Accepted Manuscript

Title: Exogenous Gibberellin Weakens Lipid Breakdown by

Increasing Soluble Sugars Levels in Early Germination of
Zanthoxylum Seeds

Authors: Jikang Sun, Hao Jia, Ping Wang, Tao Zhou, Yan Wu,
Zhiming Liu

PII: S0168-9452(18)30579-X
DOI: https://doi.org/10.1016/j.plantsci.2018.08.013
Reference: PSL 9929

To appear in: Plant Science

Received date: 19-5-2018

Revised date: 31-7-2018
Accepted date: 23-8-2018

Please cite this article as: Sun J, Jia H, Wang P, Zhou T, Wu Y, Liu Z,
Exogenous Gibberellin Weakens Lipid Breakdown by Increasing Soluble Sugars
Levels in Early Germination of Zanthoxylum Seeds, Plant Science (2018),
https://doi.org/10.1016/j.plantsci.2018.08.013

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Exogenous Gibberellin Weakens Lipid Breakdown by Increasing Soluble Sugars Levels in
Early Germination of Zanthoxylum Seeds

Jikang Sun1 2, Hao Jia1, Ping WANG3*, Tao Zhou1, Yan Wu1, Zhiming Liu4*

1 College of Life Science and Technology, Central South University of Forestry and Technology, Changsha, Hunan, China
2 Hunan Provincial Key Laboratory for Forestry Biotechnology, Central South University of Forestry and Technology, Changsha,
Hunan, China
3 College of Environmental Science and Engineering, Central South University of Forestry and Technology, Changsha, Hunan,
China

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4 Department of Biology, Eastern New Mexico University, Portales, NM88130, USA.

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*
Correspondence to wangping@csuft.edu.cn; Zhiming.Liu@enmu.edu

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Research highlights

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 Our results indicated that the exogenous GA weakened lipid

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breakdown by increasing soluble sugars levels in early germination
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of zanthoxylum seeds. Our study provided substantial evidence on
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the role of GA on lipid metabolism in promoting seed germination

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and extends our knowledge on the importance of sugar metabolis

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during early germination.

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Abstract
Zanthoxylum is a precious medicinal woody plant with a very low seed germination rate in China.
The gibberellin (GA) treatment extremely increased the germination rate of zanthoxylum seeds.
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Our previous transcriptome data showed that exogenous GA played a negative role in the
expression levels of genes involved in lipid metabolism during imbibition. Our present data
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indicated that compared with the GA-treated seeds, the soluble sugars were more quickly
consumed and lipid breakdown was prematurely and actively initiated in the water-treated seeds
during the early germination. However, the application of sucrose could improve the germination
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of water-treated seeds and significantly inhibit lipid breakdown. Both the application of sucrose
and exogenous GA could significantly reduce the catalytic activities of sugar–dependent 1 (SDP1)
and isocitratelyase (ICL), the expressions of SDP1 and ICL genes, and decrease the products of
lipid breakdown as well during the early germination. We suggested that exogenous GA might
enhance starch hydrolysis by promoting the catalytic activity of ɑ–amylase to supplement
metabolically consumed soluble sugars, thus the increased sugars levels would help to inhibit the
lipid breakdown to mitigate oxidative damages in the early germination of zanthoxylum seeds. In
the end, we summarized the possible molecular mechanism on the exogenous GA weakening lipid
breakdown by increasing soluble sugars levels in the early germination of zanthoxylum seeds.

Keywords: storage oil; sugars; seed germination; lipid peroxidation; zanthoxylum.

1. Introduction
Zanthoxylum dissitum Hemsl. (zanthoxylum), is a precious, perennial woody medicinal plant in
china, with a very low germination rate of seeds under natural conditions. Presently, the natural
reserve of zanthoxylum is very inadequate [1]. Previous investigations suggested some causes for
inhibiting germination of zanthoxylum seeds under natural conditions. First, the episperm of

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zanthoxylum seed is wax–rich, which reduces the permeability of water and air through the seed

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coat, while its endopleura is composed of a hard, thick horny outer–layer [2]. Secondly, there are
some germination inhibitors found within zanthoxylum seeds [3]. After 3 months of

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low–temperature stratification, the germination rate of the GA-treated seeds with shells removed
significantly increased. The radicle and hypocotyl of the GA-treated seeds broke through the seed

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coat and had a visible stretch in the third day of germination, while the water-treated seeds
decayed in the third day of germination [4].
Many plant hormones are involved in the germination of seeds, such as gibberellin (GA), abscisic

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acids (ABA), auxin and brassinolide. GA plays a significant role in promoting germination by
antagonizing ABA. It is frequently mentioned that GA induces expression of α–amylase in early
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germination of barley aleuronic layers [5]. Two types of functions for GA have been proposed in
promoting germination in Arabidopsis. The first function is to weaken the tissue that surrounds the
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embryo during the induction of radicle protrusion [6]. The second function is to increase the growth
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potential of the embryo, as demonstrated by the reduced growth rate of GA–deficient embryos [7].
Triacylglycerol (TAG) is stored within oil bodies in the seeds. TAG mobilization in oil bodies is
initiated by the activity of TAG lipases, which release fatty acids. The free fatty acids are
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converted to acetyl–CoA units by β–oxidation in peroxisomes. The glyoxylate cycle functions on

the conversion of acetyl–CoA to succinate for synthesis of carbohydrates [8]. Sugar–dependent 1
(SDP1) has been verified as the major triacylglycerol lipase involved in TAG mobilization in
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seedlings of Arabidopsis thaliana, since sdp1 mutants showed the lags of post-germinative growth
and exogenous sugar was able to rescue the phenotype [9]. Recently, the enhanced interaction
between oil bodies and peroxisomes was observed in sdp1 seedlings of Arabidopsis thaliana
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compared with the wild type, but was significantly suppressed by exogenous sucrose [10]. A
description of the mechanism for lipase transport in eukaryotic cells was reported that SDP1 was
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initially localized to the peroxisome membrane in the early growth of seedling, and then
transported to the oil bodies’ surface probably by peroxisome tubulations [11]. The lipase activity
was found to be already present before imbibition and involved in lipid degradation during
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germination in coffee (Coffea arabica L.) seeds [12]. However, the function of TAG mobilization is
controversial during germination. In Arabidopsis, TAG hydrolysis was less ~5% in the double
mutant of sdp1 and sdp1L than wild type, while the rate of seed germination in mutants just
slightly decreased and post-germinative seedling growth was strongly retarded. This study
suggested that TAG mobilization was not essential for seed germination.
Production of hydrogen peroxide (H2O2), a type of reactive oxygen species (ROS), is induced by
GA but inhibited by ABA, and the H2O2 induced by GA is a signal molecule that antagonizes ABA
signaling in aleuronic cells [13]. Fatty acids β–oxidation is an important source of H2O2 in
peroxisomes, which is usually more active during germination [14]. ROS excess can result in
detrimental effects, causing lipid peroxidation in cellular membranes, DNA damage, protein
denaturation, carbohydrate oxidation and pigment breakdown [15]. The final products of lipid
peroxidation are reactive aldehydes, such as MDA and 4–hydroxynonenal (HNE). MDA is as a
lipid peroxidation marker [16,17]. Furthermore, the presence of MDA can poison cell membrane
systems, proteins and DNA, leading to cell membrane degradation and loss of cellular function [18].
The lower MDA levels correlate to a smaller degree of membrane injury [19].
Our previous transcriptome study showed that the exogenous GA played inhibitory roles in
transcriptional levels of genes related to oil metabolism during imbibitions [4]. However, there

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were no direct data confirming the negative regulation of storage oil breakdown caused by the

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exogenous GA. We attempted to determine the relationship between the exogenous GA and
storage oil breakdown using some important biochemical markers, besides catalytic activities and

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transcripts of enzymes involved in storage lipid breakdown and anti-oxidation in the initial stage
of germination of zanthoxylum seeds.

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2. Materials and methods
2.1. Plant material

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The seeds of zanthoxylum were collected in Zhangjiajie, Hunan province, in December 2016.
After cold-stratification for three months the seeds were treated with 80% sulfuric acids for three
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minutes to remove shells. These seeds were considered the 0 hours (0 h) sample, also labeled as
C0. Next, the seeds were germinated and cultivated according to Sun et al [4]. The water-treated
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seeds were incubated for 1 - 2 days and were labeled as W1 and W2, respectively. The GA-treated
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seeds were incubated for 1 - 5 days, labeled as GA1, GA2, GA3, GA4 and GA5, respectively; and
collected for tissue sections according to Sun et al [4]. In addition, some seeds were soaked in
sterile water for three and a half hours on two sterile circles of filter paper. They were saturated
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with 5.0 mM.L-1 sucrose solution and incubated, in 9 cm Perspex Petri dishes. The sucrose
solution was replaced daily. The seeds were labeled as suc-treated seeds. Samples were collected
at time markers: 12 hours (12 h), 24 hours (24 h), 36 hours (36 h) and 48 hours (48 h) when the
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water-treated seeds, the GA-treated seeds and suc-treated seeds samples were also collected in 72
hours (72 h) and 96 hours (96 h) increments. The water-treated seeds, the GA-treated seeds, and
suc-treated seeds then began to cultivate in the YRG-250 growth chamber (Shanghai Heheng
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Instrument and Equipment Co.,Ltd, China).

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2.2. Enzyme catalytic activity assays

Lipase (SDP1, EC 3.1.1.3) catalytic activity was measured as described by Vorderwuelbecke et al
[20] and Suznki et al [21]. First, 0.03 mg/ml P–NP was diluted into a series of 5 ml gradients. Then, 5
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ml of 0.5 M trichloroacetic acids was added to each gradient while keeping the pH steady with
5.15 ml 0.5 M NaOH. Absorption was measured at λ = 405 nm. The correlation between the
absorption and concentration was calculated. The seeds were homogenized in 50 mM Tris–HCl
buffer (pH 8.0) supplemented with 0.1% Triton X–100. The homogenate was incubated for 30 min
and centrifuged. The reaction mixture containing 0.09 mg/ml P–NPP was preheated for 5 min then
1 ml supernatant was added to the reaction mixture (for a total volume of 4 ml). The reaction was
stopped by addition of 5 ml 0.5 M TCA after 10 min. The pH was then adjusted with 5.15 ml 0.5
M NaOH. Absorption was measured at λ = 405 nm. For lipase, the amount that catalyzes the
conversion of 1 μmol of P–NP per minute is one U.

Isocitratelyase (ICL, EC 3.1.1.3) catalytic activity was measured using isocitrate lyase kit
(Solarbio, China). The seeds were homogenized with extraction solution (1 ml) in ice bath. The
homogenate was centrifuged at 15,000 g at 4 ℃ for 10 min and then the collected supernatant was
prepared according to the kit instructions. Absorption was measured at λ = 340 nm while the
reaction mixture reacted for 20 s and 2 min 20s, respectively. One U for ICL is the amount that
consumes 1 nmol of NADH per minute.

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Catalase (CAT, EC 1.11.1.6) catalytic activity was measured according to Hao and Kang [22]. The

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seeds were homogenized in 0.2 M phosphate buffer (pH 7.0) supplemented with 1% PVP. The
homogenate was incubated for 10 min at 4 ℃; the supernatant was centrifuged at 4000 g for 15

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min and then collected. The reaction mixture (2.7 ml total) contained 0.2 M phosphate buffer (pH
7.8) 1.5 ml, 0.2 ml supernatant and 1 ml distilled water. Next, 0.3 ml 0.1 M H2O2 was preheated to

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25 °C and added to the reaction mixture. Absorption was measured at λ = 240 nm every 1 min for
4 minutes. The control sample was the reaction mixture containing 0.2 ml supernatant which had
been inactivated by boiling. One U for CAT is the amount that A240 was reduced by 0.1 per
minute.
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ɑ–amylase catalytic activity was measured according to Wang [23]. A gradient of maltose was
performed to calculate the correlation between the absorption and concentration. 0.5 g seeds were
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homogenized in 8 ml 0.05 M Tris-HCl buffer supplemented with 0.1% PMSF and 1% PVP. The
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homogenate was incubated at room temperature for 15 min and then centrifuged at 3000 g for 10
min. The supernatant was the solution of amylase. Six tubes with 1 ml supernatant were heated in
70 ℃ water bath for 15 min to inactive β–amylase for the measurement of ɑ–amylase activity.
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Three tubes were selected as control and others were used as sample tubes. 2 ml
3,5-dinitrosalicylic acid was added to each control tube. The reaction mixture was incubated at
40 ℃ water bath for 5 min and then 1 ml 1% starch solution was added to each tube. After
incubated at 40 ℃ in a water bath for 5 min, 2 ml 3,5-dinitrosalicylic acid was supplemented to
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each sample tube. All of tubes was heated in boiling water bath for 5 min and then cooled.
Absorption was measured at λ = 540 nm.
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2.3. Biochemical assays

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MDA was measured according to Shi and Sun [24]. The seeds were homogenized in 5% TCA. The
homogenate was centrifuged at 8,000 g for 10 min. The reaction mixture (4.8 ml total) contained 3
ml 5% TBA and 1.8 ml supernatant. A control sample contained 3 ml 5% TBA and 1.8 ml distilled
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water. The reaction mixture was incubated for 15 min at 100 oC and then cooled rapidly before
being centrifuged. Absorption of the collected supernatant was measured at λ = 532 nm, 600 nm
and 450 nm, respectively.

Relative electronics conductivity (REC) was measured according to Cornelissen et al [25]. The
seeds germinating for 36 hours were chilled at -5 ℃ for 2.5 hours. Randomly selected seeds were
washed 3 times with deionized water and blotted dry with filter paper. The seeds were then
immersed in 100 ml deionized water at 20 ℃ for 12 hours and the first conductivity measurement,
R1, was taken. The sample was then treated in a boiling bath for 15min and conductivity was
measured again, R2. REC can be calculated as: REC (%) = (R1/R2)*100.

Soluble sugars were measured according to Wang [23]. First, 100 ug/L exogenous sucrose was
diluted into a series of gradients, with each gradient 2 ml. Then, 5 ml 9% phenol and 5 ml sulfuric
acids were added successively to each gradient. The reaction mixture was incubated at
room temperature for 30 min to allow for a color–reaction. Absorption was measured at λ = 485
nm and the correlation between the absorption and concentration was calculated. The seeds were
homogenized in distilled water; the homogenate was incubated at 100 ℃ for 30 min and then

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filtrated. The filtrate was diluted to 25 ml. Then, 1.5 ml distilled water, 5 ml 9% phenol and 5 ml

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sulfuric acids were added successively to the 0.5 ml dilution. The reaction mixture was incubated
at room temperature for 30 min to allow for a color–reaction. Absorption was measured at λ = 485

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nm.

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GSH/TSH measured: TSH included glutathione and oxidized glutathione. 0.5g seeds were
homogenized in 5 ml reagent 4. The reaction mixture was centrifuged at 3500 g for 10 min. Then,
absorption was measured at λ = 405 nm after fulfilling the following steps according to the

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glutathione kit A061-1 of Biological Engineering Institute of Nanjing (www.njjcbio.com).
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Fatty acids measured: Fatty acids were measured according to the free fatty acids kit (FFA-1-W,
Suzhou Coming Biotechnology Co.,Ltd, China). Randomly selected seeds were washed 3 times
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with distilled water and blotted dry with filter paper. The reaction mixture contained 0.1 g mashed
seeds and 1.2 ml reagent 1 and was incubated for 3 h, then was centrifuged at 8000 g at 4 ℃ for 10
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min. The supernatant was measured at λ = 715 nm according to the kit.

Seed oil measured: Seeds were dried in an oven at 60 °C for 72 hours, milled and stored at 4 ℃
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prior to use. Dried seed powder (10.0 g) was wrapped with filter paper and placed inside a soxhlet
apparatus, then extracted with petroleum ether (40 – 60 °C) for 6 hours using the soxhlet extractor.
The solvent was evaporated under reduced pressure and the residual oil was placed inside the oven
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at 40 °C to dry to constant weight. The oily extract was weighed and counted as oil content
(mg/g).
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2.4. Expression profiles of enzyme genes

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Primers for full length cds of lipase (SDP1), ICL and CAT were designed according to the
transcriptome data of zanthoxylum seeds. After sequencing (Sangon Biotech Co., Ltd, Shanghai),
the protein sequences were analyzed via BLAST and DNAMAN 7.0 to check for homologous
alignment. The amino acids sequences alignments of lipase (SDP1), ICL and CAT are shown in
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Figure S1-S3. The RNA extracted from the seeds was converted into cDNA using PrimeScript™
One Step RT–PCR Kit Ver. 2 (Takara, China). The 10× diluted cDNA was used as a template for
qPCR. Primers for qRT–PCR were designed using Primer Premier 5.0 software and synthesized
by Sangon Biotech Co., Ltd (Shanghai). Zanthoxylum actin primers were used as a constitutive
control [26]. The above primers are shown in Table S1. The qRT–PCR was performed on an
Applied Biosystems 7500 Real–Time PCR System (ABI, USA) using a SYBR Green based PCR
assay. Each reaction mixture was 10μL in total, containing 3μL of diluted, first–strand cDNAs and
125nM of each primer, followed by 5μL SYBR Green PCR Master Mix (TaKaRa, China). The
two–step qRT–PCR protocol was run using the following parameters: 95°C for 30s, followed by
40 cycles of 95°C for 5s and 57°C for 30s; then the melting curve profile was then determined.
Expression levels of target, normalized to an endogenous reference and relative to a calibrator,
were determined by CT values and calculated by 2–△△Ct.

2.5. Statistical analysis

Experiments on the catalytic activity of enzymes, along with each qRT-PCR reaction and the
levels of MDA, REC, soluble sugars, GSH, fatty acids, and oil were carried out in 3 biological
replicates. The results were the mean ± SD. The significance analysis of differences was

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calculated with the Student’s t–test.

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3. Results

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3.1. Tissue sections during the germination of zanthoxylum seeds.
The water-treated seeds rotted by the third day of germination and the GA-treated seeds

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successfully germinated in 5-6 days. We collected the water-treated seeds incubated for 1-2 days
and the GA-treated seeds incubated for 1-5 days to perform tissue sections (Fig 1). There was not
apparent difference between C0 and W1. Compared with W1a, GA1 was more similar W2. So, it

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seemed that the GA-treated seeds had the more quick germination. Furthermore, GA1-GA5
showed the germination process of the GA-treated seeds. Interestingly, a classic vascular bundle
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was observed at the every stage of germination, which clearly showed that cotyledon nutrients
were transported through the vascular bundle to the radicle to support radicle growth. Those
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indicated that the development of vascular bundle played an important role for the early
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germination of seeds.
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Fig .1. The tissue sections during the germination of the water-treated seeds and the GA-treated seeds.
C0: the seeds cold-stratification for three months; W1-W2: the water-treated seeds incubated for 1 - 2 days; W1a:
zoomed part in W1; GA1-GA5: the GA-treated seeds incubated for 1 - 5 days. In W1a, sc: inner seed coat (outside
the sc, there is a layer of oily hard black shell) ; en: endosperm; co: cotyledons; vbt: vascular bundle; g: germ ; h:
hypocotyls ; r: radical; one millimetre (mm) per unit scale bar.

3.2. Compared with the GA-treated seeds, more acute oxidative stress and serious oxidative
damages occured in the water-treated seeds, and more catalytic activity of α–amylase and higher
levels of soluble sugars were observed in the GA-treated seeds.
MDA is a lipid peroxidation marker. The MDA levels of the water-treated and GA-treated seeds

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within 3 days of germination were shown in Fig 2A. Within 36 hours of germination MDA levels

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in the water-treated seeds were significantly higher than that in the GA-treated seeds, especially in
24-hours-old and 36-hours-old seeds, which indicated that lipid peroxidation was severer in the

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early germination of the water-treated seeds. Further, to assess the effect of lipid peroxidation on
membrane injury, REC of 36-hours-old seeds was measured. More serious membrane injuries

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resulted in lower resistance to cold, so the effects of membrane injuries increased when the seeds
were chilled for the suitable time. As shown in Fig 2B, REC of the water-treated seeds was
significantly higher than that of the GA-treated seeds, which indicated that the membrane injury

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was more serious and the cold resistance was lowered in the water-treated seeds, leading to a
significant increase of REC. Therefore, it was obvious that severe oxidative damages occurred in
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the water-treated seeds, and the exogenous GA helped to reduce the oxidative damages.
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Fig .2. The Levels of MDA (A) and REC (B) in the GA-treated seeds (GA) and the water-treated seeds (W) in the
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early germination of zanthoxylum seeds. Statistical analyses of the MDA and REC data were based on the
comparison between W and GA during the same germination times. Statistical significance at P ≤ 0.05 (*).
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It was reported that the less GSH content might lead to oxidative stress, which stimulated the
expression of H2O2 scavenging enzymes, and led to a significant increase of oxidative stress
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markers such as MDA during germination [27]. The catalytic activity of CAT and GSH content
during the early germination of seeds were measured. As shown in Fig 3A, GSH content was
much lower in the water-treated seeds than that in the GA-treated seeds during 48 hours of
germination, and more MDA were observed in the water-treated seeds within 36 hours of
germination, which meant that more acute oxidative stress occurred in the water-treated seeds. The
catalytic activity of CAT in the water-treated seeds was significantly higher than that in the
GA-treated seeds within 24 hours of germination, and its catalytic activity significantly reduced in
the water-treated seeds but only slightly decreased in the GA-treated seeds following 24 hours of
germination, as shown in Fig 3B. Besides, the expression of CAT gene in the water-treated seeds
were higher compared to the GA-treated seeds within 24 hours of germination, as shown in Fig 3C.
The higher catalytic activity of CAT and expressions of CAT gene might be related to the acute
oxidative stress in the water-treated seeds. Both the catalytic activity of CAT and expression of
CAT gene noticeably decreased in the water-treated seeds after 24 hours of germination, which
might be related to the irreversible and fatal oxidative damages happening in the water-treated
seeds. In addition, the higher GSH level, moderate catalytic activities of CAT, less MDA were
observed in the GA-treated seeds, which indicated that the exogenous GA was conductive to
alleviating oxidative stress and reducing oxidative damages.

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Fig .3. The GSH content (A), catalytic activities of CAT (B) and expression level of CAT gene (C) in the
GA-treated seeds (GA) and the water-treated seeds (W) in the early germination of zanthoxylum seeds. Statistical
analyses were based on the comparison between W and GA in the identical period. Statistical significance at
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P≤0.05 (*). Statistical significance of genes expressions at P≤0.05 (*) and more than 2–fold difference.
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Some investigations showed the increased CAT activity was related to the fatty acids β–oxidation
and its up–regulation could be treated as indirect evidence of intensification of the fatty acids
β–oxidation in sugar starved tissues [28,29]. Therefore, we inferred that the high catalytic activity of
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CAT indirectly reflected the lack of sugar in sugar starved tissues, as shown in Fig 3B.
As shown in Fig 4A, the levels of sugars in the GA-treated seeds was significantly higher than that
in the water-treated seeds within 24 hours of germination. It was worth noting that the levels of
soluble sugars were noticeably decreased in 12-hours-old water-treated seeds compared with
12-hours-old GA-treated seeds, which should be associated with the exogenous GA that promoted
the expression of α–amylase that hydrolyzed starch into soluble sugars. The soluble sugars from
hydrolysis of starch supplemented the consumption of sugars in the GA-treated seeds. As shown
in Fig 4B, the catalytic activity of α–amylase increased rapidly and was much higher in the
GA-treated seeds than that in the water-treated seeds during 48 hours of germination. The
α-amylase is one of the key enzymes involved in degradation of starch and the only enzyme that
initiates hydrolysis of starch granules during germination of seeds [30,31]. It was obviously that
more catalytic activity of α–amylase helped to convert storage starch into soluble sugars so as to
supplement the consumption of sugar metabolism.
The above results indicated that the exogenous GA contributed to increasing the levels of soluble
sugars, mitigating oxidative stress and reducing the oxidative damages during the early
germination of seeds. It was also reported that the deficit of soluble sugars in tissues stimulated
the activities of lipase and catalase [32]. All those together, we hypothesized that insufficient

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soluble sugars might lead to untimely mobilization of storage oil, which would result in active

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lipid metabolism and glyoxylate cycle to convert lipid into sugars. However, the exogenous GA
promoted the hydrolysis of starch into soluble sugars, which helped to inhibit oil mobilization and

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attenuate lipid catabolism to reduce peroxide products during the early germination.

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Fig .4. The levels of soluble sugars (A) and catalytic activities of α–amylase (B) in the GA-treated seeds (GA) and
the water-treated seeds (W) in the early germination of zanthoxylum seeds. Statistical analyses were based on the
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comparison between W and GA in the identical period. Statistical significance at P≤0.05 (*).

3.3. Both exogenous GA and application of sucrose could decrease the catalytic activities of SDP1
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and ICL, the expressions of SDP1 and ICL genes, and inhibit lipid breakdown as well.
The levels of sugars were involved in the regulation of the transcription and activities of some
enzymes, such as lipase, catalase, acyl–CoA oxidase, isocitratelyase, peroxisomal and
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mitochondrial aconitase, cytosolic and mitochondrial isocitrate dehydrogenase and

phosphoenolpyruvate carboxykinase; all of which were involved in lipid metabolism in
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germinating seeds [32,33,34,35]. In order to verify the hypothesis that the exogenous GA weaken lipid
breakdown by soluble sugars-dependent way during the early germination of seeds, the
germination experiment of suc-treated seeds was performed. By application of sucrose during the
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early germination of seeds, the catalytic activities of key enzymes related to lipid metabolism and
oil content of suc-treated seeds were measured and compared with the water-treated and the
GA-treated seeds. Our experiments showed that the germination rate of suc-treated seeds was
about (41.6% ± 3.7%), and the radicle completely broke through its seed coat in 7-8 days of
germination. In contrast, about (82.5% ± 4.1%) of GA-treated seeds successfully germinated, and
the radicle completely broke through its seed coat in 5-6 days of germination. However, the
germination rate of water-treated seeds was never over 8.0% before.
As shown in Fig 5A, there was no significant difference of oil content between the GA-treated
seeds and the water-treated seeds. However, compared with the GA-treated seeds, the content of
seed oil in the water-treated seeds more rapidly decreased within 48 hours of germination. The
content of seed oil in the suc-treated seeds was significantly higher than that in the water-treated
seeds after 36 hours of germination. As shown in Fig 5B, the fatty acids content in the
water-treated seeds was significantly higher than that in the GA-treated seeds and suc-treated
seeds after 24 hours of germination. It was obvious that the utilization of storage oil was more
active in the water-treated seeds during the early germination. Interestingly, compared with the
suc-treated seeds, the oil mobilization of the GA-treated seeds significantly accelerated after 72
hours of germination, which implied the more active development of seeds.

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The catalytic activity of SDP1 in the water-treated seeds was significantly higher than that in the
GA-treated and suc-treated seeds within 24 hours of germination, as shown in Fig 5C. Besides, the

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expression of SDP1 gene in the water-treated seeds was significantly higher than that in the
GA-treated and suc-treated seeds in 12 hours of germination, as shown in Fig 5D. It was clear that

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the exogenous GA and application of sucrose played the similar roles in reducing the catalytic
activity of SDP1 and expression of SDP1 gene.
ICL is the key enzyme of glyoxylate cycle [36]. Lipids can be converted into carbohydrates by the

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glyoxylate cycle [8]. Thus, the ICL catalytic activity is conducive to understanding the metabolic
transformation of storage oil. The catalytic activity of ICL reached the peak in the 24-hour-old
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water-treated seeds, and was also significantly higher than that in the GA-treated seeds and
suc-treated seeds. The catalytic activity of ICL in the GA-treated seeds was noticeably increased
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after 36 hours of germination, but that in the suc-treated seeds kept low catalytic activity within 72
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hours of germination, as shown in Fig 5E. The expression of ICL gene in the water-treated seeds
was significantly higher than that in the GA-treated and suc-treated seeds in 24 hours of
germination, and the expression of ICL gene in the GA-treated seeds was noticeably increased
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after 36 hours of germination. The higher catalytic activity of ICL and expression of ICL gene in
the water-treated indicated that the glyoxylate cycle converting lipid into carbohydrates was more
active within 24 hours of germination. In addition, the application of sucrose was able to
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continually reduce the expression of ICL gene and catalytic activity of ICL during the early
germination. Therefore, we postulated that the shortage of soluble sugars stimulated the glyoxylate
cycle to convert storage oil into carbohydrates in the water-treated seeds.
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In summary, because of the acute lack of soluble sugars in the water-treated seeds during the early
germination, the TAG mobilization and glyoxylate cycle were more active so as to efficiently
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convert storage oil into sugars. β–oxidation related to lipid metabolism in peroxisomes would
generate excess ROS that caused severe oxidative damages to germinating seeds. However, the
application of sucrose supplemented the soluble sugar and weakened the TAG mobilization and
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glyoxylate cycle, and the similar inhibitory roles were made by exogenous GA. Therefore, we
proposed that exogenous GA enhanced catalytic activity of ɑ–amylase that promoted starch
hydrolysis so as to supplement soluble sugars, and the increased soluble sugars helped to inhibit
lipid breakdown during the early germination of zanthoxylum seeds.
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Fig .5. The oil content (A), fatty acids content (B), catalytic activity of lipase SDP1 (C), expression level of SDP1
gene (D), catalytic activity of ICL (E), expression level of ICL gene (F) among the GA-treated seeds (GA), the
water-treated seeds (W) and suc-treated seeds (Suc) in the early germination of zanthoxylum seeds. Statistical
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analyses were based on the comparison between GA and W or between Suc and W in the identical period.
Statistical significance at P≤0.05 (*). Statistical significance of genes expressions at P≤0.05 (*) and more than
2–fold difference.
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4. Discussion
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TAG is an efficient energy store because its output is more than twice the energy of protein or
carbohydrate during complete oxidation-decomposition [37], and it also provides carbon skeletons
that support seedling growth immediately following seed germination and enable seedling
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establishment [38,39]. The amount of storage oil (TAG) in fresh zanthoxylum seeds was reported as
18.35% [40]. Our data showed that the sequence similarity of SDP1 lipase between zanthoxylum
and Arabidopsis thaliana is 86% (Figure S1), and the catalytic activity of lipase was low in the
early germination of zanthoxylum seeds. Usually, storage sugars are firstly metabolized, and then
oil is mobilized and converted to carbohydrates by the glyoxylate cycle. It is frequently reported
that GA plays an active role in inducing the expression of ɑ–amylse that hydrolyzes insoluble
starch into soluble sugars.
The lower oil content, and higher fatty acids content were observed in the water-treated seeds
within 36 hours of germination, which implied that TAG was mobilized during the early
germination. The higher catalytic activities of SDP1 and expression of SDP1 gene in the
water-treated seeds indicated that more active TAG mobilization happened within 24 hours of
germination. The higher catalytic activities of ICL and expression of ICL gene in the water-treated
seeds also indicated that more products of fatty acids β–oxidation were converted to carbohydrates
by the glyoxylate cycle within 24 hours of germination. Meanwhile, the lower levels of soluble
sugars and catalytic activity of α–amylase in the water-treated seeds were observed during the
early germination, which indicated that the starch was not able to be timely hydrolyzed into
soluble sugars. It was clear that the catabolized sugars were not able to be effectively

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supplemented because of the insufficiency of starch hydrolysis. Therefore, it can be reasonably

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inferred that storage oil was prematurely mobilized to converte lipid into sugars in the early
germination of water-treated seeds.
Fatty acids were catabolized into acetyl–CoA by β–oxidation, and then acetyl–CoA was converted

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into sugar by the glyoxylate cycle in peroxisomes [8]. It was reported that fatty acids β–oxidation

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was more active during germination and was an important source of H2O2 in peroxisomes [14]. It
was obvious that the more fatty acids, the higher catalytic activity of ICL and expression of ICL
gene implied that β–oxidation was more active in the water-treated seeds compared with the

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GA-treated seeds during the early germination. The active β–oxidation of fatty acids in
peroxisomes yielded excess ROS that led to ROS-induced lipid peroxidation, which was proved
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because the more higher levels of MDA and REC in the water-treated seeds were observed in the
early germination of zanthoxylum seeds (Fig 2A, 2B). An investigation reported that lipid
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peroxidation was an important factor that caused the deterioration of seeds due to excess free
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radicals that led to severe membrane injury [41]. All those together, it was clear that premature
TAG mobilization should be an important causation for germination failure of the water-treated
seeds. In addition, it was also observed that the expressions of genes including CAT, SDP1 and
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ICL significantly decreased after 36 hours of germination in the water-treated seeds, which might
be related to the irreversible oxidation damages of germinating seeds caused by excess peroxides.
Another research reported that exogenous sucrose (60mM) added to the medium of in vitro culture
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of embryo axes and cotyledons of lupin decreased activity of lipase, and the change of the lipase
activity caused by exogenous sucrose was based on modifications in gene expression because of
the corresponding change in the enzyme activity and in the mRNA level [33]. It had been also
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reported that the lipase activity was controlled by sugars levels at the stage of transcription [32].
Our data showed that compared with the water-treated seeds, the application of sucrose not only
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inhibited the catalytic activities of SDP1 and ICL, but also reduced the expressions of SDP1 and
ICL genes. Both SDP1 and ICL were the key enzymes involved in lipid catabolism. Those
indicated that exogenous sucrose had significantly inhibitory effects on lipid breakdown during
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the early germination.

It was reported that storage lipid breakdown was significantly enhanced under sugar starvation
conditions [42]. Recently, an investigation also showed that sugar deficiency in tissues noticeably
increased storage lipid breakdown in 96–hour–old lupin seedlings as well as in excised and
cultured in vitro cotyledons [35]. Our results showed that compared with the GA-treated seeds, the
sugars levels in the water-treated seeds was lower seeds within 24 hours of germination.
Meanwhile less oil, more fatty acids and higher activities of SDP1 and ICL were observed in the
water-treated seeds within 36 hours of germination as well. So, the effects of the application of
sucrose on inhibiting lipid breakdown, coupled with the more sugar content in the GA-treated
seeds compared with the water-treated seeds, we proposed that exogenous GA induced the
expression of α-amylase that hydrolyzed starch to soluble sugars, and the increased sugars
inhibited lipid breakdown during the early germination of zanthoxylum seeds.
Storage oil was hydrolyzed to release fatty acids in oil bodies, then the free fatty acids are
converted to acetyl–CoA by β–oxidation. Subsequently, acetyl–CoA units were converted to
carbohydrates by the glyoxylate cycle in peroxisomes during the germination of seeds. Recently, it
was reported that sugar played a key role in the dissociation of oil bodies and peroxisomes, and
sugar may negatively affect the interaction of oil bodies and peroxisomes to fine–tune lipolysis

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catalyzed by SDP1 during the germination of Arabidopsis [10]. We supposed that sugars weakened

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lipid breakdown by inhibiting the interaction between oil bodies and peroxisomes, maybe the
effect of which was not negligible in the GA-treated and suc-treated seeds during the early

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germination.
Based on our results and some literatures, we proposed a possible molecular mechanism on the

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exogenous GA weakening lipid breakdown by increasing soluble sugars levels in early
germination of zanthoxylum seeds (Fig 6). Exogenous GA promoted the catalytic activity of
ɑ–amylase, then the storage starch would be efficiently hydrolyzed into soluble sugars to

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supplement metabolically consumed sugars. The increased sugars helped to reduce the catalytic
activities of SDP1 and ICL, the expressions of SDP1 and ICL genes, thereby weakening lipid
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breakdown and glyoxylate cycle.
Compared with the water-treated seeds, less fatty acids were measured in the GA-treated seeds,
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which meant that the substrate of β–oxidation was reduced. In addition, low catalytic activity of
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ICL meant that the glyoxylate cycle was weakened. Thus, acetyl-CoA, the product of β–oxidation,
also as initial substrate for glyoxylate cycle, its consumption was declined, which contributed to
feedback inhibition of β-oxidation of fatty acids. Therefore, it was reasonable to speculate that
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β–oxidation of fatty acids was attenuated in the GA-treated seeds compared with the water-treated
seeds. The weakened β–oxidation would be conductive to the reduction of ROS, thereby helping
to maintain the well redox state to attenuate oxidative damages during the early germination of
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seeds. Thusly, the higher GSH, less MDA and lower REC could be observed in the GA-treated
seeds compared with the water-treated seeds. Finally, some literatures provided the cue: sugars
might be involved in the inhibition of interaction between oilbodys and peroxisomes, thereby
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contributing to the inhibition of TAG mobilization in the early germination of zanthoxylum seeds.
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A
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Fig .6. the mode that exogenous GA inhibits lipid breakdown by increasing soluble sugars levels.
GA: gibberellin; TAG: triacylglycerol; FFA: free fatty acids; AcCoA: Acetyl CoA; GAC: glyoxylate cycle; ICL:

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isocitratelyase; ROS: reactive oxygen species.
①The enhanced interaction between oil bodies and peroxisomes was observed in sdp1 seedlings of Arabidopsis
thaliana compared with the wild type, but was significantly suppressed by exogenous sucrose (Cui et al., 2016).
②SDP1 was initially localized to the peroxisome membrane in the early growth of seedling, and then transported

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to the oil bodies’ surface probably by peroxisome tubulations (Thazar–Poulot et al., 2015).
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5. Conclusions
The prematurely initiated TAG mobilization in the water-treated seeds might be related to the low
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levels of sugars caused by shortage of sugars supplement. The application of sucrose could
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improve the germination of water-treated seeds and significantly inhibit lipid breakdown. Both the
application of sucrose and exogenous GA could significantly reduce the catalytic activities of
SDP1 and ICL, the expressions of SDP1 and ICL genes, and decrease the products of lipid
ED

breakdown as well during the early germination. We hypothesized that exogenous GA might
enhance starch hydrolysis by promoting the catalytic activity of ɑ–amylase to supplement
metabolically consumed soluble sugars, and then the increased sugars levels would help to inhibit
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the lipid breakdown to mitigate oxidative damages in the early germination of zanthoxylum seeds.

Author Contributions
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P.W. and J.S. conceived the original screening and research plans; J.S., H.J., T.Z. and Y.W.
performed most of the experiments; J.S. provided technical assistance to H.J., T.Z. and Y.W.; J.S.
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designed the experiments and analyzed the data; J.S. conceived the project and wrote the article
with contributions of all the authors; P.W. and Z.L. supervised and complemented the writing.
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Competing interests
The authors declare that the research was conducted in the absence of any commercial or financial
relationships that could be construed as a potential conflict of interest.

Acknowledgments
This study was financially supported by National Natural Science Foundation of China (Project
Number: 31370612).
References
[1] M.A. Ying-Zi, P. Wang, Evaluation of Threatened Degree and Sustainable Utilization
Countermeasures of Zanthoxylum Dissitum Resource, Nonwood Forest Research, (2008).
[2] X. Chen, H.R. Pen, Study on the morphology and tissue of Peel and seeds in Zanthoxylum
planispinure var.dintanensis, Seeds, 28(2009) 18–20.
[3] Y.Z. Ma, B.H. Yang, P. Wang, Study on seed dormancy mechanism and germination of
Zanthoxylum dissitum Hemsl, Journal of Natural Science of Hunan Normal University, (2009).
[4] J. Sun, P. Wang, T. Zhou, J. Rong, H. Jia, Z. Liu, Transcriptome Analysis of the Effects of
Shell Removal and Exogenous Gibberellin on Germination of Zanthoxylum Seeds, Sci Rep, 7

T
(2017) 8521.

IP
[5] A. Gómezcadenas, R. Zentella, M.K. Walkersimmons, T.H.D. Ho, Gibberellin/Abscisic Acid
Antagonism in Barley Aleurone Cells: Site of Action of the Protein Kinase PKABA1 in Relation

R
to Gibberellin Signaling Molecules, Plant Cell, 13 (2001) 667-679.
[6] M. Ogawa, A. Hanada, Y. Yamauchi, A. Kuwahara, Y. Kamiya, S. Yamaguchi, Gibberellin

SC
biosynthesis and response during Arabidopsis seed germination, Plant Cell, 15 (2003) 1591-1604.
[7] S.P.C. Groot, C.M. Karssen, Gibberellins regulate seed germination in tomato by endosperm
weakening: a study with gibberellin-deficient mutants, Planta, 171 (1987) 525-531.

U
[8] F.A. Kondrashov, E.V. Koonin, I.G. Morgunov, T.V. Finogenova, M.N. Kondrashova,
Evolution of glyoxylate cycle enzymes in Metazoa: evidence of multiple horizontal transfer events
N
and pseudogene formation, Biology Direct, 1 (2006) 31.
[9] P.J. Eastmond, SUGAR-DEPENDENT1 Encodes a Patatin Domain Triacylglycerol Lipase
A
That Initiates Storage Oil Breakdown in Germinating Arabidopsis Seeds, Plant Cell, 18 (2006)
M

665.
[10] S. Cui, Y. Hayashi, M. Otomo, S. Mano, K. Oikawa, M. Hayashi, M. Nishimura, Sucrose
production mediated by lipid metabolism suppresses physical interaction of peroxisomes and oil
ED

bodies during germination of Arabidopsis thaliana, Journal of Biological Chemistry, 291 (2016)
19734-19745.
[11] N. Thazar-Poulot, M. Miquel, I. Fobis-Loisy, T. Gaude, Peroxisome extensions deliver the
PT

Arabidopsis SDP1 lipase to oil bodies, Proceedings of the National Academy of Sciences of the
United States of America, 112 (2015) 4158.
[12] S. Patui, L. Clincon, C. Peresson, M. Zancani, L. Conte, T.L. Del, L. Navarini, A. Vianello, E.
E

Braidot, Lipase activity and antioxidant capacity in coffee (Coffea arabica L.) seeds during
germination, Plant Science An International Journal of Experimental Plant Biology, 219-220
CC

(2014) 19.
[13] Y. Ishibashi, T. Tawaratsumida, K. Kondo, S. Kasa, M. Sakamoto, N. Aoki, S.H. Zheng, T.
Yuasa, M. Iwayainoue, Reactive Oxygen Species Are Involved in Gibberellin/Abscisic Acid
A

Signaling in Barley Aleurone Cells, Plant Physiology, 158 (2012) 1705-1714.

[14] M.A. Hooks, Molecular Biology, Enzymology, and Physiology of β-Oxidation, Springer
Netherlands, 2002.
[15] I.M. Møller, P.E. Jensen, A. Hansson, Oxidative modifications to cellular components in
plants, Annu.rev.plant Biol, 58 (2007) 459-481.
[16] S. Gaweł, M. Wardas, E. Niedworok, P. Wardas, [Malondialdehyde (MDA) as a lipid
peroxidation marker], Wiad Lek, 57 (2004) 453-455.
[17] D.D. Rio, A.J. Stewart, N. Pellegrini, A review of recent studies on malondialdehyde as toxic
molecule and biological marker of oxidative stress, Nutrition Metabolism & Cardiovascular
Diseases Nmcd, 15 (2005) 316.
[18] Z.M. Wang, Y. Niu, Q.L. Tang, Effects of paclobutrazol on physiological characteristics of
test–tube seedlings of ginger, Journal of Southwest University (Natural Science), 31(2009) 39–42.
[19] M.A. Shu-Yan, L.I. Ji-Yue, Z.D. Peng, Study on the Activity Changes of Enzyme in the Seeds
of Pinus flexilis James During Artificial Aging, Seed, 30 (2011) 9-14.
[20] T. Vorderwülbecke, K. Kieslich, H. Erdmann, Comparison of lipases by different assays,
Enzyme & Microbial Technology, 14 (1992) 631-639.
[21] T. Suzuki, T. Nakayama, T. Kurihara, T. Nishino, N. Esaki, A cold-active esterase with a

T
substrate preference for vinyl esters from a psychrotroph, Acinetobacter sp. strain no. 6: gene

IP
cloning, purification, and characterization, Journal of Molecular Catalysis B Enzymatic, 16 (2002)
255-263.

R
[22] J.J. Hao, Z.L. Kang, Plant Physiology Experiment Technology, Chemical Industry Press,
Beijing, (2007).

SC
[23] X.K. Wang, The Principles and Techniques of Plant Physiological and Biochemical
Experiments, Higher Education Press, Beijing, (2015).
[24] S.D. Shi, Y.Q. Sun, The Guidance of Plant Physiology Experiments, China Forestry press,
Beijing, (2011).
U
[25] J.H.C. Cornelissen, S. Lavorel, E. Garnier, N. Buchmann, D.E. Gurvich, P.B. Reich, H.T.
N
Steege, H.D. Morgan, M.G.A.V.D. Heijden, et al. 2003. Handbook of protocols for standardised
and easy measurement of plant functional traits worldwide, Australian Journal of Botany, 51 (2003)
A
335-380.
M

[26] J.K. Sun, P. Wang, H. Jia, T. Zhou, Y. Wu, Selection and validation of reference genes for
quantitative real-Time PCR during seed germination of Zanthoxylum dissitum Hemsl, guihaia,
(2017) DOI: 10.11931/guihaia.gxzw201710025
ED

[27] H. Cohen, H. Israeli, I. Matityahu, R. Amir, Seed-Specific Expression of a

Feedback-Insensitive Form of CYSTATHIONINE-γ-SYNTHASE in Arabidopsis Stimulates
Metabolic and Transcriptomic Responses Associated with Desiccation Stress, Plant Physiology,
PT

166 (2014) 1575-1592.

[28] M. Dieuaide, P. Raymond, Increased Fatty Acid β-Oxidation after Glucose Starvation in
Maize Root Tips, Plant Physiology, 99 (1992) 595-600.
E

[29] M. Dieuaide, I. Couée, A. Pradet, P. Raymond, Effects of glucose starvation on the oxidation
of fatty acids by maize root tip mitochondria and peroxisomes: evidence for mitochondrial fatty
CC

acid beta-oxidation and acyl-CoA dehydrogenase activity in a higher plant, Biochemical Journal,
296 ( Pt 1) (1993) 199-207.
[30] H. Kato-Noguchi, F.A. Macã-As, Effects of 6-methoxy-2-benzoxazolinone on the
A

germination and alpha-amylase activity in lettuce seeds, Journal of Plant Physiology, 162 (2005)
1304-1307.
[31] P. Mishra, R.S. Dubey, Effect of aluminium on metabolism of starch and sugars in growing
rice seedlings, Acta Physiologiae Plantarum, 30 (2008) 265.
[32] S. Borek, W. Ratajczak, L, Ultrastructural and enzymatic research on the role of sucrose in
mobilization of storage lipids in germinating yellow lupine seeds, Plant Science, 170 (2006)
441-452.
[33] S. Borek, K. Nuc, Sucrose controls storage lipid breakdown on gene expression level in
germinating yellow lupine (Lupinus luteus L.) seeds, Journal of Plant Physiology, 168 (2011)
1795-1803.
[34] S. Borek, S. Kubala, S. Kubala, Diverse regulation by sucrose of enzymes involved in storage
lipid breakdown in germinating lupin seeds, Acta Physiologiae Plantarum, 35 (2013) 2147-2156.
[35] S. Borek, E. Paluchlubawa, S. Pukacka, M. Pietrowskaborek, L. Ratajczak, Asparagine slows
down the breakdown of storage lipid and degradation of autophagic bodies in sugar-starved
embryo axes of germinating lupin seeds, Journal of Plant Physiology, 209 (2016) 51-67.
[36] V.N. Popov, E.A. Moskalev, M.U. Shevchenko, A.T. Eprintsev, Comparative Analysis of
Glyoxylate Cycle Key Enzyme Isocitrate Lyase from Organisms of Different Systematic Groups,

T
Journal of Evolutionary Biochemistry & Physiology, 41 (2005) 631-639.

IP
[37] F.L. Theodoulou, P.J. Eastmond, Seed storage oil catabolism: a story of give and take,
Current Opinion in Plant Biology, 15 (2012) 322.

R
[38] J.D. Bewley, M. Black, Physiology of Development and Germination, Ed 2. Plenum Press,
New York, (1994).

SC
[39] I.A. Graham, Seed storage oil mobilization, Annual Review of Plant Biology, 59 (2008)
115-142.
[40] J.Y. Chen, S.L. Luo, Q.H. Liu, S.Q. Lin, Study on Vegetation oil in Guangxi Ⅱ–The

U
ingredients of storage oil of seeds in 110 species plant, Guihaia, 2(1981) 18–31.
[41] C.P. Song, X.L. Wang, Plant Physiology, Science Press, Beijing, (2009).
N
[42] I. Morkunas, S. Borek, M. Formela, L. Ratajczak, Plant Responses to Sugar Starvation, 2012.
A
M
ED
E PT
CC
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