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Article

Exogenous Salicylic Acid Alleviates Freeze-Thaw Injury of Cabbage (Brassica oleracea L.) Leaves

by
Kyungwon Min
and
Sang-Ryong Lee
*
Department of Biological and Environmental Science, Dongguk University, Seoul 04620, Korea
*
Author to whom correspondence should be addressed.
Sustainability 2021, 13(20), 11437; https://doi.org/10.3390/su132011437
Submission received: 7 September 2021 / Revised: 5 October 2021 / Accepted: 13 October 2021 / Published: 16 October 2021
(This article belongs to the Special Issue Sustainable Assessment of Agro-Environmental Impacts)
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Abstract

:
Freezing tolerance and physiological/biochemical changes were investigated for cabbage (Brassica oleracea L. ‘Myeong-Sung’) leaves treated with 0.5 mM salicylic acid (SA) by sub-irrigation. SA treatment did not interfere with leaf-growth (fresh/dry weight, and leaf-area), rather promoted growth (leaf-area) as compared to the control. Temperature-controlled, laboratory-based freeze-thaw assays revealed that SA-treated leaves were more freeze-tolerant than controls as evident by less ion-leakage as well as malondialdehyde content after freeze-thaw stress treatments (−2.5 and −3.5 °C). SA treatment also significantly alleviated freeze-induced oxidative stress as evidenced by the lower accumulation of O2•− and H2O2, concomitant with higher activities of antioxidant enzymes (ascorbate peroxidase and superoxide dismutase) relative to the control. Specifically, SA-treated leaves had a greater abundance of compatible solute (proline) and secondary metabolites (phenolic/flavonoid contents). These changes, together, may improve freezing tolerance through protecting membranes against freeze-desiccation and mitigating freeze-induced oxidative stress.

1. Introduction

Freeze-thaw is one of the major abiotic stresses adversely affecting plant performance and yield. Despite the mean global temperature rising annually, the occurrence of erratic frost episodes during spring/fall has been increasing in recent years; these episodes are predicted to keep increasing in frequency in the future which can result in severe frost damage to this economically important horticultural crops [1,2,3,4]. Accordingly, we are motivated to develop, at least transiently, an intervention strategy to improve the plants’ freezing tolerance (FT) which bears remarkable importance to providing sustainable agriculture. In recent years, exogenous application of various chemical compounds before a predicted freezing temperature has received substantial attention as a potential course of action for improving plant FT [5].
Salicylic acid (SA), considered as a phenolic compound, is well-known to act as a signaling molecule, regulating plant defense mechanisms against pathogen attack via the development of systemic acquired resistance and a hypersensitive response [6,7]. Numerous studies have also noted that SA treatment enhanced plant stress tolerance against abiotic stresses such as ozone, heat, salinity, chilling, and drought [7,8,9,10]. Moreover, few other studies have tested the effect of SA application on FT such as in winter-wheat leaves [11], potato plantlets [12], wheat leaves [13], and spinach leaves [14,15]; however, most of these studies, other than our previous two studies [14,15], are somewhat insufficient in their experimental freeze-thaw protocols, that is, the exactly controlled freezing and thawing after ice-nucleation. Ice-nucleation is an especially essential step when evaluating plant FT since some plant tissues, without ice-nucleation, remain supercooled at a given freezing temperature whereas others are frozen, hence possibly providing heterogenous samples used for physiological/biochemical analysis and resulting in misinformation of FT. Thus, further studies are needed to determine the positive effect of SA application on FT under precisely controlled freeze-thaw protocol.
Cabbage (Brassica oleracea L.) is a crucial horticultural crop with substantial economic value. Though slightly freeze-tolerant [16], it is sensitive to frost damage from erratic frost episodes in the spring. The main goal of this study was to explore whether exogenous SA application could alleviate frost damage in cabbage leaves when subjected to the freeze-thaw cycle. In order to gain insight into the SA-induced alleviation against freeze-thaw stress, various physiological/biochemical parameters were evaluated such as measurement of leaf growth, total ion-leakage (an indication of membrane damage), malondialdehyde (MDA) accumulation (an indication of lipid peroxidation), histochemical detection of reactive oxygen species (ROS) (O2•− and H2O2), antioxidant enzyme activity (i.e., superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX)), and determination of proline/phenolic content/flavonoid contents.

2. Materials and Methods

2.1. Plant Material

Seeds of Brassica oleracea L. ‘Myeong-Sung’ (Kyoungshin seeds, Inc., Kyungbuk, Uiseong, Korea) were germinated on plug flats filed with growth media (Heuksalim Lab., Chungbuk, Goesan, Korea) and then transferred to a plant growth chamber at 20/18 °C (D/N) with a 12 h photoperiod. The average photosynthetically active radiation (PAR) was ~300 μmol m−2 s−1, and seedlings were watered as needed (~3-d interval). Two weeks from sowing, seedlings were sub-irrigated with tap water (i.e., control), or 0.5 mM SA dissolved in tap water (i.e., 0.5 mM SA). About 21day-old seedlings (i.e., 7 days after SA application) were used for studies as described below.

2.2. Leaf-Growth Measurement

Leaf growth parameters including fresh weight (FW), dry weight (DW), and area were evaluated to compare between control and SA-treated leaves. Ten pairs of leaves (total 20) per treatment were employed to determine leaf area employing LI-3100 Area Meter (LI-COR, Inc., Lincoln, NE, USA), and then measure FW on the same leaves. Subsequently, DW was determined after oven-drying leaves at 75 °C for 3 d. Data for leaf-growth from three biological replications (20 leaves per biological replicates) were used to calculate the representative treatment means with standard errors. The mean differences among treatments were compared by a Student t-test (p < 0.05).

2.3. Determination of Leaf Freezing Tolerance

Leaf FT was estimated employing ion-leakage based laboratory freeze-thaw protocol [17]. Briefly, a pair of cabbage leaves was transferred to a 2.5 × 20 cm glass test tube containing 150 uL deionized water and gradually cooled (−1 °C h−1) in a glycol bath (JSCR-30C; JS Research Inc., Chung-Nam, Gongju, Korea) to several freezing treatment temperatures; ice-nucleation was ensured by dropping an ice-chip into each test tube at −1 °C. Samples were then continuously frozen for 30 min at each test temperature and then thawed on ice overnight. Unfrozen control (UFC) leaves were held at 0 °C throughout the freeze-thaw cycle.
To select the freezing treatment temperatures, a LT50, i.e., a lethal temperature at which plants are killed 50%, was first determined for control plants by freezing the leaves at −1 to −10 °C. Samples were taken out of a glycol bath at −1 °C intervals. This test was independently repeated thrice with each including five replications per temperature. Percent ion leakage data were used to calculate percent injury values; a LT50, defined as FT, was calculated from injury vs. temperature sigmoid curve fitting the Gompertz function [18]. Based on this sigmoid curve, two treatment temperatures, −2.5 and −3.5 °C, were selected for comparing FT of control vs. 0.5 mM SA-treated leaves.
Samples were taken out of glycol bath after freezing at designed temperatures and thawed as explained above. FT tests were independently repeated thrice with each including five replications per treatment per temperatures. Percent injury data from these independent experiments were used to obtain the representative treatment means with standard errors. The mean differences were compared by least significant difference (LSD test) (p < 0.05).

2.4. Measurement of Malondialdehyde (MDA) Content

Frozen-thawed leaf tissues excised from control and SA-treated seedlings that were exposed to −2.5 and −3.5 °C, and their corresponding UFC leaves were employed to estimate MDA content as detailed by [19,20], with some modifications. Briefly, samples were ground into fine powder with liquid nitrogen, and ~100 mg tissue was homogenized with 1.5 mL cold 10% trichloroacetic acid. Samples were then vigorously mixed and then centrifugation at 10,000× g for 20 min at 4 °C. Supernatants were mixed with 0.5% 2-thiobarbituric acid. Samples were then boiled at 95 °C for 30 min followed by cooling on ice for 5 min and centrifugation at 10,000× g for 5 min at 20 °C. The supernatant was measured using a spectrometer at 450, 532, and 600 nm.

2.5. ROS Staining (O2•− and H2O2)

ROS distribution of superoxide (O2•−) and hydrogen peroxide (H2O2) was visualized via nitroblue tetrazolium (NBT) and 3,3′-diaminobenzidine (DAB) staining, respectively, as explained by [17]. Staining intensities were visually compared between control and 0.5 mM SA-treated leaves after freeze-thaw stress at −2.5 and −3.5 °C. This experiment was independently repeated twice with two replications (two leaves per replicate) per temperature per treatment.

2.6. Antioxidant Enzyme Activities

Frozen-thawed leaf tissues from control and SA-treated seedlings that were exposed to −2.5 and −3.5 °C, and their corresponding UFC leaf-tissues were employed to estimate the activity of SOD, CAT, and APX as detailed by [21,22]. Briefly, samples were ground into fine powder with liquid nitrogen, and 150 mg tissue per treatment per temperature was homogenized using 100 mM potassium phosphate buffer (1 mL; pH 7.0). The samples were then centrifuged at 10,000 g for 20 min at 4 °C and the resultant supernatants were employed as the enzyme extract for SOD, CAT, and APX. The activity of three antioxidants enzyme was estimated as described by [22].

2.7. Proline Analysis

Leaf tissues excised from control and SA-treated seedlings were used to determine the proline content as described by [23]. Briefly, ground frozen leaf tissue (100 mg) was mixed with 3% sulfosalicylic acid (1 mL) and then incubation at 80 °C for 15 min. Samples were then centrifugated at 16,000 g at room temperature for 20 min, and resultant supernatant (500 uL) was diluted with an equal amount of distilled water. Subsequently, the samples were mixed with 1 mL of ninhydrin and 1 mL of glacial acetic acid before being heated at 100 °C for 60 min. After cooling down, the solutions were vigorously mixed with 4 mL of toluene, followed by reading the absorbance of samples at 520 nm using a spectrophotometer.

2.8. Secondary Metabolite Analysis

2.8.1. Preparation of Methanolic Extracts

Leaves excised from the control and SA-treated seedlings were dehydrated followed by pulverization. The dried powders (1 g) were mixed with MeOH (10 mL) for 24-h at ~20 °C with frequent agitation. The extracts were filtered through a Buckner funnel and Whatman No. 2 filter paper, followed by concentration by drying filtrate at room temperature for 4 d, which was then stored at 4 °C until further analysis as explained below.

2.8.2. Total Phenolic Contents

The total phenolic content was determined by Folin-Ciocalteu assay as detailed by [24] with some modifications. Briefly, 1.0 mL of extract (200 μg/mL) was homogenized with 2.5 mL of 10% Folin-Ciocalteu reagent. Subsequently, the sample was homogenized with 2.0 mL of 20% Na2CO3 and then incubation at 50 °C for 10 min. After cooling down, the sample was measured using a UV spectrophotometer at 765 nm. The data were calculated as mg/g of gallic acid equivalent in milligrams per gram (mg GAE/g) of dry extract.

2.8.3. Total Flavonoid Contents

The total flavonoid content was determined as described by [24] with some modifications. Briefly, 1.0 mL of extract (200 μg/mL) was homogenized with 0.2 mL of 10% AlCl3 and 0.2 mL of sodium acetate (1 M), followed by incubation at room temperature for 45 min. Samples were then measured utilizing a UV spectrophotometer at 415 nm and resultant data were calculated as mg/g of quercetin equivalents in milligrams per gram (mg QE/g) of dry extract.

2.9. Statistical Analysis

All biochemical analyses were independently repeated thrice, each with three technical replications/treatment/temperature. The data were analyzed by Student t-test (p < 0.05) to determine any statistically significant differences, and treatments were compared using the LSD test (p < 0.05).

3. Results

3.1. Effect of Exogenous SA on Plant Growth under Ambient Condition

Leaf area of SA-treated seedlings was ~11% larger than that of control seedlings (Table 1). However, water content for leaves excised from SA-treated seedlings was significantly less than control, though only by ~1.7%. Furthermore, a significant difference in DW/FW ratio was found between control vs. SA-treated leaves.
All biochemical analyses were hereafter calculated based on DW basis since water content as well as DW/FW ratio were statistically different between control and 0.5 mM SA treatment.

3.2. LT50 and SA-Induced Freezing Tolerance

The response curve to freeze-thaw stress of control leaves (−1 to −10 °C) is shown in Figure 1A. Minimum and maximum injury percentages were 0.5% and 92%, respectively, with −3.0 °C corresponding to the mid-point of percent injury (46.5%). Thus, −3.0 °C was regarded as LT50 at which plants are killed 50% or FT for ‘Myeong-Sung’ cabbage leaves.
SA-treated leaves were statistically more freeze-tolerant than non-treated ones as evident by less freeze-thaw injury by ~22 and ~33% at −2.5 and −3.5 °C, respectively (Figure 1B).

3.3. MDA Content

Data for MDA content in UFC as well as freeze-thaw injured tissues are presented in Figure 2. MDA contents in control and SA-treated leaves accumulated after freeze-thaw stress at −2.5 and −3.5 °C as compared to corresponding UFCs. Furthermore, MDA content were more accumulated at −3.5 °C than at −2.5 °C in both treatments. MDA content in SA-treated leaves were ~59 and ~43% less than control after freeze-thaw stress at −2.5 and −3.5 °C, respectively.

3.4. ROS Staining (O2•− and H2O2) and Activity of Three Antioxidant Enzymes

Visual distribution of O2•− and H2O2 in UFC and freeze-thaw injured tissues that had been exposed to −2.5 and −3.5 °C is shown in Figure 3. Increase in O2•− and H2O2 was obvious in freeze-thaw stressed tissues relative to UFCs in both treatments, as evident by the higher abundance of blue color (A and B; superoxide) and reddish color (C and D; hydrogen peroxide), respectively. Besides, staining magnitudes for the two ROS were visually higher in control as compared to SA-fed leaves after freeze-thaw at −2.5 and −3.5 °C.
Antioxidant enzyme activity for CAT, APX, and SOD in UFC and freeze-thaw injured tissues are presented in Figure 4. All enzyme activities in both treatments were repressed following exposure to −2.5 and −3.5 °C as compared to corresponding UFCs. In addition, activities for all enzymes were more decreased at −3.5 °C, as compared to at −2.5 °C in both treatments. CAT activity in SA-treated leaves were ~6.7 and ~11.1% less than control at −2.5 and −3.5 °C, respectively, whereas APX activity in same leaves was ~26 and ~30% higher than control at both stress levels. SOD activity was also ~6.5 and 29.7% higher than control at −2.5 and −3.5 °C, respectively.

3.5. Proline, Total Phenolic and Total Flavonoid Contents

Proline, total phenolic and total flavonoid contents of SA-treated leaves were significantly higher (~1.5-, ~1.5- and ~1.6-fold, respectively) than those of control leaves (Figure 5A–C).

4. Discussion

Numerous studies have been reported on the positive effects of exogenous SA treatment on plant stress tolerance in order to withstand abiotic and biotic stresses (references in [7]). However, its effect on FT of various plant species remains elucidated. Few studies reporting an enhancement in plant FT by exogenous SA have provided various mechanistic insight into the SA-induced FT ranging from changes in activity in apoplastic proteins [11], improvement in antioxidant enzymes’ activity [12,13], expression of cold-responsive genes [25], to the accumulation of compatible solutes (osmolytes) and non-enzymatic antioxidants [14,15]. Most of these studies, however, did not consider exactly controlled cooling and thawing along with ice-nucleation when evaluating FT of plants. Above all, such temperature-controlled freeze-thaw protocols are crucial especially under laboratory conditions to collect identical freeze-thaw injured samples used for various physiological and biochemical analysis, thereby providing reliable experimental data. Accordingly, we have evaluated the effect of exogenous SA on FT as determined through various physiological parameters using the exactly controlled freezing and thawing following ice-nucleation.

4.1. The Influence of SA Treatment on Leaf Growth

The effect of exogenous SA on plant growth and stress tolerance relies on the its concentration [7,9]. Therefore, based on literature survey, two SA concentrations, i.e., 0.5 and 1.0 mM, were first tested as sub-irrigation treatments. Cabbage seedlings treated with 1.0 mM showed almost killed compared to control whereas 0.5 mM SA treatment did not show any negative effect. Accordingly, 1.0 mM SA was not utilized for further experiments (data not exhibited for these comparisons). In support of our selection of 0.5 mM SA used in the present study, [26] also noted that optimal SA concentration for most plants’ improvement of stress tolerance is less than 0.5 mM.
It has been reported that the SA treatment affects plant growth in a dose dependent manner. For example, the relatively lower SA concentration (i.e., 0.1 and 0.5 mM) promoted the growth of mung bean whereas 1.0 mM SA treatment showed inhibited growth [27]. In another example, Matricaria chamomilla treated with 50 and 250 μm SA was shown to promote and retard growth [28]. In the present study, cabbage seedlings sub-irrigated with 0.5 mM SA exhibited ~11% higher leaf area as compared to control (Table 1), indicating 0.5 mM SA concentration promotes the leaf-growth of cabbage seedlings. The reason for SA-induced leaf-growth is beyond the scope of this study, but it may be associated with increase in photosynthetic rate induced by SA application [28,29]. The marginal reduction of water content and higher DW may indicate improved FT in leaves treated with SA (Table 1) since FT induction (as in plant cold acclimation) generally involves decrease in cellular hydration as well as DW accumulation [30,31].

4.2. Exogenous SA Improves FT by Alleviating Freeze-Induced Membrane Injury

The cell membrane has been regarded as the primary site of freezing injury due to freeze-desiccation associated with extracellular freezing, as evident by ion-leakage [1,32]. Hence, ion-leakage assay is routinely used to determine FT of plants [17]. In the present study, SA-treated tissues were significantly less injured relative to controls as evident by less ion-leakage at all stress levels (Figure 1A), indicating exogenous SA is beneficially effective at mitigating freeze-induced membrane injury, hence, improves FT. Evidence also exists for a lower ion-leakage with exogenous SA application against freezing [14,15], salt [33], drought [34], and chilling [35].

4.3. Exogenous SA Bolsters Antioxidant System

It has been noted that plants subjected to freeze-thaw stress experience oxidative stress due to overproduction of ROS, by which can damage various cellular components including membrane [1,36,37,38]. Lipid peroxidation of membrane is one of such damage effects due to accumulated ROS, and its level is routinely determined by measuring MDA (i.e., a marker of oxidative lipid injury). Our data from MDA determination (Figure 2) and histochemical detection of ROS (O2•− and H2O2) (Figure 3) are in accordance with these reports. Moreover, accumulation of MDA content as well as ROS were lower in SA-treated tissues relative to control at both stress levels, indicating a possibly enhanced antioxidant capacity of SA-treated tissues. Indeed, activities of antioxidant enzymes, other than CAT, were higher in SA-treated leaves than controls at both stress levels; higher activity of APX could more efficiently remove accumulated H2O2 whereas more active SOD could scavenge excess O2•−. Several studies have also been reported for a less accumulation of MDA content concomitant with improved activity of antioxidant enzymes in SA-treated plant tissues against various abiotic stresses [39,40,41,42,43,44].
Why do SA-treated leaves have a lower activity of CAT (Figure 3A) than control leaves, another antioxidant enzyme responsible for scavenging H2O2? Based on literature survey, SA particularly bind to iron-containing enzymes such as catalase, thereby leading to repression of their activity [45]. Indeed, evidence exists for exogenous SA to reduce CAT activity, which can potentially cause H2O2 accumulation [46,47]. Conceivably, a less accumulation of H2O2 in SA-treated leaves at both stress levels (compare Figure 3C vs. Figure 3D) may be due, mainly, to upregulation of APX activity in order to compensate for diminished CAT activity. This assumption could be further supported by reports noting a specific accumulation of ascorbic acid (i.e., a substrate for APX) by SA application [14,15].

4.4. Exogenous SA Accumulates Proline, Total Phenolic and Flavonoid Compounds

Proline, known as one of compatible solutes, has been known to accumulate in response to abiotic stresses. Due to its hydrophilic characteristics, proline has a crucial role in cellular osmotic adjustment and stabilization of membrane and proteins [48,49]. Its accumulation has been also frequently reported in cold-acclimated plants, associated with improved FT [14,15,50]. In the same context, the present study also showed that the more freeze-tolerant SA-treated leaves had higher proline content than control (Figure 5A), indicating that exogenous SA increases proline content, thereby improving FT. Our results could be more supported by others noting increased proline content and concomitant alleviation of abiotic stresses in SA-treated Torreya grandis [51] and legume [33].
Phenolic and flavonoid compounds are among plant-derived secondary metabolites, widely implicated in acting as a hydrogen donor, thus functioning as effective antioxidants [52,53]. As shown in Figure 5B,C, SA-treated tissues (i.e., more freeze-tolerant) also accumulated ~55 and ~57% higher total phenolic and flavonoid compounds, respectively, compared to control, suggesting a potential linkage between SA and phenolic/flavonoid biosynthesis. No experiment was performed in our study to reveal why exogenous SA induces phenolic/flavonoid accumulation. However, increased activity and gene expression of phenylalanine ammonia-lyase (PAL, an important enzyme involved in metabolism of several secondary metabolites including phenolic compounds) has been reported in saffron treated with SA [54]; this group also reported higher amount of various phenolic contents (e.g., gallic acid, caffeic acid, and cinnamic acid) as well as flavonoid compounds in SA-treated tissues as compared to control. Several studies have also reported accumulation of phenolic and/or flavonoid compounds by SA application and concomitant amelioration of various abiotic stresses such as drought [55,56] and salt [57].

5. Conclusions

The present study offers evidence that exogenous SA application (0.5 mM), as sub-irrigation treatment, improved FT (reduced membrane leakage, lower MDA content and less accumulation of ROS) of cabbage leaves without any detrimental effect on leaf-growth. Our data indicates that SA-induced FT may be linked to enhanced antioxidant enzyme activities (i.e., APX and SOD) as well as greater accumulation of proline (a compatible solute) and phenolic/flavonoid compounds (Figure 6). Another unique finding of this study was the decreased CAT activity in the SA-treated leaves, i.e., more freeze-tolerant tissues than non-treated ones, suggesting a SA-specific response; this decreased CAT activity in SA-treated leaves may be compensated by higher APX activity to scavenge excess H2O2 caused by freeze-thaw stress. This research may provide a new insight into mechanism on SA-induced FT of cabbage, aiming to contribute sustainable yield in the face of climate change.

Author Contributions

S.-R.L. and K.M. jointly conceived the idea and designed experiments. K.M. conducted the experiments and analyzed the data. K.M. and S.-R.L. jointly wrote the paper. All authors have read and agreed to the published version of the manuscript.

Funding

This work was carried with the support of Korea Environment Industry & Technology Institute (KEITI) through “Measurement and Risk assessment Program for management of Microplastics Project (RE2021014390)” funded by Korea Ministry of Environment (MOE), Rural Development Administration (RDA) “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01429701)”, and Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through “Technology Development Program for Agriculture and Forestry” funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (Project No. 318014), Republic of Korea.

Institutional Review Board Statement

Not applicable

Informed Consent Statement

Not applicable

Data Availability Statement

The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy concern.

Conflicts of Interest

The authors declare no conflict of interests.

References

  1. Arora, R. Mechanism of freeze-thaw injury and recovery: A cool retrospective and warming up to new ideas. Plant Sci. 2018, 270, 301–313. [Google Scholar] [CrossRef]
  2. Gu, L.; Hanson, P.J.; Post, W.M.; Kaiser, D.P.; Yang, B.; Nemani, R.; Pallardy, S.G.; Meyers, T. The 2007 eastern US spring freeze: Increased cold damage in a warming world? Bioscience 2008, 58, 253–262. [Google Scholar] [CrossRef]
  3. Xiao, L.; Liu, L.; Asseng, S.; Xia, Y.; Tang, L.; Liu, B.; Cao, W.; Zhu, Y. Estimating spring frost and its impact on yield across winter wheat in China. Agric. For. Meteorol. 2018, 260–261, 154–164. [Google Scholar] [CrossRef]
  4. Zohner, C.M.; Mo, L.; Renner, S.S.; Svenning, J.C.; Vitasse, Y.; Benito, B.M.; Ordonez, A.; Baumgarten, F.; Bastin, J.F.; Sebald, V.; et al. Late-spring frost risk between 1959 and 2017 decreased in North America but increased in Europe and Asia. Proc. Natl. Acad. Sci. USA 2020, 117, 1–9. [Google Scholar] [CrossRef] [PubMed]
  5. Savvides, A.; Ali, S.; Tester, M.; Fotopoulos, V. Chemical Priming of Plants Against Multiple Abiotic Stresses: Mission Possible? Trends Plant Sci. 2016, 21, 329–340. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  6. Dempsey, D.A.; Vlot, A.C.; Wildermuth, M.C.; Klessig, D.F. Salicylic Acid Biosynthesis and Metabolism. Arab. B. 2011, 9, e0156. [Google Scholar] [CrossRef] [Green Version]
  7. Khan, M.I.R.; Fatma, M.; Per, T.S.; Anjum, N.A.; Khan, N.A. Salicylic acid-induced abiotic stress tolerance and underlying mechanisms in plants. Front. Plant Sci. 2015, 6, 1–17. [Google Scholar] [CrossRef] [Green Version]
  8. Sharma, Y.K.; León, J.; Raskin, I.; Davis, K.R. Ozone-induced responses in Arabidopsis thaliana: The role of salicylic acid in the accumulation of defense-related transcripts and induced resistance. Proc. Natl. Acad. Sci. USA 1996, 93, 5099–5104. [Google Scholar] [CrossRef] [Green Version]
  9. Miura, K.; Tada, Y. Regulation of water, salinity, and cold stress responses by salicylic acid. Front. Plant Sci. 2014, 5, 1–12. [Google Scholar] [CrossRef] [Green Version]
  10. Venegas-Molina, J.; Proietti, S.; Pollier, J.; Orozco-Freire, W.; Ramirez-Villacis, D.; Leon-Reyes, A. Induced tolerance to abiotic and biotic stresses of broccoli and Arabidopsis after treatment with elicitor molecules. Sci. Rep. 2020, 10, 1–17. [Google Scholar] [CrossRef]
  11. Taşgín, E.; Atící, Ö.; Nalbantoğlu, B. Effects of salicylic acid and cold on freezing tolerance in winter wheat leaves. Plant Growth Regul. 2003, 41, 231–236. [Google Scholar] [CrossRef]
  12. Mora-Herrera, M.E.; López-Delgado, H.; Castillo-Morales, A.; Foyer, C.H. Salicylic acid and H2O2 function by independent pathways in the induction of freezing tolerance in potato. Physiol. Plant. 2005, 125, 430–440. [Google Scholar] [CrossRef]
  13. Wang, W.; Wang, X.; Huang, M.; Cai, J.; Zhou, Q.; Dai, T.; Jiang, D. Alleviation of field low-temperature stress in winter wheat by exogenous application of salicylic acid. J. Plant Growth Regul. 2021, 40, 811–823. [Google Scholar] [CrossRef]
  14. Shin, H.; Min, K.; Arora, R. Exogenous salicylic acid improves freezing tolerance of spinach (Spinacia oleracea L.) leaves. Cryobiology 2018, 81, 192–200. [Google Scholar] [CrossRef] [PubMed]
  15. Min, K.; Showman, L.; Perera, A.; Arora, R. Salicylic acid-induced freezing tolerance in spinach (Spinacia oleracea L.) leaves explored through metabolite profiling. Environ. Exp. Bot. 2018, 156, 214–227. [Google Scholar] [CrossRef]
  16. Manley, R.C.; Hummel, R.L. Mefluidide does not consistently enhance the freezing tolerance of cabbage. HortScience 1996, 31, 402–404. [Google Scholar] [CrossRef]
  17. Min, K.; Chen, K.; Arora, R. Effect of short-term versus prolonged freezing on freeze-thaw injury and post-thaw recovery in spinach: Importance in laboratory freeze-thaw protocols. Environ. Exp. Bot. 2014, 106, 124–131. [Google Scholar] [CrossRef] [Green Version]
  18. Lim, C.C.; Arora, R.; Townsend, E.C. Comparing Gompertz and Richards functions to estimate freezing injury in Rhododendron using electrolyte leakage. J. Am. Soc. Hortic. Sci. 1998, 123, 246–252. [Google Scholar] [CrossRef]
  19. Dhindsa, R.S.; Plumb-dhindsa, P.; Thorpe, T.A. Leaf senescence: Correlated with increased levels of membrane permeability and lipid peroxidation, and decreased levels of superoxide dismutase and catalase. J. Exp. Bot. 1981, 32, 93–101. [Google Scholar] [CrossRef]
  20. Shi, Q.; Zhu, Z.; Xu, M.; Qian, Q.; Yu, J. Effect of excess manganese on the antioxidant system in Cucumis sativus L. under two light intensities. Environ. Exp. Bot. 2006, 58, 197–205. [Google Scholar] [CrossRef]
  21. Chen, K.; Arora, R. Dynamics of the antioxidant system during seed osmopriming, post-priming germination, and seedling establishment in Spinach (Spinacia oleracea). Plant Sci. 2011, 180, 212–220. [Google Scholar] [CrossRef]
  22. Chen, K.; Arora, R. Understanding the cellular mechanism of recovery from freeze-thaw injury in spinach: Possible role of aquaporins, heat shock proteins, dehydrin and antioxidant system. Physiol. Plant. 2014, 150, 374–387. [Google Scholar] [CrossRef]
  23. Oh, S.; Shin, H.; Arora, R.; Kim, K.; Kim, D. Proline accumulation and related gene expression during spring regrowth in three rosaceae species. Hortic. Environ. Biotechnol. 2017, 58, 21–26. [Google Scholar] [CrossRef]
  24. Aryal, S.; Baniya, M.K.; Danekhu, K.; Kunwar, P.; Gurung, R.; Koirala, N. Total Phenolic content, Flavonoid content and antioxidant potential of wild vegetables from western Nepal. Plants 2019, 8, 96. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  25. Wang, W.; Wang, X.; Huang, M.; Cai, J.; Zhou, Q.; Dai, T.; Cao, W.; Jiang, D. Hydrogen peroxide and abscisic acid mediate salicylic acid-induced freezing tolerance in wheat. Front. Plant Sci. 2018, 9, 1–13. [Google Scholar] [CrossRef] [PubMed]
  26. Hara, M.; Furukawa, J.; Sato, A.; Mizoguchi, T.; Miura, K. Abiotic Stress and Role of Salicylic Acid in Plants; Abiotic Stress Responses in Plants; Ahmad, P., Prasad, M.N.V., Eds.; Springer-Verlag: Berlin, Germany, 2012; pp. 235–251. ISBN 978-1-4614-0634-1. [Google Scholar]
  27. Nazar, R.; Iqbal, N.; Syeed, S.; Khan, N.A. Salicylic acid alleviates decreases in photosynthesis under salt stress by enhancing nitrogen and sulfur assimilation and antioxidant metabolism differentially in two mungbean cultivars. J. Plant Physiol. 2011, 168, 807–815. [Google Scholar] [CrossRef]
  28. Kováčik, J.; Klejdus, B.; Hedbavny, J.; Bačkor, M. Salicylic acid alleviates NaCl-induced changes in the metabolism of Matricaria chamomilla plants. Ecotoxicology 2009, 18, 544–554. [Google Scholar] [CrossRef]
  29. Zhang, Y.; Xu, S.; Yang, S.; Chen, Y. Salicylic acid alleviates cadmium-induced inhibition of growth and photosynthesis through upregulating antioxidant defense system in two melon cultivars (Cucumis melo L.). Protoplasma 2015, 252, 911–924. [Google Scholar] [CrossRef]
  30. Wanner, L.A.; Junttila, O. Cold-induced freezing tolerance in arabidopsis. Plant Physiol. 1999, 120, 391–399. [Google Scholar] [CrossRef] [Green Version]
  31. Xin, Z.; Browse, J. Cold comfort farm: The acclimation of plants to freezing temperatures. Plant Cell Environ. 2000, 23, 893–902. [Google Scholar] [CrossRef]
  32. Uemura, M.; Tominaga, Y.; Nakagawara, C.; Shigematsu, S.; Minami, A.; Kawamura, Y. Responses of the plasma membrane to low temperatures. Physiol. Plant. 2006, 126, 81–89. [Google Scholar] [CrossRef]
  33. Misra, N.; Saxena, P. Effect of salicylic acid on proline metabolism in lentil grown under salinity stress. Plant Sci. 2009, 177, 181–189. [Google Scholar] [CrossRef]
  34. La, V.H.; Lee, B.R.; Zhang, Q.; Park, S.H.; Islam, M.T.; Kim, T.H. Salicylic acid improves drought-stress tolerance by regulating the redox status and proline metabolism in Brassica rapa. Hortic. Environ. Biotechnol. 2019, 60, 31–40. [Google Scholar] [CrossRef]
  35. Kang, H.M.; Saltveit, M.E. Chilling tolerance of maize, cucumber and rice seedling leaves and roots are differentially affected by salicylic acid. Physiol. Plant. 2002, 115, 571–576. [Google Scholar] [CrossRef] [Green Version]
  36. Kendall, E.J.; McKersie, B.D. Free radical and freezing injury to cell membranes of winter wheat. Physiol. Plant. 1989, 76, 86–94. [Google Scholar] [CrossRef]
  37. Mittler, R. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 2002, 7, 405–410. [Google Scholar] [CrossRef]
  38. Gill, S.S.; Tuteja, N. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol. Biochem. 2010, 48, 909–930. [Google Scholar] [CrossRef]
  39. Ma, X.; Zheng, J.; Zhang, X.; Hu, Q.; Qian, R. Salicylic acid alleviates the adverse effects of salt stress on dianthus superbus (Caryophyllaceae) by activating photosynthesis, protecting morphological structure, and enhancing the antioxidant system. Front. Plant Sci. 2017, 8, 1–13. [Google Scholar] [CrossRef]
  40. Mei, L.; Daud, M.K.; Ullah, N.; Ali, S.; Khan, M.; Malik, Z.; Zhu, S.J. Pretreatment with salicylic acid and ascorbic acid significantly mitigate oxidative stress induced by copper in cotton genotypes. Environ. Sci. Pollut. Res. 2015, 22, 9922–9931. [Google Scholar] [CrossRef]
  41. Belkhadi, A.; Hediji, H.; Abbes, Z.; Nouairi, I.; Barhoumi, Z.; Zarrouk, M.; Chaïbi, W.; Djebali, W. Effects of exogenous salicylic acid pre-treatment on cadmium toxicity and leaf lipid content in Linum usitatissimum L. Ecotoxicol. Environ. Saf. 2010, 73, 1004–1011. [Google Scholar] [CrossRef]
  42. Moustafa-Farag, M.; Mohamed, H.I.; Mahmoud, A.; Elkelish, A.; Misra, A.N.; Guy, K.M.; Kamran, M.; Ai, S.; Zhang, M. Salicylic acid stimulates antioxidant defense and osmolyte metabolism to alleviate oxidative stress in watermelons under excess boron. Plants 2020, 9, 724. [Google Scholar] [CrossRef]
  43. Yüzbaşıoğlu, E.; Dalyan, E. Salicylic acid alleviates thiram toxicity by modulating antioxidant enzyme capacity and pesticide detoxification systems in the tomato (Solanum lycopersicum Mill.). Plant Physiol. Biochem. 2019, 135, 322–330. [Google Scholar] [CrossRef]
  44. Tahjib-Ul-Arif, M.; Siddiqui, M.N.; Sohag, A.A.M.; Sakil, M.A.; Rahman, M.M.; Polash, M.A.S.; Mostofa, M.G.; Tran, L.S.P. Salicylic Acid-Mediated Enhancement of Photosynthesis Attributes and Antioxidant Capacity Contributes to Yield Improvement of Maize Plants Under Salt Stress. J. Plant Growth Regul. 2018, 37, 1318–1330. [Google Scholar] [CrossRef]
  45. Rüffer, M.; Steipe, B.; Zenk, M.H. Evidence against specific binding of salicylic acid to plant catalase. FEBS Lett. 1995, 377, 175–180. [Google Scholar] [CrossRef] [Green Version]
  46. Chen, Z.; Silva, H.; Klessig, D.F. Active oxygen species in the induction of plant systemic acquired resistance by salicylic acid. Science 1993, 262, 1883–1886. [Google Scholar] [CrossRef]
  47. Horváth, E.; Janda, T.; Szalai, G.; Páldi, E. In vitro salicylic acid inhibition of catalase activity in maize: Differences between the isozymes and a possible role in the induction of chilling tolerance. Plant Sci. 2002, 163, 1129–1135. [Google Scholar] [CrossRef]
  48. Szabados, L.; Savouré, A. Proline: A multifunctional amino acid. Trends Plant Sci. 2010, 15, 89–97. [Google Scholar] [CrossRef] [PubMed]
  49. Hayat, S.; Hayat, Q.; Alyemeni, M.N.; Wani, A.S.; Pichtel, J.; Ahmad, A. Role of proline under changing environments: A review. Plant Signal. Behav. 2012, 7, 1–11. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  50. Kaplan, F.; Kopka, J.; Haskell, D.W.; Zhao, W.; Schiller, K.C.; Gatzke, N.; Sung, D.Y.; Guy, C.L.; Molecular, P.; Program, C.B.; et al. Exploring the Temperature-Stress Metabolome. Plant Physiol. 2004, 136, 4159–4168. [Google Scholar] [CrossRef] [Green Version]
  51. Li, T.; Hu, Y.; Du, X.; Tang, H.; Shen, C.; Wu, J. Salicylic acid alleviates the adverse effects of salt stress in Torreya grandis cv. merrillii seedlings by activating photosynthesis and enhancing antioxidant systems. PLoS ONE 2014, 9, 1–9. [Google Scholar] [CrossRef]
  52. Ali Ghasemzadeh Flavonoid compounds and their antioxidant activity in extract of some tropical plants. J. Med. Plants Res. 2012, 6, 2639–2643.
  53. Dai, J.; Mumper, R.J. Plant phenolics: Extraction, analysis and their antioxidant and anticancer properties. Molecules 2010, 15, 7313–7352. [Google Scholar] [CrossRef]
  54. Tajik, S.; Zarinkamar, F.; Soltani, B.M.; Nazari, M. Induction of phenolic and flavonoid compounds in leaves of saffron (Crocus sativus L.) by salicylic acid. Sci. Hortic. 2019, 257. [Google Scholar] [CrossRef]
  55. Awate, P.D.; Gaikwad, D.K. Influence of Growth Regulators on Secondary Metabolites of Medicinally Important Oil Yielding Plant Simarouba glauca DC. under Water Stress Conditions. J. Stress Physiol. Biochem. 2014, 10, 222–229. [Google Scholar]
  56. Chavoushi, M.; Najafi, F.; Salimi, A.; Angaji, S.A. Effect of salicylic acid and sodium nitroprusside on growth parameters, photosynthetic pigments and secondary metabolites of safflower under drought stress. Sci. Hortic. 2020, 259. [Google Scholar] [CrossRef]
  57. Shamili, M.; Esfandiari Ghalati, R.; Samari, F. The Impact of Foliar Salicylic Acid in Salt-Exposed Guava (Psidium Guajava L.) Seedlings. Int. J. Fruit Sci. 2021, 21, 323–333. [Google Scholar] [CrossRef]
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Figure 1. (A) Sigmoid curve of freeze-thaw injury in cabbage (Brassica oleracea L. cv. Myeong-sung) leaves, obtained from injury% at each treatment temperature via Gompertz function: LT50, a mid-injury (46.5% corresponding to −3.0 °C) between the minimum (0.5%) and maximum (92.0%) injury is determined as the temperature causing 50% injury; (B) Effect of exogenous SA on freezing tolerance of cabbage (Brassica oleracea L. cv. Myeong-Sung) leaves sub-irrigated with water only (control) and water + 0.5 mM SA (0.5 mM SA); injury percent (means ± S.E.) determined by ion-leakage from excised-leaves subjected to freeze-thaw stress at −2.5 and −3.5 °C. Same letter indicates no significant differences between treatments (p < 0.05) according to LSD test (mean ± S.E.). UFC, unfrozen control.
Figure 1. (A) Sigmoid curve of freeze-thaw injury in cabbage (Brassica oleracea L. cv. Myeong-sung) leaves, obtained from injury% at each treatment temperature via Gompertz function: LT50, a mid-injury (46.5% corresponding to −3.0 °C) between the minimum (0.5%) and maximum (92.0%) injury is determined as the temperature causing 50% injury; (B) Effect of exogenous SA on freezing tolerance of cabbage (Brassica oleracea L. cv. Myeong-Sung) leaves sub-irrigated with water only (control) and water + 0.5 mM SA (0.5 mM SA); injury percent (means ± S.E.) determined by ion-leakage from excised-leaves subjected to freeze-thaw stress at −2.5 and −3.5 °C. Same letter indicates no significant differences between treatments (p < 0.05) according to LSD test (mean ± S.E.). UFC, unfrozen control.
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Figure 2. MDA (malondialdehyde) content in unfrozen controls (UFCs) and freeze-thaw stressed cabbage (Brassica oleracea L. cv. Myeong-Sung) leaves that were sub-irrigated with water only (control) and water + 0.5 mM SA (0.5 mM SA) before exposure to freeze-thaw stress. Same letter indicates no significant differences between treatments (p < 0.05) according to LSD test (mean ± S.E.).
Figure 2. MDA (malondialdehyde) content in unfrozen controls (UFCs) and freeze-thaw stressed cabbage (Brassica oleracea L. cv. Myeong-Sung) leaves that were sub-irrigated with water only (control) and water + 0.5 mM SA (0.5 mM SA) before exposure to freeze-thaw stress. Same letter indicates no significant differences between treatments (p < 0.05) according to LSD test (mean ± S.E.).
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Figure 3. Histochemical detection of superoxide (O2•−) (A,B) and hydrogen peroxide (H2O2) (C,D) in unfrozen controls (UFCs) and freeze-thaw stressed cabbage (Brassica oleracea L. cv. Myeong-Sung) leaves that were sub-irrigated with water only (control) and water + 0.5 mM SA (0.5 mM SA) before freeze-thaw stress.
Figure 3. Histochemical detection of superoxide (O2•−) (A,B) and hydrogen peroxide (H2O2) (C,D) in unfrozen controls (UFCs) and freeze-thaw stressed cabbage (Brassica oleracea L. cv. Myeong-Sung) leaves that were sub-irrigated with water only (control) and water + 0.5 mM SA (0.5 mM SA) before freeze-thaw stress.
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Figure 4. The activity of CAT, APX, and SOD (A–C) in unfrozen controls (UFCs) and freeze-thaw stressed cabbage (Brassica oleracea L. cv. Myeong-Sung) leaves that were sub-irrigated with water only (control) and water + 0.5 mM SA (0.5 mM SA) before exposure to freeze-thaw stress. Same letter indicates no significant differences between treatments (p < 0.05) according to LSD test (mean ± S.E.).
Figure 4. The activity of CAT, APX, and SOD (A–C) in unfrozen controls (UFCs) and freeze-thaw stressed cabbage (Brassica oleracea L. cv. Myeong-Sung) leaves that were sub-irrigated with water only (control) and water + 0.5 mM SA (0.5 mM SA) before exposure to freeze-thaw stress. Same letter indicates no significant differences between treatments (p < 0.05) according to LSD test (mean ± S.E.).
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Figure 5. Proline (A), total phenolic (B), and total flavonoid (C) contents of cabbage (Brassica oleracea L. cv. Myeong-Sung) leaves sub-irrigated with water only (control) and water + 30 mM GB (30 mM GB). *, p < 0.05, analyzed by Students t-test. TPC, total phenolic contents; TFC, total flavonoid content.
Figure 5. Proline (A), total phenolic (B), and total flavonoid (C) contents of cabbage (Brassica oleracea L. cv. Myeong-Sung) leaves sub-irrigated with water only (control) and water + 30 mM GB (30 mM GB). *, p < 0.05, analyzed by Students t-test. TPC, total phenolic contents; TFC, total flavonoid content.
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Figure 6. Illustrative summary of the effect of exogenous SA on physiological/biochemical changes vis-à-vis improved freezing tolerance (FT) of cabbage (Brassica oleracea L. cv. Myeong-Sung); for explanation, refer to “Conclusions”. SA, salicylic acid; APX, ascorbate peroxidase; SOD, superoxide dismutase; ROS, reactive oxygen species.
Figure 6. Illustrative summary of the effect of exogenous SA on physiological/biochemical changes vis-à-vis improved freezing tolerance (FT) of cabbage (Brassica oleracea L. cv. Myeong-Sung); for explanation, refer to “Conclusions”. SA, salicylic acid; APX, ascorbate peroxidase; SOD, superoxide dismutase; ROS, reactive oxygen species.
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Table
Table 1. Leaf growth parameters of cabbage (Brassica oleracea L. cv. Myeong-Sung) seedlings sub-irrigated with only tap water (control) and 0.5 mM SA + tap water (0.5 mM SA). FW, fresh weight; DW, dry weight.
Table 1. Leaf growth parameters of cabbage (Brassica oleracea L. cv. Myeong-Sung) seedlings sub-irrigated with only tap water (control) and 0.5 mM SA + tap water (0.5 mM SA). FW, fresh weight; DW, dry weight.
Growth ParametersTreatment
Control0.5 mM SA
Water content (%) y93.7 ± 0.192.1 ± 0.2 z
DW/FW (g) y0.07 ± 0.0010.08 ± 0.002 z
Leaf area (cm2) y8.2 ± 0.19.1 ± 0.3 z
y Pooled means ± SE obtained from three independent experiments, each with 10 plants. Two leaves per plant were employed leading to a total 20 leaves per biological replications, analyzed by Student t-test. z p < 0.05.
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Min, K.; Lee, S.-R. Exogenous Salicylic Acid Alleviates Freeze-Thaw Injury of Cabbage (Brassica oleracea L.) Leaves. Sustainability 2021, 13, 11437. https://doi.org/10.3390/su132011437

AMA Style

Min K, Lee S-R. Exogenous Salicylic Acid Alleviates Freeze-Thaw Injury of Cabbage (Brassica oleracea L.) Leaves. Sustainability. 2021; 13(20):11437. https://doi.org/10.3390/su132011437

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Min, Kyungwon, and Sang-Ryong Lee. 2021. "Exogenous Salicylic Acid Alleviates Freeze-Thaw Injury of Cabbage (Brassica oleracea L.) Leaves" Sustainability 13, no. 20: 11437. https://doi.org/10.3390/su132011437

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