South African Journal of Botany 105 (2016) 279–287

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South African Journal of Botany

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

Photosynthetic performance of soybean plants to water deficit under

high and low light intensity
J. Zhang a,1, J. Liu b,c,⁎,1, C. Yang c, S. Du d, W. Yang c,⁎⁎
a
College of Horticulture, Sichuan Agricultural University, Chengdu 611130, China
b
Institute of Ecological Agriculture, Sichuan Agricultural University, Chengdu 611130, China
c
College of Agronomy, Sichuan Agricultural University, Chengdu 611130, China
d
College of Applied Mathematics, Sichuan Agricultural University, Ya’an 625014, China

a r t i c l e i n f o a b s t r a c t

Article history: The two major challenges to relay strip intercropping soybean production in Southwest China are drought and
Received 11 August 2015 low light intensity. This study tests whether the impact of drought on the photosynthetic performance of soybean
Received in revised form 12 April 2016 plants is different between low and high light intensity conditions. To investigate this, soybean plants were
Accepted 15 April 2016
grown in pots in a factorial experiment at two irrigation regimes (75 ± 2% and 45 ± 2% of soil field capacity)
Available online 8 May 2016
and two light intensity treatments (100% and 65% light intensity) in 2011. In 2012, soybean plants were grown
Edited by KI Ananieva in two irrigation regimes (75 ± 2% of soil field capacity vs. progressive soil drying) and two light intensity treat-
ments (sole cropping soybean and relay strip intercropping soybean). Photosynthetic performance was assessed
Keywords: by measuring parameters such as net photosynthetic rate (Pn), stomatal conductance (Gs), water use efficiency
Water deficit (WUE), which were decreased significantly in drought stressed plants. We also observed differences in the pho-
Shaded soybean tosynthetic responses of soybean plants to drought depending on the light intensity treatment the plants were
Exposed soybean subjected to. Shaded soybean plants in response to drought conditions had increased chlorophyll a (Chl a), chlo-
Chlorophyll rophyll b (Chl b), chlorophyll (Chl), carotenoid (Car), ratio of Car/Chl, leaf relative water content (RLWC), leaf
Photosynthetic rate
area per plant, specific leaf area (SLA), Pn, Gs, intercellular CO2 concentration (Ci), transpiration rate (Tr), photo-
chemical quenching (qP) and electron transport rate (ETR). The above-mentioned photosynthetic changes may
play an important role in determining how shaded soybean plants adjust their photosynthetic rate when
experiencing drought conditions.
© 2016 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction
As a result of the increasing human population, the demand for food
has been growing steadily over the last century (Rosegrant and Cline,
2003; Godfray et al., 2010). Meanwhile, food producers are experienc-
ing greater competition for land, water and energy, while balancing
the negative effects of food production (Tilman et al., 2001; Rosegrant
and Cline, 2003). Multiple cropping systems using crop rotations or
intercropping (two or more crops grown simultaneously) can maximize
resource use, and produce greater yield on a given piece of land (Tilman
Abbreviations: Chl a, chlorophyll a; Chl b, chlorophyll b; Car, carotenoid; RLWC, leaf
relative water content; Pn, net photosynthetic rate; Gs, stomatal conductance; Ci,
intercellular CO2 concentration; Tr, transpiration rate; WUE, water use efficiency; Fv/Fm,
maximum efficiency of PSII photochemical reaction; ΦPSII, quantum yield of PSII; qP, pho-
tochemical quenching; NPQ, non-photochemical quenching; ETR, apparent photosynthet-
ic electron transport rate.
⁎ Correspondence to: J. Liu, College of Agronomy, Sichuan Agricultural University,
Chengdu 611130, China. Tel.: +28 86290960; fax: +28 86290870.
⁎⁎ Corresponding author. Tel.: +28 86290960; fax: +28 86290870.
E-mail addresses: jiangliu@sicau.edu.cn (J. Liu), mssiyangwy@sicau.edu.cn (W. Yang).
1
These authors contributed equally to this work.
et al., 2002). Some of the benefits of intercropping are increase in yield,
improved efficiency different environmental resources, pest and disease
suppression and biological nitrogen fixation. As a result, multiple
cropping systems such as the legume/non-legume intercropping
system (Li et al., 2011), grain multiple cropping (Tong, 1994) and
wheat-corn/soybean relay strip intercropping system (Yan et al.,
2010), are becoming popular in China. In the multiple cropping systems,
plants are typically exposed to several stressors simultaneously. For ex-
ample, soybean crops grown along maize in a relay strip intercropping
system can experience limited light intensity from the shade of maize,
and limited water availability.
Physiological responses of evergreen and deciduous tree leaves to
various sunlight-drought scenarios have shown that shading could
ameliorate, or at least not aggravate, the impact of drought. This is
because the performance of leaves under drought stress depends on
how much light the leaves receive (Quero et al., 2006). Shade by
the tree canopy has indirect effects, such as reducing leaf and air tem-
peratures. Shade can also reduce the understory temperatures, and af-
fect vapor pressure deficits and oxidative stress to alleviate the impact
of drought on plants and seedlings in the understory (Holmgren,
2000). Shading conditions can allow olive trees to maintain high

http://dx.doi.org/10.1016/j.sajb.2016.04.011
0254-6299/© 2016 SAAB. Published by Elsevier B.V. All rights reserved.
280 J. Zhang et al. / South African Journal of Botany 105 (2016) 279–287
photosynthetic activity at low values of stomatal conductance (Sofo
et al., 2009). In contrast, exposed plants can experience reductions in
photosynthetic efficiency and intrinsic water efficiency due to differ-
ence in the activity of non-stomatal components of photosynthesis.
Additionally, the decrease in photosynthetic activity and the increase
in photoinhibition during drought are more marked in exposed plants
than in shaded plants (Sofo et al., 2009).
In the soybean plant, short-term shading can reduce photosynthesis,
leaf temperature, stomatal conductance, transpiration and water use
efficiency and increase intercellular CO2 partial pressure, which leads
to carbon gain and water loss (Fay and Knapp, 1995). Photosynthetic
rate, stomatal conductance and transpiration rate of soybean plants
significantly decline under water stress, while the intercellular CO2 con-
centration changes only slightly at the initiation of the stress treatment
(Ohashi et al., 2006). Excessive energy in LHC, reaction center of PSII or
PSI can cause pigment bleaching in sun leaves, the excessive energy can
induce photoinhibition, thereby damaging pigments through oxidative
stress (Kim et al., 2011). Shade reduces the chloroplast coupling factor
and shifts light-harvesting capacity in soybean plants (Burkey and
Wells, 1996). The low level of Chl contents in grapevine leaves at high
photosynthetic photon flux density (PPFD) largely results from the
decay of Chl that is likely enhanced by chlorophyllase activity
(Bertamini and Nedunchezhian, 2003). However, less is known about
whether differences in light intensity can influence the impact of
drought on photosynthetic performance of the soybean plant. To better
understand this, we investigated the impact of temporary shade and
water shortage on the photosynthetic performance of soybean plants.
We designed our experiments to (1) determine photosynthetic and
chlorophyll fluorescence characteristics as affected by drought, low
light intensity stresses and their combination; and (2) elucidate the
relationships between them.
2. Materials and methods
2.1. Plant material and growth conditions
Soybean cultivar Gongxuan No. 1, a major component of southwest-
ern indeterminate soybean cultivars was tested in the experiments per-
formed in 2011 and 2012. Each seed was weighed individually and
sown in cylindrical pots of 14-L volume (23 cm high × 28 cm diameter).
The pots contained 13 kg soil composed of 50% sand, 47.5% clay and 2.5%
organic matter. The soil was mixed with fertilizer consisting of N =
0.355 g, P2O5 = 0.556 g and K2O = 0.406 g. Fertilizers were applied
after emergence, with 3 g single super phosphate, 1 g potassium sulfate
and 1.5 g of urea per pot. The experiment was carried out in a glasshouse
of the Sichuan Agricultural University (29°59′N, 103°00′E; at an altitude
of 500 m), and the greenhouse had an upper ceiling automatic closure
system that was utilized when it rained.
Soybean plants were subjected to two light intensity levels: (1) high
light intensity treatment (HI), where the soybean plants received nor-
mal light intensity from the sun when it was sunny, with additional
light intensity inside the glasshouse when it was rainy (2011, 2012);
(2) low light intensity treatment (LI), where the soybean plants were
covered by a shade cloth (YaanNongzhi Co., China, 2011) or were
under the shade of corn (2012). These experimental light intensity
treatments were used to simulate field conditions in the relay strip
intercropping system, distinguishing two types of microhabitats: sole
cropping soybean (HI) and relay strip intercropping soybean (LI). In
the experiment conducted in 2011, the light intensity that penetrated
through the shade cloth to the soybean plants was 65%. In 2012, the
light intensity that penetrated through the maize canopy to the soybean
plants was 80% when the soybean was sown, 65% at the vegetative stage
(V5), 72% at the reproductive stage (R1) and 70% when the soybean
plant was in the reproductive stage (R2) and the maize was at maturity.
Maize (var. Chuandan 418) is 2.6 m in height, and the whole growth
period is around 109 days. Each of the watering treatments were set
up within each shade frame and replicated four times, each by one
plant in a single pot.
Pots were watered every two days during the first stage of the exper-
iment. Once the soybean seedlings reached V5 stage (at the end of July
2011/2012), two months after sowing, two separate water treatments
were applied. Half of the pots were kept continuously moist (high-
water treatment, HW, 75 ± 2% of the field water capacity, FWC), and
the other half were maintained at moderate drought conditions (low-
water treatment, LW, 45 ± 2% of FWC) in 2011. In 2012, half of the
pots were not watered (LW), while the other half was kept continuously
moist (HW, 75 ± 2% of FWC). The 2012, LW treatment simulated a typ-
ical climate situation of seasonal drought in Southwestern China, as
compared to a continuously moist treatment (HW) (Table 1). During
the experiment, we measured soil moisture in volumetric water content
(VWC) along the first 20 cm depth with a TRIME-PICO (German) on a
daily basis, in a subsample of five pots under different light intensity
and water treatments. We did this because the water content changes
were different in pots under LW treatments for the two light intensity
treatments (Zhang et al., 2011).
2.2. Microenvironment measurements
A micro-meteorological machine that included sensors for air tem-
perature, relative humidity and light intensity (Hobo, Onset, Pocasset,
MA) was used to measure microenvironmental parameters. Readings
from each sensor were recorded every 6°min with a Hobo data logger.
Two additional data loggers were installed to record air temperature
measured with sensors attached to the abaxial side of leaves of four
plants in each light intensity treatment. From the data, we could see
that the light intensity and air temperature of the LI soybean group
were lower than the HI soybean group, while relative humidity was
opposite (Fig. 1).
2.3. Relative water content
RLWC of leaves was calculated using the standard formula [(FW −
DW)/HydW − DW)°×°100] (Farrant, 2000). FW, HydW and DW
stand for the leaf fresh weight, hydrated (full turgor) and dry weights,
respectively. The hydrated weight was determined by weighing the
leaf after 24 h of immersion in distilled water in a sealed flask at room
temperature. Dry weight was determined gravimetrically after drying
to steady weight at 70 °C in an oven. Soybean leaves were harvested
daily during the V5 stage (at the end of July 2011/2012). Five plants
were randomly chosen, and one of the most recently expanded leaves
was selected from each plant. The beginning point of the non-
hydraulic root signals (nHRS) were determined depending on when
there was a significant lowering of leaf stomatal conductance (Gs)
without change in leaf RWC (compared with Gs in 75 ± 2% FWC). The
hydraulic root signal (HRS) was judged to begin when there were
significant differences for both of the above leaf parameters (Gowing
et al., 1990).
Table 1
Soil water content (measured with TDR) at the beginning of progressive drying and
hydraulic root signals (mean ± SE) in a subsample of pots under different light intensity
and water combinations in 2012.
Treatments Date of the commencement Soil relative water
of signal point (d) content (% of FWC)
HI LI
HW 1 80 ± 1.2 80 ± 1.1
LW 9 53.7 ± 2.1 53.3 ± 0.3
HI represents high light intensity; LI represents low light intensity, under the shade of
maize. LW represents low-water treatment; HW represents high-water treatment.
 J. Zhang et al. / South African Journal of Botany 105 (2016) 279–287 281

800 100

700
80
Lght intensity(lum sqf -1) 600

Relative Humidity(%)
500
60

400

40
300

200
20
100

0 0
0 20 40 60 80 100 120 0 20 40 60 80 100 120

45
HI
40 LI

35
Temperature (0C)

30

25

20

15

10
0 20 40 60 80 100 120
Time after sowing (d)

Fig. 1. Light intensity (integrated over the entire day and night), air temperature and relative humidity in exposed and shaded soybean plants in 2012.

2.4. Stomatal aperture
Epidermal replicas of leaflets were made by coating the adaxial sur-
faces with clear fingernail polish (Johnson and Brun, 1966). Then, the
dried films were peeled and mounted on slides. Images were observed
using Nikon eclipse 50i under 40 × magnification. A Nikon Digital
Sight DS-U microscope camera controller was used to transfer images
to a PC computer. Percentage of the open stomata was determined for
each surface by randomly counting open stomata and total stomata
numbers for 1 mm2 (measured by micrometer) in 10 different fields.
The percentage of open stomata was calculated as the ratio of open sto-
mata to total stomata numbers, which was used to calculate the aver-
age. Stomatal aperture length was measured by identifying the widest
aperture using the Motic Image Plus 2.0 Digital Microscopy Software.
Leaves were sealed in plastic bags and kept on ice or refrigerated until
further processed.
leaves. The amounts of pigments were calculated according to
established equations (Lichtenthaler and Wellburn, 1983).
2.6. Photosynthetic parameters
The net photosynthesis rate (Pn), stomatal conductance (Gs) and
transpiration (Tr) were measured with a Portable Photosynthesis Sys-
tem (Model LI-6400, LI-COR Inc., Lincoln, NE). Water use efficiency
(WUE) was calculated as Pn/Tr. The parameters were measured daily,
after water stress was applied from 8:00 am to 12:00 am. Five plants
were randomly chosen, and one of the most recently expanded leaves
was selected from each plant four times. The photosynthetically
active radiation (PAR), provided by an LED light source, was set to
1200 μmol m-2 s-2. The flow rate of air through the sample chamber
was set at 500 μmol-1 s− 1, and the leaf temperature was maintained
at 25 ± 0.8 °C by thermoelectric coolers. The CO2 concentration of the
chamber was adjusted to 400 μl l−1 with the system's CO2 injector.

2.5. Pigment analysis 2.7. Chlorophyll fluorescence measurements

Photosynthetic pigments were extracted according to the Arnon
method (Arnon, 1949). Measurements were taken from the most re-
cently expanded leaf of five randomly chosen plants (n = 4). In 2011,
leaf samples avoiding the veins (0.2 g) were used. In 2012, 10 leaf
discs (2 cm2) avoiding the veins were cut from the centre of each leaf.
The developed color was measured at three wavelengths 470, 646 and
663 nm (TU-1901 UV–spectrophotometer), after leaves were immersed
in 10 ml 80% acetone for 24 h until no green color was present in the
Chlorophyll fluorescence was measured by a fluorescence monitor-
ing system (MINI-PAM, WALZ Co., Germany) on randomly selected
leaves (third fully expanded leaf) of plants at 0:00–4:00 am. Following
30 min of dark adaptation, the minimum chlorophyll fluorescence
(Fo) was determined using a measuring beam of 0.2 μmol m−2 s−1
light intensity. A saturating pulse (1 s white light with a PPFD of
7500 μmol m−2 s− 1) was used to obtain the maximum fluorescence
(Fm) in the dark-adapted state. Maximum quantum yield of PSII
282 J. Zhang et al. / South African Journal of Botany 105 (2016) 279–287

Table 2 (Fv/Fm) was calculated as (Fm−Fo)/Fm (Maxwell and Johnson, 2000).

Percentage of the open stomata (%) and stomatal aperture length (μm) of soybean leaves Following from this, an actinic light (PPFD of 300 μmol m−2 s−1) was
in 2012.
applied, subsequently, further saturating flashes were applied at
Treatments Percentage of the open Stomatal aperture length appropriate intervals to measure the Fm′ (maximum fluorescence in a
stomata light-saturated stage). Ft is the steady-state fluorescence in the light-
HI LI HI LI adapted state. Three seconds after the removal of actinic light, Fo′
HW 62.3 ± 0.6a 60.6 ± 0.4a 3.2 ± 0.1a 3.6 ± 0.2a (minimum chlorophyll fluorescence in a light-adapted state) was
LW 59.6 ± 0.4b 56.8 ± 0.2b 1.5 ± 0.1b 1.7 ± 0.1b measured using a far-red light of 5 W m− 2. Quantum yield of PSII
HI represents high light intensity; LI represents low light intensity, under the shade of
(ФPSII) is calculated as ФPSII = (Fm′−Ft)/Fm′ (Genty et al., 1989). Pho-
maize. LW represents low-water treatment; HW represents high-water treatment. Within tochemical quenching (qP) was calculated as qP = (Fm′–Ft)/(Fm′–Fo′).
columns and rows, means of the same parameter followed by the same small letters are Non-photochemical quenching (NPQ) was calculated as NPQ =
not significantly different according to the LSD test (p b 0.05). (Fm − Fm′)/Fm′ according to Maxwell and Johnson (Maxwell and

a
2.00 1.8

HW
1.95 LW 1.6

Chlorophyll b content
Chlorophyll a content

1.90 1.4

(mg g-1DW)
(mg g-1DW)

1.85 1.2

1.80 1.0

1.75 .8

1.70 .6
3.6 3.5

Total carotenoid content

3.4 3.0
Total Chlorophyll content

(mg g-1DW)
3.2 2.5
(mg g-1DW)

3.0 2.0

2.8 1.5

2.6 1.0

2.4 .5
2.6 1.2

2.4
1.0
The Car/Chl ratios

2.2
The Chl a/b ratios

2.0 .8
1.8
.6
1.6

1.4
.4
1.2

1.0 .2
100% 65% 100% 65%

Light intensity

Fig. 2. (a) Photosynthesis parameters of soybean leaves under water deficit conditions plated in high and low light intensity in 2011. Photosynthesis parameters measured include:
Chlorophyll a (Chl a), Chlorophyll b (Chl b), total chlorophyll (Chl), total carotenoid (Car) concentration (mg g-1 DW), Chl a/b ratio and Car/Chl ratio. Closed circles indicate soybeans
under continuous irrigation treatment (high-water treatment), and open circles indicate soybeans subjected to drought (low-water treatment). (b) Photosynthesis parameters
of soybean leaves under water deficit conditions plated in high and low light intensity in 2012. Parameters measured include: Chl a, Chl b, Chl and Car concentration (mg g-1 DW), Chl
a/b ratio, and Car/Chl ratio. Closed circles denote soybeans under continuous irrigation treatment (high-water treatment), and open circles denote soybeans subjected to drought
(low-water treatment).
 J. Zhang et al. / South African Journal of Botany 105 (2016) 279–287 283

b
5.8 8
5.6 HW
LW
7

Chlorophyll b content
5.4
Chlorophyll a content

( mg g DW )
5.2
( mg g DW )

6
5.0
-1

-1
4.8 5
4.6
4.4 4
4.2
3
4.0
3.8 2
13 7.0

6.5

Total carotenoid content

Total Chlorophyll content

12
6.0

( mg g DW )
( mg g DW )

11 5.5

-1
-1

5.0
10
4.5
9
4.0

8 3.5

1.8 .8

1.6
.7

The Car/Chl ratios

The Chl a/b ratios

1.4
.6
1.2
.5
1.0

.4
.8

.6 .3
100% 65-80% 100% 65-80%

Light intensity

Fig. 2 (continued).

Johnson, 2000). Apparent photosynthetic electron transport rate was cal-
culated as ETR = ФPSII × PAR × 0.5 × 0.84 (PAR = 350 μmol m−2 s−1)
(Subrahmanyam et al., 2006). Transport of one electron requires ab-
sorption of two quanta, as two photosystems are involved (factor 0.5).
It is assumed that 84% of the incident quanta are absorbed by the leaf
(factor 0.84).
2.8. Specific leaf area and leaf area per plant
The leaf area was measured using a scanner with a leaf area calcula-
tion program. Following this measurement, all leaves were pooled and
dried at 70 °C to constant mass before weighing. The specific leaf area
(SLA) was calculated as the ratio of the leaf area to leaf dry weight.
Leaf area of each plant was calculated using the standard formula
SLA × dry weight of the corresponding plant. Samples were harvested
14 days after drought stress in 2011, and nine days after receiving no
water in 2012 (when the RLWC was significantly decreased).
2.9. Statistical analysis
The experiments were organized as a factorial design, in which light
intensity treatments were the main-plot factors, and water treatments
were the subplot factors. Results were analyzed by two-way analysis
of variance (LSD), and means were compared by Duncan's multiple
range tests at P b 0.05 or P b 0.01. All data were organized in Excel
(Microsoft) spreadsheets, and processed by the software Statistical
Package for the Social Sciences (SPSS) version 11.5.
3. Results
3.1. Percentage of the open stomata and stomatal aperture length
We first measured the percentage of open stomata and stomatal
aperture length to understand the effects of water deficit and shade
on stomata of the soybean plant (Table 2). Water deficiency resulted
284 J. Zhang et al. / South African Journal of Botany 105 (2016) 279–287

High light intensity Low light intensity

14 14
HW 2011 2011
LW
12 12

10 10

Photosynthetic rate (umol CO2 m s )

Photosynthetic rate (umol CO2 m s )
-1

8 8
-2

6 6

4 4

2 2

14 14
2012 2012

-2
12 12

-1
10 10

8 8

6 6

4 4

2 2
100 200 600 800 1000 1200 1400 100 200 600 800 1000 1200 1400

PAR (umol photon m-2 s-1)

Fig. 3. Photosynthetic rate with increasing radiation (light curves) of soybean plants cultivated in high (left) and low (right) light intensity. Close circles denote soybeans under continuous
irrigation treatment (high-water treatment), and open circles denote soybeans subjected to drought (low-water treatment).

in a 4.33% decrease in open stomata on the upper leaf surface under high
light intensity (HI), and a 6.27% decrease under low light intensity (LI).
We observed significant reductions in stomata size in the drought-
stressed plants, but stomatal aperture length was higher under shade
treatment when compared to high light intensity treatment.
3.2. Pigment composition
The pigment contents of soybean plants for different light intensities
and water treatments are summarized in Fig. 2. Chl a and Car content,
ratios of Chl a/b and Car/Chl were significantly reduced under low
water (LW) conditions compared to the high water (HW)-treated
plants. But Chl b and Chl content increased when plants suffered from
water deficit. Under LW conditions, Chl a, Chl b, Chl, Car and ratio of
Car/Chl were higher in the LI group compared to the HI group. Con-
versely, the ratio of Chl a/b was lower under LW treatment in LI com-
pared to HI.
3.3. Photosynthetic parameters
The reduction in the availability of light intensity and water resulted
in structural changes to the soybean leaves, and affected their photosyn-
thetic performance (Fig. 3; Table 3). Reduction in the availability of water
resulted in a decrease in the light saturation point (LSP). Under LI treat-
ment, the LW-induced reduction of LSP was alleviated. We observed a re-
duction in photosynthetic rate (Pn) under water stress. The reduction of
Pn by water stress was 98.77% in the HI group and 96.55% in the LI group.
Stomatal conductance (Gs), transpiration rate (Tr) and water use effi-
ciency (WUE) were also reduced. The decrease in Gs was 98.79% in the
HI group and 88.81% in the LI group. Tr was reduced by 97.84% in the
HI group and 92.81% in the LI group. When plants were under water
stress, intercellular CO2 concentration (Ci) increased by 53.64% for HI
treatment and 209.7% for LI treatment. WUE was reduced by 43.41% in
the HI group and 51.63% in the LI group. Under LI treatment, the reduc-
tion in Pn, Gs, Tr by LW were alleviated compared to HI treatment.

Table 3
Soybean leaf photosynthetic rate (Pn), stomatal conductance (Gs), intercellular carbon dioxide concentration (Ci), transpiration rate (Tr) and water use efficiency (WUE) under different
water and light intensity treatments in 2012.

Treatments Pn (μmol CO2 m-2 s-1) Gs (mmol m-2 s-1) Ci (mg kg-1) Tr (mmol m-2 s-1) WUE

HI HW 17.99 ± 1.27aA 0.45 ± 0.01aA 259.24 ± 21.11bB 6.10 ± 0.04aA 2.95 ± 0.01aA
LW 0.22 ± 0.01bB 0.01 ± 0.00bB 398.31 ± 17.21aA 0.13 ± 0.00bB 1.67 ± 0.01bB
LI HW 14.79 ± 1.17aA 0.21 ± 0.00aA 196.63 ± 10.17bB 4.69 ± 0.02aA 3.15 ± 0.01aA
LW 0.51 ± 0.02bB 0.02 ± 0.00bB 609.08 ± 24.27aA 0.33 ± 0.00bB 1.53 ± 0.01bB

HI represents high light intensity; LI represents low light intensity, under the shade of maize. LW represents low-water treatment; HW represents high-water treatment. Values followed
by a different small (p b 0.05) and capital letter (p b 0.01) that indicate the existence of statistically significant differences based on the LSD analysis.
 J. Zhang et al. / South African Journal of Botany 105 (2016) 279–287 285

Table 4
Maximum quantum yield of PSII (Fv/Fm), quantum yield of PSII (ΦPSII), photochemical quenching (qP), non-photochemical quenching (NPQ) and apparent photosynthetic electron
transport rate (ETR) under different water and light intensity treatments in 2011.

Treatments Fv/Fm ΦPSII qP NPQ ETR

HW 0.798 ± 0.07aA 0.53 ± 0.07aA 0.83 ± 0.01aA 2.02 ± 0.07aA 31.79 ± 1.12aA
HI
LW 0.786 ± 0.05aA 0.13 ± 0.01bB 0.58 ± 0.01bB 3.36 ± 0.12aA 15.23 ± 0.71bB
HW 0.795 ± 0.06aA 0.61 ± 0.02aA 0.99 ± 0.01aA 0.59 ± 0.08bB 53.83 ± 1.28aA
LI
LW 0.782 ± 0.04aA 0.27 ± 0.01bB 0.84 ± 0.02aA 2.99 ± 0.07aA 20.40 ± 1.34bB

HI represents high light intensity; LI represents low light intensity, under the shade of shade cloth. LW represents low-water treatment; HW represents high-water treatment. Values
followed by a different small (p b 0.05) and capital letter (p b 0.01) that indicate the existence of statistically significant differences according to LSD test.

3.4. Chlorophyll fluorescence
The drought tolerance of soybean plants was evaluated by treating
plants to seven days of drought stress, and then analyzing several fluo-
rescence parameters determined under dark-adapted and steady state
conditions (Table 4). In control leaves, maximum quantum yield of
PSII (Fv/Fm) was approximately 0.78–0.80. This parameter decreased
in response to drought stress in all leaves, but was not significantly dif-
ferent. Additionally, drought stress resulted in a reduction in quantum
yield of PSII (ΦPSII) in all leaves. The reduced ΦPSII was a result of a de-
crease in the excitation energy trapping efficiency of PSII reaction cen-
ters. A significant decrease in qP was also observed in all drought
stressed plants, indicating that there was a change in the balance be-
tween the excitation rate and the electron transfer rate. This change
may have led to a reduced state of the PSII reaction centers. We also ob-
served an increase in NPQ under drought conditions, which reflect the
non-photochemical energy dissipation in all plants, and the increase
in NPQ levels were significant compared to controls. Additionally, NPQ
levels in drought conditions were significantly higher in shaded soy-
bean plants.
3.5. Leaf relative water content, special leaf area and leaf area per plant
RLWC decreased with reduced soil water content, but was higher in
shaded soybean leaves compared with exposed soybean leaves (Fig. 4).
Special leaf area (SLA) increased under drought and low-light intensity
conditions (Fig. 5). Leaf area per plant was significantly reduced under
drought stress. The leaf area per plant was highest under the HW-LI
treatment.
3.6. Regression and correlation analysis
To assess whether the difference in photosynthetic parameters
among soil moisture gradients of exposed and shaded plants was
associated with variations in other parameters, we conducted a regres-
sion analysis between soil moisture and RWC, Pn and Gs, Tr and Gs, etc.
Regression analysis of corresponding values among eight different
treatments showed that there was a quadratic line function between
RLWC and RSWC [Y = 27.31X + 73.35, r = 0.8695 N r0.01 =
0.798(n = 7)]. This revealed that when RSWC decreased, RLWC reduced
to the threshold. Pn and Tr were quadratically correlated with Gs [Y =
0.003X2 − 0.039X + 0.028, r = 0.9980 N r0.01 = 0.990(n = 3); Y =
0.021X2 − 0.060X + 0.026, r = 0.9980 N r0.01 = 0.990(n = 3)]. This
revealed that the decrease in Gs caused the reduction in Pn and Tr to
some extent. RLWC was the reason for changes in the value of Gs.
ΦPSII, Fv/Fm and chl(a + b) were significantly correlated with Pn
[Y = −0.003X2 + 0.078X + 0.171, r = 0.9783 N r0.01 = 0.990(n = 3);
Y = 1E − 05X2 + 0.784, r = 0.9701 N r0.01 = 0.990(n =
3);Y = −0.054X2 + 1.244X + 41.21, r = 0.7987(n = 3)]. Light intensity
and water treatments had significant influences on leaf area per plant,
Chl b, Chl, Chl a/b ratio, Pn, Gs, WUE, ΦPSII, qP and NPQ. The P values
of the parameters that were listed here reached 0.01 or 0.05. We
found significant interactions between light intensity and water treat-
ments on leaf area, Chl b, Chl, Pn, Gs, Tr, WUE and qP, with the P values
lower than 0.01 or 0.05 (Table 5).
4. Discussion
Previous studies have shown that leaves subjected to drought exhib-
it large reductions in RLWC and water potential (Dekov et al., 2001;
Nayyar and Gupta, 2006). In our experimental drought conditions, the
water availability of the soil was lower, and the exposed high light in-
tensity plants experienced lower relative humidity and higher temper-
atures (Fig. 1). The lower RLWC detected in the exposed water-stressed
plants reflect the fact that both high light intensity and the soil water
deficiency resulted in the dehydration of plant tissue (Nicolás et al.,
2005). The improvement in RLWC values in the shaded plants may be
attributed to the higher relative humidity and lower temperature in

100 100
HW
Leaf water content in 2012 (%)
Leaf water content in 2011 (%)

LW
90 90

80 80

70 70

60 60

50 50
100% 65% 100% 65-80%

Light intensity (%)

Fig. 4. General variation in RLWC of soybean leaves (means and SE bars, n = 4) in response to four combinations of light intensity and water treatments. Treatment conditions are as
follows: low light intensity (LI, 65% or 65%–80%), high light intensity (HI, 100%), high water (HW, solid line) and low water (LW, dashed line).
286 J. Zhang et al. / South African Journal of Botany 105 (2016) 279–287

2500 450
Leaf area per plant (cm2 plant -1) HI and HW
a
a

Specific leaf area (cm g )

HI and LW
LI and HW
ab 400
2000 LI and LW a
b
350
a
1500 b b c b b
b
b c 300

2
c
c

-1
1000
250

500 200
2011 2012 2011 2012

Fig. 5. General variation in leaf area per plant and specific leaf area of soybeans (means and SE bars, n = 4) in response to four combinations of light intensity and water treatments.
Treatment conditions are as follows: low light intensity (LI, 65% or 65%–80%), high light intensity (HI, 100%), high water (HW, 73%–77% of FWC) and low water (LW, 43%–47% of FWC
in 2011 or 53%–55.8% of FWC in 2012).

the environment. Shading may also decrease water loss and improve absorption peaks in the red light is higher in Chl a, and the absorption
water uptake by improved root growth and root hydraulic conductance peaks in blue light are higher in Chl b (Murata et al., 1966; Bornman
(Li et al., 2005). et al., 1991). Therefore, the relative increase of Chl b (or decrease of
Stomata are sensitive to RLWC, and tend to close with decreasing Chl a/b ratio) enables plants to improve efficiency of blue-violet light
RLWC, which can result in lower Gs levels in exposed plants. This de- absorption, and adapt to a shaded environment. Studies show that chlo-
crease in Gs may be caused by the reduced open stomata ratio and sto- rophyll content of shade-tolerant plants increases under shade. In this
matal aperture size in exposed water-stressed plants (Tables 2 and 3). study, water-stressed plants had lower Chl a, Chl b and Car content,
Stomatal closure primarily causes a decline in the photosynthesis rate and the ratios of Chl a/b, Car/Chl were also lower. Shaded plants main-
(Mahajan and Tuteja, 2005). The variation in Ci can be used as a stan- tained a higher Chl a, Chl b and Car content, lower ratios of Chl a/b,
dard to estimate the reasons for decreased Pn, and whether decreases Car/Chl when under drought stress. The results are in agreement with
in Gs or reductions of mesophyll can result in changes in the cell's Cicek and Çakırlar (2008), who reported that salt stress affects the Chl
photosynthetic capacity. In this study, we observed a decrease in Gs a/b ratio in several soybean cultivars. Some of the cultivars seemed to
and increase in Ci in the water-stressed plants. These results suggest adapt to the salt stress by reducing their Chl a/b ratio, which suggests
that the strong decrease in Pn in water-stressed plants may be caused that those cultivars may have a larger antenna size. In conclusion,
by the closure of stomata, and reduction in the photosynthetic capacity shaded soybean plants show enhanced ability to capture and use light
of mesophyll cells, which in turn results in increased Ci. by increasing the chlorophyll content and reducing the impact of
Chlorophyll is the photosynthetic pigment of plants. Chlorophyll primary reactions.
content can serve as a measure for the plant's ability to use light, as chlo- Zlatev and Yordanov (2004) reported that drought stress induced an
rophyll plays a central role in the absorption and transmission of light increase in Fo and a decrease in Fm, and an associated increase in NPQ in
quantum. Absorption spectra of Chl a and Chl b are similar, but the bean plants. In our study, drought-stressed soybean plants showed in-
creased NPQ within shaded and exposed plants, but shaded soybean
plants had lower NPQ compared to exposed soybean plants. The lower
NPQ levels may cause an increase in the probability of heat emission,
Table 5
Results of the two-way ANOVA for traits, based on the light intensity and water treatment
lowering the trapping efficiency of open reaction centers for shaded
factors. soybean plants. Quantum yield of PSII (ΦPSII) is the product of the effi-
ciency of the open reaction centers (Fv′/Fm′) and the photochemical
Traits Factors Interactions R2
quenching (qP) (Genty et al., 1989; Lu and Zhang, 2000). In all plants
Light intensity Water deficit under drought treatment, we observed a decrease in qP, indicating
SLW 16.29*** 25.85*** 3.74* 45.89 that a larger percentage of the PSII reaction centers was closed at any
SLA 8.19 67.88* 6.74* 82.81 time. This in turn indicates a change in the balance between excitation
Leaf area per plant 31.15*** 51.19*** 4.35 86.69
rate and electron transfer rate. In our study, shaded soybean plants
Chla 36.05** 13.81 5.7 55.55
Chlb 51.04*** 11.96 13.7* 76.7
had higher qP, ΦPSII and ETR, as compared to the exposed soybean
Chl 44.81*** 12.54** 9.16* 66.51 plants when under drought stress. This could be because drought did
Chla/b ratio 41.16*** 5.04 17.5** 63.7 not have a serious effect on the shaded soybean plants compared to
Pn 3.97*** 89.08*** 2.36*** 95.41 exposed soybean plants.
Gs 19.26*** 48.23*** 20.94*** 88.43
Light intensity is known to be the main factor that promotes shifts in
Ci 0.18 74.69*** 1.37 76.24
Tr 21.98*** 51.9*** 20.96*** 94.84 SLA (Niinemets and Tenhunen, 1997). Water stress has also been re-
WUE 16.81*** 25.64*** 21.57*** 64.02 ported as an environmental factor that may increase SLW (Fagnano
Fo 0.25 31.37** 3.75 35.36 and Merola, 2007; Gao et al., 2010). The increase in SLA has been
Fv/Fm 2.06 27.57* 3.91 33.54 interpreted as a mechanism to optimize light harvesting under low
ΦPSII 26.06** 31.37** 0.59 58.03
qP 8.24 10.20* 9.18* 27.62
light intensity conditions. However, the higher SLA in shaded seedlings
NPQ 6.58* 13.17** 10.31 30.06 would result in reduced efficiency of controlling water losses under
drought conditions. In our study, the highest increase of SLA was ob-
The proportion of the explained variance (SSx/SStotal) and the level of significance (*, P b
0.05; **, P b 0.01; ***, P b 0.001) for each factor and the interactions are indicated.
served in shaded seedlings. This implies that there may be a higher
Values that remained not significant after controlling the false discovery rate are leaf area per unit for light harvesting, and better drought SLW elasticity
underlined. R2 is the proportion of total variance absorbed by the model. under limited light conditions.
 J. Zhang et al. / South African Journal of Botany 105 (2016) 279–287
In conclusion, the photosynthetic performance of soybean plants
was severely reduced in drought conditions, but shading (lower light
intensity) alleviated the drought impact. Results of this study suggest
that shaded soybean plants have enhanced drought tolerance due to in-
creased Gs, Tr, pigment content, qP, ΦPSII, ETR and decreased Chl a/b
ratio to maintain a higher Pn. Taking into account the economic
importance of soybean, the study is of potential importance in an ap-
plied context, especially in Southwest China where growth conditions
applied in the present study are typical for soybean cultures.
Acknowledgement
The research was supported by National Natural Science Foundation
of China (31301277 and 31401329), National Program on Key Basic Re-
search Project (2011CB100402), and Program on Industrial Technology
System of National Soybean (CARS-04-PS19).
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