Exogenous Methylglyoxal Improves Maize Yield by Alleviating Plant Diabetes and leaf senescence under Drought

Abstract

Drought-induced leaf senescence is related to high sugar levels in leaves, photosynthesis inhibition, and ultimate yield loss. This physiological phenomenon in leaves bears resemblance to the symptom of diabetes in human disease. However, the underlying mechanisms of plant diabetes on carbon imbalance in maize leaf and corresponding detoxification strategy have not been well understood. In this study, we demonstrated that foliar application of exogenous methylglyoxal (MG) delayed leaf senescence and promoted photoassimilation, retrieved 14% yield loss induced by drought stress during grain filling stage. Transcriptome and metabolite analysis revealed that drought increased sugar accumulation in leaf with inhibition of sugar transporters facilitating phloem loading. This further lead to disequilibrium of glycolysis and over-accumulation of endogenous MG. Contrarily, exogenous MG significantly upregulated glycolytic flux and glyoxalase system catabolizing endogenous MG and advanced glycation end products toxicity, ultimately alleviating plant diabetes. Besides, the genes facilitating anabolism and catabolism of trehalose- 6-phosphate were promoted and suppressed by drought, respectively, whereas exogenous MG reversed the effect. Moreover, exogenous MG activated phenylpropanoid biosynthetic pathway, likely promoting cell structural integrity. Collectively, these results suggest that exogenous MG alleviates the toxic effect from drought-induced sugar accumulation and activates the defense-related pathway, thereby maintaining leaf function and yield production.

Highlight
Exogenous methylglyoxal stimulates glycolytic flux and glyoxalase system, providing a potential insight to alleviate plant diabetes under drought condition.

Full text

Vol:.(1234567890)
Journal of Plant Growth Regulation (2023) 42:3934–3946
https://doi.org/10.1007/s00344-022-10862-4
1 3
Exogenous Methylglyoxal Ameliorates Source Strength andRetrieves
Yield Loss Under Drought Stress During Grain Filling inMaize
Yi‑HsuanLin1· Yu‑KaJin1· Zhen‑YuanChen1· Zu‑DongXiao1· SiShen1,2· Shun‑LiZhou1,2
Received: 2 August 2022 / Accepted: 22 October 2022 / Published online: 16 November 2022
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022
Abstract
In maize (Zea mays L.) production, grain-filling stage is a pivotal phase that drought occurring would irreversibly cause leaf
senescence and yield loss. Meanwhile, drought induces the disequilibrium of carbohydrates that inevitably produces the toxic
substance methylglyoxal (MG) that plays dual roles including cytotoxic metabolite or signaling molecule in plants. However,
how exogenous MG influences maize yield formation in response to drought during grain filling remains unknown. In this
study, maize plants were exposed to moderate and severe drought conditions from 15 to 28days after pollination with foliar
spraying of MG (0, 15, 25, and 35mM). Notably, MG application significantly increased kernel number and retrieved yield
loss by 14–48% under drought, demonstrating an increased resistance of the plants. Interestingly, high (25–35mM) and
low concentrations (15–25mM) of MG application under moderate and severe drought performed the highest yield output,
respectively. To investigate the mechanisms by which MG enhanced drought resistance, we confirmed that MG application
postponed leaf senescence under both drought conditions and significantly improved photosynthesis under severe drought
during filling stage. Specifically, MG application escalated the levels of soluble sugar and sucrose while suppressing endog-
enous MG accumulation by activating glyoxalase system in leaf during the early phase of drought stress and well-watered
condition. Collectively, these results demonstrate that exogenous MG application enhances drought tolerance during maize
grain filling, possibly through regulation on the homeostasis of endogenous MG and sugars. These findings provide a new
approach to secure yield against drought stress in maize production.
Keywords Methylglyoxal· Drought tolerance· Leaf senescence· Sugars· Glyoxalase· Yield
Abbreviations
MG Methylglyoxal
Endo. MG Endogenous methylglyoxal
GLY I Glyoxalase I
GLY II Glyoxalase II
DAP Days after pollination
DAI Days after drought induction
CK Well-watered
DS1 Moderate drought
DS2 Severe Drought
Introduction
Drought caused by climate change severely limits crop
development and ultimate yield. The frequency and sever-
ity of drought stress are increasing with global warming
(Trenberth etal., 2014; Dietz etal., 2021). In maize (Zea
mays L.) cultivation, the flowering and grain-filling stages
are susceptible to drought due to the severe influence on ker-
nel set (Tardieu etal., 2018; Shen etal., 2018; 2020b; 2022).
Water scarcity at grain-filling stage induces leaf senescence,
resulting in reduction of the source strength and thus hinder-
ing kernel filling and yield formation (Reynolds etal., 2021).
However, the mechanism of leaf senescence under adverse
scenarios is sophisticated and contains crosstalk between
plenty of metabolisms.
Handling Editor: Serena varatto .
* Si Shen
shensi@cau.edu.cn
* Shun-Li Zhou
zhoushl@cau.edu.cn
1 College ofAgronomy andBiotechnology, China
Agricultural University, Beijing100193, China
2 Innovation Center ofAgricultural Technology forLowland
Plain ofHebei, Wuqiao061802, China

3935Journal of Plant Growth Regulation (2023) 42:3934–3946
1 3
Sugars play important roles in the onset of leaf senes-
cence during stress conditions. Under drought stress, evident
soluble sugar, serving as signaling molecules or osmolytes,
is accumulated in leaves (Zhang and Zhou, 2013; Tardieu
etal., 2018). Meanwhile, promoted expression of senes-
cence-associated gene (SAG) and the degradation of chlo-
roplast are taking place (VanDoorn, 2008). Application
of exogenous sugar strongly induced senescence and sup-
pressed photosynthetic rate by inducing AtSAG12 expression
in Arabidopsis (Wingler etal., 2006). Moreover, fluctuation
of sugar content could regulate phytohormone synthesis and
metabolism, including abscisic acid and ethylene that cause
the senescence of leaves (León and Sheen, 2003; Pourtau
etal., 2004; Kumar etal., 2019). Excessive sugar accumu-
lation would in turn affect carbohydrate metabolism. For
instance, the activities of enzymes involved in glycolysis
were increased at early stage but decreased at later stage
due to excessive sugar (Borysiuk etal., 2018). This traffic-
jam situation in the glycolytic pathway results in an accu-
mulation of the cytotoxic metabolite methylglyoxal (MG)
in cells. Likewise, high level of sugars unavoidably gener-
ated toxic compound MG, promoting the aging process in
human beings (Schalkwijk and Stehouwer, 2020). However,
whether excessive sugar accumulation in leaves inevitably
induces excessive endogenous MG and its relationship with
drought-induced leaf senescence is unknown.
MG is a reactive α, β-dicarbonyl aldehyde compound
ubiquitously presented in bacteria (Booth etal., 2003),
humans (Allaman etal., 2015), and plants (Singla-Pareek
etal., 2020). MG is a by-product that inevitably originated
from the metabolisms of carbohydrates, fatty acids, and
proteins in cytosol, mitochondria, and chloroplast (Saito
etal., 2011; Shimakawa etal., 2014; Kaur etal., 2016). The
major pathway of MG formation is catalyzed from triose
phosphate, glyceraldehyde-3-phosphate (G3P), and dihy-
droxyacetone phosphate (DHAP) in glycolysis and Calvin
cycle (Kaur etal., 2014). Under physiological pH, G3P and
DHAP are unstable and highly toxic to cells that tend to lose
the α-carbonyl proton and form the enediolate phosphate.
These intermediates are easy for spontaneous β-elimination
of phosphate group with a low energy barrier following
the formation of MG (Richard, 1984, 1993). In fact, the
MG level was maintained at quite low concentration in the
absence of abiotic stress, accounting for 0.1–0.4% of yield in
glycolysis (Shumilina etal., 2019). However, the MG level is
boosted by several times when exposed to different environ-
mental stresses (Li, 2016). The excessive MG accumulation
causes the toxic damage of functioning and proliferation in
cells. MG alters the structures and stabilities of amino acid
and nucleic acid, which forms the advanced end glycation
products (Ahmed and Thornalley, 2007; Shumilina etal.,
2019). In animals, overproduction of MG was associated
with aging, cardiovascular disease, cancer, and diabetes
(Schalkwijk and Stehouwer, 2020). A theory of plant dia-
betes was proposed that high levels of sugar-derived MG
accumulation from glycolysis or photosynthesis would result
in protein carbonylation, ROS generation, PSII inhibition,
malfunction in plant cell development and cell death (Saito
etal., 2011; Shimakawa etal., 2014; Takagi etal., 2014). To
cope with the toxicity of MG, plant has evolutionarily devel-
oped a highly efficient glyoxalase system for detoxification,
which process are facilitated by two key enzymes, glyoxalase
I (GLY I) and glyoxalase II (GLY II). The detoxification
of MG follows two irreversible steps: MG and glutathione
(GSH) spontaneously react to form the hemithioacetal and
then transform to S-D-lactoylglutathione (SLG) in the reac-
tion catalyzed by GLY I. The GLY II hydrolyzes the SLG
to form D-lactate and release the GSH back into the sys-
tem pool (Racker, 1951; Crook and Law, 1952). Finally,
D-lactate then converted to pyruvate and entered into the
tricarboxylic acid cycle (Engqvist etal., 2009).
In addition to be toxic, MG plays dual roles in regulation
of plants in a concentration-dependent manner. For instance,
MG at low level played an important role in transduction
pathway as a signaling molecule. Recently, pretreatment of
MG was demonstrated to enhance heat tolerance and frost
hardiness in maize and wheat seedlings, respectively (Wang
etal., 2019; Majláth etal., 2020). However, these instant
stresses’ implementation only occurred at seedling and there
was no assessment on MG impact on yield performance. It
also remains unclear whether MG increases tolerance to a
continuous drought during the critical stage of grain filling
of maize.
To this end, this study focuses on the effect of exogenous
MG on drought tolerance in maize during grain filling and
its underlying mechanisms. We conducted gradient degrees
of drought stresses coupled with different concentrations of
MG application at grain-filling stage and investigated the
responses in carbohydrate metabolism, MG homeostasis,
yield performance, and thus drought resistance. By these
results, we aim to (i) enhance continuous drought toler-
ance with MG application and (ii) decipher the MG effect
on yield performance, carbohydrate, and endogenous MG
metabolisms. These findings would provide a new method
to maintain yield output in cereal production in coping with
global environmental change.
Materials andMethods
Plant Materials andCultivated Arrangement
Maize plants were grown at Wuqiao Experimental Station of
the China Agricultural University in Hebei Province, China,
116.3°E, 37.4°N. Maize hybrid Zhengdan 958 was used in
this experiment, which was generally used in production in

3936 Journal of Plant Growth Regulation (2023) 42:3934–3946
1 3
China. Maize was cultivated at a density of 80,000 plants
per hectare with fertilizing basal compound (750kg ha−1;
N 15%, P2O5 15%, K2O 15%) at seedling stage and a top
dressing of urea (245kg ha−1; N 46%) at the V13 stage.
Pesticides were applied as necessary to protect the plants
from insects and diseases.
Gradient Drought Treatments, Exogenous MG
Application, andSampling
In order to simulate drought stress at grain-filling stage,
we implemented drought treatments by artificial irrigation
for 3days after pollination (DAP) to distinguish the well-
watered and drought conditions. We continuously moni-
tored the soil relative water content with soil moisture sen-
sor (Thetaprobe ML2x, Delta-T Device, UK). Soil relative
water content of 20–30cm depth soil was measured at 3,
6, 9, 11, 14, 15, 18, 21, 24, 28, and 30 DAP, until the soil
relative water content decreased to ~ 50% and ~ 40% and was
marked as moderate (DS1) and severe drought stress (DS2),
respectively. In the control, pools were well-watered to
maintain soil relative water content at ~ 75% throughout the
growth period. The measurement of relative water content
was measured according to (Chen etal. 2022). At 30 DAP,
we re-watered the plants and maintained well-watered status
until maturity (Fig.1). Drought treatment was conducted in
pools (10.89 m2, 0.6m depth each, total in 18 independent
pools) with canvas roofs to prevent rainfall.
Each drought pool would divide into two sections by
vertically placing transparent polyethylene sheet (3m x
2m) served as shelterbelt. At 14, 17, 20, 23, 26 DAP, MG
solution (0, 15, 25, 35mM) was separately applied in each
treatment by foliar spraying (Fig.1). To ensure every indi-
vidual exposure to same volume of MG solution (Macklin),
foliar spraying (~ 30mL plant−1 of MG) attained the maxi-
mum of aqueous holding capacity. Same volume of water
was sprayed as the control.
Middle part of ear leaves were collected at 9 am on 21 and
28 DAP corresponding to 7 and 14days after drought induc-
tion (DAI). Sampled leaves were immediately frozen by liq-
uid nitrogen and stored in ultra-low freezer for physiologi-
cal analyses. Yield and yield components were measured at
physiological maturity, and kernel samples were determined
after 48h of drying at 80°C in oven.
Measurements ofTotal Green Leaf Area, Relative
Chlorophyll Content, Photosynthetic Rate,
andChlorophyll Fluorescence
With a view to evaluating the effects of treatments on phe-
notypic traits, the green leaf area was estimated with the
empirical model: LA = 0.75* L* W, where L and W served
as the length and width of green leaf, respectively (Lizaso,
etal. 2003). Relative chlorophyll content of the ear leaf
and 9th leaf was measured by SPAD values (Chlorophyll
meterSPAD-502Plus, Japan) at 7 and 14 DAI, respectively.
The photosynthetic rate was measured by using Portable
Photosynthesis System (LI-6400, LI-COR Corporation,
USA). The setting and conditions were 1200µmol m−2 s-1
inner light intensity, 30°C temperature, and 600μmol mol−1
of ambient CO2 concentration. The photosynthetic rate was
measured at 10 am, 10 DAI.
In addition, quantum efficiency of PSII was carried out
with Walz (MINI-PAM-II, Germany). The measurement of
PSII efficiency (Fv/Fm) was determined after 20min dark-
adapted on ear leaves. The chlorophyll fluorescence was
measured at 10 DAI after the photosynthetic rate estimation.
Measurements ofSoluble Sugar, Sucrose, andStarch
Content
The middle part of ear leaves were used for extracting solu-
ble sugars. Fresh samples (0.1g) were homogenized in 5mL
of deionized water and added 10mg of charcoal, then boiled
in water for 20min, centrifuged at 4500rpm for 10min, and
repeated the steps above three times until all the soluble
sugar was completely extracted (Hanft and Jones, 1986).
Quantification of soluble sugars and starch content would
use 4mL anthranone and 1mL crude extraction solution at
45°C for 15min. Quantification ofsucrosecontentwould
use0.4mL of crude extraction solution with 0.2mL of
NaOH(2N) heat up in boiling water for 5min. Afterward,
added 2mL 30% hydrochloric acid and 0.8mL 0.1% ben-
zene-1, 3-diol, completed reaction and showing color in water
bath at 80°C for 10min (Cardini etal., 1955). The content of
0510 15 20 25 30
tnetnoc evitaler retaw lioS
Days after pollination
CK
DS1
DS2
80%
60%
40%
Sampling II
(14 DAI)
Sampling I
(7 DAI)
Water control
MG MG MG MG MG
Re-watering
Fig. 1 The water dynamic during well-watered and drought treat-
ments and the timelines of water control (blue arrow), MG applica-
tion (yellow arrow), and sampling. Soil water relative content was
measured in 20cm depth soil at 2–4days of intervals. DAI, days after
drought induction; CK, well-watered; DS1, moderate drought; and
DS2, severe drought (Color figure online)

3937Journal of Plant Growth Regulation (2023) 42:3934–3946
1 3
soluble sugars, starch, and sucrose was calculated with stand-
ard curve and expressed as mg/g DW.
Determination ofGlyoxalase System
andEndogenous MG Content
Extraction of GLYI and GLYII crude proteins was as
described (Mustafiz etal., 2010) with modification. Briefly,
0.2g fresh leaf tissues was homogenized in 1mL of extract
buffer containing 50mmol Tris–HCL (pH 7.0), 16mmol
MgSO4, 0.2mmol phenylmethanesulfonyl fluoride, and 0.2%
PVP-40. Homogenates were centrifuged at 13,000g for 30min
at 4°C. Afterward, the supernatant was transferred to a new
tube incubated on ice for determination.
Glyoxalase I (EC: 4.4.1.5) activity was determined by
investigating the formation of SLG at 240nm: the enzyme
mixture included 3.5mmol MG, 1.7mmol reduced glu-
tathione, 16mmol MgSO4, and 50mmol Tris–HCL pH 7.5 in
a final volume of 0.2mL. Glyoxalase II (EC: 3.1.2.6) activity
monitors the broke down of SLG at 24 0nm: assay mixture
contained 300µmol SLG and 50mmol Tris–HCL pH 7.2 in
a final volume of 0.2mL. The molar absorption coefficient
of SLG at 240nm is 3.370 mM−1 cm−1. All enzyme activi-
ties were measured by microplate reader (Thermo Scientific
Multiskan, USA) at room temperature.
Extraction of MG content was carried out as follows
(Mustafiz etal., 2010); fresh plant samples (0.25g) were
homogenized in 1mL of 0.5M perchloric acid and incubated
on ice for 15min. Homogenates were centrifuged at 13,000g
for 15min at 4°C, splitting the supernatant into two new
tubes individually as technical replication. 10mg of charcoal
was added into each tube for decolorizing and neutralizing
the solution with 1M CK2O3, centrifuged again, and trans-
ferred supernatant into new tube and placed on ice. For MG
determination, the reaction was started by adding 7.2mmol 1,
2-diaminobenzene after 30min incubation and estimated at
336nm. MG content was calculated with standard curve and
expressed as µmol g−1 FW.
Data Analysis andStatistics
Experimental data were analyzed using IBM SPSS 25 and
Microsoft Excel 2016. All statistics were calculated at least
three to five independent biological replicates from independ-
ent plants. All the data were conducted by Student’s t test and
one-way ANOVA using Duncan’s new multiple range test. All
figures were designed using OriginPro 2021 and PowerPoint
2016.
Results
MG Application Enhanced Drought Resistance Based
onYield Performance andComponents
The agronomic traits of ears and kernels at maturity stage
in response to drought coupled with MG application were
determined. Without MG application, drought strongly
imposed the reduction of yield components and yield com-
pared with the well-watered conditions (Fig.2). Specifi-
cally, the kernel number was significantly decreased by
29% and 33%, and kernel weight was significantly declined
by 9 and 25%, but the abortion length was twofold and
1.95-fold higher, under DS1 and DS2, respectively
(Fig.2B–D). Consequently, the yield was approximately
reduced by 33 and 50% under DS1 and DS2, respectively
(Fig.2E).
Whereas dose-dependent manner of MG increased
the kernel number by 28, 29, and 34% under DS1, and
27, 45, and 13% under DS2, respectively, compared with
control (Fig.2B), the 500 kernel weight was significantly
higher by 12% than control in 15mM treatment under DS2
(Fig.2C). Compared with control, abortion length was
significantly decreased with MG applying under drought
conditions (Fig.2D). Furthermore, compared with control,
MG application increased the yield by 14, 24, and 29%
with dose-dependent manner of MG under DS1. Similarly,
MG application in 15 and 25mM treatments significantly
improved the yield by 48 and 48%, respectively, compared
with control under DS2. These results indicate that exog-
enous MG application enhanced the drought tolerance by
declining the yield penalty under drought condition.
Exogenous MG Application Postponed Leaf
Senescence Under Drought Conditions
Water control was commenced at 3 DAP, and we continu-
ously monitored the soil relative water content for reach-
ing the gradient drought. Furthermore, we distinguished
both drought conditions by the extent of leaf curling and
kernel abortion rate from our previous studies (Shen etal.
2020a; 2020b). Started from 15 DAP, strong effects of
drought on leaves were emerged, and soil relative water
content reduced to approximately 50 and 40% in accord-
ance with DS1 and DS2, respectively (Fig.1). Leaves were
seriously curved and color fading especially in the lower
part of leaves (data not shown). Therefore, in order to
monitor and quantify the leaf senescence, total green leaf
area and SPAD values were determined at 7 DAI and 14
DAI. Leaves were color-faded and curved with the gradi-
ent drought, whereas there is no clear trend or change on

3938 Journal of Plant Growth Regulation (2023) 42:3934–3946
1 3
total green leaf area and SPAD values of ear leaf, under
well-water and DS1 conditionat 7 DAI (Fig.3A, B).
However, MG application significantly increased the total
green leaf area in 15mM treatment by 11% compared with
control under DS2. Moreover, exogenous MG significantly
delayed the 9th leaf senescence by maintaining 10, 13,
and 32% higher than control with dose-dependent manner
under DS1. Likewise, SPAD values of 9th leaf with MG
application in 15 and 25mM treatments were significantly
increased by 29 and 33%, respectively, compared with con-
trol (Fig.3C).
With prolonged drought conditions, the effects of MG
were pronounced. Similarly, exogenous MG did not affect
the green leaf area and SPAD values under well-watered
conditions (Fig.3D–F). Under DS1, MG application effi-
ciently postponed leaf senescence, and the total green leaf
area was 10, 10, and 15% higher than control with dose-
dependent manner (Fig.3D). This consequence was attrib-
uted to alleviating the lower part of leaves’ senescence,
which was approximately 22, 40, and 37% higher than con-
trol (Fig.3D). Under DS2, the total green leaf area was sig-
nificantly escalated by 25% in treatment 15mM compared
with control. Lower part of leaves area was significantly
81 and 42% higher in treatments 15 and 25mM, respec-
tively, compared with control (Fig.3D). In addition, the
leaf-senescing trend of SPAD values of ear leaf consisted
of SPAD values of 9th leaves under drought conditions.
Under DS1, the SPAD values of ear leaves and 9th leaves
in treatment 35mM were 14 and 26% higher than control,
respectively (Fig.3E, F). Moreover, under DS2, the SPAD
values of ear leaves in treatments 15 and 25mM were 9 and
8% higher than control. Compared to control, SPAD values
of 9th leaves in 15 and 25mM treatments were elevated by
34 and 44%, respectively (Fig.3E, F).
Exogenous MG Improved thePhotosynthetic Rate
andFv/Fm Under Severe Drought
During the process of leaf senescence, chloroplast degrada-
tion occurred that declined the source strength in leaves.
In our study, without MG application photosynthetic rate
was significantly decreased under DS2, implying that the
functions of source leaves were severely affected by the
adverse conditions. Interestingly, the rate of photosynthesis
Fig. 2 The effects of exog-
enous MG on A phenotype of
ear, B–C yield components, D
abortion length, and E yield
under well-watered and drought
conditions at maturity. CK,
well-watered; DS1, moder-
ate drought; and DS2, severe
drought. Data are means (± SE).
Asterisks indicate significant
difference between well-watered
and drought treatments (t test,
n = 5, *p < 0.05, **p < 0.01,
***p < 0.001). One-way
ANOVA was conducted using
Duncan’s new multiple range
test, and different letters above
the bars indicate significant dif-
ferences (p < 0.05)
a
c
b
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CK DS1DS2
100
150
200
250
300
dleiY(rae g
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***
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300
450
600
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rebmun lenreK ( rae niarg
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***
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0mM 15mM 25mM 35mM
a
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120
160
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A
B
C
DE

3939Journal of Plant Growth Regulation (2023) 42:3934–3946
1 3
was slightly declined with dose-dependent manner of MG
and decreased by 25 and 10% in treatment 35mM under
well-watered conditions and DS1, respectively (Fig.4a).
In contrast, the photosynthetic rate was higher than control
approximately by 102, 310, and 134%, respectively, with
dose-dependent manner of MG under DS2 (Fig.4a). Fur-
thermore, the maximal quantum efficiency of PSII (Fv/Fm)
was related to the optimal capacity of photosynthesis. As
shown in Fig.4b, there was no significant difference in Fv/
Fm from dose-dependent manner of MG under well-watered
CK DS1 DS2
0
2500
5000
7500
10000
12500
aera fael neerg latoT (cm
2
)
NS
NS
0mM
15mM 25mM 35mM
0mM 15mM 25mM 35mM
Upper
Lower
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40
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tnetnoc llyhporolhc evitaleR
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0mM 15mM 25mM 35mM
**
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***
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ABC
D
EF
7DAI
14 DAI
Fig. 3 The effects of exogenous MG on A, D total green leaf area, B,
E SPAD values of ear leaf, and C, F SPAD values of 9th leaf under
well-watered and drought conditions at DAI 7 and DAI 14. Lower:
Represented the sum of leaf area from 1st to the ear leaf; upper:
summed up rest of the leaf area. CK, well-watered; DS1, moderate
drought; and DS2, severe drought. Data are means (± SE). Asterisks
indicate significant difference between well-watered and drought
treatments (t test, n = 5, NS non-significant difference, *p < 0.05,
**p < 0.01, ***p < 0.001). One-way ANOVA was conducted using
Duncan’s new multiple range test, and different letters above the bars
indicate significant differences (p < 0.05)
CK DS1DS2
0.6
0.7
0.8
0.9
1.0
mF/vF
aaaaaa
aaaaa
b
NS
*
a
a
b
aa
a
aaa
aa
a
CK DS1DS2
0
10
20
30
40 0mM 15mM 25mM 35mM
etar citehtnysotohP ( m lom
-2
s
-1
)
NS
**
AB
Fig. 4 The effects of exogenous MG on A photosynthetic rate and
B Fv/Fm of ear leaf under well-watered and drought conditions at
DAI 10. CK, well-watered; DS1, moderate drought; and DS2, severe
drought. Data are means (± SE). Asterisks indicate significant differ-
ence between well-watered and drought treatments (t test, n = 5, NS
non-significant difference, *p < 0.05, **p < 0.01, ***p < 0.001). One-
way ANOVA was conducted using Duncan’s new multiple range test,
and different letters above the bars indicate significant differences
(p < 0.05)

3940 Journal of Plant Growth Regulation (2023) 42:3934–3946
1 3
and DS1 conditions. In contrast, Fv/Fm was significantly
higher by 5, 6, and 4% with dose-dependent manner com-
pared with control under DS2, respectively. These results
suggest that the effect of MG application could improve the
source strength on ear leaves and maintain the relative higher
capacity of maximal PSII photochemistry than control.
MG Application Increased theSoluble Sugar
andSucrose Levels Under Well‑Watered
andDrought Conditions
The sugars accumulation was considered the onset of the
leaf senescence. The levels of intermediates in carbohydrate
metabolism were investigated. At 7 DAI, without MG appli-
cation the content of soluble sugar and sucrose was signifi-
cantly elevated by 55 and 177% under DS1, 82 and 244%
under DS2 compared with well-watered conditions (Fig.5A,
B). However, there is no difference in starch content between
treatments (Fig.5C). In 25–35mM treatments, the content
of soluble sugars and sucrose was approximately 200 and
450% higher under well-watered, around 60 and 51% higher
under DS1 compared with control, respectively (Fig.5A,
B). Moreover, the content of soluble sugars and sucrose was
approximately elevated by 31 and 28% in treatments 15 and
25mM, respectively (Fig.5A, B), compared with control
under DS2.
At 14 DAI, rapid-filling stage of kernel development,
storage accumulation relies on assimilates supplied from
the source leaves (Shen etal., 2022). In consistent with the
results from 7 DAI, without MG application the content of
soluble sugar and sucrose was significantly increased under
DS2 and both drought conditions, respectively (Fig.5D, E).
Also, there is no difference in the content of starch between
treatments (Fig.5F). Furthermore, the content of soluble
sugar and sucrose in 25 and 35mM treatments was 1.3-
and 2.2-fold higher, respectively, compared with control
under well-watered conditions (Fig.5D, E). However, under
drought conditions, the content of soluble sugar and sucrose
was slightly declined with dose-dependent manner of MG
which was totally different from the trend at 7 DAI. The
content of soluble sugar was declined by 15% in 35mM
treatment under DS1, 18 and 13% lower in 15 and 25mM
treatments under DS2, respectively, compared with control
(Fig.5D). Likewise, compared with control, the content of
sucrose was decreased by 12% in 35mM treatment under
DS1, 44% and 12% lower in 15 and 25mM treatments under
DS2, respectively (Fig.5E).
aa
a
a
a
a
a
a
a
a
a
a
CK DS1 DS2
0
20
40
60
80
NS
NS
baa
ab
aa
ab
aa
a
aa
CK DS1DS2
20
30
40
50
60
70
tnetnoc raguS ( g gm
-1
DW)
NS
*
caa
ab
a
b
bc
a
ab
a
aab
CK DS1 DS2
0
5
10
15
20
25
30
*
*
a
a
a
aa
a
a
a
a
a
a
a
CK DS
1D
S2
0
10
20
30
40
50
NS
NS
b
cb
b
bc ab
a
ab a
aa
ab
CK DS1 DS2
0
10
20
30
40
50
60
70
0mM 15mM 25mM 35mM
tnetnoc raguS (g gm
-1
DW)
*
**
b
b
b
b
ab ab
a
ab a
aa
b
CK DS1DS2
0
5
10
15
20
25
30
**
**
ABC
DE
F
7 DAI
14 DAI
Soluble sugarSucrose Starch
Fig. 5 The effects of exogenous MG on sugar content of ear leaf at
7 and 14 DAI, including A, D soluble sugar, B, E sucrose, and C, F
starch under well-watered and drought conditions. CK, well-watered;
DS1, moderate drought; and DS2, severe drought. Data are means
(± SE). Asterisks indicate significant difference between well-watered
and drought treatments (t test, n = 3, NS non-significant difference,
*p < 0.05, **p < 0.01, ***p < 0.001). One-way ANOVA was con-
ducted using Duncan’s new multiple range test, and different letters
above the bars indicate significant differences (p < 0.05)

3941Journal of Plant Growth Regulation (2023) 42:3934–3946
1 3
In this case, sugars levels were significantly higher
than well-watered under drought conditions. Moreover,
MG application elevated the content of sugars under well-
watered at 7, 14 DAI and drought conditions at 7 DAI.
MG Application Stimulated theGlyoxalase
System andPrevented Excessive Accumulation
ofEndogenous MG Under Drought Conditions
The toxic metabolites would magnificently accumulate in
cells that originated from glycolysis under abiotic stress,
leading to inhibiting cell development and functioning.
Therefore, we investigated the MG metabolisms and MG-
scavenging system as well.
Drought imposed the excessive MG accumulation, where
the content of MG was 44% higher under DS1 and 102%
higher under DS2 compared with well-watered condition
(Fig.6A), whereas the content of MG was 13, 18, and 34%
lower, respectively, compared with the control with dose-
dependent manner of MG under well-watered condition
(Fig.6A). On the other hand,MG content was significantly
declined by 44% in treatment 35mM and 17, 24% in treat-
ments 15, 25mM compared with control under DS1 and
DS2, respectively (Fig.6A). Similarly, at 14 DAI, MG
content was significantly increased with gradient degree of
drought (Fig.6D). However, MG content was significantly
decreased by 25% in 35mM treatment under DS1 and 23,
21% lower in 15 and 25mM treatments under DS2, respec-
tively, compared with control (Fig.6D).
The reduction of MG content was attributed to the activa-
tion of glyoxalase system. Both main enzymes GLYI and
GLYII eliminated excessive accumulation of MG in plants
cell. At 7 DAI, without MG application the activities of
GLYI and GLYII were enhanced by 59 and 101% under
DS1, 82 and 32% under DS2 compared with well-watered
condition, respectively (Fig.6B, C). Notably, the activities
of GLYI and GLYII in 35mM treatment were increased by
130 and 92% under well-watered, 220 and 39% under DS1
compared with control, respectively (Fig.6B, C).
Furthermore, both activities of GLYI and GLYII in
treatments 15 and 25mM were significantly induced by 52
and 32%, 47 and 48% compared with control under DS2,
respectively (Fig.6B, C). Likewise, at 14 DAI, the activity
of GLY I was increased 39% with applying 25mM of MG
compared with control, and the activity of GLY II in 25
and 35mM was elevated 44 and 60% compared with con-
trol under well-watered condition (Fig.6E, F). Moreover,
the activity of GLY I in 15mM treatment was significantly
induced by 31% compared with control under DS2 (Fig.6E).
The activity of GLY II was significantly induced by 52% in
a
a
a
a
ab
b
a
bc ab
ac
ab
CK DS1 DS2
20
40
60
80
tnetnoc GM (mg lom
-1
FW)
*
*
a
a
a
a
ab
b
aab
b
ab
b
CK DS1DS2
0
20
40
60
tnetnoc GM ( mg lom
-1
FW)
NS
**
0mM 15mM 25mM 35mM
cbb
bc
b
a
ab ab ab
a
aab
CK DS1 DS2
0
50
100
150
200
ytivitca II esalaxoylG
(mg lom
-1
nim WF
-1
)
NS
NS
b
c
b
b
bc ab
ab
a
a
a
ab
b
CK DS
1D
S2
0
50
100
150
200
250
ytivitca II esalaxoylG
(mg lom
-1
nim WF
-1
)
*
NS
a
a
bc
a
a
a
a
ab
a
ac
CK DS1 DS2
0
50
100
150
200
ytivitca I esalaxoylG
(mg lom
-1
nim WF
-1
)
NS
NS
b
b
b
b
ab
a
ab
a
a
a
a
b
CK DS1DS2
0
50
100
150
ytivitca I esalaxoyl
G
(mg lom
-1
nim WF
-1
)
***
***
D
AB
C
EF
DAI 7
DAI 14
Fig. 6 The effects of exogenous MG on endogenous MG content and
glyoxalase system at 7 and 14 DAI, including A, D MG content, B,
E activity of glyoxalase I, and C, F glyoxalase II under well-watered
and drought conditions. CK, well-watered; DS1, moderate drought;
and DS2, severe drought. Data are means (± SE). Asterisks indicate
significant difference between well-watered and drought treatments
(t test, n = 3, NS non-significant difference, *p < 0.05, **p < 0.01,
***p < 0.001). One-way ANOVA was conducted using Duncan’s new
multiple range test, and different letters above the bars indicate sig-
nificant differences (p < 0.05)

3942 Journal of Plant Growth Regulation (2023) 42:3934–3946
1 3
35mM treatment under DS1, and 59 and 38% higher in 15
and 25mM treatments under DS2, respectively, compared
with control (Fig.6F).
The results above indicate that the effect of MG applica-
tion scavenged excessive MG with higher activities of GLYI
and GLYII in leaf tissue. Moreover, our results also sug-
gest that the MG application resists higher level of sugars
in leaf tissues without accumulating excessive MG under
well-watered and drought conditions.
Correlations Between Endogenous MG, Glyoxalase,
Sugar Contents, SPAD, andYield
In order to explore the correlation between sugar content,
endogenous MG content, leaf senescence, and yield out-
put in response to drought stress without any interference
from exogenous MG effects, the Pearson correlation analysis
was performed. Drought imposed the sugar accumulation
in senescing leaves (Fig.2; Fig.5). The soluble sugar and
sucrose content was positively correlated with endogenous
MG content, and the coefficients were 0.91 and 0.71 with P
value less than 0.01 and 0.001, respectively (Fig.7A), indi-
cating significant correlations between sugar contents and
endogenous MG content. SPAD value, an indicator of leaf
strengthen, was negatively correlated with soluble sugar,
sucrose, and endogenous MG content, and the coefficients
were −0.59, −0.82, and −0.58 with P value less than 0.01,
0.001, and 0.05, respectively. Moreover, the SPAD value
was positively correlated with yield production (r = 0.97,
P < 0.001) (Fig.7A). The correlation analysis demonstrated
that SPAD was associated with sugar content, endogenous
MG content, and yield.
GLY I facilitates the reaction scavenging endogenous
MG as a substrate and thus plays an important role in MG
homeostasis (Veena etal. 1999). Importantly, the key roles
of GLYI have been demonstrated by the improved stress
tolerance and yield output via overexpression of OsGly I in
rice (Zeng etal. 2016). Thus, to further investigate exog-
enous MG impact on drought tolerance during grain filling,
a correlation between GLY I activity and endogenous MG
content was carried out under well-watered and both drought
conditions, separately. Under well-watered condition, the
endogenous MG content did not show significant correla-
tion with the GLY I (Fig.7B). However, the endogenous
MG content demonstrated significantly negative correla-
tions with the GLY I under both DS1 (R2 = 0.33, P < 0.01)
and DS2 conditions (R2 = 0.36, P < 0.01) (Fig.7B). These
results further support the key role of GLYI in elimination
of endogenous MG and implied that the retrieved yield and
postponed leaf senescence by exogenous MG application
might be attributed to the enhanced MG detoxification under
drought conditions (Figs.6 and 7).
Discussion
Considered the dual roles of endogenous MG in plant growth
regulation, previous studies explored the effect of exog-
enous MG application to deal with environmental stresses.
To date, only a few studies pointed out that exogenous MG
Fig. 7 A Pearson correlation analysis demonstrated the association
between the variables. Left bottom of the diagonal: the values of cor-
relation coefficient. Right top of the diagonal: the level of significance
and marked by asterisks. The size of the cycle represented the value
of correlation, and the colors of red and blue indicated the positive
and negative correlations, respectively. B The correlation between
endogenous MG content and GLYI activity under well-watered and
both drought conditions. The area represented the confidence inter-
vals of 95%. The asterisks indicated the significant differences at
*p < 0.05, **p < 0.01, ***p < 0.001, respectively. The dash and solid
lines indicated non-significant and significant levels, respectively.
CK, well-watered; DS1, moderate drought; DS2, severe drought; and
Endo. MG content, endogenous MG content

3943Journal of Plant Growth Regulation (2023) 42:3934–3946
1 3
applying increased the tolerance of instant stresses at seed-
ling stage including frost and heat (Li, 2016; Mostofa etal.,
2018), whereas the effects of exogenous MG on leaf senes-
cence and yield formation in response to continuous drought
tolerance during grain filling were yet reported. Thus, this
study explored the exogenous MG effect on maize growth in
response to continuous drought during the critical stage of
grain filling and the subsequent yield performance at maturity.
Physiological processes, in terms of carbohydrate metabolism
and MG homeostasis, were determined to evaluate the under-
lying mechanisms of MG regulation.
Exogenous MG Enhances Continuous Drought
Tolerance andRetrieves Drought‑Induced Yield Loss
It has been precisely described that development of kernels
was hindered resulted from the limited assimilates and dis-
equilibrium in various metabolic pathways (Zhang etal.,
2017; Shen etal., 2020a, 2020b). Grain-filling stage was
closely related to yield potential, subjected to drought at this
period caused serious apical kernel abortion and yield reduc-
tion (Zhang etal., 2017, 2018). In this study, MG application
increased the kernel number and kernel weight, but decreased
the abortion length, under drought conditions (Fig.2A–D).
Moreover, MG application significantly elevated the yield out-
put by ~ 14 and ~ 48% under DS1 (25 and 35mM MG appli-
cation) and DS2 (15 and 25mM MG application), respec-
tively (Fig.2). Similarly, overexpression of GLYI and GLYII,
encoding glyoxalase that detoxified excessive MG, enhanced
the yield or without yield penalty under various stress con-
ditions had been previously characterized. Effectively elimi-
nated cytotoxic MG in cells was able to develop the offspring
normally under stress conditions (Singla-Pareek etal., 2003;
Zeng etal., 2016; Gupta etal., 2018; Askari-Khorasgani and
Pessarakli, 2019). In our study, according to the yield perfor-
mance, we assessed the optimal concentration of MG applying
high concentration (25 and 35mM) under DS1 conditions and
low concentration (15 and 25mM) of MG applying under
DS2 (Fig.2). We consider that under DS1, the endogenous
MG level was not accumulated at level that may be hazard-
ous in leaf tissue. High concentration of MG applying could
effectively induce MG-related metabolism activation. Besides,
MG magnificent accumulation under severe drought, applying
high concentration of MG may result in over-dose symptom
and cause toxic responses, vice versa. Interestingly, the yield
and kernel number were significantly increased in treatment
15 and 35mM under DS1 and DS2, respectively (Fig.2B).
However, the kernel weight was significantly decreased in both
treatments under drought conditions (Fig.2C). Here, we point
out several hypotheses, including (i) source limitation like
reduced photosynthesis and blocked phloem loading and (ii)
sink limitation from the source–sink communication (Liang
etal., 2020). However, the underlying mechanism remains
unknown.
Exogenous MG Enhances theSource Strength Under
Drought Conditions
Leaf senescence would be accelerated under drought stress
at grain-filling stage with the orders from the lower to the
top leaves of maize (NeSmith and Ritchie, 1992). Such del-
eterious effect may decrease the leaf area and inhibit the
capabilities and photosynthesis, exacerbating competing for
the limited photo-assimilates between kernels (Shen etal.,
2018, 2020b, 2022; Ye etal., 2020).
In this study, relative chlorophyll content and green leaf
area were significantly decreased with increasing extent of
drought, especially in leaves from lower position (Fig.3).
Nevertheless, MG application with optimal concentration
under DS1 and DS2 significantly maintained the relative
chlorophyll content and delay lower part of leaf senescence
at 7 and 14 DAI. Similarly, Li etal. (2018) and Wang etal.
(2019) reported that exogenous MG application enhanced
the heat tolerance of maize seedling by stabilizing plasma
membrane and cell integrity. These results indicate that MG
application with optimal concentration elevates the toler-
ance under drought conditions by maintaining the function
of source leaf. Although the photosynthetic rate was slightly
decreased with MG applying under well-watered and DS1
conditions (Fig.4a), the increased yield suggests that photo-
assimilate was overall increased (Fig.2). Saito etal. (2011)
and Takagi etal. (2014) elucidated that MG would inevi-
tably generate and trigger the oxidative damage in chloro-
plast while intensifying the photosynthesis under high light
and high CO2 condition. MG was reduced by PSI as hill
oxidant and spontaneously produced O2−. Our results sug-
gest that MG application provoked the negative feedback
of MG homeostasis that prevented oxidative stress effect
from MG excessive accumulation under well-watered and
DS1 conditions. Furthermore, the photosynthetic rate and
Fv/Fm were significantly decreased under DS2 (Fig.4),
reflected damage to PSII system by oxidative stress. To sup-
port this, previous studies also demonstrated that declined
in Fv/Fm was as a result of damage to PSII or dissipation of
the excess excitation away from PSII (Franklin etal., 1992;
Saito etal., 2011). However, the photosynthetic rate and Fv/
Fm were significantly increased with optimal MG applying
under DS2 (Fig.4). Our results imply that MG application
triggered the protection within oxidative stress in chloro-
plast and maintained the higher efficiency of source strength
under DS2 (Hoque etal., 2012; Mostofa etal., 2018; Wang
etal., 2019).

3944 Journal of Plant Growth Regulation (2023) 42:3934–3946
1 3
MG Application Improves theCapacity ofSugar
Content Without Accumulating Excessive MG
byActivation ofGlyoxalase System
It has been found that imbalance of carbohydrate metabo-
lism affected various other regulations including onset of
senescence (VanDoorn, 2008). Exogenous application of
sugars induced the expression of senescence-associated
genes and resulted in the early reduction of Fv/Fm which
decreased prior to degradation of chlorophyll in leaf tissue.
These results indicated that hexoses accumulation could
induce leaf senescence without accelerating development
(Wingler etal., 2004; Pourtau etal., 2006). However, over-
loading of hexoses in cells enhanced the carbon metabolism
resulting in producing unavoidably cytotoxic by-product MG
from glycolysis and Calvin cycle (Takagi etal., 2014; Li,
2016). Exogenous MG precursors G3P in Arabidopsis wild
type decrease the chlorophyll content similar to pdtpi mutant
which was tended to overproduce MG (Chen and Thelen,
2010). Therefore, these negative progressions and phenom-
ena were referred to as plant diabetes in previous studies
(Shimakawa etal., 2014; Takagi etal., 2014). In the present
study, the contents of soluble sugar, MG level, and extent
of leaf senescence were all significantly increased with
gradient drought conditions (Fig.3; Fig.5A, B; Fig.6A).
Moreover, we presumed that the hexose accumulation and
sugar-derived MG overproduction were associated with the
process of leaf senescence (Fig.7A). In contrast, with opti-
mal concentration of MG applying, the content of soluble
sugar and sucrose was significantly increased in compari-
son with control under well-watered and drought conditions
(Fig.5A, B). Additionally, the MG content was significantly
reduced due to the activation of GLY I and GLY II with
optimal concentration of MG (Fig.6A–C). The results were
consistent with previous studies (Li etal., 2018; Wang etal.,
2019; Majláth etal., 2020), MG pretreatment and MG foliar
spraying could scavenge the endogenous MG resulting from
inducing the expression and activation of glyoxalase system.
Therefore, we consider that drought stress causes the sugar
accumulation in leaves and inevitably produced considerable
MG, which mainly caused the leaf senescence (Fig.7A). Our
results suggest that MG application mitigated the adverse
effect of plant diabetes, thereby delaying leaf senescence
under drought conditions. This consequence was via enhanc-
ing the capability in high concentration of sugars and with-
out accumulating excessive MG in leaf tissue by inducing
the activities of glyoxalase system (Fig.6; Fig.7B; Fig.8).
Interestingly, the contents of soluble sugar and sucrose
were significantly increased by applying high concentrations
of MG under well-watered condition; however, they were
slightly declined with MG application under both drought
conditions (Fig.5D, E). The degrees of reduction in soluble
sugar and sucrose in leaves and the escalation of starch in
kernels were in corresponding to the amounts of MG appli-
cation under drought conditions (Fig.5D, E; Fig. S1). Con-
sidering that the kernel yield was significantly promoted by
MG application during the critical stage of kernel filling
(Fig.2), when sugars were rapidly transported into kernels
(Shen etal., 2022), we assumed that this discrepancy of sug-
ars content in leaves may be because of considerable assimi-
lates allocating from the source into the kernels (Fig.5).
Therefore, we hypothesize that MG application enhances
the tolerance to plant diabetes and promotes sugar allocation
from the source to the sink.
Fig. 8 Hypothesized scheme
of exogenousMG effects on
continuous drought tolerance
and underlying mechanisms

3945Journal of Plant Growth Regulation (2023) 42:3934–3946
1 3
Conclusions
Exogenous application of MG enhances continuous toler-
ance to drought, improves the source strength, and retrieves
the yield loss under drought conditions. Moreover, MG
application increases the soluble sugar and sucrose content
without overproduction of endogenous MG by stimulation
of glyoxalase system under drought condition. Collectively,
these findings provide a new theoretical and practical bases
to maintain or promote yield in maize production in face of
drought stress.
Supplementary Information The online version contains supplemen-
tary material available at https:// doi. org/ 10. 1007/ s00344- 022- 10862-4.
Acknowledgements This research was supported by theearmarked
fund for CARS(CARS-02-16), National Natural Science Foundation of
China (32272013), and the Deutsche Forschungsgemeinschaft (DFG;
328017493/GRK 2366).
Author Contributions YHL, SS, and SLZ designed the study. YHL,
YKJ, ZYC, and ZDX performed the experiments and collected the data.
YHL analyzed and interpreted the data. YHL, SS, and SLZ wrote the
drafts. YHL, SS, and SLZ revised the manuscript. All authors approved
the final version of the manuscript.
Funding Funding was provided by MOF and MARA, and the Deutsche
Forschungsgemeinschaft, DFG, Shun-Li Zhou, 328017493/GRK
2366), Shun-Li Zhou, National Natural Science Foundation of China,
32272013, Si Shen
Declarations
Conflict of interest The authors declare that they have no conflict of
interest.
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