of the antioxidant systems, as a result of BR applications, remarkably defeat the chilling injury of the tomato species by minimizing membrane lipid peroxidation in stress conditions. Moreover, proline content increased in response to low-temperature stress. During stress, proline, as an osmolyte, plays a critical role in controlling cell turgor and stability of membranes [72]. Furthermore, proline can reduce lipid peroxidation and acts as an antioxidant to overcome the oxidative stress created by cold stress [72,73]. Application of brassinosteroid in peppermint (*Mentha piperita* L.) under salinity hampered the death of the plant even at severe stress (150 mM) and prevented negative impact of salinity stress through elevating the activities of antioxidant enzymes and reducing the lipid peroxidation [74]. Moreover, our results revealed that the content of MDA and proline were not influenced by EBR treatment. Overall, it seems that BRs work from a proline-independent pathway to increase endurance to low-temperature stress, although more detailed studies are needed.

#### *4.4. Effects of EBR on Activity of Antioxidant Enzymes*

To enhance uncontrolled free radicals, plants respond by non-enzymatic and enzymatic antioxidants to regulate cellular homeostasis and mitigate oxidants [75,76]. Moreover, maintenance and regulation of redox homeostasis seem to be crucial to elevate chilling tolerance in tomato plants [77,78]. Thus, adjustment of the antioxidant system is remarked as a significant mechanism for increasing tomato chilling tolerance. It was demonstrated that using BRs induces antioxidant enzyme activity as well as non-enzymatic antioxidants. For instance, maize seedlings treated by brassinolide (BL) increased the activities of SOD, CAT, APX, carotenoid content, and ascorbic acid [10]. Antioxidative enzymes activity and mRNA expression of Cat A, Mn-SOD, Cat B, Cu/Zn-SOD, GR, and APX remarkably enhanced with EBL treatment under heavy metal stress in *Oryza sativa* L. [79]. In the present study, the effect of EBR application on the antioxidant activity, CAT, and GPX, in the tomato species, was different under low-temperature stress. Different behaviors of CAT were observed in both cold sensitive and cold tolerant species, or, in other words, the cold stress led to an increase in CAT activity in both species. However, the enhancement was much higher in cold sensitive compared to the cold tolerant species (Figure 4). There were substantial differences based on the genotype considered in BR treated plants; the CAT activity in the cold-tolerant species was increased by EBR treatment compared with untreated plants under low-temperature stress. In fact, cold tolerant plants treated with EBR showed an increase in CAT activity, which was higher than untreated plants. In cold sensitive genotypes the EBR treatment seem to impair the CAT activity, whereas in cold-sensitive species, the GPX activity was more influenced by EBR application (Figure 4). It seems that the effect of BR on the activity of antioxidant enzymes depends on the plant species, likely depending on the amount of stress received, the tomato species uses different mechanisms to reduce the induced oxidants. Previous studies on the effect of BR on elevated tolerance of resistant and susceptible tomato species in low temperatures indicated that EBR treatment enhanced the activities of the enzymes in pepper [80], cucumber [81], and eggplant [82] in low temperatures. It seems that oxidative stress is induced by low-temperature stress in the tomato species. Antioxidant enzymes, CAT, and GPX are induced to reduce the oxidants and keep cellular redox. From these results, it could be concluded that BR treatment could play a significant role in the alleviation of ROS damage by increasing antioxidant the defense system, resulting in elevating the tolerance of the tomato species to chilling stress.

#### *4.5. Effects of Low-Temperature Stress on Cold-Related Genes*

*Ethylene responsive factor* (*ERF*) genes belong to the *AP2/ERF* gene family, known as a large gene family of transcription factors [83,84]. The ERF gene family, as a key regulator, plays an important role in response to adverse conditions, such as cold stress in plant species [85,86]. In this study, the expression level of cold-responsive genes, *ERF* genes, and *ICE1,* selected based on previous studies [41,42], was significantly induced by lowtemperature stress. *ICE1* as an upstream transcription factor can regulate cold-responsive genes, such as *CBF* genes [42]. Interestingly under EBR application in a cold-tolerant species, expression patterns of *ERF* genes and *ICE1* reversed to normal temperature conditions. However, Kagale et al. indicated that the EBL application could induce the expression of cold-related genes [87]. Extensive studies were performed on the role of *AP2/ERF* gene family in response to abiotic and biotic stresses as well as hormone treatments, but the effect of BR on this gene family has not yet been investigated. Recently, Xie et al. stated that TINY, an AP2/ERF transcription factor, may negatively affect the expression of BRresponsive genes while it positively controls drought-responsive genes in *Arabidopsis* [88]. In addition, previously, it has been revealed that EBR treatment increases the basic thermotolerance of *Brassica napus* [89]. The merit of EBR to grant tolerance in plants to different stresses was corroborated via expression analysis of a subset of cold and drought stress marker genes [87]. Brassinosteroid induced auxin-related genes and cell wall-modifying in soybeans, contrarily, transcriptome analysis demonstrated the twisted BR roles in various biological processes by suppressing some WRKY genes engaged in senescence and stress

responses [90]. Overall, our results disclosed that exogenous EBR application might interact with endogenous hormones and reduce the negative effects of low temperatures that induce the expression of cold-responsive genes, *ERFs* and *ICE1*, to return to a state similar to that without stress.

#### **5. Conclusions**

In the current study, the effect of the EBR application was investigated in two tomato species under low-temperature stress. The results depicted that low-temperature stress can create oxidative stress and reduce the content of growth-regulatory hormones, IAA and GA3. Moreover, the EBR application increased the content of endogenous ABA, and a synergetic interaction was observed between BR and ABA in response to low-temperature stress. Furthermore, our findings indicated that ABA/GA3 and ABA/IAA ratios are not affected by EBR treatment. In the current study, we found that EBR treatment could not affect the content of MDA and proline under low-temperature stress, but could increase the content of antioxidant enzymes to reduce ROS induced by low-temperature stress. Overall, we concluded that EBR diminishes the adverse effect of low-temperature stress by increasing the content of endogenous phytohormones, increasing the content of antioxidant enzymes, and controlling the gene expression. Furthermore, it seems that BR effects are dependent on the tomato species.

**Author Contributions:** P.H., M.E. and A.E. designed the study. P.H. and M.E. conducted the experiments and analyzed the data. P.H. and M.A. investigated. All authors contributed to writing the manuscript. A.V., F.P. and G.B. contributed to the interpretation, presentation, and discussion of the data. All authors contributed to writing and editing the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All datasets generated or analyzed during this study are included in the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

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