Polyamines in plasma membrane function in melatonin-mediated tolerance of apricot fruit to chilling stress
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Czech Journal of Food Sciences, 40, 2022 (4): 313–322 Original Paper
https://doi.org/10.17221/74/2022-CJFS
Polyamines in plasma membrane function
in melatonin-mediated tolerance of apricot
fruit to chilling stress
Qiyao Dong1, Huaipan Liu1,2*, Ronald Kurtenbach1
1
College of Life Science and Agronomy/Henan Key Laboratory of Crop Molecular Breeding
and Bioreactor, Zhoukou Normal University, Zhoukou, China
2
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China
*Corresponding author: liuhuaipan2013@126.com
Citation: Dong Q., Liu H.P., Kurtenbach R. (2022): Polyamines in plasma membrane function in melatonin-mediated tole-
rance of apricot fruit to chilling stress. Czech J. Food Sci., 40: 313–322.
Abstract: Polyamines are closely associated with environmental stresses and melatonin pretreatment enhances the
resistance of fruit to chilling stress. However, a mechanism underlying melatonin-mediated chilling resistance re-
mains to be answered. This research aimed to illuminate whether polyamines would be involved in melatonin-media-
ted chilling resistance. Therefore, in the experiment, the polyamines conjugated to the plasma membrane from the
melatonin-pretreated apricot flesh cell were examined under chilling stress. Chilling resistance was judged by four
parameters: fruit browning degree, plasma membrane permeability, malondialdehyde content and plasma membrane
protein sulfhydryl level. Results showed melatonin pretreatment led to obvious rises in the levels of non-covalently
conjugated spermine and spermidine, and covalently conjugated putrescine and spermidine in the plasma membrane.
Methylglyoxyl-bis (guanylhydrazone) pretreatment could inhibit the melatonin-induced increases of non-covalently
conjugated spermidine and spermine by inhibiting S-adenosylmethionine decarboxylase (SAMDC) activity and free
spermidine and spermine contents in flesh, coupled with the decrease in chilling resistance. Similarly, phenanthroline
pretreatment could inhibit the melatonin-induced increases in covalently conjugated putrescine and spermidine in the
plasma membrane through inhibiting transglutaminase (TGase) activity and simultaneously could aggravate chilling
damage. The results suggested melatonin pretreatment could enhance chilling resistance by increasing non-covalently
conjugated spermidine and spermine, as well as covalently conjugated putrescine and spermidine in the plasma mem-
brane of apricot fruit.
Keywords: postharvest; fruit quality; chilling resistance; conjugated polyamines; melatonin; mechanism
It is increasingly important and attractive to preserve and biochemical indicators, such as malondialdehyde
the nutritional quality of fruit for a long time. For ex- content, relative plasma membrane permeability, degree
ample, cold storage has been developed for maintaining of flesh browning and sulfhydryl group content in pro-
fruit quality after harvest (Cao et al. 2016), while dehy- teins, which are generally called chilling damage (Lurie
dration transforms the fresh product into a different one and Crisosto 2005; Cao et al. 2016). The main symptoms
(Karaaslan and Ekinci 2022). Low temperature storage of fruit chilling injury are changes in skin colour, gel
sometimes is regarded as one of the most effective meth- breakdown, flesh woolliness and colour, etc. (Koushesh
ods for keeping postharvest fruit fresh. However, cold Saba et al. 2012). Among the symptoms, flesh brown-
storage can bring about some changes in physiological ing is typical in apricot fruit. Therefore, some modern
Supported by the National Natural Science Foundation of China (Project No. 31271627) and the Natural Science Foundation
of Henan province (Project No. 222300420394).
313Original Paper Czech Journal of Food Sciences, 40, 2022 (4): 313–322
https://doi.org/10.17221/74/2022-CJFS
effective bio-techniques, such as treatment with an ex- mine, as well as covalently conjugated putrescine and
ogenous regulator, are applied to alleviate chilling dam- spermidine in the plasma membrane, which might
age and elevate the resistance of fruit against cold stress maintain normal conformation and function of plasma
(Palma et al. 2015; Min et al. 2020; Jiao 2021). Recently, membrane and proteins.
the application of melatonin (N-acetyl-5-methoxy- To explore the mechanism underlying melatonin-
tryptamine) has been increasingly attractive. Melatonin -induced chilling resistance, postharvest apricot fruit
pretreatment can enhance the resistance of summer was used as experimental material. Non-covalently and
fruit against low temperatures (Cao et al. 2016). How- covalently conjugated polyamines in the plasma mem-
ever, the mechanism underlying melatonin-induced brane of fruit flesh were detected and a relationship
chilling resistance remains to be answered. between these forms of conjugated polyamines and
Polyamines are plant growth regulators that mainly chilling resistance was discussed.
consist of putrescine, spermidine and spermine. Putres-
cine can be converted into spermidine and spermine MATERIAL AND METHODS
through being linked with one and two aminopropyls,
respectively. The biosynthesis of spermidine or sper- Material selection and treatment
mine is mainly regulated by S-adenosylmethionine de- In the orchard (latitude: 33°38'28'', longitude:
carboxylase (SAMDC) (Tiburcio et al. 1993). Slocum 114°40'52'') of Zhoukou Normal University, the fruit
(1991) reported that methylglyoxal-bis (guanylhydra- of apricot (Prunus armeniaca L.) cultivar 'Yuxuan 5',
zone) (MGBG) was the potent inhibitor of SAMDC. which were bred by Henan Academy of Agricultural
Besides free forms, polyamines, which carry positive Sciences, China, were selected and harvested when the
charges at physiological pH, are able to be non-co- volume of the fruit stopped increasing and the colour
valently conjugated to acidic proteins and phospho- was becoming yellow. After fruits were picked, they
lipid in cell membranes, which carry negative charges were shortly transferred to a laboratory. For uniformity
at physiological pH, through ionic and hydrogen bond in volume and colour, the fruits were selected without
(Sood and Nagar 2003). Galston and Kaur-Sawhney any damage. Then, they were divided into four groups
(1995) reported that non-covalently conjugated poly- and treated with chilling, melatonin, inhibitors MGBG
amines play key roles in maintaining cell membrane or phenanthroline.
integrity under environmental stresses. Additionally, Apricot fruit were immersed in de-ionised water
polyamine can be covalently conjugated to protein (control group), melatonin solution (0.1 mM) [treat-
glutamine residues through transglutaminase (TGase) ment 1 group (T1)], melatonin (0.1 mM) + MGBG
action, of which phenanthroline is a strong inhibitor. (0.2 mM) solution [treatment 2 group (T2)] and me-
Covalently conjugated polyamines function in modi- latonin (0.1 mM) + phenanthroline (0.1 mM) solution
fying proteins at the post-translating stage (Del Duca [treatment 3 group (T3)] for 100 min, then shifted into
et al. 1995). Even though polyamines are involved a chamber with relative humidity of 80% and suffered
in some studies on the mechanism of chilling resist- chilling storage at 4 °C for 30 days. According to our
ance (Koushesh Saba et al. 2012; Jahan et al. 2019a), preliminary experiment, the concentrations of mela-
so far, the significance of polyamines conjugated to the tonin at 0.1 mM, MGBG at 0.2 mM and phenanthro-
plasma membrane in melatonin-induced chilling re- line at 0.1 mM were appropriate, and the three reagents
sistance has not been revealed yet. were from Sigma Chemical Co. (US). During 30 days
The plasma membrane is an important site where of storage at 4 °C, the fresh fruits were sampled on the
plant cells first sense environmental stress informa- 0th, 6th, 12th, 18th, 24th, and 30th day. The experiments
tion, including a chilling stress signal. Under cold were repeated in 2019, 2020, and 2021. Every year,
conditions, the plasma membrane is damaged and the 180 fruit were selected as materials for every treatment
damage is performed by increasing the membrane per- group (including the control), so 720 fruits for four
meability and the content of malondialdehyde, which groups were used in one year. During the time-course
is a by-product of peroxidation of membranes, as well for 30 days of storage, 180 fruits were sampled 6 times.
as decreasing the content of protein sulfhydryl in the So at each time, 30 fruits were sampled for 3 replicates.
plasma membrane. Therefore, it would be anticipated
that melatonin pretreatment could enhance apricot Assessment of physiological parameters
fruit chilling resistance through increasing the levels The degree of apricot flesh browning was assessed
of non-covalently conjugated spermidine and sper- by the method of Cao et al. (2016) with modifica-
314Czech Journal of Food Sciences, 40, 2022 (4): 313–322 Original Paper
https://doi.org/10.17221/74/2022-CJFS
tions by observed browning surface with a range from putrescine, spermidine and spermine were quantified
0 to 4: degree 0, no browning area; degrees 1, 2, 3, by high performance liquid chromatography (HPLC)
and 4 meant the browning area < 20%, ≥ 20% and < 50%, (Model 2695; Waters, US).
≥ 50% and < 70%, and ≥70% of the cut surface, respec- Purification of the plasma membrane. Apricot
tively. The chilling injury index indicated the browning fruit flesh tissue (2 g) was ground (Model JJ-2; Shang-
degree and was calculated by the formula in Equation 1. hai Shuaideng Instrument Equipment Co., Ltd., China)
Plasma membrane permeability was assessed by re- in sorbitol Tris-Hepes buffer (70 mM, pH 7.5). After
ferring to the means of Jahan et al. (2019b) with a few filtering (diameter 9 cm filter paper; Shanghai Shunyou
adjustments. The fruit flesh tissue was immersed into Technology Co., Ltd., China), the sample was centri-
de-ionised water and incubated for 2 h at 25 °C. Original fuged at 15 000 × g for 15 min. The supernatant was
electrical conductivity (OEC) of the water containing then centrifuged at 75 000 × g for 40 min. After micro-
the flesh tissue was determined with a conductivity somal precipitate was suspended in sucrose Tris-Hepes
meter. Afterwards, the samples were boiled and cooled. buffer (3.0 mM, pH 7.8), it was further purified (Model
The terminal electrical conductivity (TEC) was deter- XPN-100; Beckman Culter Co., Ltd., US) through the
mined. De-ionised electrical conductivity (DEC) repre- method of Qiu and Su (1998) to obtain the isolated
sented electrical conductivity (EC) of de-ionised water. plasma membrane solution.
Relative plasma membrane permeability was assessed Isolation and detection of proteins in the plasma
by the formula: (OEC – DEC)/(TEC – DEC) × 100. membrane. The isolated plasma membrane solu-
Malondialdehyde content was determined through tion was added 10% Triton X-100 (v/v) drop-wise un-
the method of Alexieva et al. (2001) with some modifi- til the triton concentration being lowered to 1% and
cations. After the flesh sample was ground (Model JJ-2; then ultrasonicated with an ultrasonic disintegrator
Shanghai Shuaideng Instrument Equipment Co., Ltd., (Model 200-W; Shanghai Guanter Co., Ltd., China). Af-
China) in trichloroacetic acid solution and centrifuged terwards, it was centrifuged at 20 000 × g for 30 min.
(Model TGL-16; Hunan Xiangyi Instrument Equip- The supernatant consisted of the proteins in the plasma
ment Co., Ltd., China), aliquot trichloroacetic acid was membrane. Protein content was detected by the Brad-
added to the supernatant. It was boiled, cooled down and ford method of protein-dye binding (Bradford 1976);
centrifuged. The content of malondialdehyde was deter- bovine serum albumin was used as standard.
mined by detecting absorbance at 532 nm and 600 nm. Determination of non-covalently and covalently
A part of the prepared plasma membrane vesicle solu- conjugated polyamines. They were determined
tion was treated with the disulphide reagent. Sulfhydryl through the method of Sharma and Rajam (1995) with
group content in the plasma membrane of flesh cells a few modifications. Perchloric acid (w/v: 5%) was
was detected following the method described by Ellman added into the aforementioned isolated plasma mem-
et al. (1959). One unit was defined as nmol mg–1 protein. brane solution. The sample was centrifuged for 40 min
at 30 000 × g. The supernatant contained non-cova-
Detection of biochemical parameters lently conjugated polyamines. Perchloric acid (w/v: 5%)
Free polyamines in the tissue of apricot fruit flesh. was added into the aforementioned plasma membrane
Extraction and quantification of free polyamines were protein solution. The mixture was centrifuged for
conducted by the method of Sharma and Rajam (1995) 50 min at 30 000 × g at 4 °C. The precipitate was hydro-
with minor adjustments. An aliquot of 0.5 g of flesh lysed with 12 N HCl. Then, two forms of polyamines
tissue was homogenised (Model JJ-2; Shanghai Shuaid- were benzoylated and quantified by HPLC through the
eng Instrument Equipment Co., Ltd., China) in 5 mL method of Sharma and Rajam (1995).
perchloric acid (5%, v/v), and the homogenate was left Determination of SAMDC and TGase activities
to stand for 1 h in a refrigerator at 4 °C. Then, it was cen- in apricot flesh. SAMDC activity was assayed by exam-
trifuged at 21 500 × g for 30 min (Model TGL-16; Hu- ining the release of 14CO2 using substrates labelled with
nan Xiangyi Instrument Equipment Co., Ltd., China). isotope 14C, through the method of Kaur-Sawhney and
The supernatant was collected for a free polyamine Shin (1982). TGase activity was assessed via detecting
assay. After benzoylation with benzoyl chloride, free the incorporation rate of putrescine labelled with 3[H]
∑ ( browning degree ) × ( fruit number of the browning degree )
Chilling injury index = (1)
4 ( the highest browning degree ) × 10 ( fruit number of each treated group )
315Original Paper Czech Journal of Food Sciences, 40, 2022 (4): 313–322
https://doi.org/10.17221/74/2022-CJFS
into proteins according to the method of Icekson and RESULTS AND DISCUSSION
Apelbaum (1987).
Chilling damage was alleviated by melatonin and
Statistical analysis aggravated by polyamine biosynthesis inhibitors.
The data shown in the paper are averages of 9 val- The parameter of flesh browning degree showed that
ues ± standard error (SE). Microsoft Excel and SPSS 16.0 apricot fruit suffered chilling damage. After 6 days
(SPSS Inc., US) were used for analysing data. The devia- of chilling storage, apricot fruit began to develop dam-
tion of the averages was statistically evaluated by analysis age symptoms as displayed by flesh browning degree,
of variance (ANOVA) and Duncan's method was used and the damage degree increased with the prolonged
to compare multiple group means at P < 0.05 level. Signif- time of chilling storage (Figure 1A). Reactive oxygen
icant differences between multiple groups were indicated species induced by chilling stress could lead to the per-
by different letters above the columns in the figures. oxidation of lipids and proteins in the plasma membrane
(A) 1.2
control T1 T2 T3
a
1.0 control T1 T2 T3a
browning degree
b
0.8
Apricot flesh
control T1b T2 T3 b
control T1 T2 T3 b
0.6 b
c cd
0.4 d d d
e
0.2 ef
g f f ef
h
0.0
0 6 12 18 24 30
Relative plasma membrane
(B) 80
a
70
permeability (%)
ab ab
50
60 bc
b b
40
50 c
30
40 d cd
e
20
30 f
ef
10
20 g gh
0 j j j j j j j j i h
10
0 6 12 18 24 30
0
0 6 12 18 24 30
(C) 40
content
(C) 40 a
weight)
35
content
a
weight)
35
30
30 b
Malondialdehyde
25 b bc
fresh
c bc
Malondialdehyde
25 c cd c
fresh
20 c cd c d c
20 e
g–1g–1
15 d
e fg f e
15
0 e h g g g
(nmol
10 0 6 12g g g18
fg f 24 30
protein) (nmol
(D) 10
250
0 j j j j j j ij i h
5
j j0 j j j j 6ij i 12 18 24 30
content
(D) 5
250 Figure 1. Changes in apricot
0
200
0 0 6 12 18 24 30
content
(D) 250 0 6 12 18 24 30 (A) flesh browning degree,
(D) 200
250
protein)
content
150 (B) relative plasma membrane
sulfhydryl
a a
a a
content
200 a a a a a a a
protein)
a a
150 a a a permeability, (C) malondial-
–1 –1
sulfhydryl
200 a a a a a a a ab ab
protein)
a a
mg
100 a
b ab b ab b
dehyde, and (D) protein sulf-
150
sulfhydryl
b b
mg
(nmol
100
150 b
sulfhydryl
hydryl contents
Protein
c
50
–1 –1
c cd
(nmol
mgmg
cd
100 d
Protein
d cd a–j
50
100 d e
cd
d Significant differences
(nmol
0 e between multiple groups are
Protein
0 6 12 18 24 30
(nmol
50
Protein
0
50 indicated by different letters
0 6 12 time (day)
Storage 18 24 30
0 above the columns in the figure
0 0 6 Storage
12 time (day)
18 24 30 (P < 0.05); T1 – treatment 1
0 6 12 18 24 30 group; T2 – treatment 2 group;
Storage time (days)
Storage time (days) T3 – treatment 3 group
316Czech Journal of Food Sciences, 40, 2022 (4): 313–322 Original Paper
https://doi.org/10.17221/74/2022-CJFS
(Koushesh Saba et al. 2012; Cao et al. 2016, 2018), which damage degree only got to less than 50%, compared
might result in increasing the plasma membrane per- with the control. Besides the browning degree in-
meability and malondialdehyde content and decreasing dex, the changes in the other parameters collectively
the protein sulfhydryl content. Therefore, to provide showed that exogenous melatonin treatment authenti-
more evidence for chilling damage to apricot fruit, cally enhanced the resistance of apricot fruit to chilling
some latent parameters, such as plasma membrane per- stress. In the research, the roles which conjugated poly-
meability (Figure 1B), malondialdehyde (Figure 1C) and amines in the plasma membrane might play in melato-
protein sulfhydryl (Figure 1D) contents, were examined nin-mediated resistance were illuminated.
in the research. All of the changes in these parameters Free spermidine and spermine were involved
indicated that apricot fruits were suffering from chill- in melatonin-mediated resistance. To explore the
ing damage, since plasma membrane permeability and function of conjugated polyamines in the plasma mem-
malondialdehyde content increased, and sulfhydryl brane, it was necessary to examine free polyamines
content decreased. in apricot flesh. In the apricot flesh tissue, three major
However, 0.1 mM melatonin treatment led to a de- free polyamines, putrescine, spermidine and spermine
crease in browning degree and no marked browning could be detected. Under chilling conditions at 4 °C,
was found on the 6th day. On the 12th day, brown- free putrescine increased sharply during the storage
ing merely began to appear mildly and on the 30th day, for 30 days (Figure 2A). Free spermidine (Figure 2B)
50
40
50
(A) 30
350
40
50 control T1 T2 a
20
30
300
(nmol g–1 fresh weight)
40 ab
Free putrescine level
10
20
250
30
b
0 bc bc
10
20 0 6 12 18 24 30
2000 c
10 0 6 e 12 18 24 30
e
1500 ef ef
fg f
gh 0h gh 6 g 12 18 24 30
g gh
100
50
0
0 6 12 18 24 30
a
(B) 200
1600
fresh weight)
0 6 12 18 24 30
a
contentlevel
b
(D) 250
1600
120 0 6 12 18 24
b 30 c
spermidine
(D) 250
2000 cd cd
gprotein)
0 6 12
de 18 d 24 de 30
120 de c
content
de de
(D) 80
250 ef ef ef
e cd cd
200 f ef f
protein)
150 d
sulfhydryl
de de de
content
–1 –1
de de
80 ef ef ef
e
200
40 f ef
protein)
f
(nmol
150
sulfhydryl
Free
mg
100
40
–1
150
sulfhydryl
mg(nmol
0
mg
100
Protein
50 0 6 12 18 24 30
–1
(nmol
0
100
Protein
a
50 0 6 12 18 24 30
1200
(nmol
0 6 12 18 24 30
Free spermine level Protein
b a
(C) 50
150
1200 0 6 Storage
12 time (day)
bc
18 24 30
90 b a c Figure 2. The dynamic course
1200 d
Storage
12 time (day)
d d of free polyamine content:
(nmol g–1 fresh weight)
90 0 6 18
bc de de 24
b 30 c
e
60 e d
e
d (A) putrescine, (B) spermi-
90
ef ef f f
ef f Storage time (day)
bc de
de
d c
60 e d
e e d dine, and (C) spermine in apri-
30 ef ef f f
ef f de de
d
e cot flesh
60 ef f e
e
30 ef ef f f a–h
0 Significant differences
30 0 6 12 18 24 30 between multiple groups are
0 indicated by different letters
0 6 12 18 24 30
0 above the columns in the figure
0 6 12 18 24 30 (P < 0.05); T1 – treatment 1
Storage time (days) group; T2 – treatment 2 group
317Original Paper Czech Journal of Food Sciences, 40, 2022 (4): 313–322
https://doi.org/10.17221/74/2022-CJFS
and free spermine (Figure 2C) levels remained almost with the control. Secondly, MGBG treatment inhibited
unchanged throughout the whole chilling storage melatonin-induced resistance (Figure 1), coupled with
at 4 °C for 30 days. Melatonin pretreatment signifi- inhibiting melatonin-induced increases in SAMDC
cantly (P < 0.05) affected the free polyamine contents. activity (Figure 3A) and the contents of the polyamines
After chilling storage at 4 °C for 6 days, free spermidine (Figures 2B, 2C). The study of Palma et al. (2015)
and free spermine levels of the melatonin-pretreated showed free putrescine played roles in the resistance.
group increased significantly (P < 0.05), coupled with Therefore, we speculated that the increase in putres-
the marked decrease in putrescine content during the cine content might only be a premise for chilling re-
30-day storage period. sistance and a key issue was whether chilling-induced
Min et al. (2020) reported that free polyamines free putrescine could be subsequently transformed
in fruit played an important function in methyl jasmo- to free spermidine and spermine. The speculation
nate-mediated resistance of tomato fruit to chilling. was testified by our study results on the free putres-
In the present study, we found that free spermidine and cine profile (Figure 2A) in apricot flesh under chilling,
spermine might be involved in melatonin-mediated re- melatonin and MGBG treatments. Chilling stress in-
sistance. The finding was supported by melatonin and duced the increase in free putrescine and melatonin
MGBG treatments. Firstly, exogenous melatonin pre- inhibited the chilling-induced increase, while MGBG
treatment markedly (P < 0.05) enhanced chilling re- reversed the melatonin effect. Then, why could free
sistance, which was judged by the parameters of flesh spermidine and free spermine play an important func-
browning degree (Figure 1A), plasma membrane per- tion in the resistance induced by melatonin? They
meability (Figure 1B), malondialdehyde (Figure 1C) carried more positive charges and thereby could
and protein sulfhydryl (Figure 1D) contents. Mean- be non-covalently conjugated more easily to cell com-
while, exogenous melatonin enhanced the increases ponents with negative charges. The plasma membrane
in SAMDC activity (Figure 3A) and the contents of free was mainly involved in chilling stress, so non-cova-
spermidine and spermine (Figures 2B, 2C), and inhib- lently conjugated polyamines in the plasma membrane
ited the increase in putrescine (Figure 2A), compared were detected next.
(A) 180
control T1 T2
(μmol CO2 g–1 fresh weight min–1)
a
150 a
SAMDC activity
120 b
bc
bc
cd c cd
90 cd cd
de d
e ef ef ef
ef f
60
30
0
0 6 12 18 24 30 Figure 3. The dynamic course
(B) 7
8 of enzyme activity: (A) SAMDC
a
(nmol putrescine mg–1 protein h–1)
control T1 T3 b and (B) TGase in apricot flesh
6
7
b during chilling storage
5
6 c
a–g
4
5 d de Significant differences
TGase activity
c
e e between multiple groups are
3
4 d cd de
fg g fg fg fg f f f
e indicated by different letters
g e
2
3 fg fg fg f f f cd above the columns in the figure
fg fg g
1
2
g (P < 0.05); T1 – treatment 1
group; T2 – treatment 2 group;
0
1 T3 – treatment 3 group; SAMDC
0 6 12 18 24 30
0 – S-adenosylmethionine decar-
0 6 Storage
12 time (day)
18 24 30 boxylase; TGase – transgluta-
Storage time (days) minase
318Czech Journal of Food Sciences, 40, 2022 (4): 313–322 Original Paper
https://doi.org/10.17221/74/2022-CJFS
Function of spermidine and spermine non-co- spectively. Treatment with MGBG reversed the effects
valently conjugated to plasma membrane. Chilling of melatonin treatment on non-covalently conjugated
stress led to an increase in the non-covalently conju- spermidine and spermine contents markedly (P < 0.05)
gated putrescine level in the plasma membrane from the (Figures 4B, 4C). From these interesting results, it could
apricot flesh tissue, whereas non-covalently conjugated be concluded that under chilling, melatonin and MGBG
spermidine and spermine contents were maintained treatments, the changes in non-covalently conjugated
nearly unchanged throughout the entire storage period spermidine and spermine contents were in parallel with
of chilling at 4 °C (Figure 4). Melatonin pretreatment in- the change in chilling resistance (Figure 1) during the
hibited markedly (P < 0.05) the chilling-induced increase entire storage. The finding was supported by Dutra et al.
in the non-covalently conjugated putrescine level after (2013), who suggested that free spermidine and sperm-
storage for 18 days at 4 °C, and meanwhile melatonin ine, which carry more positive charges than putrescine,
pretreatment induced markedly (P < 0.05) the increases could be non-covalently conjugated to the plasma mem-
in non-covalently conjugated spermidine and spermine brane more easily and maintain the plasma membrane
levels after storage for 12 days and 18 days at 4 °C, re- configuration and function.
(A) 100
control T1 T2
a
ab
conjugated
80 ab
(A) 100 b
control T1
b T2
bc
mg–1 protein)
bc
levels levels
cd cd cd cd c
cd cd cd
conjugated
60
80 cd d cd
(A) 100
control T1 T2
mg–1 protein)
putrescine
Non-covalently
40
60
conjugated
80
(nmol
protein)
putrescine
Non-covalently
20
levels
40
60
(nmol
putrescine
0
Non-covalently conjugatedNon-covalently
20
–1
40 0 6 12 18 24 30
(nmol mg
0
20
(B) 240
0
200 a
a a
(B) 240
(nmol mg–1 protein)
160
spermidine level
200 a a
a
120 b b b b
160
c c c c
80 cd cd cd cd cd d cd
120 b b b b
40
c c c c
80 cd cd cd cd cd d cd
0
40 0 6 12 18 24 30
(C) 150
a Figure 4. The dynamic course
Non-covalently conjugated
120
b of non-covalently conjugated
(nmol mg–1 protein)
polyamine content: (A) putres-
spermine level
90
c cine, (B) spermidine, and
d
60 d (C) spermine in the membrane
e ef
f ef
fg fg a–g
fg g g g g g g Significant differences
30 between multiple groups are
indicated by different letters
0 above the columns in the figure
0 6 12 18 24 30 (P < 0.05); T1 – treatment 1
Storage time (days) group; T2 – treatment 2 group
319
40
(A) 30
350
control T1 T2 a
20
300
h weight)
ab
10
ne level
b
250
50
0 bc bc
0 6 12 18 24 30Original Paper Czech Journal of Food Sciences, 40, 2022 (4): 313–322
https://doi.org/10.17221/74/2022-CJFS
Why were non-covalently conjugated polyamines re- and covalently conjugated polyamines (Figure 5) in the
lated to plant resistance? The interaction of ion chan- plasma membrane, because melatonin pretreatment
nels and conjugated polyamines might be involved markedly (P < 0.05) increased TGase activity (Figure 3B)
in the issue. For instance, it was suggested that non-co- and the contents of covalently conjugated putrescine
valently conjugated polyamines regulated ion channels (Figure 5A) and spermidine (Figure 5B). More inter-
in bio-membranes (Williams 1997), and in guard cells, estingly, pretreatment with phenanthroline (TGase in-
a stomatal movement was modulated by the polyam- hibitor) decreased significantly (P < 0.05) not only the
ines through the KATl-like (K+1 transporter 1) chan- melatonin-mediated chilling resistance (Figure 1), but
nels (Liu et al. 2000). Additionally, the activity of the also TGase activity (Figure 3B), resulting in the drastic
proton pump in plasma membrane could be enhanced decreases in the contents of covalently conjugated pu-
by the polyamines (Athwal and Huber 2002). All the trescine and spermidine. Sulfhydryl in plasma mem-
researches have suggested that non-covalently conju- brane protein was oxidised by reactive oxygen species
gated polyamines can play an important role in main- induced by chilling stress, and transformed into disul-
taining the conformation and function of the plasma phide bonds, resulting in protein structure destruc-
membrane and membrane proteins. tion and denaturation. Free putrescine and spermidine
Significance of putrescine and spermidine co- might be covalently conjugated to proteins in the plasma
valently conjugated to the plasma membrane. Be- membrane to inhibit the oxidation induced by chilling
sides free and non-covalently conjugated polyamines, stress. Our suggestion was in good accordance with
by TGase catalysing, free polyamines might be con- the results of Del Duca et al. (1995) on covalently con-
jugated covalently to protein glutamine residues jugated polyamines in the thylakoid membrane of the
(Del Duca et al. 1995). Cell membranes are rich in pro- chloroplast. Collectively, it could be inferred that co-
teins, such as proton pumps, ion channels, transport- valently conjugated putrescine and spermidine in the
ers, and protein enzymes. So in the study, the contents plasma membrane should play a similarly important role
of the polyamines conjugated covalently to protein were to non-covalently conjugated spermidine and spermine
also assessed. The melatonin-mediated resistance was in maintaining the normal conformation and function
closely associated with TGase activity in apricot flesh of the plasma membrane and membrane proteins.
(A) 150
control T1 T3 a
(A) 150
120 control T1 T3 b a
conjugated
120
protein)
b
90
levellevel
c
conjugated
cd
protein)
d
90 d c
putrescine
60 cd
–1
e
mg–1mg
Covalently
ef d
d
putrescine
g fg fg fg fg
60 g fg fg fg fg
(nmol
e
Covalently
30 ef
g fg fg fg fg
g fg fg fg fg
(nmol
30
0
0 6 12 18 24 30
0
(B) 80
a
Figure 5. The dynamic course
b
Covalently conjugated
60 of covalently conjugated poly-
(nmol mg–1 protein)
spermidine level
amine content: (A) putrescine
c
40 and (B) spermidine in the
de
d membrane
de
e a–g
20 ef Significant differences
fg g fg f fg fg fg fg fg
g between multiple groups are
indicated by different letters
0 above the columns in the figure
0 6 12 18 24 30 (P < 0.05); T1 – treatment 1
Storage time (days) group; T3 – treatment 3 group
320Czech Journal of Food Sciences, 40, 2022 (4): 313–322 Original Paper
https://doi.org/10.17221/74/2022-CJFS
CONCLUSION Ellman G.L. (1959): Tissue sulfhydryl groups. Archives Bio-
chemistry Biophysics, 82: 70–77.
In summary, this work was the first to illuminate that Galston A.W., Kaur-Sawhney R. (1995): Polyamines as en-
melatonin pretreatment enhanced chilling resistance dogenous growth regulators. In: Davies P.J. (ed): Plant
via increasing non-covalently conjugated spermidine Hormones: Physiology, Biochemistry and Molecular
and spermine, as well as covalently conjugated putres- Biology. Dordrecht, the Netherlands, Kluwer Academic
cine and spermidine in the plasma membrane of apri- Publishers: 158–178.
cot fruit. Although the present study was just regarded Icekson I., Apelbaum A. (1987): Evidence for transglutamin-
as a modest spur, it revealed a novel mechanism un- ase activity in plant tissue. Plant Physiology, 84: 972–974.
derlying melatonin-mediated chilling resistance. Jahan M.S., Shu S., Wang Y., Chen Z., He M., Tao M., Sun J.,
In the future, the research on conjugated polyamines Guo S. (2019a): Melatonin alleviates heat-induced damage
in bio-membranes would be increasingly attractive. of tomato seedlings by balancing redox homeostasis and
By gene knockout and recombination technology, new modulating polyamine and nitric oxide biosynthesis. BMC
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ing stress. creases the heat tolerance in tomato (Solanum lycoper-
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Received: April 27, 2022
Accepted: July 25, 2022
Published online: August 25, 2022
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