Plant Science 287 (2019) 110184

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Plant Science
journal homepage: www.elsevier.com/locate/plantsci

The β-amylase PbrBAM3 from pear (Pyrus betulaefolia) regulates soluble T

sugar accumulation and ROS homeostasis in response to cold stress
Liangyi Zhaoa,1, Tianyuan Yanga,b,1, Caihua Xinga,1, Huizheng Donga, Kaijie Qia, Junzhi Gaoa,
⁎ ⁎
Shutian Taoa, Juyou Wua, Jun Wua, Shaoling Zhanga, , Xiaosan Huanga,
a
College of Horticulture, State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China
b
State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei 230036, China

A R T I C LE I N FO A B S T R A C T

Keywords: β-Amylase (BAM) is involved in sugar metabolism, but the role of BAM genes in cold tolerance remains poorly
Cold stress understood. Here, we report the identification and functional characterization of the chloroplast-localized BAM-
β-amylase (BAM) encoding gene PbrBAM3 isolated from Pyrus betulaefolia. The transcript levels of PbrBAM3 were up-regulated
Osmolyte under cold, dehydration and ABA, but repressed by maltose. Overexpression of PbrBAM3 in tobacco (Nicotiana
Starch degradation
tabacum) and pear (P. ussuriensis) conferred increased BAM activity, promoted starch degradation after chilling
Antioxidant enzyme
treatments and enhanced tolerance to cold. Under the chilling stress, the transgenic tobacco and P. ussuriensis
Reactive oxygen species (ROS)
exhibited lessened reactive oxygen species (ROS) generation, higher levels of antioxidant enzymes activity, and
greater accumulation of soluble sugars (specially maltose) than the corresponding wild type plants. Taken to-
gether, these results demonstrate that PbrBAM3 plays an important role in cold tolerance, at least in part, by
raising the levels of soluble sugars capable of acting as osmolytes or antioxidants.

1. Introduction scientific interest.

As sessile organisms, plants are inevitably exposed to environmental
stresses. Of these, cold stress is one of the most devastating abiotic
stresses: it impairs plant growth and development, reduces productivity
and limits geographical distribution. Over a long evolutionary period,
plants have evolved a set of sophisticated mechanisms to cope with and
adapt to cold stress. During the last decades, significant advancements
have been made in revealing the mechanisms underlying plant defen-
sive responses to abiotic stresses [1–6]. Such defensive responses of
plants are considerably complex processes regulated through multiple
signaling pathways. A general stress signal transduction pathway starts
with a signal perception, transduction to secondary messengers and
signal amplification (Ca2+, reactive oxygen species (ROS)), which in
turn leads to an array of biochemical and physiological modifications
allowing the plant to tolerate the stress [7–9]. In comparison to tradi-
tional breeding, genetic engineering has been shown as a more effective
and time-saving approach to generate new germplasms with improved
stress tolerance [17]. With this aim in mind, the exploitation of a large
reservoir of genes with desirable functions in cold tolerance is of utmost
Plants cope with cold stress by regulating a large number of genes at
a transcriptional level [31]. These cold-responsive genes can be clas-
sified according to their response timing into early-response genes and
delayed-response genes. The former group codes for regulatory pro-
teins, such as transcription factors (TFs) and protein kinases, which
function in stress signal transduction and gene expression regulation.
The latter group consists of genes whose products protect plant cells
against damage derived from stresses and sustain cell viability [10–12].
Under cold stress, ROS accumulate due to a disrupted balance be-
tween ROS production and ROS scavenging by scavenging enzymes and
non-enzymatic antioxidants [13,14]. They oxidize lipids, proteins and
nucleic acids, which in turn activates further stress (possibly leading to
cell membrane damage and osmotic potential unbalance [15–17], but
also counter-acting defense pathways. Contributing to the latter,
modulated metabolism-related genes lead to the production and accu-
mulation of “compatible solutes”, i.e., osmolytes or osmoprotectants
such as proline and soluble sugars. It is becoming increasingly clear that
soluble sugars play a key role in ROS scavenging and mediating the cold
tolerance [18–20].

⁎
Corresponding authors.
E-mail addresses: 2016104019@njau.edu.cn (L. Zhao), yangtianyuan2008@163.com (T. Yang), 2015104018@njau.edu.cn (C. Xing),
2017104017@njau.edu.cn (H. Dong), qikaijie@njau.edu.cn (K. Qi), 2017804152@njau.edu.cn (J. Gao), toast@njau.edu.cn (S. Tao), juyouwu@njau.edu.cn (J. Wu),
wujun@njau.edu.cn (J. Wu), slzhang@njau.edu.cn (S. Zhang), huangxs@njau.edu.cn (X. Huang).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.plantsci.2019.110184
Received 29 December 2018; Received in revised form 3 July 2019; Accepted 6 July 2019
Available online 09 July 2019
0168-9452/ © 2019 Elsevier B.V. All rights reserved.
L. Zhao, et al.
Fig. 1. Phylogenetic analysis of β-amylases from five different plants. A phylogenetic tree was constructed based on the deduced amino acid sequences of β-amylases
in Arabidopsis thaliana, Malus x domestica, Populus trichocarpa, Poncirus trifoliata, Oryza sativa and Pyrus betulaefolia.
A large number of genes encode enzymes involved in the synthesis
or degradation of soluble sugars in plants [21]. β-amylase (BAM) is an
exohydrolase hydrolyzing α-1,4-linked glucan chains and functions in
converting starch into maltose [22]. The maltose can be exported from
chloroplast into cytosol, and then it is converted into glucose through
disproportionating glucosyltransferases [23–26]. BAM is the mediator
of starch degradation into the downstream sugars and its role in cold
stress has been characterized in a range of plant species. For example,
cold stress increased the BAM activity of potato more than 4-fold [27].
Nine BAM homologues have been identified in the Arabidopsis genome
[25]. Knock down of AtBAM3 by RNA inference (RNAi) led to the de-
crease of soluble sugars levels [28] and silencing of AtBAM3 resulted in
starch accumulation [29]. In contrast, silencing of AtBAM1 did not
result in starch accumulation [30], and AtBAM4 did not code for an
active BAM enzyme [26]. Two other BAM genes, AtBAM7 and AtBAM8,
have also been associated with plant growth and development, via a
crosstalk with brassinosteroid signaling [22], a pathway different from
that of AtBAM3. Collectively, these findings suggest that AtBAM3 is the
dominant hydrolase involved in starch breakdown.
Having originated in Northern China, Pyrus betulaefolia is extremely
cold-hardy [32], making it an ideal source for genes of agronomical
importance with potential use for genetic engineering of cold tolerance.
Although the BAMs of Arabidopsis have been characterized, the roles of
BAM3 orthologs in stress tolerance remain poorly characterized in non-
model plants, especially in perennial fruit trees. In this study, we report
the isolation and functional characterization of PbrBAM3 from Pyrus
betulaefolia. Transgenic overexpression of PbrBAM3 in tobacco and P.
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Plant Science 287 (2019) 110184
ussuriensis confers cold tolerance, concurrent with an increased BAM
activity and higher accumulation of maltose and other soluble sugars.
Overall, our data suggest that PbrBAM3 enhances cold tolerance, at
least in part, by enhancing sugar metabolism.
2. Materials and methods
2.1. Plant material, growth conditions and stress treatments
Pyrus betulaefolia seedlings were grown in the experimental base of
the National Center of Pear Engineering Technology Research, Nanjing
Agricultural University. To examine PbrBAM3 expression pattern, uni-
form and healthy shoots of Pyrus betulaefolia from grown 45-day-old
seedlings were subjected to various stress treatments. For cold treat-
ment, the seedlings were kept in the growth chamber at 4 °C for 0, 6, 24,
72 and 144 h. For dehydration treatment, the seedlings were placed on
dry filter paper above a work bench and dried at 25 °C (in autumn, with
relative humidity of 60–70 %) for 0, 0.5, 1, 3, and 6 h. For the ABA
treatment was performed based on a previous method [33,34]. The
seedlings were incubated with their roots dipped in solution containing
100 μM ABA for 0, 1, 3, 6, 12, 24, and 72 h. In addition, maltose
treatment was performed by incubating the seedlings in 200 mM mal-
tose solution for 0, 6, 12, 24, 48 and 72 h. For each treatment, at least
40 seedlings shoots were harvested at the indicated time points, im-
mediately frozen in liquid nitrogen and stored at -80 °C until further
use.
L. Zhao, et al.
2.2. Quantitative real-time RT-PCR analysis of PbrBAM3 expression
Total RNA was extracted from the shoot samples using TRIZOL re-
agent (Invitrogen, United States). Approximately 1 μg of total RNA was
reversely transcribed into cDNA using the PrimeScript™ RT reagent kit
(Takara, China) according to manufacturer’s instructions. QRT-PCR was
performed on a BioRad CFX96 real-time system using an SYBR® Green
Master Mix kit (Takara) according to the manufacturer’s instructions as
described previously [35]. The primers used are listed in Supplemen-
tary Table S1. Each sample was analyzed in four replicates, and the
2−ΔΔCt method [36] was applied to calculate relative expression levels
of each gene. Expression of Ubiquitin (DQ830978) was used as an in-
ternal reference for tobacco, while Tubulin (AB239681) was used for
Pyrus betulaefolia. The expression analysis at each time point was re-
peated at least three times, and the data are shown as the mean va-
lues ± SE.
2.3. Isolation and sequence analysis of PbrBAM3
Based on the sequence a pair of primers was designed for RT-PCR to
amplify the ORF of BAM3 gene in Pyrus betulaefolia. RT-PCR assays
were performed as described earlier [37]. The multiple alignments of
the deduced amino acid sequence were performed using the ClustalW
program. A phylogenetic tree of PbrBAM3 and other plant species was
constructed by the Maximum Likelihood (ML) method using MEGA
(version 6.0) software [38]. The analysis of PbrBAM3 theoretical iso-
electric point and molecular weight was performed using the online
website ExPASy (http://www.align.genome.jp/). In addition, multiple
alignments of the BAM sequences containing the core glucosyl hydro-
lase domains were identified by ClustalX program with default settings
and presented by Jalview (http://www.jalview.org/).
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Plant Science 287 (2019) 110184
Fig. 2. Alignment of the core glucosyl hydro-
lase domains of PbrBAM3 (line 3) and its nine
Arabidopsis orthologs, AtBAM1–9. Identical
and similar amino acid residues among the
aligned sequences are indicated by black and
grey shading, respectively. Substrate binding
sites and two catalytic sites (Glu186 and
Glu380) are marked with large black arrow-
heads. The residues forming flexible loop and
inner loop are indicated with solid lines above
the sequences.
2.4. Subcellar localization of PbrBAM3
The complete ORF of PbrBAM3 was amplified by RT-PCR using
primers (GSP3; Table S1), then cloned into the pCAMBIA1302 vector
and fused in-frame to the N-terminal of GFP (Green Fluorescent
Protein), under the control of CaMV 35S promoter. After validation by
sequencing, the fusion constructs 35S pro:PbrBAM3::GFP or 35S
pro:GFP (as a control) were used for transient expression in A. thaliana
protoplast cells (about 1 μg plasmids of each) according to the poly-
ethyleneglycol method [35,39]. For the subcellular localization of
PbrBAM3 protein in A. thaliana protoplast cells, we used a confocal
laser scanning microscope (LSM410; Carl Zeiss) to observe the green
GFP and RFP fluorescence (at an excitation (Ex) wavelength of 488 nm
and emission (Em) wavelength of 525 nm) and the red autofluorescence
of chloroplasts (at Ex = 488 and Em = 587–610 nm), as previously
described [33].
2.5. Generation and identification of transgenic plants
To generate transgenic tobacco plants overexpressing PbrBAM3, the
whole ORF of PbrBAM3 was PCR-amplified with special primers GSP4
(Table S1) and inserted into binary vector pCMABIA1301 linearized
with BamH I and Xba I under the control of the CaMV 35S promoter.
The overexpression vectors were mobilized into Agrobacterium tumefa-
ciens strain EHA105 and then used to transform tobacco or P. ussuriensis
according to previous reports [40,41]. The overexpression vectors were
mobilized into a positive transformants and identified by genomic PCR
using primers specific for sequences of the hygromycin gene and
PbrBAM3 in tobacco or P. ussuriensis. The PbrBAM3 mRNA levels were
examined by RT-PCR, as described [33], using Tubulin and Ubiquitin as
internal controls for P. ussuriensis and tobacco, respectively. T2 homo-
zygous plants of tobacco and propagated plants of P. ussuriensis [40,41]
were used for the following experiments.
L. Zhao, et al. Plant Science 287 (2019) 110184

Fig. 3. Expression of PbrBAM3 in Pyrus betulaefolia in response to various treatments. Time-courses of expression levels in the shoot were analyzed by qPCR in
response to the indicated stresses. Error bars are S.E. Note the different durations of the stress treatments (Materials and Methods).

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L. Zhao, et al. Plant Science 287 (2019) 110184

Fig. 4. Subcellular localization of PbrBAM3-GFP. The fusion construct (35S:PbrBAM3-GFP, A–D) and a control vector with 35S:GFP (E–F) were transformed into
Arabidopsis protoplasts by PEG transformation. Images of fluorescence (A, B; see Materials and Methods) and transmitted-light (C and F), and merged images (D and
G). The horizontal scale bar = 20 μm.

Fig. 5. Morphological, structural and biochemical analyses of PbrBAM3 effect on starch metabolism in tobacco plants. (A, B) Starch iodine staining of tobacco leaves
from a wild-type plant (WT) and three lines (indicated by #s) of PbrBAM3-overexpressing plants, grown in the dark (A) and under light (B). Note that only in the WT
leaf did the illumination cause starch accumulation. (C) Ultra-structural partial view of the tobacco leaf cells of light-grown plants (WT and the three transgenic
lines). CW, cell wall; SG, starch grain. Note the relative scarcity of starch grains in the leaves of the trangenes. (D) β-Amylase activity, (E) maltose content and (F)
total soluble sugar content in the WT and the transgenic tobacco plants. ** and *** indicate significant differences between the WT and the three transgenic lines (at
P < 0.01 and P < 0.001, respectively).

2.6. Starch accumulation analysis and ultra-structure observation
In order to analyse starch accumulation, tobacco and P. ussuriensis
plants were kept at 25 °C in darkness for 12 h and then under constant
light for 12 h (P. ussuriensis) or 48 h (tobacco). Iodine staining was
performed to examine the in-situ starch accumulation in WT and
transgenic plants, collected from dark and light treatments as described
previously [26]. In addition, we observed the ultra-structure of the
leaves of WT and transgenic plants by an already described method
[43] with a minor modification. Briefly, the leaves were cut into ultra-
thin sections and collected onto 200-mesh PELCO® copper grids (TED
PELLA, Inc., Redding, CA, USA). After staining with uranyl acetate and
lead citrate, the air-dried grids were visualized under the HT7700
transmission electron microscope (Hitachi, Tokyo, Japan).

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L. Zhao, et al. Plant Science 287 (2019) 110184

Fig. 6. Morphological, structural and biochemical analyses of PbrBAM3 effect on starch metabolism in P. ussuriensis plants. (A, B) Starch iodine staining of P.
ussuriensis leaves from a wild-type plant (WT) and three (numbered) lines of PbrBAM3-overexpressing (OE) plants, grown in the dark (A) and under light (B). Note
that only in the WT leaf did the illumination cause starch accumulation. (C) Ultra-structural partial view of the P. ussuriensis leaf cells in light-grown plants (WT and
the three transgenic lines). CW, cell wall; SG, starch grain. Note the relative scarcity of starch grains in the leaves of the trangenes. (D) β-Amylase activity, (E) maltose
content and (F) total soluble sugar content in the WT and the transgenic P. ussuriensis plants. *, ** and *** indicate significant differences between the WT and the
three transgenic lines (at P < 0.05; P < 0.01 and P < 0.001, respectively).

2.7. Assessment of cold effects and cold tolerance hallmarks
For the chilling-tolerance assay, 15-day-old tobacco plants from
wild-type (WT) and three transgenic lines (Fig. S1F, hereafter desig-
nated as #3, #7 and #11) were chilled (at 0 °C) without cold accli-
mation, for 24 h followed by recovery at room temperature (25 °C) for
further growth. Survival rate was recorded after 10 d of recovery
growth. In addition, the 60-day-old WT and transgenic plants (#3, #7
and #11) were photographed before and after the chilling treatment
were taken at each time point. In parallel, the electrolyte leakage (EL),
and malondialdehyde (MDA) and proline content in the leaves of to-
bacco WT and transgenic (#3, #7 and #11) plants were measured as in
previous reports [40,42,44,45]. Cell death was assessed with trypan
blue staining of leaves in the WT and transgenic lines as described [46].
In another experiment, P. ussuriensis WT plants and three transgenic
lines (OE2, OE3 and OE19; Fig.S2F), were chilled (at 0 °C) for 8 h; at the
end of the treatment, their leaves were collected for measurement of EL,
MDA, and proline levels as well as for cell death assessment by trypan
blue staining. The chilling assay was repeated at least three times, with
three biological replicates for each sample Antioxidant enzymes (SOD,
POD and CAT) activity, H2O2 level, and anti-superoxide anion activity
in WT and transgenic plants, were determined by the relevant kits
(Nanjing Jiancheng Bioengineering Institute) according to the manu-
facturer’s instructions. Histochemical staining with 3,3′-diaminobenzi-
dine (DAB) and nitro-blue tetrazolium chloride (NBT) was performed to
detect the in situ accumulation of H2O2 and O2− according to [44] and
[45], respectively. The determination in the leaves of β-amylase ac-
tivity, maltose content and other soluble sugars was as described pre-
viously [46] and [47]. Antioxidant enzymes (SOD, POD and CAT) ac-
tivity, H2O2 level, and anti-superoxide anion activity in WT and
transgenic plants, were determined by the relevant kits (Nanjing Jian-
cheng Bioengineering Institute) according to the manufacturer’s in-
structions. Histochemical staining with 3,3′-diaminobenzidine (DAB)
and nitro-blue tetrazolium chloride (NBT) was performed to detect the
in situ accumulation of H2O2 and O2− according to [47] and [48], re-
spectively. The determination in the leaves of β-amylase activity, mal-
tose content and other soluble sugars was as described previously [49]
and [50].
2.8. Statistical analysis
Each treatment was repeated at least three times with consistent
results. Data are presented as means of three biological replicates ± SE
from one representative experiment. The data were analyzed by
Duncan’s multiple range tests in the ANOVA program of SPSS (IBM
SPSS 17), taking P < 0.05, P < 0.01, P < 0.001 as significantly dif-
ferent.
3. Results
3.1. PbrBAM3 cloning and sequence analysis
In an earlier work, we obtained and analyzed a cold-induced β-
amylase gene (Pbr014538.1) from a transcriptome of Pyrus betulaefolia
[51]. It contained a complete ORF of 1638 bp, flanked by an 80 bp 5’-
untranslated region (UTR) and a 168 bp 3’-UTR. The cDNA, designated
as PbrBAM3, contains an intact ORF and encodes a polypeptide of 541
amino acids with a calculated molecular mass of 60.88 kDa and an
isoelectric point of 6.58. A phylogenetic tree was constructed using a
total of 43 BAM protein sequences, 8 from P. trifoliata, 9 from Arabi-
dopsis, 12 from apple, 6 from rice and 9 from poplar. The BAMs from
different plant species are classified into four major groups, I to IV.
PbrBAM3 was grouped in the same cluster as BAM3 of Arabidopsis
thaliana (Fig. 1). Multiple alignments between PbrBAM3 and nine other
BAM proteins revealed a highly conserved glucosyl hydrolase domain
(Fig. 2). In the domain, there are 18 substrate- (amylopectin-) binding

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L. Zhao, et al. Plant Science 287 (2019) 110184

Fig. 7. The effect of PbrBAM3 overexpression on cold tolerance of tobacco plants. (A, B and C), Phenotypes of 15-d-old WT and transgenic seedlings (lines #3, #7 and
#11) before (A) and after a chilling treatment (12 h at 0 °C) (B), followed by recovery at room temperature for 10 d (C). (D), Trypan blue staining of the seedlings of B.
(E) Survival rates of the transgenic and wild-type plants assessed after recovery from the chilling treatment. (F), Phenotypes of 60-d-old WT and transgenic plants
before and after the chilling treatment. (G, H and I), biochemical hallmarks of cold stress effects measured in WT and transgenic plants; electrolyte leakage (G),
malondialdehyde (MDA) (H) and proline content (I). ** and *** indicate that the values measured in the three transgenic lines were significantly different from those
in WT (at P < 0.01 and P < 0.001, respectively.).

residues (small black and blue arrow heads) and 2 catalytic residues
(large blue arrow heads).
3.2. Time-course expression of PbrBAM3 in Pyrus betulaefolia leaves in
response to abiotic stresses
Upon exposure to cold, the transcript level of PbrBAM3 was up-
regulated gradually within 6 h, increased progressively until reaching
the highest level at 72 h (greater than 25-fold of the initial level), and
declined to half this value at 144 h (Fig. 3A). Upon exposure to dehy-
dration, the transcript level of PbrBAM3 exhibited steady up-regulation
during a 6 h period, with > 12-fold increase relative to the onset of
treatment (Fig. 3B). ABA application resulted in a slow up-regulation of
PbrBAM3 expression level, to less than 5-fold the initial value at 6 h,
peaking at over 25-fold the initial value at 24 h, followed by a decrease
to 15-fold of the initial level at 72 h (Fig. 3C). In contrast to these,
PbrBAM3 expression was repressed by maltose treatment to below 20%
of initial value at 6 h and then it fluctuated remaining below 80% of
initial value till 72 h (Fig. 3D).
3.3. Subcellular localization of PbrBAM3
Confocal microscopy of Arabidopsis protoplast cells showed a per-
fect overlap between the green fluorescence of the PbrBAM3-fused GFP
and the red auto-fluorescence of chloroplasts (Fig. 4A–D), indicating
that PbrBAM3 was localized in the chloroplast. In contrast, the GFP-
alone green fluorescence was found in the entire cytoplasm region
(Fig. 4E–G).
3.4. Analysis of amylase activity, starch and soluble sugars contents in the
transgenic plants
The strong induction by cold of PbrBAM3 expression in Pyrus betu-
laefolia (Fig. 3) suggested that a critical role for PbrBAM3 in the reg-
ulation of cold response. To test this hypothesis, we analyzed three
independent overexpression lines of tobacco with the highest transcript
levels of PbrBAM3 (lines #3, #7 and #11; Fig. S1), along with the
corresponding untransformed wild type (WT).
Neither WT nor transgenic lines of tobacco (60-day-old) grown in
the dark for 12 h accumulated any starch, as revealed by the absence of
iodine staining (Fig. 5A), whereas only the leaves of transgenic plants
showed starch absence under constant light for 48 h in contrast to the
WT leaf (Fig. 5B). To further confirm this result, starch accumulation of
all tested plants after light exposure was examined by transmission
electron microscope. 60-day-old leaves were fixed in 2.5% (v/v) glu-
taraldehyde in 50 mM sodium cacodylate buffer adjusted to pH 7.3 for
16 h at 25 °C. Ultra-thin gold transverse sections of 90 nm were cut and
collected onto 200 mesh copper grids that were pre-filmed with 4% (v/
v) pyroxylin in amyl acetate and carbon coated. The sections were
stained with 2% (w/v) uranyl acetate for 1 h and 1% (w/v) lead citrate

7
L. Zhao, et al.
Fig. 8. Cold tolerance assay of transgenic P. ussuriensis plants overexpressing PbrBAM3. (A, B) Plant phenotypes of WT and transgenic lines (OE2, OE3 and OE19)
before (A) and after (B) a chilling stress (Materials and methods). (C, D–F) Cell death (C), proline content (D), electrolyte leakage (E) and MDA (E) determined in WT
and transgenic lines after the chilling treatment (Materials and methods). ** and *** indicate values that differ significantly between the WT and the three transgenic
lines (at P < 0.01 and P < 0.001, respectively).
for 1 min. The grids with washed in water and dried in air were viewed
in the HT7700 transmission electron microscope (Hitachi, Tokyo,
Japan).
Consistent with the previous result, transgenic lines exhibited fewer
starch grains in comparison with the WT (Fig. 5C). As can be seen in
Fig. 5D, the BAM activity in three transgenic lines was 60%–160%
higher than in the WT. Consistent with the higher BAM activity, the
leaves of PbrBAM3-overexpressing tobacco plants contained more
maltose (Fig. 5E) and more total soluble sugars (Fig. 5F) in comparison
with WT.
PbrBAM3 was also transferred into P. ussuriensis to further char-
acterize its function (Fig.S2). Three transgenic P. ussuriensis lines (OE2,
OE3 and OE19; 21-day-old) exhibiting a higher abundance of PbrBAM3
mRNA, along with untransformed plants (WT), were subjected to dark
and light treatments (Materials and methods). The results resembled
those of tobacco: no starch accumulation was seen in the leaves of all
the tested plants grown in the dark (Fig. 6A). However, much deeper
staining was visualized in the WT after exposure to the light as com-
pared with the transgenic plants (Fig. 6B).The SGs (starch granules)
were very abundant in the WT chloroplasts, whereas in the chloroplasts
of the three transgenic plants their numbers were dramatically reduced
(Fig. 6C), again indicating that PbrBAM3 overexpression led to reduced
starch accumulation. BAM activity in the three transgenic lines was
significantly higher than that in the WT (Fig. 6D). In parallel, the
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Plant Science 287 (2019) 110184
transgenic lines stored lower levels of maltose (Fig. 6E) and total so-
luble sugars (Fig. 6F) as compared to WT.
3.5. PbrBAM3 overexpression increased the cold tolerance of the transgenic
plants
Under normal growth conditions (Materials and methods), there
was no apparent difference in plant morphology between the transgenic
plants and the wild type (Fig. 7A). When 15-d-old seedlings were sub-
jected to a freezing temperature (0 °C) for 12 h, the WT plants suffered
more serious cold damage compared with the transgenic plants
(Fig. 7B), and all of them were dead by the end of the recovery period,
whereas most of three transgenic plants recovered and grew well
(Fig. 7C). The same contrasting outcome in another experiment was
confirmed by trypan blue staining directly after the chilling treatment
(strikingly dark in the dead tissues and almost absent in the live seed-
lings; Fig. 7D). At the end of the recovery period, the transgenic seed-
lings exhibited significantly higher survival rates (3# (50%), #7
(41.6%) and #11 (66.7%)) plants than that of the WT seedlings (0%;
Fig. 7E). An enhanced cold tolerance relative to the WT was also seen in
60-d-old transgenic tobacco plants (Fig. 7F).
After the chilling stress, EL and MDA levels in the transgenic lines
were significantly lower relative to the WT after chilling stress (Fig. 7G
and H). Proline (Pro) is considered as “a compatible solute” which helps
L. Zhao, et al. Plant Science 287 (2019) 110184

Fig. 9. Oxidative stress status in WT and transgenic plants after a chilling treatment. Representative images of leaves stained in situ by the H2O2 and O2− respective
probes DAB and NBT in: (A), 15-d-old seedlings of tobacco WT and transgenics (lines #3, #7 and #11), (B), 60-d-old tobacco WT and transgenics (lines #3, #7 and
#11), (C), P. ussuriensis WT and transgenics (lines OE2, OE3 and OE19). (D, E), Levels of H2O2 (D) and O2− (E) quantified (Materials and methods) in chilled tobacco
seedlings; (F, G), Levels of H2O2 (F) and O2− (G) quantified in chilled P. ussuriensis. ** and *** indicate that the values measured in the three transgenic lines in
tobacco and P. ussuriensis were significantly different from those in their respective WT (at P < 0.01 and P < 0.001, respectively).

plants to overcome abiotic stress. Therefore, Pro contents in the tested
plants were measured. After the chilling stress, the Pro content on three
transgenic lines was significantly higher than in the WT (Fig. 7I).
The growth performance of WT pear and pear plants overexpressing
PbrBAM3 did not differ before the chilling treatment (Fig. 8A). How-
ever, a chilling treatment (Materials and methods) caused more pro-
nounced leaf drooping in the WT than in the transgenic lines (Fig. 8B).
Trypan blue staining was considerably more intense in the WT leaves
relative to the transgenic lines, attesting to more sever chilling stress
due cell damage in the WT (Fig. 8C). After the chilling treatment, the
Pro content of the transgenic lines was significantly higher than that of
the WT (Fig. 8D) and the EL of OE2 (36.5%), OE3 (31.4%) and OE19
(43.5%) plants was remarkably lower than that of the WT (87.7%)
(Fig. 8E). In addition, the transgenic lines displayed significantly lower
MDA content compared with the WT (Fig. 8F).
3.6. Analysis of ROS accumulation by histochemical staining
Under chilling treatments, the leaves of the WT plants accumulated
H2O2 and O2− to a larger extent than the leaves of the transgenic
plants, as revealed by in-situ histochemistry using the respective probes,
DAB and NBT (Materials and methods) showing more intense staining
in the leaves of WT plants (Fig. 9A–C). This result was consistent across
all the types of the chilling assays: in the 15-day-old seedlings chilled
for 12 h (Fig. 9A), in the 60-d-old tobacco plants chilled for 24 h
(Fig. 9B), and in the chilled pear leaves (Fig. 9C). In confirmation of the
eye-balled in-situ the histochemical staining results, the quantitation of
H2O2 and O2− (Materials and methods), also showed that following a
chilling treatment, the levels of these ROS were significantly lower in
the transgenic plants, than in WT, both in the tobacco seedlings (Fig. 9D
and E) and in P. ussuriensis (Fig. 9F and G).
3.7. Overexpression of PbrBAM3 increased antioxidant enzyme activities in
transgenic plants
Since, after the chilling treatment, the transgenic lines contained
lower ROS levels relative to the WT, we expected there higher activities
of the three significant antioxidant enzymes (SOD, CAT, and POD).
Indeed, activities of all three enzymes were significantly higher in the
transgenic than in the WT plants, both in the tobacco (Fig. 10A–C) and
in P. ussuriensis plants (Fig. 10D and E).

9
L. Zhao, et al.
Fig. 10. Analysis of enzyme activities in chilled tobacco seedlings and in P. ussuriensis (Materials and methods). (A, B and C), Activity of CAT (A), POD (B), and SOD
(C) in tobacco WT and transgenic lines (#3, #7 and #11). (D, E and F), Activity of CAT (D), POD (E), and SOD (F) in P. ussuriensis WT and transgenic lines (OE2, OE3
and OE19). ** and *** indicate that the values measured in the three transgenic lines of tobacco and P. ussuriensis were significantly different from those of their
respective WTs (at P < 0.01 and P < 0.001, respectively).
3.8. Overexpression PbrBAM3 promoted the accumulation of soluble sugars
To gain further insight into the physiological mechanism underlying
the enhanced cold tolerance of the PbrBAM3 overexpressors, the β-
amylase activity and soluble sugar contents were determined in trans-
genic lines and WT plants. As shown in Fig. 11A, the β-amylase activ-
ities in the three transgenic tobacco lines were much higher than those
of WT after cold treatment. Maltose levels in the three transgenic to-
bacco lines were significantly higher than that of WT (Fig. 11B) ranging
from 1.6 to 1.8 folds. Consistent with the accumulation of maltose,
overexressing PbrBAM3 induced accumulation of soluble sugar levels
under chilling treatment (Fig. 11C). Similarly, after chilling stress the β-
amylase activity in the three P. ussuriensis transgenic lines (OE2, OE3
and OE19) were significantly higher than that of the WT (Fig. 11D).
Meanwhile, the maltose and soluble sugar levels were higher in the
transgenic lines than WT (Fig. 11E and F). These results suggest that
overexpression PbrBAM3 could enhance the accumulation of compa-
tibles solutes such as maltose and soluble sugar in transgenic plant.
4. Discussion
Over a long evolutionary period, plants have evolved a range of
complex mechanisms to tolerate harsh environmental stresses [52]. For
example, sugar metabolism and carbon partitioning are two effective
ways to sustain the balance required for growth and energy supply in
cold acclimation [53]. Because of carbohydrates produced mainly by
10
Plant Science 287 (2019) 110184
starch degradation, the genes encoding enzymes leading to starch de-
gradation are crucial for sugar metabolism [54]. Previous studies sug-
gested that in the beginning the SGs is mainly catalysed by BAM en-
zymes [55], therefore, BAM genes might hold a great potential for
modulating sugar homesostasis under abiotic stresses.
According to their relationship and gene structure, PbrBAMs can be
classified into Group IIII category, suggesting that BAMs are evolutio-
narily conserved among different plants, the reasonable fact plants
share commonality on sugar metabolism for providing an array of su-
gars required for growth development and stress response. It is note-
worthy that not all BAMs are responsive for starch degradation. In
Arabidopsis, only AtBAM3 deletion caused significantly diminished
starch degradation whereas down-regulation of the other family did not
[56,57]. Multiple alignments revealed that PbrBAM3 shared the same
set of amino acids related to SG binding and hydrolytic catalysis in the
core glucosyl hydrolase domain with AtBAM3, indicating that PbrBAM3
is a putative BAM3 homologue of Pyrus betulaefolia. Therefore, we hy-
pothesized that PbrBAM3 functions in Pyrus betulaefolia as does AtBAM3
in Arabidopsis, i.e., degrades starch in SGs. Indeed, PbrBAM3-over-
expressing lines tobacco and P. ussuriensis plants exhibited a higher
BAM activity, a reduced accumulation of starch and elevated level of
maltose. These results confirmed the crucial role of PbrBAM3 in starch
degradation, allowing it as a desirable target of interest for modulation
of sugar levels.
Given that the greatest induction of PbrBAM3 transcript level by
cold treatment, indicating that PbrBAM3 plays an important role in
L. Zhao, et al.
Fig. 11. Analysis of β-amylase activity and soluble sugars in transgenic tobacco and P. ussuriensis after chilling treatment. Activity of β-amylase activity (A), maltose
content (B), and soluble sugar content (C) in tobacco WT and transgenic lines (#3, #7 and #11) after chilling treatment. (D, E and F) Activity of β-amylase (D),
maltose content (E), and soluble sugar content (F) in P. ussuriensis WT and transgenic lines (OE2, OE3 and OE19) after chilling stress.** and *** indicate that the
values measured in the three transgenic lines of tobacco and P. ussuriensis were significantly different from those of their respective WTs (at P < 0.01 and
P < 0.001, respectively).
response to cold tolerance. To this end, transgenic tobacco (pear) plants
were produced via Agrobacterium-mediated transformation of PbrBAM3
under the control of CaMV 35S promoter. The three selected transgenic
lines (OE2, OE3 and OE19 lines of pear, and #3, #7 and #11 lines of
tobacco) exhibited better phenotypic morphology, concomitant with
higher levels of soluble sugars than wild type under chilling stress,
suggesting that the significance of sugars derived from PbrBAM3
-mediated starch hydrolysis in the stress tolerance.
Cold-stress-inflicted damage – depending on its severity and dura-
tion – consists of membrane disruption, metabolism dysfunction, loss of
turgor or even cell death [57]. Since cold stress is frequently mediated
by osmotic stress, their damaging effects may countered by accumula-
tion of osmolytes, such as soluble sugars, restoring osmotic balance
[28,58,59,61–65]. Sugar metabolism is a complex enzymatic process, in
which several of soluble sugars may be involved, such as sucrose, glu-
cose, fructose etc [60]. The higher soluble sugar levels (especially for
maltose) in the cold- stressed PbrBAM3-overexpressors in our study
suggested these transgenic plants are more capable of osmotic adjust-
ment than the WT plants. In parallel, the PbrBAM3 overexpressors (both
tobacco and P. ussuriensis) had lower EL and showed better re-growth
during the recovery period as compared with WT under cold stress
(Figs. 7 and 8), which correlated well with their increased capacity for
osmotic adjustments due to elevated soluble sugars. Therefore, over-
expressing-PbrBAM3 plants produced higher soluble sugars functioning
as osmolytes or compatible solutes, which is one of the adapted me-
chanisms contributing to the enhanced cold tolerance in transgenic
plants.
Soluble sugars have been shown to act as antioxidants ameliorating
ROS-derived oxidative stresses [19,20,63]. Here, we found that ROS
accumulation was obviously reduced in PbrBAM3 -overexpressing lines
compared with wild-type plants under chilling stress, consistent with
11
Plant Science 287 (2019) 110184
the higher level of soluble sugars, implying that the production of ROS
in these lines under cold stress may be scavenged in a more powerful
manner. Although ROS could be generated in different cellular com-
partments, chloroplast is the major site of ROS generation [66–68].
Interestingly, PbrBAM3 is localized in the chloroplast. Therefore, we
speculate that the PbrBAM3-OE overexpressors contained more maltose
and its metabolites in the chloroplast, which work alone or in concert
with other antioxidants scavenging the ROS produced in this apparatus.
Moreover, the ROS produced in the cytosol can also be scavenged by
the soluble sugars [18]. Thus, the ROS spread from the production site
to other places in cell may be significantly decreased, resulting in lower
ROS accumulation, which is consistent with the dramatic reduction of
ROS level in these lines. Taken together, our results indicate that the
promotion of ROS scavenging due to greater accumulation of soluble
sugars is a feasible strategy for increasing cold tolerance of the trans-
genic plants.
5. Summary
In conclusion, our data demonstrate that PbrBAM3 is a stress re-
sponsive β-amylase and plays a positive role in cold, oxidative stress
tolerance by participating in starch degradation and the mobilization of
soluble sugar capable of cell osmo-protection and ROS-counteraction.
Additional work is needed to decipher the details of the links between
soluble sugars and cold responses, to allow deeper insight into the
molecular mechanisms underlying PbrBAM3 function in cold tolerance.
Author contributions
HX designed and conceived the research. ZL, XC, HS, DH, GJ per-
formed the experiments and YT wrote and revised the manuscript with
L. Zhao, et al.
help of HX. HX, ZS, TS, WJ and QK contributed to proofreading and
critical review of this manuscript. All the authors reviewed the results
and approved the final version of the manuscript.
Acknowledgments
We sincerely appreciate Professor Nava Moran from Hebrew
University of Jerusalem for giving the constructive comments and
language editing to this work. This work has been supported by the
Excellent Youth Natural Science Foundation of Jiangsu Province (Grant
No. BK20170086to HX), the partly supported by the open funds of the
State Key Laboratory of Crop Genetics and Germplasm Enhancement
(Grant No. ZW201908 to YT), the National Key Research and
Development Program of China (Grant No. 2018YFD1000300 to HX),
the National Science Foundation of China (Grant No. 31872070 to HX),
the Jiangsu Agriculture Science and Technology Innovation Fund
(Grant No. CX(18)3065 to HX), the Fundamental Research Funds for
the Central Universities (Grant No. KYZ201607 to HX), and also sup-
ported in part by the National Natural Science Foundation of China
(Grant No. 31800584 to YT), Anhui Provincial Natural Science
Foundation (Grant No. 1808085QC75 to YT), Anhui Provincial
Postdoctoral Science Foundation (Grant No. 2017B158 to YT), and
Science Foundation for Anhui Province (Grant No. KJ2018A0129 to
YT).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.plantsci.2019.110184.
References
[1] W. Wang, B. Vinocur, A. Altman, Plant responses to drought, salinity and extreme
temperatures: towards genetic engineering for stress tolerance, Planta 218 (2003)
1–14.
[2] F. Kaplan, J. Kopka, D.Y. Sung, W. Zhao, M. Popp, R. Porat, C.L. Guy, Transcript and
metabolite profiling during cold acclimation of Arabidopsis reveals an intricate
relationship of cold‐regulated gene expression with modifications in metabolite
content, Plant J. 50 (2007) 967–981.
[3] J. Zhu, C. Dong, J. Zhu, Interplay between cold-responsive gene regulation, meta-
bolism and RNA processing during plant cold acclimation, Curr. Opin. Plant Biol. 10
(2007) 290–295.
[4] K. Nakashima, Y. Ito, K. Yamaguchi-Shinozaki, Transcriptional regulatory networks
in response to abiotic stresses in Arabidopsis and grasses, Plant Physiol. 149 (2009)
88–95.
[5] F. Qin, K. Shinozaki, K. Yamaguchi-Shinozaki, Achievements and challenges in
understanding plant abiotic stress responses and tolerance, Plant Cell Physiol. 52
(2011) 1569–1582.
[6] C. Jin, K. Li, X. Xu, H. Zhang, H. Chen, Y. Chen, J. Hao, Y. Wang, X. Huang,
S. Zhang, A novel NAC transcription factor, PbeNAC1, of Pyrus betulifolia confers
cold and drought tolerance via interacting with PbeDREBs and activating the ex-
pression of stress-responsive genes, Front. Plant Sci. 8 (2017) 1049.
[7] K. Nakashima, K. Yamaguchi-Shinozaki, Regulons involved in osmotic stress‐re-
sponsive and cold stress-responsive gene expression in plants, Physiol. Plantarum.
126 (2006) 62–71.
[8] P. Achard, F. Gong, S. Cheminant, M. Alioua, P. Hedden, P. Genschik, The cold-
inducible CBF1 factor-dependent signaling pathway modulates the accumulation of
the growth-repressing DELLA proteins via its effect on gibberellin metabolism, Plant
Cell 20 (2008) 2117–2129.
[9] J. Liu, T. Peng, W. Dai, Critical cis-acting elements and interacting transcription
factors: key players associated with abiotic stress responses in plants, Plant Mol.
Biol. Rep. 32 (2014) 303–317.
[10] J. Zhu, Salt and drought stress signal transduction in plants, Annu. Rev. Plant Biol.
53 (2002) 247–273.
[11] K. Yamaguchi-Shinozaki, K. Shinozaki, Organization of cis-acting regulatory ele-
ments in osmotic-and cold-stress-responsive promoters, Trends Plant Sci. 10 (2005)
88–94.
[12] K. Shinozaki, K. Yamaguchi-Shinozaki, Gene networks involved in drought stress
response and tolerance, J. Exp. Bot. 58 (2007) 221–227.
[13] R. Mittler, Oxidative stress, antioxidants and stress tolerance, Trends Plant Sci. 7
(2002) 405–410.
[14] G. Miller, N. Suzuki, S. Ciftci Yilmaz, R. Mittler, Reactive oxygen species home-
ostasis and signalling during drought and salinity stresses, Plant Cell Environ. 33
(2010) 453–467.
[15] C.O. Silva-Ortega, A.E. Ochoa-Alfaro, J.A. Reyes-Agüero, G.A. Aguado-Santacruz,
J.F. Jiménez-Bremont, Salt stress increases the expression of p5cs gene and induces
12
Plant Science 287 (2019) 110184
proline accumulation in cactus pear, Plant Physiol. Biochem. 46 (2008) 82–92.
[16] A. Theocharis, C. Clément, E.A. Barka, Physiological and molecular changes in
plants grown at low temperatures, Planta 235 (2012) 1091–1105.
[17] J.V. Cabello, A.F. Lodeyro, M.D. Zurbriggen, Novel perspectives for the engineering
of abiotic stress tolerance in plants, Curr. Opin. Plant Biol. 26 (2014) 62–70.
[18] I. Couée, C. Sulmon, G. Gouesbet, A. El Amrani, Involvement of soluble sugars in
reactive oxygen species balance and responses to oxidative stress in plants, J. Exp.
Bot. 57 (2006) 449–459.
[19] M.R. Bolouri Moghaddam, K. Le Roy, L. Xiang, F. Rolland, W. Van den Ende, Sugar
signalling and antioxidant network connections in plant cells, FEBS J. 277 (2010)
2022–2037.
[20] E. Keunen, D. Peshev, J. Vangronsveld, W. Van Den Ende, A. Cuypers, Plant sugars
are crucial players in the oxidative challenge during abiotic stress: extending the
traditional concept, Plant Cell Environ. 36 (2013) 1242–1255.
[21] K. Maruyama, Y. Sakuma, M. Kasuga, Y. Ito, M. Seki, H. Goda, Y. Shimada,
S. Yoshida, K. Shinozaki, K. Yamaguchi Shinozaki, Identification of cold-inducible
downstream genes of the Arabidopsis DREB1A/CBF3 transcriptional factor using two
microarray systems, Plant J. 38 (2004) 982–993.
[22] H. Reinhold, S. Soyk, K. Šimková, C. Hostettler, J. Marafino, S. Mainiero,
C.K. Vaughan, J.D. Monroe, S.C. Zeeman, β-Amylase–like proteins function as
transcription factors in Arabidopsis, controlling shoot growth and development,
Plant Cell (2011) 110–81950.
[23] Y. Lu, T.D. Sharkey, The importance of maltose in transitory starch breakdown,
Plant Cell Environ. 29 (2006) 353–366.
[24] T. Niittylä, G. Messerli, M. Trevisan, J. Chen, A.M. Smith, S.C. Zeeman, A previously
unknown maltose transporter essential for starch degradation in leaves, Science 303
(2004) 87–89.
[25] S.C. Zeeman, D. Thorneycroft, N. Schupp, A. Chapple, M. Weck, H. Dunstan,
P. Haldimann, N. Bechtold, A.M. Smith, S.M. Smith, Plastidial α-glucan phos-
phorylase is not required for starch degradation in Arabidopsis leaves but has a role
in the tolerance of abiotic stress, Plant Physiol. 135 (2004) 849–858.
[26] D.C. Fulton, M. Stettler, T. Mettler, C.K. Vaughan, J. Li, P. Francisco, M. Gil,
H. Reinhold, S. Eicke, G. Messerli, β-AMYLASE4, a noncatalytic protein required for
starch breakdown, acts upstream of three active β-amylases in Arabidopsis chlor-
oplasts, Plant Cell 20 (2008) 1040–1058.
[27] T.H. Nielsen, U. Deiting, M. Stitt, A β-amylase in potato tubers is induced by storage
at low temperature, Plant Physiol. 113 (1997) 503–510.
[28] F. Kaplan, C.L. Guy, β-Amylase induction and the protective role of maltose during
temperature shock, Plant Physiol. 135 (2004) 1674–1684.
[29] A. Scheidig, A. Fröhlich, S. Schulze, J.R. Lloyd, J. Kossmann, Downregulation of a
chloroplast-targeted β-amylase leads to a starch-excess phenotype in leaves, Plant J.
30 (2002) 581–591.
[30] M. Zanella, G.L. Borghi, C. Pirone, M. Thalmann, D. Pazmino, A. Costa, D. Santelia,
P. Trost, F. Sparla, β-amylase 1 (BAM1) degrades transitory starch to sustain proline
biosynthesis during drought stress, J. Exp. Bot. 67 (2016) 1819–1826.
[31] J. Zhang, X. Li, H. Lin, K. Chong, Crop Improvement through temperature resi-
lience, Annu. Rev. Plant Biol. 70 (2019) 753–780.
[32] L.H. Xian, P.P. Sun, S.S. Hu, J. Wu, J.H. Liu, Molecular cloning and characterization
of CrNCED1, a gene encoding 9-cis-epoxycarotenoid dioxygenase in Citrus reshni,
with functions in tolerance to multiple abiotic stresses, Planta 239 (2014) 61–77.
[33] X.Q. Gong, J.Y. Zhang, J.B. Hu, W. Wang, H. Wu, Q.H. Zhang, J.H. Liu, FcWRKY70,
a WRKY protein of Fortunella crassifolia, functions in drought tolerance and
modulates putrescine synthesis by regulating arginine decarboxylase gene, Plant
Cell Environ. 38 (2015) 2248–2262.
[34] K. Li, X. Xu, X. Huang, Identification of differentially expressed genes related to
dehydration resistance in a highly drought-tolerant pear, Pyrus betulaefolia, as
through RNA-Seq, PLoS One 11 (2016) e149352.
[35] T. Yang, S. Zhang, Y. Hu, F. Wu, Q. Hu, G. Chen, J. Cai, T. Wu, N. Moran, L. Yu, The
role of OsHAK5 in potassium acquisition and transport from roots to shoots in rice
at low potassium supply levels, Plant Physiol. 166 (2014) 945–959.
[36] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using real-
time quantitative PCR and the 2-ΔΔCT method, Methods 25 (2001) 402–408.
[37] C. Jin, X. Huang, K. Li, H. Yin, L. Li, Z. Yao, S. Zhang, Overexpression of a bHLH1
transcription factor of P. ussuriensis confers enhanced cold tolerance and increases
expression of stress-responsive genes, Front. Plant Sci. 7 (2016) 441.
[38] A. Dereeper, V. Guignon, G. Blanc, S. Audic, S. Buffet, F. Chevenet, J. Dufayard,
S. Guindon, V. Lefort, M. Lescot, Phylogeny. fr: robust phylogenetic analysis for the
non-specialist, Nucleic Acids Res. 36 (2008) 465–469.
[39] Y. Zhang, J. Su, S. Duan, Y. Ao, J. Dai, J. Liu, P. Wang, Y. Li, B. Liu, D. Feng, A
highly efficient rice green tissue protoplast system for transient gene expression and
studying light/chloroplast-related processes, Plant Methods 7 (2011) 30.
[40] X. Huang, J. Liu, X. Chen, Overexpression of PtrABF gene, a bZIP transcription
factor isolated from Poncirus trifoliata, enhances dehydration and drought tolerance
in tobacco via scavenging ROS and modulating expression of stress-responsive
genes, BMC Plant Biol. 10 (2010) 230.
[41] Y.J. Yang, D.F. Wang, C.S. Wang, X.H. Wang, J.N. Li, R. Wang, Construction of high
efficiency regeneration and transformation systems of P. ussuriensis Maxim, Plant
Cell Tissue Organ Cult. 131 (2017) 139–150.
[42] K. Li, C. Xing, Z. Yao, X. Huang, PbrMYB21, a novel MYB protein of Pyrus betu-
laefolia, functions in drought tolerance and modulates polyamine levels by reg-
ulating arginine decarboxylase gene, Plant Biotechnol. J. 15 (2017) 1186–1203.
[43] P. Derbyshire, K. Findlay, M.C. McCann, K. Roberts, Cell elongation in Arabidopsis
hypocotyls involves dynamic changes in cell wall thickness, J. Exp. Bot. 58 (2007)
2079–2089.
[44] L. Zhao, F. Liu, W. Xu, C. Di, S. Zhou, Y. Xue, J. Yu, Z. Su, Increased expression of
OsSPX1 enhances cold/subfreezing tolerance in tobacco and Arabidopsis thaliana,
L. Zhao, et al.
Plant Biotechnol. J. 7 (2009) 550–561.
[45] X. Fu, E.U. Khan, S. Hu, Q. Fan, J. Liu, Overexpression of the betaine aldehyde
trifoliate orange (Poncirus trifoliata L. Raf.), Environ. Exp. Bot. 74 (2011) 106–113.
[46] M. Pogány, J. Koehl, I. Heiser, E.F. Elstner, B. Barna, Juvenility of tobacco induced
by cytokinin gene introduction decreases susceptibility to Tobacco necrosis virus
and confers tolerance to oxidative stress, Physiol. Mol. Biol. Plants 65 (2004) 39–47.
[47] M. Orozco-Cardenas, C.A. Ryan, Hydrogen peroxide is generated systemically in
plant leaves by wounding and system in via the octadecanoid pathway, Proc. Acad.
Sci. India Sect. B 96 (1999) 6553–6557.
[48] H.L. Wong, R. Pinontoan, K. Hayashi, R. Tabata, T. Yaeno, K. Hasegawa, C. Kojima,
H. Yoshioka, K. Iba, T. Kawasaki, Regulation of rice NADPH oxidase by binding of
Rac GTPase to its N-terminal extension, Plant Cell 19 (2007) 4022–4034.
[49] R.J. Laby, D. Kim, S.I. Gibson, The ram1 mutant of Arabidopsis exhibits severely
decreased β-amylase activity, Plant Physiol. 127 (2001) 1798–1807.
[50] P.S. Chow, S.M. Landhäusser, A method for routine measurements of total sugar and
starch content in woody plant tissues, Tree Physiol. 24 (2004) 1129–1136.
[51] T. Yang, X. Huang, Deep sequencing-based characterization of transcriptome of P.
ussuriensis in response to cold stress, Gene 661 (2018) 109–118.
[52] K. Shinozaki, K. Yamaguchi-Shinozaki, Molecular responses to dehydration and low
temperature: differences and cross-talk between two stress signaling pathways,
Curr. Opin. Plant Biol. 3 (2000) 217–223.
[53] V. Hurry, N. Druart, A. Cavaco, P. Gardeström, Å. Strand, Photosynthesis at low
temperatures A case study with Arabidopsis, Plant Cold Hardiness (2002) 161–179.
[54] A.M. Smith, S.C. Zeeman, S.M. Smith, Starch degradation, Annu. Rev. Plant Biol. 56
(2005) 73–98.
[55] S.C. Zeeman, S.M. Smith, A.M. Smith, The diurnal metabolism of leaf starch,
Biochem. J. 401 (2007) 13–28.
[56] F. Kaplan, C.L. Guy, RNA interference of Arabidopsis beta-amylase8 prevents
maltose accumulation upon cold shock and increases sensitivity of PSII photo-
chemical efficiency to freezing stress, Plant J. 44 (2005) 730–743.
13
Plant Science 287 (2019) 110184
[57] J. Dreier, A. Bermpohl, R. Eichenlaub, Southern hybridization and PCR for specific
detection of phytopathogenic Clavibacter michiganensis subsp. michiganensis,
Phytopathology 85 (1995) 462–468.
[58] R. Datta, M.T. Selvi, N. Seetharama, R. Sharma, Stress-mediated enhancement of β-
amylase activity in pearl millet and maize leaves is dependent on light, J. Plant
Physiol. 154 (1999) 657–664.
[59] T. Peng, X.F. Zhu, Q.J. Fan, P.P. Sun, J.H. Liu, Identification and characterization of
low temperature stress responsive genes in Poncirus trifoliata by suppression sub-
tractive hybridization, Gene 492 (2012) 220–228.
[60] Z. Chen, T.A. Cuin, M. Zhou, A. Twomey, B.P. Naidu, S. Shabala, Compatible solute
accumulation and stress-mitigating effects in barley genotypes contrasting in their
salt tolerance, J. Exp. Bot. 58 (2007) 4245–4255.
[61] H.J. Bohnert, R.G. Jensen, Strategies for engineering water-stress tolerance in
plants, Trends Biotechnol. 14 (1996) 89–97.
[62] J. Krasensky, C. Jonak, Drought, salt, and temperature stress-induced metabolic
rearrangements and regulatory networks, J. Exp. Bot. 63 (2012) 1593–1608.
[63] L. Xiong, J.K. Zhu, Molecular and genetic aspects of plant responses to osmotic
stress, Plant Cell Environ. 25 (2002) 131–139.
[64] H. Chen, J. Jiang, Osmotic adjustment and plant adaptation to environmental
changes related to drought and salinity, Environ. Rev. 18 (2010) 309–319.
[65] R. Valluru, W. Van den Ende, Myo-inositol and beyond–emerging networks under
stress, Plant Sci. 181 (2011) 387–400.
[66] K. Asada, Production and scavenging of reactive oxygen species in chloroplasts and
their functions, Plant Physiol. 141 (2006) 391–396.
[67] D. An, J. Yang, P. Zhang, Transcriptome profiling of low temperature-treated cas-
sava apical shoots showed dynamic responses of tropical plant to cold stress, BMC
Genomics 13 (2012) 64.
[68] S.E. Weise, A.P. Weber, T.D. Sharkey, Maltose is the major form of carbon exported
from the chloroplast at night, Planta 218 (2004) 474–482.