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The Antioxidant Defense System in Chinese Jujube Is Triggered to Cope with

Phytoplasma Invasion

Article in Tree Physiology · June 2020

DOI: 10.1093/treephys/tpaa067

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Tree Physiology 00, 1–13
doi:10.1093/treephys/tpaa067

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Research paper

The antioxidant defense system in Chinese jujube is triggered

to cope with phytoplasma invasion

Chaoling Xue 1,2 , Zhiguo Liu3 , Lihu Wang4 , Hongtai Li 1,2 , Weilin Gao1,2 , Mengjun Liu3 ,
Zhihui Zhao3,5 and Jin Zhao 1,2,5

1
College of Life Science, Hebei Agricultural University, Baoding 071000, China; 2 Key Laboratory of Hebei Province for Plant Physiology and Molecular Pathology, Hebei
Agricultural University, Baoding 071000, China; 3 Research Center of Chinese Jujube, Hebei Agricultural University, Baoding 071000, China; 4 College of Landscape and
Ecological Engineering, Hebei University of Engineering, Handan 056038, China; 5 Corresponding author (zhaojinbd@126.com,lyzhihuizhao@126.com)

Received September 21, 2019; accepted May 27, 2020; handling Editor Hans-Peter Mock

Reactive oxygen species (ROS) in plants increase dramatically under pathogen attack, and the antioxidant defense system
is then triggered to protect the plant against the ROS. Jujube witches’ broom disease (JWB), caused by phytoplasma,
is a destructive disease of Chinese jujube. The results of fluorescence-based measurement revealed that ROS were
overproduced within jujube leaves after phytoplasma invasion. Furthermore, analysis based on mRNA and metabolite
levels revealed that ascorbic acid (AsA) metabolism was strengthened under phytoplasma stress. The high expression of
genes involved in the AsA/glutathione (GSH) cycle and thioredoxin (Trx) synthesis in diseased leaves indicated that
GSH and Trx actively respond to phytoplasma infection. Moreover, higher activities of enzymatic antioxidants and
the upregulated expression of related genes were confirmed in diseased tissues. Both nonenzymatic and enzymatic
antioxidants in the host jujube were strongly stimulated to cope with ROS caused by phytoplasma stress. Compared with
that in the susceptible variety, the activities of glutathione S-transferase and peroxidase in the resistant variety at the
earlier infection stage were higher, indicating that enzymes might be involved in the resistance to phytoplasma. These
results highlight the roles of the antioxidant defense system of the host plant in the tolerance to phytoplasma invasion.

Keywords: antioxidant defense system, AsA/GSH, Chinese jujube, phytoplasma, reactive oxygen species.

Introduction by programmed cell death (Gao et al. 2018). However, the

Overproduction of reactive oxygen species (ROS) in plants
under stress conditions is a common phenomenon. In gen-
eral, high ROS production is triggered when host cells are
infected with pathogenic bacteria (Asada 1999, Møller 2001,
Imlay 2003). Enhanced ROS production in plants is termed an
‘oxidative burst’ (Trivedi et al. 2013), and the ROS derived
from the oxidative burst can directly damage bacteria (Anjum
et al. 2015, Doke et al. 1996, Molina-Cruz et al. 2008) and
function as signaling molecules. For example, ROS activate
a variety of defense responses, including an increase in cell
wall strength, the synthesis of phytoalexins and pathogenesis-
related proteins, and the suppression of pathogen growth
accumulation of ROS will significantly damage cell structures and
functions.
Correspondingly, plants tend to neutralize ROS by synthe-
sizing nonenzymatic and enzymatic antioxidants (Akram et al.
2017, Lee and Lee 2000, Oidaira et al. 2000). The antioxidant
defense machinery protects cells and tissues against ROS-
induced oxidative damage. Ascorbic acid (AsA) is known to
protect organelles and cells from the adverse effects of ROS,
which overaccumulate due to stress-induced oxidative damage
(Kuźniak 2010, Latif et al. 2016, Naz et al. 2016). Ascorbic
acid can act in coordination with glutathione (GSH) in the
AsA/GSH cycle to provide the appropriate redox environment

†
Chaoling Xue and Zhiguo Liu contributed equally to the work.
© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.
 2 Xue et al.
for regulating diverse defense pathways and determine the
tolerance of plants to various stresses (Trivedi et al. 2013).
Thioredoxin (Trx) peroxidase is also an antioxidant protein that
limits the activity of ROS (Ichimiya et al. 1997). In the redox
environment of a cell, thioredoxin h-type (Trxh) can activate
the nonexpressor of pathogenesis-related genes 1 (NPR1) to
induce the expression of resistant genes (Mou et al. 2003, Pas-
tori et al. 2003, Tada et al. 2008). Antioxidant enzymes, such
as superoxide dismutase (SOD, EC 1.15.1.1), peroxidase (POD,
EC 1.11.1.7) and laccase, are the most important components
of the ROS scavenging system (Haque et al. 2014). Laccase
and POD have been widely recognized as antioxidants involved
in ROS scavenging.
Chinese jujube (Ziziphus jujuba Mill.), one of the major
dry fruit crops, is the most economic value species in the
family Rhamnaceae. Jujube witches’ broom disease (JWB), which
is associated with ‘Candidatus Phytoplasma ziziphi’ (‘Ca. P.
ziziphi’), is a typical phytoplasma disease. Typical symptoms
of JWB include the witches’ broom, yellowing and phyllody
(PL). This disease causes large economic losses and destructive
death in the Chinese jujube industry. The interaction between
phytoplasma and host plant is a multifaceted process. For
the host plant, such an interaction might derail the fate of a
developing meristem at every possible stage during vegetative
growth, floral transition and flower development (Wei et al.
2019). For the pathogen, previous reports have suggested that
various symptoms may be induced by different virulence factors,
while onion yellows (OY) phytoplasma TENGU and aster yellow
witches’ broom (AYWB) phytoplasma SAP11 were attributed to
witches’ broom symptom, AYWB phytoplasma SAP54 and OY
phytoplasma phyllogen were implicated in floral malformation
(Hoshi et al. 2009, MacLean et al. 2011, Maejima et al. 2014,
Sugio et al. 2014). The genome of JWB phytoplasma is crucial
for a comprehensive understanding of the interactions between
JWB phytoplasmas and jujube (Wang et al. 2018). Like other
plant pathogens, phytoplasmas induce host defense responses
(Liu et al. 2019, Nejat et al. 2012, Timm and Reineke 2014,
Xue et al. 2019). It has been reported that ROS are produced
within the plant host in response to phytoplasma infection
(Rossatto et al. 2017, Sánchez-Rojo et al. 2011). Zafari et al.
(2011) showed that antioxidant enzymes of infected lime
plant can be activated in response to phytoplasma infection.
Miura et al. (2012) showed that the OY phytoplasma works
in conjunction with SOD to eliminate O2 − . Thus far, it is
unclear whether the host antioxidant defense system copes
with phytoplasma infection and what the differences are in the
antioxidant systems of resistant and susceptible varieties. The
aim of this study was to explore the changes in the antiox-
idant systems of jujube plants before and after phytoplasma
infection. Our study will provide evidence and new perspectives
on the molecular tolerance mechanisms of the host plant to
phytoplasmas.
Tree Physiology Volume 00, 2020
Materials and methods
Plant material
Healthy and JWB-infected Z. jujuba Mill. ‘Dongzao’ trees were
used in this study. The jujube trees were obtained from the
Experimental Station of Chinese Jujube, Hebei Agricultural Uni-
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versity, Baoding, China. Three types of tissues that showed
disease symptoms of different severity, including the apparent
normal leaves (ANL), PL and witches’ broom leaves (WBL),
from diseased trees and healthy leaves (HL) from healthy trees
were collected in June, July, August and September. The samples
were used in ROS detection. All treatments were conducted in
triplicate.
The JWB-resistant and JWB-susceptible varieties from Z. jujuba
Mill. ‘Junzao’ were used as scions. The rootstock cultivar was
‘Dongzao’. The scions were grafted onto JWB-diseased and
healthy rootstocks in May (Figure 1). All grafting inoculation
treatments were conducted in three replicates each. The mature
leaves in the middle of the bearing shoot were collected from
sprouted scions at five stages (June, July, August, September and
October). The diseased leaves of susceptible variety were mixed
with ANL, PL and WBL. All samples were frozen in liquid nitrogen
rapidly and stored at −80 ◦ C for DNA and RNA extraction. The
subsequently tested sample was derived from 10 g of finely
powdered leaves.
Detection of JWB phytoplasma
The thymidylate kinase gene (TMK, KC493615) of JWB phyto-
plasma was used for pathogen detection (Xue et al. 2018).
The expression of the TMK gene in jujube leaves exhibiting
disease characteristics of different severity was analyzed by
qRT-PCR with ZjACT as the internal control (Bu et al. 2016).
All treatments were conducted with three biological replicates.
Detection of ROS
Leaves collected from healthy and infected jujubes and suscep-
tible and resistant varieties (in June) were soaked in phosphate-
buffered saline (0.01 mM) for 30 min. Then, the liquid was
removed and replaced with 2,7-dichlorodihydrofluorescein diac-
etate (H2DCFDA, 10 μM, Invitrogen). The leaves were placed
under vacuum for 40 min and then shocked before being put on
slides. A confocal microscope (LSM880NLO, Zeiss, Germany)
was used to observe ROS fluorescence (Zhuang et al. 2019).
Measurement of the AsA content
The AsA content of jujube leaves was measured by HPLC
method (Gao et al. 2011). The process is as follows: 0.25 g
of leaves was finely powdered and dissolved in 50 ml of 0.1%
H3 PO4 (pH 2.3) while avoiding light for 20 min. The solution
was filtered through a 0.45-μm water film and then loaded onto
a Venusil MP-C18 column (4.6 × 250 mm, 5 μm), eluting at 1 ml
min at 25 ◦ C. For the mobile phase, 0.02 M phosphoric acid
buffer was used. The wavelength of detection was 260 nm. An
 The antioxidant defense system in Chinese jujube 3

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Figure 1. The symptoms of Chinese jujube infected by phytoplasma. (A) Healthy flowers, (B) the flowers with elongated peduncle, (C) phyllody, (D)
witches’ broom with tiny leaves. Both the JWB-resistant (G) and susceptible varieties (E) grafted on the diseased trees behaved elongated peduncle
in June, but then the JWB-resistant varieties resumed the normal growth in July (H), while the susceptible ones directly developed more severe
symptoms, i.e., phyllody and witches’ broom with tiny leaves (F), (I) the grafting diagram: the scions of JWB-resistant and susceptible varieties were
simultaneously grafted onto diseased and healthy rootstocks.

AsA standard was accurately weighed in a 25-ml brown bottle,
dissolved in 0.1% H3 PO4 , fixed and used to prepare six gradient
dilutions with which to generate a standard curve. All samples
were measured in triplicate.
Measurement of antioxidant enzyme activity
The enzyme activity of SOD, POD and laccase in jujube leaves
was examined by ultraviolet spectrophotometry (Haque et al.
2014): 0.25 g of fresh leaves was finely powdered and
dissolved in 5 ml of 0.05 M phosphoric acid buffer. The mixture
was centrifuged for 20 min at 12,000 r.p.m. The supernatant
was used as the crude enzyme extract.
The activity of SOD was determined by the nitro blue tetra-
zolium (NBT) photochemical reduction method (Rossatto et al.
2017) in a reaction medium containing 0.1 mM NBT, 15 mM
methionine, 0.003 mM EDTA, 0.1 mM riboflavin and enzyme
extract; buffer was used to bring the final reaction volume to
4.2 ml. The absorbance was recorded at 560 nm. One unit of
SOD equaled the amount of enzyme capable of inhibiting NBT
photoreduction by 50%.
The activity of POD was determined by the guaiacol method
in a reaction medium containing 0.1% guaiacol, 0.08% H2 O2
and enzyme extract; buffer was used to bring the final reaction
volume to 3.5 ml. The absorbance was recorded at 470 nm
(Allahveran et al. 2018). The laccase activity was determined
by the method of Pizzocaro et al. (1993). The absorbance was
recorded at 420 nm. The OD value changed by 0.01 per minute
for one unit of enzyme activity.
RNA extraction and qRT-PCR analysis
Total RNA was extracted using an RNAprep Pure Plant Kit
(TIANGEN , Beijing, China) according to the manufacturer’s

Tree Physiology Online at http://www.treephys.oxfordjournals.org

 4 Xue et al.
protocol, and genomic DNA was removed by RNase-free DNase
I (TIANGEN). The DNA and RNA concentration and purity were
checked using a NanoDrop 2000 spectrophotometer. First-
strand cDNA was synthesized by reverse transcribing 500 ng
of the total RNA with a FastQuant RT Super Mix Kit (TIANGEN).
cDNA that had been diluted 10-fold was used as the template
for qRT-PCR.
The expression levels of the genes related to antioxidant sys-
tem metabolism and the content of phytoplasma were detected
by qRT-PCR. The primers used in this study are listed in Table S1
available as Supplementary Data at Tree Physiology Online,
and the amplicon sizes were within 80–250 bp. PCR products
were amplified in triplicate using Bio-Rad iQTM 5 with TransStart
Top Green qPCR SuperMix AQ131 (TransGen Biotech, China)
in 20-μl reactions. The reaction system contained 10 μl of
2× TransStart® Top Green qPCR SuperMix, 0.4 μl each of
10 μM primers, 8.2 μl of ddH2 O and 1 μl of diluted DNA or
cDNA. The temperature program for qRT-PCR was as follows:
preincubation for 30 s at 95 ◦ C; 40 cycles of 5 s at 95 ◦ C; 10 s
at 50–60 ◦ C (varied depending on the specific primers); and
10 s at 72 ◦ C. Three biological replicates were performed for
each sample. To normalize the total amount of cDNA present
in each reaction, the ZjACT gene was co-amplified as an
endogenous control for calibration of the relative expression
(Bu et al. 2016). Relative expression levels were calculated
according to the 2−CT method (Livak and Schmittgen 2001).
Healthy jujube plants were used as controls.
Statistical analysis
The data are presented as the mean ± SD of at least three inde-
pendent experiments. Statistical analyses were conducted using
one-way ANOVA with SPSS. Statistically significant differences
are indicated by lowercase letters (P < 0.05).
Results
The symptoms of Chinese jujube infected by phytoplasma
Once infected by JWB phytoplasma, the jujube trees behaved
preliminary symptoms, such as elongated peduncle (Figure 1A
and B) and some yellowing leaves. With the aggravation of the
disease, the typical symptoms including phyllody (Figure 1C)
and witches’ broom (Figure 1D) with tiny leaves appeared in
the diseased branches. Both the JWB-resistant and -susceptible
varieties grafted on the diseased trees exhibited elongated
peduncle in June (Figure 1E and G), but then the JWB-resistant
variety resumed normal growth in July (Figure 1H), while the
susceptible one directly developed more severe symptoms, i.e.,
phyllody and witches’ broom with tiny leaves (Figure 1F).
Meanwhile, the successful infection by phytoplasmas of dif-
ferent samples was measured and confirmed at different stages,
and as shown in Figure S1 available as Supplementary Data at
Tree Physiology Online, the expression of thymidylate kinase
Tree Physiology Volume 00, 2020
(TMK) increased gradually in the diseased leaves and the
susceptible variety from June to September and was highest in
September (diseased leaves) and October (susceptible variety),
respectively. The high level of phytoplasma was detected during
the whole infected period in the susceptible variety (Figure S1
available as Supplementary Data at Tree Physiology Online). On
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the contrary, the lower content of phytoplasma was detected at
early infected stage (June) in resistant variety, and then almost
no phytoplasma was found at later stages (Figure S1 available
as Supplementary Data at Tree Physiology Online).
In three types of diseased leaves, the expression levels of
TMK were much lower in July than other periods (Figure S1
available as Supplementary Data at Tree Physiology Online).
However, the expression level of TMK in the susceptible variety
was much lower in August. The results in the two kinds of
condition did not conflict with each other. The susceptible and
resistant varieties were infected in the current year by grafting
inoculation; therefore, the scions sprouted 1 month later than
other branches without grafting, and their diseased stages were
also 1 month later than those of the three types of diseased
leaves. In the susceptible variety, the expression level of TMK in
July should compare with that of diseased leaves in June. Their
changing trends were in accordance with each other.
The phytoplasma invasion clearly induces overproduction
of ROS in host jujube
In order to determine whether the phytoplasma invasion could
induce the ROS in jujube leaves, a fluorescence-based measure-
ment was performed to detect the formation of ROS (Meng and
Ma 2018). As observed in Figure 2, phytoplasma invasion stim-
ulated the production of ROS in diseased leaves, and the ROS
level gradually increased with the development of the disease
severity (Figure 2A, Figure S2 available as Supplementary Data
at Tree Physiology Online). ROS levels were significantly higher
in phyllody (PL) and WBL than in the HL. Meanwhile, we found
that ROS level in susceptible varieties was higher than in the
resistant one (Figure 2B, Figure S2 available as Supplementary
Data at Tree Physiology Online). This result demonstrated that
the production of ROS in jujube leaves was clearly induced by
phytoplasma invasion.
Phytoplasma invasion affects the AsA profile in the host
jujube
The synthesis of AsA plays an important role in oxidative stress,
and AsA metabolism might also be involved in phytoplasma
attack in Chinese jujube. Thus, the contents of AsA were tested
in four types of jujube tissues and in susceptible and resistant
varieties.
Next, the AsA content in the above samples was detected by
HPLC (Figure 3). The results showed that the AsA content of
diseased leaves (such as in ANL and WBL) was higher than
that of HL in July and lower than that of HL in August and
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Figure 2. Detection of ROS. Observation of ROS fluorescence (stained with H2DCFDA, ROS) in the healthy and diseased jujube leaves of ‘Dongzao’
(A) and in resistant and susceptible varieties of ‘Junzao’ grafted on healthy and diseased jujube (B). The green speckles show the distribution of
ROS. Bars, 100 μm. DS, susceptible varieties grafted on diseased jujube; HS, susceptible varieties grafted on healthy jujube; DR, resistant varieties
grafted on diseased jujube; HR, resistant varieties grafted on healthy jujube.

September (Figure 3A). On the other hand, the AsA contents
of infected susceptible and resistant varieties were higher than
that of noninfected controls in August and September, and then
decreased markedly in October (Figure 3B). All these results
illustrated that compared with HL, the AsA contents in diseased
leaves were increased in early disease stages and decreased
significantly with the accumulation of phytoplasma.
the expression level of ZjGLDH increased considerably in both
susceptible and resistant varieties in September and decreased
significantly in October, which was in accordance with the
patterns of the AsA content (Figure 3B). These results indicated
that the synthesis, regeneration and decomposition pathways of
AsA were all strengthened in jujube leaves under phytoplasma
stress (Figure 3).
L -Gulonolactone oxidase (GULO, EC 1.1.3.8) is another cru-

Phytoplasma invasion triggers the expression
of AsA-associated genes
Based on the obvious change of AsA content in jujube after
phytoplasma infection, the AsA-associated genes were further
analyzed. As shown in Figure 3, the expression levels of key AsA
synthesis (ZjGLDH, L-galactono-1,4-lactone dehydrogenase;
EC 1.3.2.3) and regeneration (ZjDHAR (dehydroascorbate
reductase; EC 1.8.5.1) and ZjMDHAR (monodehydroascorbate
reductase; EC 1.6.5.4)) genes were all increased in the
diseased leaves, which might have promoted the increase in
AsA. However, the expression of ZjAO (ascorbate oxidase, EC
l.10.3.3), which is related to AsA decomposition, was extremely
upregulated in July and September in the diseased leaves, which
might have contributed to the lower AsA content from August to
September compared with that in the healthy control leaves, but
the expression levels of ZjAPX, ZjGPP and ZjGME showed no
significant differences among the diseased and HL. In addition,
cial enzyme that can convert aldonolactone into AsA in many
animals at the final step of AsA synthesis, and this enzyme is
replaced by L-galactono-1,4-lactone dehydrogenase (GLDH) in
plants (Wheeler et al. 2015). Interestingly, during the interaction
between jujube and phytoplasmas, the expression level of
ZjGULO4, in particular, dramatically increased in the diseased
jujube leaves and the susceptible variety (Figure 3), whereas
the expression levels of all the GULO genes were not detected
or extremely low in healthy organs (root, stem, leaf, flower
and fruit) (Figure S3 available as Supplementary Data at Tree
Physiology Online). The abnormally high expression of ZjGULO
indicated that the pathway from aldonolactone to AsA was
triggered in diseased jujube tissues.
Phytoplasma infection enhances the expression
of GSH-associated genes
In addition to the important roles of AsA metabolism, the
AsA/GSH cycle plays a crucial role under biotic stress and is

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Figure 3. (A) The AsA content and the expression of genes related to AsA metabolism in four types of jujube leaves. MDHAR (monodehydroascorbate
reductase), DHAR (dehydroascorbate reductase), GLDH (L-galactono-1,4-lactone dehydrogenase), AO (L-ascorbate oxidase), APX (L-ascorbate
peroxidase), GPP (L-galactose phosphorylase), GME (GDP-D-mannose 3 ,5 -epimerase) and GULO4 (L-gulonolactone oxidase). (B) The AsA content
and the expression of genes related to AsA metabolism in the susceptible and resistant varieties. Significant differences were compared between the
different cultivars at each time point; the same is true below.

involved in the production of AsA. For example, GSH peroxidase the function of the AsA/GSH cycle in the accumulation of
(GSH-Px, EC 1.11.1.9), which is a key enzyme in the AsA/GSH AsA in diseased leaves, the expression levels of GSH-related
circulatory system, oxidizes reduced GSH to oxidized glu- genes were detected. In diseased tissues, higher expression
tathione (GSSG) with the production of AsA. Thus, to elucidate of γ -glutamylcysteine synthase (GCL, Figure 4), which can

Tree Physiology Volume 00, 2020


Figure 4. The expression of GSH-associated genes in four types of jujube leaves (A) and in the susceptible and resistant varieties under phytoplasma
stress (B). GCL (γ -glutamate cysteine ligase) and GSH-Px (glutathione peroxidase).
synthesize a precursor of GSH, was observed. In addition,
the expression of ZjGSH-Px increased significantly from August
to September in the diseased leaves, indicating that GSH
actively responds to phytoplasma infection and might lead to
the accumulation of AsA. However, the expression levels of
GSH transferase genes (ZjGSTs) showed no obvious changes
in the diseased leaves but were higher in the resistant variety
(Figure 4B). This result indicated that glutathione S-transferases
(GSTs) might be involved in the resistance to JWB.
Phytoplasma invasion affects the expression
of Trx-associated genes
Trx proteins are antioxidants that catalyze thiol-disulfide inter-
change in the target protein. H-type Trx proteins are involved
in the regulation of cellular redox and defense responses (Sun
et al. 2010). Thus, the expression levels of three Trxh genes
were evaluated in the four types of jujube tissues and the
susceptible and resistant varieties. ZjTrxh2 and ZjTrxh4 expres-
sion was upregulated in the diseased leaves compared with
that in the healthy controls (Figure 5A), and ZjTrxh4 expression
The antioxidant defense system in Chinese jujube 7
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increased significantly in the susceptible variety compared with
that in the resistant variety. This result indicated that ZjTrxh4
actively responded to phytoplasma infection, whereas ZjTrxh2
showed slightly higher expression in the resistant variety than
in the susceptible variety from June to September (Figure 5B).
The distinct expression pattern of ZjTrxhs in the susceptible
and resistant varieties might contribute to the difference in
antioxidant capacity of these two varieties.
Phytoplasma invasion increases the activity of antioxidant
enzymes
In addition to the important function of nonenzymatic antioxi-
dants in response to ROS production during pathogenic infec-
tion, antioxidant enzymes are also critical molecules for scaveng-
ing toxic ROS, which could protect host plants from pathogenic
attack. Therefore, the activities of SOD, POD and laccase in
jujube leaves and the expression of their related genes were
examined to evaluate the effect of JWB phytoplasma on antiox-
idant enzymes. As shown in Figure 6, the trend in SOD activity
was similar in diseased jujube and the healthy controls and was
Tree Physiology Online at http://www.treephys.oxfordjournals.org
 8 Xue et al.
Figure 5. The expression of three ZjTrxh genes in four types of jujube leaves (A) and in the susceptible and resistant varieties (B) under phytoplasma
stress.
Figure 6. The activity of SOD, POD and laccase in four types of jujube leaves (A) and in the susceptible and resistant varieties (B) under phytoplasma
stress.
not significantly different in the infected susceptible and resistant
varieties. While the activity of laccase was increased in slightly
diseased leaves (ANL) and decreased in severely diseased
leaves (WBL) at the early stage of infection, its activity in both
resistant and susceptible varieties showed no significant differ-
ences from June to October. However, at the early stage of phy-
toplasma infection (June–July), the POD activity in diseased trees
was significantly higher than that in healthy trees. The activity of
POD increased significantly in July in the susceptible trees and
was activated earlier in resistant trees (June) than in susceptible
trees. These results indicated that POD might play more impor-
tant roles than the other two enzymes in the ROS response and
functions at the early stage (June–July) of infection.
As POD might be the key enzyme during phytoplasma
infection, the expression levels of POD synthesis-related genes
were tested. ZjPOD4 was expressed at continuously high
Tree Physiology Volume 00, 2020
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levels in diseased leaves from June to September (Figure 7A),
which might have led to the high activity of POD. Compared
with their expression in the susceptible variety, ZjPOD2 and
ZjPOD4 were expressed at a higher level in the resistant
variety at the early stage of infection (June) (Figure 7B),
which might have contributed to their different patterns of POD
enzyme activity. Moreover, there were no significant differences
in the expression levels of the six genes encoding SOD
between diseased and HL, whereas their expression levels were
markedly higher in the resistant variety than in the susceptible
variety (Figure 7B). The expression levels of the four genes
encoding POD in diseased leaves were markedly increased
at the middle stage of infection (July) (Figure 7A). Overall,
phytoplasma infection had a more significant impact on the
POD activity in jujube tissues than the other two antioxidant
enzymes.
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Figure 7. The expression of antioxidant genes in jujube leaves under phytoplasma stress. (A) Heat map of qRT-PCR data for antioxidant genes in
four types of jujube leaves. Scaled log2 expression values are shown from green to red, indicating low to high expression. (B) The expression of
antioxidant genes in the susceptible and resistant varieties under phytoplasma stress.

Discussion molecule for sustained photosynthesis (Torres et al. 2006).

Plants have a complex and sophisticated antioxidant system AsA biosynthesis takes place in the mitochondria; however, its
whose function is to protect cells from the damage caused prime requirement is in the chloroplast (Foyer 2015, Foyer and
by ROS (Desikan et al. 2001, Musetti et al. 2005). As one Noctor 2016, Torres et al. 2006). In this study, lower AsA
of the most powerful antioxidants, AsA is a key antioxidant contents were confirmed in diseased jujube leaves in the later

Tree Physiology Online at http://www.treephys.oxfordjournals.org

 10 Xue et al.
Figure 8. Model proposed for the antioxidant defense system of jujube in response to phytoplasma infection.
infection stages. Previous results also showed that the chloro-
phyll contents decreased markedly and that the structure of
chloroplasts was destroyed in diseased jujube leaves (Xue et al.
2018). In Arabidopsis, the chlorophyll content also decreased
more rapidly in vitamin C-deficient (vtc1) mutants than in wild-
type plants (Zhang 2013). The above results suggested that
there is a link between the inhibition of photosynthesis and AsA
biosynthesis in the chloroplast, which also occurs during the
interaction between the host plant and phytoplasma.
For AsA metabolism, the L-galactose pathway is the dominant
synthetic pathway in Chinese jujube (Liu et al. 2014). In this
study, it was proven that this pathway is significantly strength-
ened under phytoplasma stress. The D-mannose pathway, a neg-
ligible pathway in AsA biosynthesis by healthy jujube trees, was
triggered by an extremely high expression of ZjGULOs in the dis-
eased trees. All wild-type and vtc mutant lines expressing GULO
showed an increased AsA content compared with that in controls
(Radzio et al. 2003), suggesting that GULOs could rescue vtc
mutants. This result indicated that the high expression of ZjGU-
LOs should increase the AsA content in diseased jujube plants;
thus, the lower content observed is a result of accelerating
decomposition. However, in further work, it will be worthwhile
to clarify how ZjGULOs are triggered by phytoplasma.
The AsA/GSH cycle in diseased jujube leaves was compre-
hensively stimulated by phytoplasma, a cycle that has been
confirmed to be advantageous for plants under stress because
Tree Physiology Volume 00, 2020
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it can limit the deleterious effects of oxidative stress and
reset redox homeostasis (Noctor and Foyer 2016, Zechmann
2014). In addition, this cycle might protect against biotic stress
by activating defense mechanisms through redox signaling. A
previous study suggested that the AsA redox state in the leaf
apoplast was regulated by L-ascorbate oxidase (AO) and that
it played an important role in modulating the ROS tolerance
of plant tissue (Fotopoulos et al. 2006); therefore, the higher
expression of ZjAO in jujube leaves under phytoplasma stress
might have a similar function.
Laccase can catalyze the formation of lignin and other
phenolic oxides to form a protective barrier against invasion by
pathogens. The laccase-catalyzed formation of quinone is toxic
and plays a direct role in disease resistance. In this study, the
expression levels of laccase genes were markedly increased in
jujube tissues after infection by phytoplasma, which indicated
that laccase positively responds to phytoplasma infection. In
addition, the higher expression of ZjPOD2 and ZjPOD4 in the
resistant variety at the early infection stage suggested that these
genes might participate in the resistance to phytoplasma.
In the present study, both nonenzymatic and enzymatic
antioxidants were strongly stimulated in the host jujube,
indicating that a strong and comprehensive antioxidant defense
system was triggered (Figure 8). Overall, phytoplasma invasion
induces the overproduction of ROS in jujube, and the ROS
trigger an enhanced antioxidant defense system that neutralizes

the ROS and re-establishes cellular redox balance. At the same
time, the ROS can also function as signaling molecules to
induce a variety of defense responses (Figure 8). Therefore,
ROS reduction in the host jujube is beneficial not only for
the host jujube cells but also for the survival and propagation
of the phytoplasma. In July, the POD activity was significantly
higher, and the expression of some genes encoding antioxidants
(such as ZjAO, ZjMDHAR, ZjDHAR and ZjGCL) was also clearly
upregulated in diseased leaves compared with the activity and
expression in healthy controls, indicating that the enhanced
antioxidant defense system in diseased tissues contributed
to the temporary decrease in the phytoplasma concentration
during the same period (Figure S1 available as Supplementary
Data at Tree Physiology Online). With the extension of the
infection time and the accumulation of phytoplasma, the damage
to the structure and function of jujube cells became increasingly
severe. In general, the diseased jujube trees survived for
3–5 years after phytoplasma infection; thus, the antioxidant
defense system should function as an important weapon in the
confrontation between jujube and phytoplasma. Phytoplasma
encounter strong resistance from host jujube trees, which should
contribute to the host survival for several years after infection.
This study improves our understanding of the crucial function
of the antioxidant defense system involved in phytoplasma
tolerance.
The phytoplasma diseases are very difficult to control because
the pathogens exist in plant phloem tissue. Phytoplasma infec-
tion causes a series of biochemical changes in the plant and
in the recovery process (Musetti 2010). Screening resistant
genotypes can prevent the disease effectively. In this study, the
JWB-resistant variety could recover to the normal growth from
slight diseased symptoms. The similar recovery processes were
also reported in other phytoplasma diseases (Seemüller et al.
1992, Jarausch et al. 1999, Bisognin et al. 2008, Jarausch
et al. 2011). In their breeding program, they found that the
resistance to phytoplasma could be inherited. Thus, the JWB-
resistant variety can be used in jujube breeding programs to
produce offspring with resistance. In the other side, further
studies should focus on the molecular mechanism of resistance
to phytoplasma, which would provide some novel insights on
the plant–phytoplasma interaction.
Supplementary Data
Supplementary Data for this article are available at Tree Physiol-
ogy Online.
Acknowledgments
The authors owe deep gratitude to Dr Lixin Wang for his critical
reading and revising of the manuscript.
The antioxidant defense system in Chinese jujube 11
Data and materials availability
All data and materials are presented in the main paper and
additional supporting file.
Downloaded from https://academic.oup.com/treephys/article-abstract/doi/10.1093/treephys/tpaa067/5849838 by Columbia University user on 06 July 2020
Authors’ contributions
J.Z. designed the research; C.X., Z.L. and J.Z. performed the
experiments, analyzed the data and wrote the paper. Others
participated in the data analysis. All authors read and approved
the final the manuscript.
Funding
This work was supported by grants from the National Key R&D
Program Project Funding (2018YFD1000607), the Foundation
for 100 Innovative Talents of Hebei Province (SLRC2019031),
the Hebei Distinguished Young Scholar (C2016204145) and
the National Natural Science Foundation of China (31772285).
These funding bodies had no role in the design of the study,
sample collection, analysis or interpretation of data, or in writing
the manuscript.
Conflict of interest
The authors declare that they have no competing interests.
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