Plant Physiology and Biochemistry 151 (2020) 188–196

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Plant Physiology and Biochemistry

journal homepage: www.elsevier.com/locate/plaphy

Research article

Photochemistry and proteomics of ginger (Zingiber officinale Roscoe) under T

drought and shading
Yao Lva, Yanyan Lia, Xiaohui Liub, Kun Xua,∗
a
College of Horticulture Science and Engineering, Shandong Agricultural University, Taian, 271018, China
b
School of Environment, Tsinghua University, Beijing, 100084, China

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

Keywords: Drought has become an increasingly serious ecological problem that limits crop production. However, little is
Drought known about the response of ginger (Zingiber officinale Roscoe) to drought and shading, especially with respect to
Ginger photosynthetic electron transport. Here, differential proteomics was used to study the response of ginger to four
iTRAQ experimental treatments: control, drought, 50% shading, and the combination of 50% shading and drought.
Shading
Proteomic analysis suggested that ginger increased cyclic electron flow under drought stress by enhancing the
expression of proteins related to photosystem I and cytochrome b6f. Shading significantly increased the ex-
pression of proteins related to the light harvesting complex, even under drought stress. In addition, shading
increased the expression of proteins related to the oxygen evolution complex, plastocyanin, and
ferredoxin–NADP+ reductase (FNR), thereby enhancing the efficiency of photosynthetic electron utilization. The
shading and drought combination treatment appeared to enhance ginger's drought tolerance by reducing the
expression of FNR and enhancing cyclic electron flow. Photosynthetic and fluorescence parameters showed that
drought stress caused non-stomatal limitation of photosynthesis in ginger leaves. Drought stress also significantly
reduced the quantum efficiency of photosystem II (Fv/Fm), the non-cyclic electron transfer efficiency of pho-
tosystem II (ϕPSII), and photochemical quenching (qP), while simultaneously increasing nonphotochemical
quenching (NPQ). The addition of shading improved photosynthetic efficiency under drought. These results
provide important baseline information on the photosynthetic mechanisms by which ginger responds to drought
and shading. In addition, they provide a theoretical basis for the study of shade cultivation during the arid
season.

1. Introduction producer of ginger, but statistics from the China Meteorological

Increasing global atmospheric CO2 concentrations are associated
with reductions in average precipitation in mid-latitude and subtropical
regions (Zandalinas et al., 2018). Drought-induced fires are expected to
further increase atmospheric CO2 concentrations (Aragão et al., 2018),
resulting in a vicious cycle. Drought can affect the normal growth of
crops by inhibiting root development (Hasibeder et al., 2015), reducing
stomatal opening (Martin StPaul et al., 2017), altering water use effi-
ciency (Yang et al., 2016), producing superoxide anions (Anjum et al.,
2017), degrading key enzymes (Carmo-Silva et al., 2010) and, in severe
cases, damaging the photosynthetic apparatus (Silva et al., 2003).
Drought is an ecological problem that must be confronted without
delay.
As an important industrial crop in China, ginger has important ap-
plications in food, industry and other fields. China is the world's main
Administration (http://www.cma.gov.cn) indicate that annual pre-
cipitation in the important ginger-producing regions of Shandong and
Henan has been less than 1000 mm/year on average for the past five
years, causing ginger to experience drought stress frequently.
Photosynthesis is the main process by which carbohydrates are as-
similated and accumulated, and the effects of drought stress on crops
are first reflected in photosynthesis. Early in drought stress, leaves in-
itiate a stress response by closing their stomata (Anjum et al., 2011; Ji
et al., 2018) to reduce transpirational water loss (Miyashita et al.,
2005), and the resulting decline in CO2 uptake leads to a decline in
photosynthetic rate, mainly as a result of stomatal limitation (Liu et al.,
2017). As drought stress increases, the activity of photosynthetic en-
zymes decreases (Dias and Brüggemann, 2010), as do photopho-
sphorylation and ATP synthesis (Ji et al., 2012), leading to further
decreases in CO2 assimilation due to non-stomatal limitation (Ji et al.,

∗
Corresponding author.
E-mail address: xukun@sdau.edu.cn (K. Xu).

https://doi.org/10.1016/j.plaphy.2020.03.021
Received 25 October 2019; Received in revised form 17 March 2020; Accepted 17 March 2020
Available online 20 March 2020
0981-9428/ © 2020 Elsevier Masson SAS. All rights reserved.
Y. Lv, et al.
2012). Under severe drought, superoxide anions are produced in large
quantities (Tian et al., 2012), causing damage to the photosynthetic
system (Yan et al., 2010). The degradation of the D1 protein of pho-
tosystem II (PSII) directly abolishes photosynthetic electron transport
(Batra et al., 2014), ultimately leading to crop death. However, a series
of protective mechanisms also exist to mitigate photosynthetic damage,
including the Mehler reaction for scavenging superoxide anions
(Mehler, 1951; Carvalho, 2018), and photorespiration and xanthophyll
cycle for the dissipation of excess light energy (Wang et al., 2010; Voss
et al., 2013; Zhou et al., 2015b).
Under drought stress, plants may reduce photochemical damage by
the natural folding of their leaves, and the resultant shading can reduce
damage by lowering photon flux density (Thomas and Turner, 2001).
Shading can mitigate the adverse effects of drought stress, especially for
crops, such as ginger, that have low light saturation points. On the other
hand, shading is ineffective for crops, such as coffee, that have higher
light saturation points (Cavatte et al., 2012). The alleviation of drought
stress by shading arises mainly from decreased stomatal limitation,
reduced photochemical inactivation of the photosystems, maintenance
of higher water use efficiency, and, as a result, higher photosynthetic
electron transfer efficiency (Montanaro et al., 2009). Shading can also
protect the antioxidant enzyme activities in ginger, underscoring the
necessity of shade in cultivation under dry, sunny conditions during the
spring and summer (Zhang et al., 2013).
Proteomics provides insights into gene regulation and has been
widely used to study plant responses to drought stress (Budak et al.,
2013; Rollins et al., 2013; Alvarez et al., 2014; Jedmowski et al., 2014;
Lyon et al., 2016; Wu et al., 2019). The proteins that are differentially
expressed in response to drought stress are involved in many cellular
functions and differ among plant organs. For example, proteins related
to osmotic regulation, defense signals and programmed cell death play
an important role in the drought adaptation of roots (Alam et al., 2010;
Faghani et al., 2015; Hao et al., 2015), while proteins related to cell
wall biosynthesis and photosynthesis are differentially expressed in
response to drought in leaves (Faghani et al., 2015; Hao et al., 2015).
Comparative proteomics has also been used to demonstrate increases in
metabolism-related proteins and decreases in energy- and translation-
related proteins under drought and to identify methionine synthetase as
a drought response protein (Mohammadi et al., 2012). Comparative
proteomics at the cellular level demonstrated that drought stress in-
duced ribosome-related functions and upregulated an oxidative phos-
phorylation protein. Furthermore, an endocytosis protein was also
significantly enriched under drought, consistent with an increase in
active transport and circulation of membrane proteins under abiotic
stress (Alqurashi et al., 2018). When combined with plant genetic
transformation, proteomics can also provide insights into protein in-
teractions (Meng et al., 2019).
Although the physiological responses of ginger to drought stress
have been studied, the molecular mechanisms by which ginger re-
sponds to drought and shading remain unclear. Therefore, it is parti-
cularly important to characterize the expression and metabolic path-
ways of proteins whose abundance changes in response to drought and
shading. Ours is the first study to use an iTRAQ tandem mass spectro-
metry approach to analyze the response of ginger proteins to drought
and shading. In particular, the molecular mechanisms of drought and
shading response were investigated by measuring protein levels of
photosynthetic electron transport chain components. This work lays a
foundation for further studies of photosynthetic electron transport
under stress in ginger and provides a theoretical basis for the alleviation
of drought stress during ginger production.
2. Methods and materials
Experiments were performed at the experiment centre of Shandong
Agricultural University (36°160′N, 117°156′E) in China. A soil-free
culture system was used in order to minimize the effects of extraneous
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Plant Physiology and Biochemistry 151 (2020) 188–196
variables. The ginger cultivar ‘Shannong 1’ was sown in pots (25 cm
diameter, 30 cm height) filled with acid-cleaned quartz sand on 10 May
2018, and Hoagland's nutrient solution (Sarwar et al., 2018) was added
once every three days. When seedlings had five or six leaves and three
branches, four experimental treatments were initiated: control (CK),
drought (D), 50% shading (S), and 50% shading and drought (SD).
Drought was simulated using 5% PEG-6000. The degree of shading was
gradually increased by increasing the number of layers of yarn net. The
number of layers required to create a 50% shading of the natural light
was determined by measuring light intensity with a model 3415f
quantum radiometer (LI-COR Biosciences). Each treatment was re-
plicated three times and five gingers were planted for each replicate.
The average day and night temperatures were 36 °C and 26 °C, re-
spectively, the day length was 14 h, and the light intensities are shown
in Fig. S1. The roots, stems, leaves, and rhizomes were collected 7 d
after treatment, carefully washed in running water to remove adhering
soil and debris, and then packed individually and stored at −80 °C for
subsequent analysis.
2.1. Photosynthesis and chlorophyll fluorescence
The photosynthetic rate (Pn), stomatal conductance(Gs), and in-
tercellular CO2 concentration (Ci) were measured using a portable
photosynthesis system (Ciras-3, PP SYSTEMS, USA). Inside the IRGA
chamber, the light was 1200 μmol m−2 s−1 PPD (Natural light) and
600 μmol m−2 s−1 PPD (Shading), the leaf temperature was 27 °C, and
the vapor-pressure deficit (VPD) was 1.8 ± 0.2 KPa and CO2 partial
pressure of 38 Pa. All measurements were taken between 10:50 and
11:10 a.m.
The photochemical quenching coefficient (qP), non-photochemical
quenching coefficient (NPQ), quantum efficiency of PS II (ΦPS II), and
variable fluorescence/fluorescence maximum (Fv/Fm) were measured
using FMS-Ⅱ (Hansatech, UK). In order to determine Fv/Fm, ginger was
dark-adapted for 30 min with the aid of a dark clip adapter.
2.2. Protein extraction
Equal amounts (1g each) of root, stem and leaf of ginger were fully
mixed. Then they were grind to powder in liquid nitrogen. 10 mL of
lysis buffer (40 mM Tris-HCl, pH 8.5, 7 M urea, and 4% CHAPS (3-((3-
Cholamidopropyl)dimethylammonium)-1-propanesulfonate)) con-
taining 1 mM PMSF (Phenylmethanesulfonyl fluoride) and 2 mM EDTA
(Ethylenediaminetetraacetic) were added. After 5 min, DTT (DL-
Dithiothreitol) was added to 10 mM final concentration. The suspension
was sonicated for 15 min and then centrifuged 25000 ×g for 20 min.
The supernatant was reduced with 10 mM DTT at 56 °C for 1 h. The
cysteine was blocked with 55 mM iodoacetamide in dark room for
45 min. Five volumes chilled acetone were added into the supernatant
for 2 h at −20 °C. After centrifugation 25,000 g for 20 min, the su-
pernatant was discarded, and the pellet was dried in the air for 5 min,
dissolved in 200 μL 0.5 M TEAB (Triethylammonium bicarbonate), and
sonicated at 200 W for 15 min. Finally, samples were centrifuged
25,000 ×g for 20 min. The supernatant was transferred to a new tube.
Protein concentration was determined using a series of concentrations
of BSA (Albumin from bovine serum) as the standard according to
Bradford (1976), and SDS-PAGE was performed to verify the protein
quality and concentration. The proteins in the supernatant were stored
at −80 °C for further analysis.
2.3. Protein digestion
Total protein (100 μg) was taken out of each sample solution. The
protein was digested with trypsin gold with the ratio of protein:
trypsin = 20:1 at 37 °C for 4 h. Then, trypsin gold was added with the
ratio of protein: trypsin = 20:1 once more for 8 h.
Y. Lv, et al.
2.4. iTRAQ labeling
After trypsin digestion, the peptides were lyophilized. They were
dissolved with 0.5 M TEAB, the iTRAQ labeling of peptide samples were
performed using iTRAQ Reagent 8-plex Kit according to the manufac-
turer's protocol (AB Sciex Inc., MA, USA) (113 for CK; 114 for S; 115 for
D; 116 for SD). The peptides were labeled with the isobaric tags and
incubated at room temperature for 2 h.
2.5. SCX chromatography
SCX chromatography was performed with a LC-20AB HPLC Pump
system (Shimadzu, Kyoto, Japan), the peptide from digestion was re-
constituted with 4 mL buffer A (25 mM NaH2PO4 in 25% ACN
(Acetonitrile), pH2.7) and loaded onto a 4.6 × 250 mm Ultremex SCX
column containing 5 μm particles (Phenomenex). The peptides were
eluted at a flow rate of 1 mL min−1 with a gradient of buffer A for
10 min, 5–35% buffer B (25 mM NaH2PO4, 1 M KCl in 25% ACN,
pH2.7) for 11 min, 35–80% buffer B for 1 min. The system was then
maintained in 80% buffer B for 3 min before equilibrating with buffer A
for 10 min prior to the next injection. Elution was monitored by mea-
suring absorbance at 214 nm, and fractions were collected every 1 min.
The eluted peptides were pooled as 20 fractions (Table S1), desalted by
Strata X C18 column (Phenomenex) and vacuum-dried.
2.6. LC-ESI-MS/MS analysis based on triple TOF 5600
Each fraction was resuspended in buffer A (5% ACN, 0.1% FA
(Fomic acid)) and centrifuged at 20000 ×g for 10 min, the final con-
centration of peptide was about 0.5 μg μL−1 on average. 10 μL super-
natant was loaded on a LC-20AD nanoHPLC (Shimadzu, Kyoto, Japan)
by the autosampler onto a 2 cm C18 trap column. Then, the peptides
were eluted onto a 10 cm analytical C18 column (inner diameter 75)
packed in-house. The samples were loaded at 8 μL min−1 for 4 min,
then the 41 min gradient was run at 300 nL min−1 starting from 5 to
35% B (95% ACN, 0.1% FA), followed by 5 min linear gradient to 80%,
and maintenance at 80% B for 5 min, and finally return to 5% in 1 min.
Data acquisition was performed with a TripleTOF 5600 System (AB
SCIEX, Concord, ON) fitted with a Nanospray III source (AB SCIEX,
Concord, ON) and a pulled quartz tip as the emitter (New Objectives,
Woburn, MA). Data was acquired using an ion spray voltage of 2.5 kV,
curtain gas of 30 PSI, nebulizer gas of 15 PSI, and an interface heater
temperature of 150 °C. The MS was operated with a resolving power
(R.P.) of ≥30000 FWHM for TOF MS scans. For information dependent
data acquisition (IDA), survey scans were acquired in 250 ms and as
many as 30 product ion scans were collected if exceeding a threshold of
120 counts/s and with a 2+ to 5+ charge-state. Total cycle time was
fixed to 3.3 s. Q2 transmission window was 100 Da for 100%. Four time
bins were summed for each scan at a pulser frequency value of 11 kHz
through monitoring of the 40 GHz multichannel TDC detector with
four-anode channel detect ion. A sweeping collision energy setting of
35 ± 5 eV coupled with iTRAQ adjust rolling collision energy was
applied to all precursor ions for collision-induced dissociation. Dynamic
exclusion was set for 1/2 of peak width (15 s), and then the precursor
was refreshed off the exclusion list.
2.7. Data analysis
Raw data files acquired from the Orbitrap were converted into MGF
files using Proteome Discoverer software (PD 1.2, Thermo) and the
MGF file were searched. Proteins identification was performed by using
Mascot software (Matrix Science, Boston, USA, version 2.3.02) against
the NCBI database containing 72852 sequences (http://www.ncbi.nlm.
nih.gov/protein/?term = txid3847 [Organism: noexp]). For protein
identification, a mass tolerance of 20 ppm was permitted for intact
peptide masses and 0.05 Da for fragmented ions, with allowance for one
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Plant Physiology and Biochemistry 151 (2020) 188–196
missed cleavages in the trypsin digests. Gln- > pyro-Glu (N-term Q),
Oxidation (M), Deamidated (NQ) were the potential variable mod-
ifications, and Carbamidomethyl (C), iTRAQ 8 plex (N-term), iTRAQ 8
plex (K) were fixed modifications. The charge states of peptides were
set to +2 and + 3. Specifically, an automatic decoy database search
was performed in Mascot by choosing the decoy checkbox in which a
random sequence of database is generated and tested for raw spectra as
well as the real database. To reduce the probability of false peptide
identification, only peptides with significance scores (≥20) at the 99%
confidence interval by a Mascot probability analysis greater than
“identity” were counted as identified. For protein quantitation, it was
required that a protein contains at least two unique peptides. The
quantitative protein ratios were weighted and normalized by the
median ratio in Mascot. Ratios with p-values < 0.05, and only fold
changes ≥1.2 or ≤0.83 was considered as significant.
Functional annotations of the proteins were conducted using
Blast2GO program (http://www.geneontology.org) against the non-re-
dundant protein database (NR; NCBI). The KEGG database (http://
www.genome.jp/kegg/) and the COG database (http://www.ncbi.nlm.
nih.gov/COG/) were used to classify and group these identified pro-
teins.
2.8. qRT-PCR
Total RNA was extracted using a RNA Isolation Kit (TianGen). A
total of 400 ng RNA was reverse transcribed to cDNA using a cDNA
synthesis kit (TianGen). qPCR was performed using the ABI Q6 Real-
Time PCR system. The target gene primers were designed using Primer
Express 5.0, and the 28s gene was selected as the reference gene. Every
primer was used 3 times. The relative expression levels were calculated
using 2−ΔΔCt method, and the primers are shown in Supplementary
Table (Table S2).
3. Result and discussion
3.1. Effects of drought and shading on photosynthetic efficiency of ginger
3.1.1. Analysis of H2O–CO2 exchange parameters
Abiotic stress can inhibit photosynthetic electron transport, re-
sulting in a lower photosynthetic rate (Berla et al., 2015; Zhang and Liu,
2016) due to stomatal and/or non-stomatal limitations (Chandra et al.,
2018). In a previous study, the photosynthetic rate of ginger decreased
significantly under high tetracycline concentration, and measurements
of stomatal conductance and intercellular CO2 suggested that the de-
crease resulted primarily from non-stomatal limitation (Liu et al.,
2018). This study had similar results: under drought stress, the stomatal
conductance of ginger leaves was significantly lower than that of con-
trols, but the intercellular CO2 concentrations were higher (Table 1,
P < 0.05). These results suggest that non-stomatal limitation was the
main reason for the decreased photosynthetic rate. The photosynthetic
rate of shaded plants was higher than that of controls (Table 1,
P < 0.05), probably because of increased photosynthetic electron
transfer efficiency. Although the photosynthetic rate of plants in the
drought and shading treatment was significantly lower than that of
controls, it was higher than that of plants that experienced drought
stress alone, indicating that shading could alleviate drought stress. The
stomatal conductance and intercellular CO2 concentration showed that
drought stress was alleviated and non-stomatal limitation was reduced.
3.1.2. Analysis of chlorophyll fluorescence parameters
Chlorophyll fluorescence parameters can accurately characterize
the extent of abiotic stress in plants. Fv/Fm is the efficiency index of PSII
(Giorio, 2011) and is often used as a reference for screening the degree
of plant abiotic stress tolerance. The higher the Fv/Fm, the greater the
abiotic stress tolerance (Zhou et al., 2015a; Sharma et al., 2017). Kalaji
et al. (2014) pointed out that Fv/Fm of C3 plants is typically 0.83–0.84.
Y. Lv, et al.
Table 1
Effects of shading and drought on photosynthesis and chlorophyll fluorescence.
Treatment Control Drought
2 −1 b d
Pn (umol (m ·s) ) 11.77 ± 0.34 3.00 ± 0.22
Gs (mmol (m2·s)−1) 317.00 ± 10.80a 95.00 ± 5.72b
Ci (μg mL−1) 240.33 ± 4.50c 278.67 ± 8.18a
Fv/Fm 0.72 ± 0.01b 0.57 ± 0.02d
ϕPSⅡ 0.42 ± 0.02b 0.28 ± 0.03c
qP 0.62 ± 0.01b 0.41 ± 0.01c
NPQ 0.85 ± 0.02c 1.29 ± 0.03a
Note: average ± standard deviation (n = 3). The different letters indicate significant differences at level of p < 0.05.
However, Fv/Fm is often significantly reduced under stress, and it can
therefore be used as an indicator of PSII sensitivity to photoinhibition
(Kalaji et al., 2017). The Fv/Fm of the control plants in this study was
0.72, which may reflect the fact that measurements were made at about
11:00 a.m. when Fv/Fm may have been reduced by chlorophyll de-
gradation and lipid accumulation under high light intensities (He et al.,
2015). Other studies have pointed out that Fv/Fm decreases with in-
creasing light intensity and often reaches its lowest value at noon
(Demmig-Adams et al., 2012; Desotgiu et al., 2013; Kalaji et al., 2017).
Ginger Fv/Fm may therefore have been inhibited by excess light at
noon, and indeed the Fv/Fm of ginger was significantly increased by
shading (P < 0.05). Hosseinzadeh et al. (2016) showed that drought
stress could significantly reduce the Fv/Fm of peas, mainly due to the
degradation of the D1 protein. Similar findings in this study showed
that Fv/Fm decreased significantly under drought stress (Table 1,
P < 0.05) and that the Fv/Fm of the shading and drought combination
treatment was significantly higher than that of drought alone
(P < 0.05). Kalaji et al. (2017) pointed out that the Fv/Fm of crops was
relatively stable and that Fv/Fm was significantly reduced only after the
leaves had become dehydrated, suggesting that drought stress in our
experiment may have damaged the leaf structure of ginger. However,
the proteomic results showed that drought stress had no significant
effect on PSII-related protein levels (Table S4, P > 0.05), indicating
that the proportion of inactive PSII reaction centers increased without
any change in the expression of PSII-related proteins.
The term ϕPSII denotes the non-cyclic electron transfer efficiency of
PSII. We found that ϕPS II decreased significantly under drought stress
and increased under shading (Table 1, P < 0.05). A similar study
showed that light inhibition decreased and ϕPSII increased with de-
creased light intensity (Fu et al., 2012). The proteomics results of the
present study showed that drought stress significantly increased the
expression of PSI-related proteins (Table S4), suggesting a drought-re-
lated decrease in non-cyclic electron transfer efficiency and an increase
in cyclic electron transfer around PSI. Previous studies have shown that
CEF increases under drought stress, effectively protecting the photo-
synthetic system (Zivcak et al., 2013; Singh et al., 2014; Lima Neto
et al., 2017). A similar protective mechanism may have been at work in
this study.
The term qP represents the degree of reduction of QA in the PSII
reaction center; it can be significantly reduced in response to drought
and strong light (Bączek-Kwinta et al., 2011; Hazrati et al., 2016). In
this study, we found that the qP of ginger leaves decreased significantly
under drought stress (Table 1, P < 0.05), suggesting that drought
stress reduced photosynthetic electron transfer efficiency by closing
PSII reaction centers, which is consistent with the study of Chen et al.
(2016). Shading significantly increased qP, possibly because shading
increased the abundance of LHCs and produced enough excited elec-
trons to promote the opening of QA. There was no significant difference
in qP between the shading and drought and control treatments
(P > 0.05), indicating that shading could mitigate the adverse effects
of drought stress on QA response centers and that QA opening was re-
lated to the energy of the photochemical reaction.
NPQ represents nonphotochemical energy dissipation in the form of
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Plant Physiology and Biochemistry 151 (2020) 188–196
Shading Shading and Drought
a
12.80 ± 0.24 5.37 ± 0.25c
316.67 ± 4.99a 108.33 ± 3.86b
246.33 ± 3.3bc 258.33 ± 3.3b
0.75 ± 0.00a 0.68 ± 0.00c
0.55 ± 0.01a 0.38 ± 0.02b
0.78 ± 0.02a 0.61 ± 0.01b
0.82 ± 0.02c 1.17 ± 0.05b
heat and usually increases under abiotic stress (Wang et al., 2016). We
found that NPQ increased significantly under drought stress (Table 1,
P < 0.05), indicating that ginger dissipated a large amount of ab-
sorbed light energy as heat and protected the photosynthetic system
from being damaged, with a concomitant decrease in photosynthetic
rate. Previous studies have shown that NPQ increases with the in-
creasing duration of drought stress (Dahal et al., 2015) and that abiotic
stress increases NPQ at the expense of photochemistry (Wang et al.,
2015). The NPQ of the drought and shading treatment was lower than
that of drought alone (Table 1, P < 0.05). Shading may have reduced
the damage caused by drought stress, thereby reducing heat dissipation.
More electrons were used for photosynthetic electron transfer, im-
proving the photosynthetic performance of ginger leaves.
3.2. Protein identification
In this study, a total of 6523 proteins and 20,423 peptides were
identified, of which 18,544 were unique peptides (Fig. S2A). Relative
molecular mass statistics indicated that 9.38% of the proteins were
0–10 kD, 18.44% were 10–20 kD, and 4.77% were > 100 kD (Fig. S2B).
There were 2024, 2256, 1433, and 224 proteins with 1, 2–5, 6–40,
and > 41 peptide, respectively (Fig. S2C). The percentage of proteins
with different coverage was 40%–100%, 30%–40%, 20%–30%,
10%–20%, and < 10%, respectively, which accounted for 4.63%,
6.81%, 14.51%, 27.44%, and 46.61% of the identified total proteins
(Fig. S2D). This result demonstrates that most of the protein sequences
had good coverage.
3.3. Differential expression analysis of proteins
Four comparisons were performed: S/CK (shading versus control),
D/CK (drought versus control), SD/S (shading and drought versus
shading alone), and SD/D (shading and drought versus drought alone).
In the shading treatment, 72 proteins were up-regulated and 67 pro-
teins were down-regulated compared to the control. In the drought
treatment, 57 proteins were up-regulated and 41 proteins were down-
regulated compared to the control. In the shading and drought treat-
ment, 58 proteins were up-regulated and 56 were down-regulated
compared with the shading treatment, 61 proteins were up-regulated
and 51 were down-regulated compared with the drought treatment.
(Table S3).
Analysis of the number of differentially expressed proteins and the
four pathways with the lowest P-values indicated that photosynthesis
and phenylalanine metabolism were associated with more differen-
tially-expressed proteins, while ABC transporters were associated with
fewer differentially-expressed proteins and had a higher P-value. The P-
value for photosynthesis was < 0.05, and differential expression was
found among the four comparison groups. In addition, although the
number of differentially expressed proteins among the photosynthetic
antennae proteins was small, the corresponding P-value was low
(Fig. 1). Therefore, proteins related to photosynthesis and the photo-
synthesis antenna were selected for further study of the effects of
drought and shading on the photosynthetic electron transport chain.
Y. Lv, et al.
Fig. 1. Number of proteins enriched and P-value of different pathways. Note: The graphics size indicates the number of enriched proteins.
Many studies have found that photosynthesis-related proteins are
among the most important differentially expressed proteins under
drought stress (Zhou et al., 2015b; Qin et al., 2016; Su et al., 2019).
3.4. Analysis of differentially expressed photosynthesis-related proteins
A heat map analysis of differentially expressed proteins in the
photosynthetic electron transport chain indicated that shading sig-
nificantly increased the expression of various photosynthesis-related
proteins in ways that appeared to facilitate photosynthetic electron
transport. Drought stress reduced the expression of some photo-
synthetic proteins but increased the expression of proteins related to
defense mechanisms, a result consistent with that of Zhou et al.
(2015b). The combination of shading and drought significantly reduced
the expression of various photosynthetic proteins and aggravated
photoinhibition compared with shading alone (Fig. 2).
In terms of photosynthesis-related proteins (P < 0.05), 22 sig-
nificant differences were found among the four comparison groups,
including three proteins related to cytochrome b6f, two proteins related
to electron transport, one protein related to ATPase, six proteins related
to the light harvesting complex (LHC), six proteins related to PSII and
four proteins related to photosystem I (PSI) (Table S4).
3.4.1. Analysis of LHC-related protein expression
Light intensity has a significant effect on chla/b, mainly owing to
changes at the LHC level (Fujita et al., 1989). The expression of LHC
chlorophyll a/b binding (cab) protein genes is also significantly corre-
lated with light duration (Kellmann et al., 1990). In this study, we
found that shading directly increased the abundance of cab protein
(Table S4), perhaps because of ginger's low light saturation point.
Shading created an appropriate light intensity for ginger growth, and
this was directly reflected in the increased expression of LHC-related
proteins, a finding also reported by Fan et al. (2019). Different stresses
have contrasting effects on the expression of LHC proteins in different
crops (Jiang et al., 2014). Several studies have shown that the de-
gradation of LHCII-related proteins in wheat leaves under severe
drought stress is related to the degradation of the D1 protein (Barbato
et al., 1992; Barber, 1998; Wei et al., 2000). However, in our study,
drought stress had no significant effect on the expression of LHC-related
proteins (Table S4). It may because that the degree of stress in our
experiment was not sufficient to cause photodamage in ginger, a hy-
pothesis supported by the fact that PSII-related protein expression was
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Plant Physiology and Biochemistry 151 (2020) 188–196
also unaffected. Under the combination of shading and drought, the
abundance of cab protein was again increased, further highlighting the
sensitivity of LHC protein levels to light.
3.4.2. Analysis of oxygen evolution complex (OEC)-related protein
expression
The oxygen evolution complex (OEC) is an important component of
PSII and is responsible for the cleavage and oxidation of H2O (Roose
et al., 2009). Reduced levels of the OEC-related protein PsbP are as-
sociated with reduced photosynthetic capacity (Du et al., 2014). Ex-
pression of oxygen-evolving enhancer 1 (a component of the OEC
complex) increased under salt stress and helped to sustain PSII activity
(Momonoki et al., 2009). The expression of PsbO, PsbP and PsbQ in
Vaccinium uligiuosum was significantly down-regulated under low tem-
peratures and short days, but the expression of PsbO and PsbP was
stable under low temperatures and long days (Wu, 2018), indicating
that the effect of abiotic stress on OEC-related proteins depends upon
the type of stress and the crop species. In this study, shading sig-
nificantly increased the expression of OEC-related proteins (Table S4).
The increased expression of LHC may have provided more excitation
energy for the reaction center, requiring OEC to provide more electrons
and thereby driving its increased expression level. However, neither
drought nor shading plus drought had a significant effect on the ex-
pression of OEC-related proteins (Table S4). Constitutive OEC levels
under drought stress may permit electron flow and protect the photo-
synthetic apparatus from photodamage (Downs et al., 1999).
3.4.3. Analysis of protein expression related to cytochrome b6f and PSI
The chloroplast cytochrome b6f complex (cyt b6f) is located be-
tween PSII and PSI; it is also known as plastoquinone (PQ)/plastocyanin
(PC) oxidoreductase because it catalyzes the transfer of electrons from
PQ to PC (Cramer et al., 2011). Salt stress has been shown to sig-
nificantly increase the expression of cyt b6f (Joaquin-Ramos et al.,
2014). Under drought stress, cyt b6f may limit linear electron transfer
(Li and Ma, 2012) and increase cyclic electron flow around PSI (Zhu
et al., 2001; Iwai et al., 2010). Similar results were obtained in this
study: the abundance of cyt b6f was significantly increased under
drought stress (Table S4). Cytochrome b6f may accelerate the oxidation
rate of reduced plastoquinone and increase the proton concentration in
the thylakoid lumen, thus increasing the transmembrane proton motive
force and ATP production. However, under some circumstances, cyt b6f
can participate in cyclic electron flow (CEF) with PSI, which generates
Y. Lv, et al.
Fig. 2. Clustering analysis of photosynthetic related differentially expressed proteins.
ATP without the accumulation of NADPH and plays an important role
in photoprotection. Here, drought increased the expression of PSI-re-
lated proteins (Table S4), consistent with an enhancement of CEF
(Zhong et al., 2019). The response of PSI-related proteins was similar to
that of cyt b6f proteins, further suggesting a correlation between them.
Mou et al. (2013) reported that non-photochemical quenching (NPQ)
and the lutein cycle were mainly related to the thylakoid proton gra-
dient. In this study, cyt b6f enhanced the transmembrane proton motive
force to promote NPQ and prevent damage to the photosynthetic ap-
paratus from excess electrons. The combination of shading and drought
had no significant effect on cyt b6f, perhaps because shading improved
photosynthetic electron transfer efficiency and alleviated the effects of
drought.
3.4.4. Analysis of ATPase-related protein expression
ATPase is the key enzyme of photophosphorylation and plays an
important role in the process of energy conversion in photosynthesis
(Kartashov et al., 2015). NaCl stress can increase ATPase activity, but
specific salt tolerance mechanisms differ among plant varieties (Huang
et al., 2012). Several studies have shown that ATPase significantly
decreases under drought stress (Lawlor, 2002; Kohzuma et al., 2009),
while Chen and Liu (2001) found that drought stress promoted ATPase
activity. In this study, drought stress reduced the expression of ATPase
(Table S4). Although the expression of ATPase decreased, enhanced
CEF and increased transmembrane proton electromotive force under
drought stress may have increased ATP production.
3.5. Analysis of key gene expression with qRT-PCR
To evaluate the correlation between mRNA abundance and protein
abundance, genes encoding 8 of the 28 differentially expressed proteins
were selected for qRT-PCR analysis. They included proteins associated
with LHC, OEC, cyt b6f, PC, PSI, ferredoxin (Fd), and FNR. We mea-
sured the expression of genes encoding these eight proteins by qRT-
PCR, and their expression was broadly consistent with the corre-
sponding protein abundance measurements (Fig. 3).
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Plant Physiology and Biochemistry 151 (2020) 188–196
3.6. Model of the response of photosynthetic electron transfer proteins to
drought and shading
A model of the response of photosynthetic electron transfer proteins
to drought and shading was developed by measuring the expression of a
series of photosynthetic proteins and analyzing associated photo-
synthetic parameters (Fig. 4). The LHC of naturally grown ginger re-
ceives light energy: part is lost to fluorescence, part is dissipated as
heat, and most is used for photochemistry. The light energy received by
LHC activates the PSII reaction center, and the OEC decomposes water
and shuttles electrons to cyt b6f via PSII and PQ. PC accepts electrons
from cyt b6f and transports them to the PSI reaction center. Electrons
are transferred to Fd via PSI, at which point they can take one of several
paths: (1) linear electron flow to NADP+ via FNR to produce NADPH,
which enters the Calvin cycle, (2) cyclic electron flow, whereby elec-
trons are transferred back to the PQ shuttle to generate a circulating
electron flow around the PSI reaction center, or (3) transfer to O2 to
form superoxide anions, which are subsequently detoxified through the
Mehler reaction. The PQ shuttle and water photolysis move large
numbers of protons to the thylakoid lumen where they constitute a
transmembrane proton motive force that drives ATPase to produce
ATP, which enters the Calvin cycle.
In our study, drought stress increased NPQ, which reduced photo-
chemistry; it also closed QA (PS II reaction center), which reduced
photosynthetic electron transport. Drought stress had no significant
effect on OEC but enhanced cyt b6f and PSI, which increased the CEF
around PSI and enhanced the tolerance of ginger to drought. Drought
stress also increased SOD formation and enhanced the Mehler reaction
as detailed in previous studies. Although ATPase was down-regulated
under drought stress, enhanced CEF counteracted the down-regulation
of ATPase, reducing the effect of drought on ATP production. Shading
significantly increased the expression of LHC, OEC, PC and FNR, pro-
moted the utilization of light energy and linear electron transfer, in-
creased the production of NADPH and ATP, promoted the Calvin cycle,
and improved photosynthetic electron transfer efficiency. The combi-
nation of shading and drought appeared to increase the utilization of
Y. Lv, et al. Plant Physiology and Biochemistry 151 (2020) 188–196

Fig. 3. The transcript levels of the differentially abundant proteins.

Fig. 4. The models of photosynthetic electron transport pathways affected by drought and shading. Note: The orange arrow represents the linear electron flow, the
red arrow represents the cyclic electron flow, the green arrow represents the Mechler reaction, and the purple arrow represents the common pathway. Three
rectangles in the protein complex from left to right represent shading, drought, shading and drought respectively; red indicates up-regulation, blue indicates down-
regulation, and gray indicates no significant change. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of
this article.)

194
Y. Lv, et al.
light energy by increasing the expression of LHC proteins and to in-
crease CEF by reducing the expression of FNR, thereby improving the
tolerance of ginger to drought.
4. Conclusion
In conclusion, our data suggest that ginger alleviates drought-re-
lated damage by increasing CEF around PSI at the cost of reduced
electron transfer efficiency and photosynthetic rate. Heat dissipation is
also increased under drought stress. Shading significantly improves
photosynthetic electron transport efficiency, enhances the photosynth-
esis of ginger leaves, and alleviates some of the effects of drought,
further highlighting the necessity of shade in the cultivation of ginger
under the dry, high-light conditions of spring and summer.
Declaration of competing interest
The authors declare that they have no conflict of interest.
Acknowledgement
We appreciate the linguistic assistance provided by TopEdit (www.
topeditsci.com) during the preparation of this manuscript. This study
was supported by China Agricultural Research System (Grant No.
CARS-24-A-09), Taishan Industrial Experts Programme, China (Grant
No. tscy20190105), and Shandong Province's Dual-class Discipline
Construction Project, China (Grant No. SYL2017YSTD06).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.plaphy.2020.03.021.
Authors’ contributions
YL, XHL, and KX designed the experiments, YL, YYL and KX selected
the material, YL and YYL performed the experiments, YL and XHL
analyzed the data, YL, XHL, and KX wrote the paper. All authors read
and approved the final manuscript.
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