- Email: [email protected]
Brassinosteroid-induced changes of lipid composition in leaves of Pisum sativum L. during senescence
Accepted Manuscript Br assinoster oid-induced changes of lipid composition in leaves of Pisum sativum L . dur ing senescence Evgenia Fedina, Andrey Yarin, Faimya Mukhitova, Alexander Blufard, Ivan Chechetkin PII: DOI: Reference:
S0039-128X(16)30161-1 http://dx.doi.org/10.1016/j.steroids.2016.10.009 STE 8049
To appear in:
Steroids
Received Date: Revised Date: Accepted Date:
19 May 2016 21 October 2016 26 October 2016
Please cite this article as: Fedina, E., Yarin, A., Mukhitova, F., Blufard, A., Chechetkin, I., Br assinoster oid-induced changes of lipid composition in leaves of Pisum sativum L . dur ing senescence, Steroids (2016), doi: http:// dx.doi.org/10.1016/j.steroids.2016.10.009
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Brassinosteroid-induced changes of lipid composition in leaves of Pisum sativum L. during senescence Evgenia Fedina, Andrey Yarin, Faimya Mukhitova, Alexander Blufard, Ivan Chechetkin Kazan Institute of Biochemistry and Biophysics, Kazan Scientific Center, Russian Academy of Sciences, 420111, 2/31 Lobachevsky str., Kazan, Russian Federation
Email addresses: Dr. Evgenia Fedina; [email protected] Dr. Andrey Yarin; [email protected] Dr. Faimya Mukhitova; [email protected] Dr. Alexander Blufard; [email protected] Dr. Ivan Chechetkin; [email protected]
The name and address of the corresponding author: Dr. Evgenia Fedina; Kazan Institute of Biochemistry and Biophysics, Kazan Scientific Center, Russian Academy of Sciences, 2/31 Lobachevsky str., Kazan, 420111, Russian Federation, Email: [email protected]; phone number: +7 8432319047
2
Abstract
The effect of steroid phytohormone 24-epibrassinolide (EBR) on the composition of some lipid classes (free fatty acids, triacylglycerols and galactolipids) in detached pea leaves was studied for the first time. EBR (0.1 µM) promoted senescence and increased the content of 14:0, 16:0 and 18:1 free fatty acids as well as 18:2 and 18:3 bound to triacylglycerols in the detached leaves in contrast to mock-treated leaves. The content of all identified fatty acids bound to galactolipids decreased in the detached leaves treated with EBR compared to that in mocktreated leaves. These findings suggest that free fatty acids are liberated from polar lipids and then undergo esterification to neutral lipids in the detached leaves upon EBR treatment. We propose that steroid phytohormones may be involved into regulation of leaf senescence via alteration of cell lipid composition.
3 Keywords: Pisum sativum, 24-epibrassinolide, leaf senescence, fatty acids, glycerolipids, triacylglycerols Highlights: Brassinosteroids (BRs ) promote the leaf senescence in concentration dependent manner
BRs-induced senescence is accompanied by the alteration of cell lipid metabolism BRs increase triacylglecerol and free fatty acid content while decrease galactolipids
Abbreviations: BRs – brassinosteroids, DGDG – digalactosyldiacylglycerol, FA – fatty acids, FW – fresh weight, MGDG –
monogalactosyldiacylglycerol, GC-MS – gas chromatography – mass
spectrometry, EBR – 24-epibrassinolide, AzA – azelaic acid, SbA – sebacic acid, SubA – suberic acid
4 1. Introduction Brassinosteroids (BRs) comprise a group of steroid phytohormones widespread in the Kingdom Plantae. Research on BRs has a long history, starting with the study of oil substance from the pollen of rape and eller that exhibited plant growth promoting effects at low concentrations [1]. The first member of BRs, brassinolide, was isolated from rape pollen and identified as (22R,23R,24S)-2α,3α,22,23-tetrahydroxy-24-methyl-B-homo-7-oxa-5α-cholestan6-one [2]. Later over 70 structurally related BRs were isolated from different plants, including angiosperms, gymnosperms, ferns and algae. Common structural characteristic of BRs is 5αcholestan skeleton. The structural variations of BRs come from the kind and orientation of oxygenated functions in rings A and B [3]. BRs were detected in all parts and organs of plants [4]. BR level is usually higher in the young growing tissues (1-100 ng/g FW) than in mature ones (0.01-0.1 ng/g FW) [5]. The most abundant BRs detected in plants are castasterone, brassinolide, typhasterol, 6-deoxocastasterone, teasterone and 28-norcastasterone [5]. The greatest variety of BRs (25 free and 2 conjugated forms) was found in the unripe bean (Phaseolus vulgaris) seeds [5]. Other legumes possess lesser amount of BR members detected in pollen, seeds and shoots and their quantity is in the range of 0.007 – 628 µg/g FW [6]. Castasterone and brassinolide are the most important BRs in nature due to their wide distribution in plants and higher biological activity. However, 24epibrassinolide (EBR) is the most popular BR to study physiological effects of exogenous steroid phytohormones on plants due to its commercial availability. BRs play multiple roles in plants and function at extremely low concentrations (10-12-10-7 М). They control such plant developmental processes as seed germination, cell division and elongation,
flowering,
vascular-differentiation,
reproduction,
root
development,
and
photomorphogenesis [7]. They have recently been considered as stress hormones since their level increases under adverse environmental conditions and that leads to improvement of plant
5 resistance to different stressors such as heavy metals, pesticides, salt, high and low temperature, drought and pathogens [8, 9]. Plants deficient in the enzymes participating in BR biosynthesis and perception have a phenotype of prolonged leaf lifespan and so they are notable for the delayed senescence [10]. BR also was shown to promote chlorophyll degradation and decrease anthocyanin content in leaves [11, 12]. Based on these observations, BR are thought to regulate the plant senescence processes [13, 14]. Other phytohormones were shown to play a significant role in the regulation of leaf senescence [15]. Plant hormones such as ethylene, abscisic acid, salicylic acid, and jasmonic acid promote senescence, whereas auxin, gibberellic acids, and cytokinins retard it [16]. Plant leaf senescence is a complex process of the final stage of plant life, which is characterized by dramatic changes in metabolism in all cellular compartments followed by modification of macromolecules as well as lipids. One of the earliest manifestations of senescence is the loss of selective permeability of membranes due to their molecular disassembly. The onset of membrane leakage is a result of changes in the composition and molecular organization of the lipid bilayers due mainly to de-esterification of the major membrane lipids [17]. The synthesis of triacylglycerols from de-esterified free fatty acids (FAs) in parallel with dismantling of galactolipids is the most dramatic change observed in membrane lipid composition with progression of foliar senescence [18, 19]. Further on, triacylglycerols can be metabolized by β-oxidation to acetyl CoA, which is the final step of lipid catabolism in senescing tissues. Triacylglycerols may be a transient intermediate that prevents detrimental levels of free FA accumulation, or depletion of the free CoA-SH pool. Though lipid metabolism in the senescent plant leaves was thoroughly studied, mechanisms underlying the regulation of plant senescence by BR need to be revealed. The accelerated leaf senescence is known to occur in the detached leaves and so the latter are often used as a model system for studying senescence [20]. In the present work, the system was used to study the effect of 24-epibrassinolide (EBR), one of physiologically active BRs, on
6 the composition of some lipid classes (free fatty acids, triacylglycerols and galactolipids) in detached pea leaves.
2. Experimental
2.1 Plant material
Pisum sativum L. (cv. Truzhenik) was from the Tatar Research Institute of Agriculture of the Russian Academy of Sciences (Kazan, Russian Federation). Pea seeds were sterilized by soaking in 40% NaOCl (v/v) for 10 min, washed with distilled water and then were put to germinate in Petri dishes filled with solution containing (mM): 1,25 mМ KNO3, 0,25 mМ KH2PO4, 0,25 mМ MgSO4, 1,25 mМ Ca(NO3)2 (approximately ¼ classical Hoagland & Arnon’s nutrient medium I; рН 6,5). 2-Day-old seedlings were transferred to sterile test tubes filled with the sterile agar nutrient medium and were grown under 12-h photoperiod at the 23±1°C and 200 µmol m-2 s-1 irradiance for 10 days. Leaves of 10-day-old seedlings were detached and placed in Petri dishes on the sterile nutrient medium containing no phytohormone (mock-treated leaves) or 0.1 µM EBR and were incubated under constant temperature and irradiance conditions (see above) for 15 days. Leaves of 10-day-old intact plants grown on the sterile nutrient medium without EBR were used as a control. The length of detached leaves placed in Petri dishes was 1.40±0.03 cm, was not significantly changed during the experiments and corresponded to the length of control leaves. Nutrient solutions were renewed daily. 24-epibrassinolide was a generous gift from Prof. Vladimir Khripach (Laboratory of Steroid Chemistry, Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Minsk).
2.2 The extraction and separation of lipids
7
Total lipids were extracted from pea leaves with boiling propan-2-ol and separated by the silicic acid column chromatography as described before [21] to afford galactolipid and neutral lipid fractions. Neutral lipids were eluted from the column with chloroform/methanol (9:1, v/v) and galactolipids with acetone/methanol (9:1, v/v), successively. Then neutral lipids and galactolipids were separated by thin-layer chromatography (TLC), solvent hexane/diethyl ether/acetic acid (70:30:1, v/v) and acetone/benzene/water (91:30:8, v/v), respectively, using TLC plates Silica gel 60 (Merck, Germany). Zones with Rf 0.18, 0.41 and 0.74 containing DGDG, MGDG and triacylglycerols were scraped from the plates and corresponding lipids were eluted from silica with ethanol. Free fatty acids were extracted from the fraction of neutral lipids and purified using Supelclean LC-NH2 (3 ml) and LC-Si (1 ml) cartridges (Supelco, USA) as described previously [22]. Free fatty acids were methylated with CH2N2 while triacylglycerols and galactolipids were transesterified with sodium methoxide. The resulting methyl esters of fatty acids (derived from free fatty acids and acyl residues of triacylglycerols, MGDG and DGDG) were analyzed by GC-MS as described before [23]. Methyl ester of hexadecanoic acid (17:0) was used as an internal standard. The content of fatty acids was expressed as nmol per g of fresh weight.
2.3 Leaf senescence evaluation
Leaf senescence was evaluated by measurements of chlorophyll content of pea leaves as follows. Leaf tissue (0.1 g) was homogenized in 1 mL of 80% acetone (v/v), the extract was added to 25 mL of 80% acetone and then centrifuged at 12,000 g for 10 min. The absorbance of the supernatant was measured at 663 nm and 646 nm, respectively, on a Lambda 25 spectrophotometer (Perkin Elmer, USA). The total chlorophyll a and b content (FChl а+b)
8 was calculated according to Porra method [24]. Chlorophyll content data were presented in mg per g of fresh weight.
2.4 Statistics.
All experiments were in triplicate. Difference significance was assessed by the Student’s tcriterion. The mean values and standard deviations are given.
3. Results The yellowing of detached leaves due to the decrease in chlorophyll content of cells is an obvious symptom of their senescence. The decrease in chlorophyll content of pea leaves was noticeable 10 days after leaf detachment (Fig. 1). On day 15, chlorophyll loss in the detached leaves treated with EBR (0.1 µM) was more dramatic than in mock-treated leaves (Fig. 1). These findings are consistent with the data on EBR-stimulated chlorophyll degradation in senescent plant leaves [12-14]. It should be noted that other examined concentrations of phytohormone had no statistically significant influence on chlorophyll content compared to one in mock-treated leaves ( Fig.1 ). Since EBR effect on chlorophyll content of pea leaves was most obvious on day 15, we analyzed lipid composition of the leaves 15 days after their detachment. GC-MS analysis revealed decrease in the content of palmitic (16:0), linoleic (18:2), α-linolenic (18:3) and stearic (18:0) acids bound to galactolipids of ageing pea leaves compared to control (Fig. 2). The decrease was more dramatic in EBR-treated leaves than in mock-treated leaves (Fig. 2). These data are consistent with chlorophyll degradation in ageing leaves and its stimulation by EBR (Fig. 1). GC-MS analysis of free FA revealed the presence of myristic acid (14:0), 16:0, 18:0 and oleic acid (18:1) as well as oxylipins such as suberic (SubA), azelaic (AzA) and sebacic (SbA) acids in the leaves of control pea plants (see Supplementary Data, Fig. S1). The profile of free
9 FA that were isolated from the mock-treated leaves showed no oxylipins and drastic decrease in the content of 14:0, 16:0, 18:0 and 18:1 (Fig. 3). EBR-treated leaves contained only traces of AzA and the increased amounts of 14:0, 16:0 and 18:1 compared to control (Fig. 3). EBR was shown to increase the content of 16:0 and 18:0 FA as well as 18:2 and 18:3 bound to triacylglycerols in the detached leaves in contrast to mock-treated leaves (Fig. 4).
4. Discussion The effect of BRs on FA profiles of plants was demonstrated earlier. EBR (0.1 µM) decreased the content of saturated FA (16:0 and 18:0) and increased the content of unsaturated FA (18:1, 18:2 and 18:3) bound to MGDG in rape callus under cold acclimation. The impact of EBR on the fatty acid composition of DGDG was different, i.e. 18:0 content was significantly increased, whereas 18:2 diminished to a trace level [25]. Changes in the ratio of saturated to unsaturated FA bound to polar lipids of plasma membranes in mango fruits stored at 5°C were also induced by 10 µM brassinolide [26]. EBR application to the plants subjected to salt stress has also affected content of several fatty acids. For instance, EBR application led to overaccumulation of 18:2 in the salt-stressed rape seeds and to partial reversion of 18:1 and 20:1 content in the seeds of salt-stressed Arabidopsis [27]. Our data demonstrate the effect of EBR on FA composition in detached pea leaves for the first time. Unlike above mentioned observations in the plants under cold acclimation and salt stress, EBR did not affect the ratio of saturated to unsaturated FA in the senescent pea leaves. Most changes were seen in FA content of different lipid classes. EBR increased the content of free FA as well as FA bound to triacylglycerols and decreased the content of FA bound to galactolipids. These findings suggest that FA are liberated from polar lipids and then undergo esterification to neutral lipids in the detached leaves upon EBR treatment. Similar changes in FA composition of polar and neutral lipids occur in plants under various unfavorable conditions and are associated with leaf senescence and diacylglycerol acyltransferase action [28].
10 Degradation of the chloroplast membranes is known as one of the major features of the leaf senescence [29]. Moreover, the decrease in content of FA bound to DGDG and MGDG indicate the destruction of the chloroplast membranes in senescent leaves [28]. In our experiment, EBR in a concentration of 0.1 µM accelerated this process (Fig. 2). We also detected that EBR decreased the chlorophyll content of detached leaves on the 15th day after treatment compared to mock-treated and control leaves (Fig.1). Our data are consistent with the results obtained by other researchers [12-14, 30]. There is no data on mechanisms underlying the regulation of lipid metabolism in senescent plant leaves by BRs in the current literature. Steroid phytohormones were shown to induce expression of hydroxy steroid dehydrogenase AtHSD1, one of the lipid droplet proteins [31]. Plant lipid droplets are traditionally considered as inert organelles that play role in the storage of lipids, mainly triacylglycerols, as a source of metabolic energy. On the other side, there is growing evidence that these organelles, like their counterparts in yeast and mammals [3], are highly dynamic and are involved in various physiological processes, including the regulation of plant senescence [17]. Based on our data, steroid phytohormones may be involved into regulation of leaf senescence via alteration of cell lipid composition.
Declaration of interest We declare that there is no conflict of interest.
Acknowledgments This work was financially supported by the Russian Foundation for Basic Research (No.16-04-01553).
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15
Figure captures (legends) Figure 1. Effect of 24-epibrassinolide on the leaf senescence (expressed as changes in chlorophyll content) in pea leaves: control (native) leaves (grey column), mock-treated leaves (black columns), leaves treated with 0.1 µM 24-epibrassinolide (white columns); leaves treated with 10 nM 24-epibrassinolide (columns with horizontal stripes); leaves treated with 1 nM 24epibrassinolide (columns with diagonal stripes). Figure 2. Effect of 24-epibrassinolide on the content of fatty acids bound to pea leaf galactolipids (DGDG and MGDG). Control (native) leaves (grey column), mock-treated leaves (black columns), leaves treated with 24-epibrassinolide (0.1 µM; white columns). Figure 3. Effect of 24-epibrassinolide on the content of free fatty acids (FA) in pea leaves. Control (native) leaves (grey column), mock-treated leaves (black columns), leaves treated with 24-epibrassinolide (0.1 µM; white columns). Figure 4. Effect of 24-epibrassinolide on the content of fatty acids bound to pea leaf triacylglycerols. Fatty acid content in control (native) leaves (grey column), mock-treated leaves (black columns), leaves treated with 24-epibrassinolide (0.1 µM; white columns).
Chlorophyll content, mg/g FW
16
Fig.1
2
1
0 0 5 10
Time, days 15
17
Fig. 2
FA content, nmol/g FW
600 DGDG
MGDG
400
200
0 16:0 18:2 18:3
18:0
16:0
18:2
18:3 18:0
18
Fig. 3 .
FA content, nmol/ g FW
0,75
0,5
0,25
0
Sub A AzA Sb A
14:0
16:0
18:1
18:0
19
Fig. 4
FA content, nmol/ g FW
120
60
0 14:0
16:1
16:0
18:2
18:3
18:0
20
Supplementary Data Figure S1. GC-MS analysis of free fatty acids (as methyl esters) isolated from control leaves (from native plants). 750e3 TIC
AzA
500e3
16:0
250e3 SubA
18:0 SbA 14:0
0e3 5.0
7.5
15:0 10.0
18:1 12.5
Retention time, min
15.0
17.5
S0039-128X(16)30161-1 http://dx.doi.org/10.1016/j.steroids.2016.10.009 STE 8049
To appear in:
Steroids
Received Date: Revised Date: Accepted Date:
19 May 2016 21 October 2016 26 October 2016
Please cite this article as: Fedina, E., Yarin, A., Mukhitova, F., Blufard, A., Chechetkin, I., Br assinoster oid-induced changes of lipid composition in leaves of Pisum sativum L . dur ing senescence, Steroids (2016), doi: http:// dx.doi.org/10.1016/j.steroids.2016.10.009
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Brassinosteroid-induced changes of lipid composition in leaves of Pisum sativum L. during senescence Evgenia Fedina, Andrey Yarin, Faimya Mukhitova, Alexander Blufard, Ivan Chechetkin Kazan Institute of Biochemistry and Biophysics, Kazan Scientific Center, Russian Academy of Sciences, 420111, 2/31 Lobachevsky str., Kazan, Russian Federation
Email addresses: Dr. Evgenia Fedina; [email protected] Dr. Andrey Yarin; [email protected] Dr. Faimya Mukhitova; [email protected] Dr. Alexander Blufard; [email protected] Dr. Ivan Chechetkin; [email protected]
The name and address of the corresponding author: Dr. Evgenia Fedina; Kazan Institute of Biochemistry and Biophysics, Kazan Scientific Center, Russian Academy of Sciences, 2/31 Lobachevsky str., Kazan, 420111, Russian Federation, Email: [email protected]; phone number: +7 8432319047
2
Abstract
The effect of steroid phytohormone 24-epibrassinolide (EBR) on the composition of some lipid classes (free fatty acids, triacylglycerols and galactolipids) in detached pea leaves was studied for the first time. EBR (0.1 µM) promoted senescence and increased the content of 14:0, 16:0 and 18:1 free fatty acids as well as 18:2 and 18:3 bound to triacylglycerols in the detached leaves in contrast to mock-treated leaves. The content of all identified fatty acids bound to galactolipids decreased in the detached leaves treated with EBR compared to that in mocktreated leaves. These findings suggest that free fatty acids are liberated from polar lipids and then undergo esterification to neutral lipids in the detached leaves upon EBR treatment. We propose that steroid phytohormones may be involved into regulation of leaf senescence via alteration of cell lipid composition.
3 Keywords: Pisum sativum, 24-epibrassinolide, leaf senescence, fatty acids, glycerolipids, triacylglycerols Highlights: Brassinosteroids (BRs ) promote the leaf senescence in concentration dependent manner
BRs-induced senescence is accompanied by the alteration of cell lipid metabolism BRs increase triacylglecerol and free fatty acid content while decrease galactolipids
Abbreviations: BRs – brassinosteroids, DGDG – digalactosyldiacylglycerol, FA – fatty acids, FW – fresh weight, MGDG –
monogalactosyldiacylglycerol, GC-MS – gas chromatography – mass
spectrometry, EBR – 24-epibrassinolide, AzA – azelaic acid, SbA – sebacic acid, SubA – suberic acid
4 1. Introduction Brassinosteroids (BRs) comprise a group of steroid phytohormones widespread in the Kingdom Plantae. Research on BRs has a long history, starting with the study of oil substance from the pollen of rape and eller that exhibited plant growth promoting effects at low concentrations [1]. The first member of BRs, brassinolide, was isolated from rape pollen and identified as (22R,23R,24S)-2α,3α,22,23-tetrahydroxy-24-methyl-B-homo-7-oxa-5α-cholestan6-one [2]. Later over 70 structurally related BRs were isolated from different plants, including angiosperms, gymnosperms, ferns and algae. Common structural characteristic of BRs is 5αcholestan skeleton. The structural variations of BRs come from the kind and orientation of oxygenated functions in rings A and B [3]. BRs were detected in all parts and organs of plants [4]. BR level is usually higher in the young growing tissues (1-100 ng/g FW) than in mature ones (0.01-0.1 ng/g FW) [5]. The most abundant BRs detected in plants are castasterone, brassinolide, typhasterol, 6-deoxocastasterone, teasterone and 28-norcastasterone [5]. The greatest variety of BRs (25 free and 2 conjugated forms) was found in the unripe bean (Phaseolus vulgaris) seeds [5]. Other legumes possess lesser amount of BR members detected in pollen, seeds and shoots and their quantity is in the range of 0.007 – 628 µg/g FW [6]. Castasterone and brassinolide are the most important BRs in nature due to their wide distribution in plants and higher biological activity. However, 24epibrassinolide (EBR) is the most popular BR to study physiological effects of exogenous steroid phytohormones on plants due to its commercial availability. BRs play multiple roles in plants and function at extremely low concentrations (10-12-10-7 М). They control such plant developmental processes as seed germination, cell division and elongation,
flowering,
vascular-differentiation,
reproduction,
root
development,
and
photomorphogenesis [7]. They have recently been considered as stress hormones since their level increases under adverse environmental conditions and that leads to improvement of plant
5 resistance to different stressors such as heavy metals, pesticides, salt, high and low temperature, drought and pathogens [8, 9]. Plants deficient in the enzymes participating in BR biosynthesis and perception have a phenotype of prolonged leaf lifespan and so they are notable for the delayed senescence [10]. BR also was shown to promote chlorophyll degradation and decrease anthocyanin content in leaves [11, 12]. Based on these observations, BR are thought to regulate the plant senescence processes [13, 14]. Other phytohormones were shown to play a significant role in the regulation of leaf senescence [15]. Plant hormones such as ethylene, abscisic acid, salicylic acid, and jasmonic acid promote senescence, whereas auxin, gibberellic acids, and cytokinins retard it [16]. Plant leaf senescence is a complex process of the final stage of plant life, which is characterized by dramatic changes in metabolism in all cellular compartments followed by modification of macromolecules as well as lipids. One of the earliest manifestations of senescence is the loss of selective permeability of membranes due to their molecular disassembly. The onset of membrane leakage is a result of changes in the composition and molecular organization of the lipid bilayers due mainly to de-esterification of the major membrane lipids [17]. The synthesis of triacylglycerols from de-esterified free fatty acids (FAs) in parallel with dismantling of galactolipids is the most dramatic change observed in membrane lipid composition with progression of foliar senescence [18, 19]. Further on, triacylglycerols can be metabolized by β-oxidation to acetyl CoA, which is the final step of lipid catabolism in senescing tissues. Triacylglycerols may be a transient intermediate that prevents detrimental levels of free FA accumulation, or depletion of the free CoA-SH pool. Though lipid metabolism in the senescent plant leaves was thoroughly studied, mechanisms underlying the regulation of plant senescence by BR need to be revealed. The accelerated leaf senescence is known to occur in the detached leaves and so the latter are often used as a model system for studying senescence [20]. In the present work, the system was used to study the effect of 24-epibrassinolide (EBR), one of physiologically active BRs, on
6 the composition of some lipid classes (free fatty acids, triacylglycerols and galactolipids) in detached pea leaves.
2. Experimental
2.1 Plant material
Pisum sativum L. (cv. Truzhenik) was from the Tatar Research Institute of Agriculture of the Russian Academy of Sciences (Kazan, Russian Federation). Pea seeds were sterilized by soaking in 40% NaOCl (v/v) for 10 min, washed with distilled water and then were put to germinate in Petri dishes filled with solution containing (mM): 1,25 mМ KNO3, 0,25 mМ KH2PO4, 0,25 mМ MgSO4, 1,25 mМ Ca(NO3)2 (approximately ¼ classical Hoagland & Arnon’s nutrient medium I; рН 6,5). 2-Day-old seedlings were transferred to sterile test tubes filled with the sterile agar nutrient medium and were grown under 12-h photoperiod at the 23±1°C and 200 µmol m-2 s-1 irradiance for 10 days. Leaves of 10-day-old seedlings were detached and placed in Petri dishes on the sterile nutrient medium containing no phytohormone (mock-treated leaves) or 0.1 µM EBR and were incubated under constant temperature and irradiance conditions (see above) for 15 days. Leaves of 10-day-old intact plants grown on the sterile nutrient medium without EBR were used as a control. The length of detached leaves placed in Petri dishes was 1.40±0.03 cm, was not significantly changed during the experiments and corresponded to the length of control leaves. Nutrient solutions were renewed daily. 24-epibrassinolide was a generous gift from Prof. Vladimir Khripach (Laboratory of Steroid Chemistry, Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Minsk).
2.2 The extraction and separation of lipids
7
Total lipids were extracted from pea leaves with boiling propan-2-ol and separated by the silicic acid column chromatography as described before [21] to afford galactolipid and neutral lipid fractions. Neutral lipids were eluted from the column with chloroform/methanol (9:1, v/v) and galactolipids with acetone/methanol (9:1, v/v), successively. Then neutral lipids and galactolipids were separated by thin-layer chromatography (TLC), solvent hexane/diethyl ether/acetic acid (70:30:1, v/v) and acetone/benzene/water (91:30:8, v/v), respectively, using TLC plates Silica gel 60 (Merck, Germany). Zones with Rf 0.18, 0.41 and 0.74 containing DGDG, MGDG and triacylglycerols were scraped from the plates and corresponding lipids were eluted from silica with ethanol. Free fatty acids were extracted from the fraction of neutral lipids and purified using Supelclean LC-NH2 (3 ml) and LC-Si (1 ml) cartridges (Supelco, USA) as described previously [22]. Free fatty acids were methylated with CH2N2 while triacylglycerols and galactolipids were transesterified with sodium methoxide. The resulting methyl esters of fatty acids (derived from free fatty acids and acyl residues of triacylglycerols, MGDG and DGDG) were analyzed by GC-MS as described before [23]. Methyl ester of hexadecanoic acid (17:0) was used as an internal standard. The content of fatty acids was expressed as nmol per g of fresh weight.
2.3 Leaf senescence evaluation
Leaf senescence was evaluated by measurements of chlorophyll content of pea leaves as follows. Leaf tissue (0.1 g) was homogenized in 1 mL of 80% acetone (v/v), the extract was added to 25 mL of 80% acetone and then centrifuged at 12,000 g for 10 min. The absorbance of the supernatant was measured at 663 nm and 646 nm, respectively, on a Lambda 25 spectrophotometer (Perkin Elmer, USA). The total chlorophyll a and b content (FChl а+b)
8 was calculated according to Porra method [24]. Chlorophyll content data were presented in mg per g of fresh weight.
2.4 Statistics.
All experiments were in triplicate. Difference significance was assessed by the Student’s tcriterion. The mean values and standard deviations are given.
3. Results The yellowing of detached leaves due to the decrease in chlorophyll content of cells is an obvious symptom of their senescence. The decrease in chlorophyll content of pea leaves was noticeable 10 days after leaf detachment (Fig. 1). On day 15, chlorophyll loss in the detached leaves treated with EBR (0.1 µM) was more dramatic than in mock-treated leaves (Fig. 1). These findings are consistent with the data on EBR-stimulated chlorophyll degradation in senescent plant leaves [12-14]. It should be noted that other examined concentrations of phytohormone had no statistically significant influence on chlorophyll content compared to one in mock-treated leaves ( Fig.1 ). Since EBR effect on chlorophyll content of pea leaves was most obvious on day 15, we analyzed lipid composition of the leaves 15 days after their detachment. GC-MS analysis revealed decrease in the content of palmitic (16:0), linoleic (18:2), α-linolenic (18:3) and stearic (18:0) acids bound to galactolipids of ageing pea leaves compared to control (Fig. 2). The decrease was more dramatic in EBR-treated leaves than in mock-treated leaves (Fig. 2). These data are consistent with chlorophyll degradation in ageing leaves and its stimulation by EBR (Fig. 1). GC-MS analysis of free FA revealed the presence of myristic acid (14:0), 16:0, 18:0 and oleic acid (18:1) as well as oxylipins such as suberic (SubA), azelaic (AzA) and sebacic (SbA) acids in the leaves of control pea plants (see Supplementary Data, Fig. S1). The profile of free
9 FA that were isolated from the mock-treated leaves showed no oxylipins and drastic decrease in the content of 14:0, 16:0, 18:0 and 18:1 (Fig. 3). EBR-treated leaves contained only traces of AzA and the increased amounts of 14:0, 16:0 and 18:1 compared to control (Fig. 3). EBR was shown to increase the content of 16:0 and 18:0 FA as well as 18:2 and 18:3 bound to triacylglycerols in the detached leaves in contrast to mock-treated leaves (Fig. 4).
4. Discussion The effect of BRs on FA profiles of plants was demonstrated earlier. EBR (0.1 µM) decreased the content of saturated FA (16:0 and 18:0) and increased the content of unsaturated FA (18:1, 18:2 and 18:3) bound to MGDG in rape callus under cold acclimation. The impact of EBR on the fatty acid composition of DGDG was different, i.e. 18:0 content was significantly increased, whereas 18:2 diminished to a trace level [25]. Changes in the ratio of saturated to unsaturated FA bound to polar lipids of plasma membranes in mango fruits stored at 5°C were also induced by 10 µM brassinolide [26]. EBR application to the plants subjected to salt stress has also affected content of several fatty acids. For instance, EBR application led to overaccumulation of 18:2 in the salt-stressed rape seeds and to partial reversion of 18:1 and 20:1 content in the seeds of salt-stressed Arabidopsis [27]. Our data demonstrate the effect of EBR on FA composition in detached pea leaves for the first time. Unlike above mentioned observations in the plants under cold acclimation and salt stress, EBR did not affect the ratio of saturated to unsaturated FA in the senescent pea leaves. Most changes were seen in FA content of different lipid classes. EBR increased the content of free FA as well as FA bound to triacylglycerols and decreased the content of FA bound to galactolipids. These findings suggest that FA are liberated from polar lipids and then undergo esterification to neutral lipids in the detached leaves upon EBR treatment. Similar changes in FA composition of polar and neutral lipids occur in plants under various unfavorable conditions and are associated with leaf senescence and diacylglycerol acyltransferase action [28].
10 Degradation of the chloroplast membranes is known as one of the major features of the leaf senescence [29]. Moreover, the decrease in content of FA bound to DGDG and MGDG indicate the destruction of the chloroplast membranes in senescent leaves [28]. In our experiment, EBR in a concentration of 0.1 µM accelerated this process (Fig. 2). We also detected that EBR decreased the chlorophyll content of detached leaves on the 15th day after treatment compared to mock-treated and control leaves (Fig.1). Our data are consistent with the results obtained by other researchers [12-14, 30]. There is no data on mechanisms underlying the regulation of lipid metabolism in senescent plant leaves by BRs in the current literature. Steroid phytohormones were shown to induce expression of hydroxy steroid dehydrogenase AtHSD1, one of the lipid droplet proteins [31]. Plant lipid droplets are traditionally considered as inert organelles that play role in the storage of lipids, mainly triacylglycerols, as a source of metabolic energy. On the other side, there is growing evidence that these organelles, like their counterparts in yeast and mammals [3], are highly dynamic and are involved in various physiological processes, including the regulation of plant senescence [17]. Based on our data, steroid phytohormones may be involved into regulation of leaf senescence via alteration of cell lipid composition.
Declaration of interest We declare that there is no conflict of interest.
Acknowledgments This work was financially supported by the Russian Foundation for Basic Research (No.16-04-01553).
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15
Figure captures (legends) Figure 1. Effect of 24-epibrassinolide on the leaf senescence (expressed as changes in chlorophyll content) in pea leaves: control (native) leaves (grey column), mock-treated leaves (black columns), leaves treated with 0.1 µM 24-epibrassinolide (white columns); leaves treated with 10 nM 24-epibrassinolide (columns with horizontal stripes); leaves treated with 1 nM 24epibrassinolide (columns with diagonal stripes). Figure 2. Effect of 24-epibrassinolide on the content of fatty acids bound to pea leaf galactolipids (DGDG and MGDG). Control (native) leaves (grey column), mock-treated leaves (black columns), leaves treated with 24-epibrassinolide (0.1 µM; white columns). Figure 3. Effect of 24-epibrassinolide on the content of free fatty acids (FA) in pea leaves. Control (native) leaves (grey column), mock-treated leaves (black columns), leaves treated with 24-epibrassinolide (0.1 µM; white columns). Figure 4. Effect of 24-epibrassinolide on the content of fatty acids bound to pea leaf triacylglycerols. Fatty acid content in control (native) leaves (grey column), mock-treated leaves (black columns), leaves treated with 24-epibrassinolide (0.1 µM; white columns).
Chlorophyll content, mg/g FW
16
Fig.1
2
1
0 0 5 10
Time, days 15
17
Fig. 2
FA content, nmol/g FW
600 DGDG
MGDG
400
200
0 16:0 18:2 18:3
18:0
16:0
18:2
18:3 18:0
18
Fig. 3 .
FA content, nmol/ g FW
0,75
0,5
0,25
0
Sub A AzA Sb A
14:0
16:0
18:1
18:0
19
Fig. 4
FA content, nmol/ g FW
120
60
0 14:0
16:1
16:0
18:2
18:3
18:0
20
Supplementary Data Figure S1. GC-MS analysis of free fatty acids (as methyl esters) isolated from control leaves (from native plants). 750e3 TIC
AzA
500e3
16:0
250e3 SubA
18:0 SbA 14:0
0e3 5.0
7.5
15:0 10.0
18:1 12.5
Retention time, min
15.0
17.5