Apple (Malus
domestica Rehd.) is one of the
important cash crops and is mostly cultivated in hills and mountains in China.
Its growth and development are vulnerable to various stresses, such as drought
and heat stress, due to its special grown environments. Previous studies showed
that under drought stress, apple growth is reduced due to leaf wilt, direction
changed and angle changed and branch thickening (Wang 2017). Moreover, under
drought stress, the net photosynthetic rate of plants decreased, and the
ultra-structure of chloroplasts is also damaged (Hussain et al. 2018). Moreover, under high temperature stress,
denaturation, degradation, synthesis and assembly of membrane proteins to be
blocked, and cell membrane lipid peroxidation take place leading to reduced CO2
fixation (Mathur et al. 2014; Hussain et al. 2018).
According to statistics, from 1952 to 2006, only the drought caused a loss of
380 million yuan in apple-producing areas of Xianyang City (Ma et al. 2008). Meanwhile, due to the
greenhouse effect, the number of high temperature days increased significantly
(Zhou et al. 2011). The decrease of
photosynthesis caused by drought stress or heat stress is an important reason
for crop yield reduction. With global warming, summer drought and high
temperature has become the limiting factors of apple development in China (Wang
2017).
In recent years,
the development of plant stress resistance research has played an important
role in enhancing crop yield under stress. Studies have found that plants
respond differently to drought, heat stress and their combination.
Transcription mode, metabolic mode and molecular reaction are obviously
different (Rizhsky et al. 2002, 2004;
Shulaeva et al. 2008). However, most
of these studies are single-stress adaptation studies under laboratory control
conditions, while field stresses are more complex than indoors where often
multiple stresses occur at the same time such as drought and heat stress, or
drought and cold stress (Mittler et al.
2001; Moffat 2002). Mittler (2006) proposed that in order to improve the
tolerance of plants to natural environmental conditions, studies should focus
on the tolerance of environmental conditions coexisting with multiple
adversities, especially the simulation of field environment.
Table 1: The growth information of PYTC seeds used in this
study
|
Category |
Information |
|
Sapling height (cm) |
250-300 |
|
Sapling diameter
(cm) |
15-20 |
|
Collection location |
Shuohang Fruit Professional Cooperative of Mengyin County, Shandong Province, China |
|
Planting area (km2) |
26680 |
|
Total growth amount of plants
(tree) |
5000 |
|
Average temperature (°C) |
8.6-12.8 |
|
Sunshine time (h) |
>7 |
|
Shading rate (%) |
40-50 |
Note: PYTC: Pingyi Tiancha (Chinese name
of M. hupehensis var. pinyiensis Jiang)
Glycine
betaine (GB) is considered as a very effective osmotic adjusting substance and
stress-resistance factor (Hussain et al.
2008; Kurepin et al. 2015). It plays
an important role in improving plant resistance to single stress such as
drought or heat stress (Hussain et al.
2008; Anjum et al. 2011). However,
these studies indicated that GB might play an important role in enhancing plant
tolerance to individual drought and heat stress, and these research on GB
stress resistance mainly focuses on annual plants and a few woody plants (Ahmad
et al. 2013; Li et al. 2013, 2014). There is still limited data on GB’s improvement
of plant resistance to the combination of drought and heat stress.
Moreover,
what is the content of GB in apple (Malus)? Is GB also synthesized and
accumulated in apple under drought, heat and other environmental stresses? What
is the relationship between GB and stress resistance of Malus? So far, there is little research. The PYTC is a widely used
rootstock variety for apple cultivars in China. Understanding the resistance of
PYTC to stress and improving its resistance to stress are important for the
development of apple industry. Therefore, this study was conducted to evaluate
the impact of drought and heats stress applied alone and in combination on
photosynthesis of PYTC seedlings.
Moreover, GB was applied to find out the possible physiological and
biochemical mechanisms of GB in improving the resistance of PYTC seedlings to the three stresses.
PYTC seeds were
laminated mixed with fine sand in a refrigerator at 4°C for 20 days after being
saturated with water. Then, the germinating seeds were planted orderly into
plastic pots (height 8 cm, diameter 10 cm) containing quartzite, with a density of four plants per pot. The
plants were grown in an artificial chamber at 25/20°C with a photon flux
density of 300-400 μmol m-2 s-1,
a relative humidity of 65~70%, and a photoperiod of 14/10-h
light/dark. The plants were first cultured with distilled water to the
three-leaf-and-one-leaflet period, then distilled water was replaced with 1/2
Hoagland nutrient solution. Post-1 week, 1/2 Hoagland nutrient solution were
replaced with whole Hoagland nutrient solution. When the 6th leaf
was expanded fully, the plants were used for experiment treatment.
Treatments
details
Before stresses were imposed, an aqueous solution
of 10.0 mM GB (+GB)
(optimal concentration according to pre-test) (Wang et al. 2014) and 0.1% (v/v) Tween 20 (1 mL
of Tween 20 was added to 1 L of distilled water) (control, -GB) were sprayed on leaves until runoff was detected. The
solution was sprayed twice a day at 6:00 a.m. and 6:00 p.m. for 3 days. After
GB application, some of the GB treated and control treated plants were
subjected to drought, heat stress and the combination of the drought and heat
stress. Drought was induced with 30% (w/v) PEG-6000 (osmotic potential of about
-1.85 MPa) until reaching relative water content (RWC) of 80 to 87% (to
moderate drought stress level, 96 h; control (normal watering and normal temperature):
94.1 to 94.3%). A combination of drought and heat stress was performed by
subjecting drought-stressed plants to a high temperature 42°C for 3 h
(illumination 300-400 µmol/(m2·s),
humidity 65~70%), Single heat stress was applied by raising the temperature in
artificial chamber to 42°C for 3 h at the same time when stress combination was
executed. Thus, all treatments included single drought stress (DS), single heat
stress (HS), the combination of drought and heat stress (DS+HS) and normal
watering and normal temperature (WW). All the experiments were performed in
triplicate parallels and all the determinations were repeated three times. The
physiological parameters involved in stress tolerance were observed as below.
Table 2: Effects of foliar-applied
glycinebetaine (GB) on the GB contents in leaves of PYTC seedlings subjected to drought stress, heat stress and
their combination
|
Treatment |
+GB |
-GB |
||||||
|
WW |
DS |
HS |
DS+HS |
WW |
DS |
HS |
DS+HS |
|
|
75.2 ± 4.6d |
130.6 ± 5.2b |
110.6 ± 3.5c |
151.3 ± 4.3a |
28.2 ± 2.3gh |
41.8 ± 3.6ef |
36.7 ± 5.4c |
52.4 ± 3.3e |
|
Note: DW: dry
weight of the leave; WW: well watered control; DS: drought stress; HS: heat
stress; DS+HS: combination of drought stress and heat stress. Values are the
means ± standard errors (S.E.) of three replicates. Means in
a row followed by the different letters indicate significant difference at P < 0.05
Table 3: Chlorophyll (Chl) and carotenoid (Car) contents in leaves of PYTC
subjected to drought stress, heat stress and their combination
|
Treatments |
+GB |
-GB |
||||||
|
WW |
DS |
HS |
DS+HS |
WW |
DS |
HS |
DS+HS |
|
|
Chl
(mg·g-1DW) |
8.75 ± 0.38a |
8.61 ± 0.18a |
8.07 ± 0.53ab |
7.82 ± 0.25b |
8.67 ± 0.27a |
8.49 ± 0.20a |
5.93 ± 0.26c |
5.45 ± 0.21cd |
|
Car
(mg·g-1DW) |
1.43 ± 0.10a |
1.43 ± 0.04a |
1.39 ± 0.01ab |
1.25 ± 0.11c |
1.46 ± 0.06a |
1.26 ± 0.03c |
1.04 ± 0.04d |
0.73 ± 0.03e |
Note: DW: dry weight of the leave; WW: well watered control; DS: drought
stress; HS: heat stress; DS+HS: combination of drought and heat stress. Values
are the means ± standard errors (S.E.) of three replicates. Means
in a row followed by the different
letters indicate significant difference at P
< 0.05
Table 4: Net photosynthetic rate (Pn), transpiration
rate (Tr), stomatal conductance(Gs), intercellular CO2 concentration
(Ci), the apparent quantum yield (AQY) and the carboxylation efficiency of
photosynthesis (CE) of PYTC
seedlings subjected to drought stress, heat stress and their combination
|
Treatments |
+GB |
-GB |
||||||
|
WW |
DS |
HS |
DS+HS |
WW |
DS |
HS |
DS+HS |
|
|
Pn (µmol . m-2.s-1) |
13.9 ± 0.4a |
11.9 ± 0.b5 |
9.3 ± 0.2c |
3.3 ± 0.1f |
13.7 ± 0.1a |
8.6 ± 0.3cd |
6.3 ± 0.1e |
1.0 ± 0.3g |
|
Tr (mmol. m-2.s-1) |
3.2 ± 0.0a |
2.3 ± 0.3bc |
2.5 ± 0.1b |
1.6 ± 0.1f |
3.1 ± 0.0a |
2.0 ± 0.2d |
2.3 ± 0.2bc |
1.2 ± 0.1e |
|
Gs(mmol.
m-2.s-1) |
137.0 ± 9.4a |
68.0 ± 4.6d |
102.0 ± 5.6bc |
46.5 ± 3.6e |
143.0 ± 8.0ab |
99.0 ± 6.8be |
124.0 ± 5.5d |
60.0 ± 9.0f |
|
Ci(µmol . mol-1) |
193.0 ± 12.5bc |
134.0 ± 14.3d |
185.0 ± 9.5c |
196.0 ± 10.3bc |
214.0 ± 11.4ab |
142 ± 11.9d |
226.0 ± 10.6ab |
261.0 ± 18.0a |
|
AQY(CO2.photon-1) |
0.032 ± 0.0003a |
0.026 ± 0.0006c |
0.016 ± 0.0004e |
0.012 ± 0.0001f |
0.030 ± 0.0005ab |
0.020 ± 0.0006d |
0.010 ± 0.0005fg |
0.003 ± 0.0001h |
|
CE(µmol. m-2.s-1) |
0.041 ± 0.006a |
0.028 ± 0.005c |
0.019 ± 0.004d |
0.012 ± 0.003e |
0.039 ± 0.004ab |
0.020 ± 0.003d |
0.011 ± 0.003e |
0.005 ± 0.002f |
Note: WW: well watered control; DS: drought stress; HS: heat stress; DS+HS:
combination of drought and heat stress. Values are the means ± standard errors
(S.E.) of three replicates. Means in a row followed by the different letters indicate significant difference at P < 0.05
Table 5: Changes in maximal
efficiency of PSII photochemistry (Fv/Fm,) , actual
efficiency of PSII (ФPSII), the activities of Ca2+-ATPase
and Mg2+-ATPase in the thylakoid membrane of PYTC seedling leaves subjected to drought stress, heat stress
and their combination
|
Treatments |
+GB |
-GB |
||||||
|
WW |
DS |
HS |
DS+HS |
WW |
DS |
HS |
DS+HS |
|
|
Fv/Fm |
0.840 ± 0.008a |
0.832 ± 0.035ab |
0.697 ± 0.021d |
0684 ± 0.033de |
0.849 ± 0.012a |
0.799 ± 0016c |
0.635 ± 0.013ef |
0.611 ± 0.017f |
|
ФPSII |
0.475 ± 0.022a |
0.326 ± 0.006b |
0.314 ± 0.015b |
0.249 ± 0.027d |
0.476 ± 0.027a |
0.298 ± 0.011c |
0.247 ± 0.022d |
0.208 ± 0.007e |
|
Ca2+-ATPase |
127.7 ± 7.7a |
115.3 ± 7.0b |
92.2 ± 3.4d |
82.4 ± 8.8de |
126.2 ± 7.2a |
106.0 ± 2.5c |
73.1 ± 9.7e |
55.0 ± 5.1f |
|
Mg2+-ATPase |
42.7 ± 0.9a |
30.1 ± 0.7c |
22.0 ± 1.5d |
20.8 ± 2.5de |
40.6 ± 1.7ab |
21.3 ± 1.5d |
13.9 ± 2.0f |
9.1 ± 2.2g |
Note: WW: well watered control; DS: drought stress; HS: heat stress; DS+HS:
combination of drought and heat stress. Values are the means ± standard errors
(S.E.) of three replicates. Means in a row followed by the different letters indicate significant difference at P < 0.05
Photosynthetic gas-exchange parameters: The net photosynthetic rate (Pn),
stomatal conductance (Gs), transpiration rate (Tr), intercellular CO2
concentration (Ci), apparent quantum yield (AQY) and carboxylation efficiency
of photosynthesis (CE) were carried out with 6th fully expanded
attached leaves using a portable infrared gas analyzer (Ciras-2, PP Systems, Norfolk, U.K.). The
light-saturating photosynthetic rate was recorded at a CO2
concentration of 360 μL L-1
and temperature of 25°C with relative humidity of 80% and saturating light (800
μmol m-2 s-1).
The initial slope of the reaction curve was obtained with the linear regression
of data with light intensity below 250 µmol·m-2·s-1 in
the Pn-PPFD reaction curve as the AQY; and the initial slope
obtained by linear regression of data below 250 ppm CO2 in the Pn-Ci
reaction curve was the CE. The photosynthetic parameters were measured for
about 10 min, during which no significant recovery was detected.
Chl a fluorescence analysis: Photosystem (PSII) chlorophyll a fluorescence measurements were performed using an FMS-2 portable pulse modulated
fluorometer (Hansatech, U.K.). The
leaves were first adapted to darkness for 20 min, the fluorescence parameters
under dark adaptation were measured, where Fo was the initial
fluorescence, Fm was the maximum fluorescence, and Fv/Fm=（Fm-Fo）/Fm.
Thereafter, the leaves were exposed to light intensity of 300 µmol·m-2·s-1, and
the fluorescence parameters such as maximum fluorescence (Fm') and
steady-state fluorescence (Fs) under light adaptation were measured,
the actual PSII efficiency under irradiance (ΦPSII) were
calculated according to the formula: ΦPSII = (Fm' –Fs)/
Fm'.
Fig. 1: Effect of GB application on relative leaf
conductivities (A), MDA content (B), the production rate of O2– (C)
and H2O2 content (D)
of PYTC plants subjected to lone and combined drought and heat stress
Means in a row
followed by the different letters indicate significant difference at P < 0.05 (Values are the means ±
standard errors (S.E.) of three replicates)
DW: Leaf dry weight; WW:
Well-watered; DS: Drought stress; GB: Glycine betaine
Fig. 2: Effect of GB application on activities of superoxide
dismutase (SOD, A), catalase (CAT, B), peroxidase (POD, C), and ascorbate peroxidase (APX, D) in leaves of PYTC subjected to alone and
combined drought and heat stress
Means in a row
followed by the different letters indicate significant difference at P < 0.05 (Values are the means ±
standard errors (S.E.) of three replicates)
DW: Leaf dry weight; WW:
Well-watered; DS: Drought stress; GB: Glycine betaine
Water status, free proline and soluble sugar contents of the leaves: The
relative water content (RWC) of the leaves Table 6: Leaf relative water content (RWC), osmotic adjustment
(OA) of PYTC seedlings subjected to
drought stress, heat stress and their combination Treatments +GB -GB WW DS HS DS+HS WW DS HS DS+HS RWC (%) 94.1 ± 2.1a 86.9 ± 1.2bc 92.9 ± 2.3a 82.4 ± 1.8d 94.3 ± 2.1a 80.1 ± 1.1d 89.4 ± 1.1ab 70.1 ± 1.1e OA (Mpa) 0.0 ± 0.0d 0.19 ± 0.01de 0.15 ± 0.00cd 0.22 ± 0.01a 0.0 ± 0.0d 0.15 ± 0.00 c 0.13 ± 0.01f 0.16 ± 0.01c Note: WW: well watered control; DS: drought stress; HS: heat stress;
DS+HS: combination of drought and heat stress. Values are the means ±
standard errors (S.E.) of three replicates. Means in a row followed
by the different
letters indicate significant difference at P < 0.05 Table 7: Leaf total soluble sugars and proline contents of PYTC Seedlings subjected to drought stress, heat stress
and their combination Treatments +GB -GB WW DS HS DS+HS WW DS HS DS+HS soluble sugar content (mg.g-1 DW) 11.0 ± 1.0fg 33.6 ± 1.5b 14.6 ± 1.3e 39.7 ± 4.2a 10.5 ± 0.9fg 25.0 ± 2.0d 12.3 ± 2.3ef 30.3 ± 1.9be Proline content (µg.g-1 DW) 15.8 ± 1.8g 34.3 ± 2.0b 25.5 ± 2.7d 50.7 ± 3.9a 14.2 ± 1.0gh 22.6 ± 1.8de 19.7 ± 1.8ef 30.9 ± 2.6c Note: WW: well watered control; DS: drought stress; HS: heat stress;
DS+HS: combination of drought and heat stress. Values are the means ±
standard errors (S.E.) of three replicates. Means in a row followed
by the different
letters indicate significant difference at P < 0.05
Ion leakage and MDA levels: The
ion leakage from the cellular membrane was determined via conductivity
measurement according to Fan et al.
(1997). Malondialdehyde (MDA) level was assayed according to the method that was described by Zhao et al. (2002b).
Superoxide anion radical (O2-)
production rate and H2O2 contents: The
production rate of O2- was
measured according to the method described by Wang and Luo (1990). The H2O2
contents were measured according to the method of Sairam and Srivastava
(2002).
All experiments were
repeated 3 times, the presented values were the means ± standard errors (S.E.) of three replicates. The test
results were plotted with Sigmaplot 12.0 and the statistical analysis was
conducted using Data Processing System (DPS; Zhejiang University, China). Differences among the GB-pretreated
and the control treated plants or treatments were compared
using Duncan’s multiple range tests at 0.05 probability levels.
Results
Chlorophyll, carotenoid and GB
contents
The contents of
chlorophyll (Chl) and carotenoid (Car) in the leaves of GB-pretreated plants
and control treated plants were determined (Table 2). DS had only caused a
decrease in Car content in the control treated plants. HS significantly
decreased the contents of Chl and Car in the leaves of PYTC, DS + HS further redused the Chl and Car contents in the
leaves of PYTC (P < 0.05). Foliar application of GB alleviated
the decreases of Chl and Car contents under the three stresses. GB content of
the GB pre-treated plants (+GB) was significantly (P < 0.05) higher than that of the
control treated plants (-GB) when no stress treatment was given (Table 2) due
to absorption of exogenous GB by leaves. When subjected to DS, HS and DS+HS, GB
contents in leaves of GB pre-treated and control treated plants increased
significantly (P < 0.05)
than that of WW, DS+HS induced a greater level of GB than individual DS or HS,
and the DS induced the greater level of GB than HS. GB pre-treated plants,
showing that the PYTC plants sensitively responded to these stresses by accumulating
more GB in their leaves (Table 2).
The effects of foliar-applied GB and the stresses on the CO2 assimilation
and other gas exchange parameters were significant (Table 3). The net
photosynthetic rates (Pn) of the leaves of PYTC were significantly inhibited by DS and HS and were
dramatically decreased upon exposure to DS+HS. The degree of inhibition in HS
was more than that in DS, and the degree in co-stress of DS and HS was more
serious than that in single DS or HS. Similar results were observed in apparent
quantum yield (AQY) and carboxylation efficiency (CE) were essentially the same
as the Pn. However, the effects of diferent stress on transpiration
rate (Tr) and stomatal conductance (Gs) were different from the Pn,
AQY and CE. The degree of inhibition of Tr and Gs in DS
was more than that in HS, .and when HS was accompanied by DS, the
degree of inhibition decreased again. Additionally, DS alone decreased Ci in the plants, when the plants
were subjected to a combination of DS and HS; however, Ci was increased significantly. The data in Table 4 also revealed that foliar-applied GB improved Pn and various gas exchange parameters
under all stresses. In particular, the most evident effect of GB is on Gs, then on AQY, and then on CE
(Table 3).
Individual DS had little effect on Fv/Fm, however, HS
and DS+HS decreased
it significantly; all three stress decreased ФPSII, the DS+HS resulted in a more drastic decline of Fv/Fm
and ФPSII than each stress alone. Compared to control treated plants, foliar-applied GB alleviated the decrease of both Fv/Fm and ФPSII under stress conditions. At the same time, the ATP activity of the
thylakoid membrane was also detected (Table 4). DS and HS had significantly
reduced the activity of Ca2+-ATPase and Mg2+-ATPase in
thylakoid membrane, the combined stress had a more drastic decline of these
enzyme activities than with application of each stress type alone. Foliar-applied GB reduced the decrease of the ATP
activity under all three stresses (Table 4).
Data given in
Table 4 showed that HS had no significant effect on the RWC of PYTC leaves. DS caused a significant
decrease in RWC, and DS+HS deteriorated the RWC further. Foliar-applied GB significantly alleviated the decrease
in RWC of PYTC leaves compared to the control treated plants under DS
and DS+HS (P < 0.05). Compared
with WW, under DS and DS + HS, the RWC of the GB-pretreated plants decreased by
7.7 and 12.5% respectively, while that of the control treated plants decreased
by 15.1 and 25.7% respectively. Under stress, the PYTC leaves showed
higher osmotic adjustment ability (Table 5). The response of OA to DS was
greater than that to HS, and the greatest OA was observed under DS+HS. The OAs
of GB-pretreated plants were greater than that in the control treated plants
under the three stresses (Table 4). Soluble sugar and proline contents were increased significantly under DS, HS
and DS+HS (Table 7). The
contents of soluble sugar and proline were highest under DS+HS, than under the
DS, and then under the HS. Moreover, the levels of them in the GB-pretreated
group were dramatically higher than in the control treated plants, this was consistent
with OA levels in Table 4.
Both DS and HS
increased ion leakage significantly (P<0.05), with
the increase being slightly greater under HS than DS, and the greatest increase
was observed under DS+HS (P < 0.05) (Fig.
1A). Similar results were observed with MDA levels (Fig. 1B). All stresses increased O2·–
and H2O2 productions. Compared to DS, HS increased them to a lesser extent,
and the greatest increase was observed under stress combination. However, all these
levels, including O2· – and H2O2 as well as MDA, were
lower in GB-pretreated plants than that in the control treated plants (Fig.
1C). Further, the activities of
several key antioxidant enzymes, namely superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and ascorbate
peroxidase (APX) under different stress conditions were investigated (Fig. 2). The responses
of SOD and APX to DS were observed as increased activity, but that of CAT and
POD were detected as decreased activity. When they were subjected to HS,
however, almost all the antioxidant enzyme activities were decreased except for
APX. A stress combination of drought and heat inhibited SOD and APX activities
further, but pretreatment of DS increased the tolerance of CAT and POD to HS.
Foliar-applied GB increased activities of all four antioxidant enzymes,
especially POD, followed by CAT and APX (Fig. 2).
Results confirmed that PYTC synthesizes and accumulates GB in vivo
naturally; the amount of natural synthesis GB
in PYTC is very
low, which was substantially lower than that of resistant plants such as
Chenopodiaceae and Poaceae (McCue and Hanson 1990; Ma et al. 2003). Foliar-applied GB was
absorbed and accumulated in PYTC leaves; this was consistent with findings in other crop
plants (Ma et al. 2006, 2007). Furthermore,
stress-induced GB was significantly higher in GB-pretreated
plants than that in the control treated plants. GB content reached 75.2–151.3 µmol.g-1 DW in GB
pre-treated plants, but in GB pre-untreated plants it is 28.2-52.4µmol·g-1
DW (Table 2).
The
responses of plants to different stress stimulation maybe different, especially, when they
occur together, different stresses might accrue conflicting or antagonistic
responses (Mittler
2006). In the present study, different responses were observed and interrogated
the underlying mechanisms of Pn to drought, heat, and the
combination in PYTC leaves.
First, the stress combination resulted in a greater decrease in Pn
of PYTC leaves than single
drought stress or heat stress, and heat stress reduced Pn more than
drought stress (Table 3). The negative effect of drought on Pn was
due to decreased Gs that resulted in the decreased Ci; while heat stress and
the combination of drought and heat stress decreased Gs and increased Ci
(except for the HS stressed GB-pretreated plants), suggesting that there are
different mechanisms underlying the decrease in Pn caused by drought
stress and heat stress. It means that the decrease of Pn may result
from the stomatal factors under drought stress, but that it may be due to
non-stomatal factors under heat stress. Second, drought stress limited transpiration rate more seriously than heat stress (Table 3), this
consistent with the higher RWC of the heat stressed plants (Table 4). However,
the heat stress decreased the activity of some enzymes and the cell membrane
integrity more effectively than the drought stress leading to reduced Pn (Fig.
2). These observations were consistent with those of Rizhsky et al. (2002).
Foliar
application of GB increased Pn of PYTC leaves under stresses, which was consistent with previous
studies on tomato and wheat crops (Zhao et
al. 2007; Li et al. 2013; Athar et al. 2015). But what was its
mechanism? Results suggested that foliar application of GB can enhance the
tolerance of PSII and ATPase activities to DS and HS (Table 4) and protect the
structures of chloroplast and thylakoid from the damage by stress (data not
shown). This was consistent with other reports as well (Mamedov et al. 1993). Furthermore, results also
suggested that the foliar application of GB on the water status (Table 6) and
the antioxidative defense system (Fig. 2) were involved in its mechanisms.
Foliar-applied GB and increase in other metabolites (such as soluble sugars and
free proline) reduced the osmotic potential, which facilitated cells’ water
absorption and stomata opening, it was speculated that foliar application of GB
may facilitate the maintenance of aquaporin activity of PYTC leaves under the
stresses, this may be beneficial to the maintenance of photosynthesis.
GB may maintain the photosynthetic capacity not only
through increasing Gs but also by maintaining Rubisco activity (Sage and Kubien
2007) under stress. Correspondingly, foliar-applied GB improved those
parameters also to a greater extent under drought than heat stress, and which
may be related to the greater osmotic adjustment in GB-pretreated plants in
drought condition. The greater OA in GB-pretreated plants under drought than
heat stress resulted from the accumulation of GB and other solutes. This
indicated that high OA value in the leaves of GB-pretreated plants may be
related to more GB content. These observations aligned with the findings
proposed by Wang et al. (2010) with
exceptions. These differences involves the proline accumulation under heat
stress which was inconsistent with that of Rizhsky et al. (2004) in Arabidopsis, and their results were that proline
was not increased under heat stress. It was speculated that it may be related
to different experimental materials and different stress conditions. In this
study, heat stress condition was treating at 42°C for 3 h, while Rizhsky et al. (2004) treated at 38°C for 6 h.
While different approaches were applied which can justify the discrepancies,
future investigations are required to clarify such differences.
Cell membrane stability is often affected by lipid
peroxidation caused by ROS under stress conditions (Sudhakar et
al. 2001; Hussain et al. 2017),
which results in the production of MDA as shown in Fig. 2B. Foliar
application of GB can help to alleviate
lipid peroxidation (Fig. 2B) and maintain the cell membrane stability (Fig. 2A)
consistent with previous studies (Saneoka et al. 2004; Lv et al. 2007). GB acts as a potential scavenger of the toxic ROS
produced during abiotic stress (Banerjee and Roychoudhury 2017). However, GB
could not directly remove reactive oxygen (Hanson et al. 1985), and our results indicated that foliar application of
GB increased antioxidant enzyme activity (Fig. 2), especially POD, then CAT and
APX. The POD can transform some carbohydrates contained in tissues into
lignin and increase the degree of lignification; this may be helpful to keep plants upright under stresses. The CAT and APX both remove H2O2,
which was consistent with the lower H2O2 content of the
GB-pretreated plants (Fig. 1D), the enhanced activities of the antioxidative
enzymes (Fig. 2) may be the major factors involved in the GB-mediated decrease
of ROS (Fig.1C and 1D). Previous studies have shown that GB can stabilize the
stereoscopic structure of enzyme to maintain enzyme activity under high salt,
cold and heat stresses (Gorham 1995). Also it has been reported that GB can
stabilize RuBP carboxylase activity (Yang et
al. 2007). Interestingly, in present study, the results (Fig. 2) have
showed that under normal conditions, the activities of POD and CAT in the
leaves of GB-pretreated plants were higher than that of the control treated
plants. Under stresses, it was speculated that GB was beneficial to stabilizing
the antioxidant enzyme protein on one hand, and may promote the expression of
relevant genes of POD and CAT to enhance their antioxidant activity on the
other hand (Park et al. 2006).
Photosynthesis in PYTC
leaves was affected by drought and heat stresses, effect was more sever under
combined stresses, in different ways. For instance, inhibitions of net
photosynthesis, apparent quantum efficiency and carboxylation efficiency were
more in heat stress while drought effects were more severe on transpiration
rate and stomatal conductance.
Foliar-applied of GB increased the tolerance of plants by alleviating the
inhibition of photosynthesis, due to improvement of water balance and antioxidant
metabolism involved in the protection of the photosynthetic machinery. Moreover,
GB, under stress conditions, application also improved osmotic adjustment
function and relative water contents.
Supported by Youth Research Fund of Shandong Institute of Pomology
(2018KY07); Shandong Provincial Natural Science Foundation, China (No.
ZR2011CM034), and Earmarked Fund for China Agriculture Research System
(CARS-27).
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