Journal of Photochemistry and Photobiology B: Biology 110 (2012) 18

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Genotypic variations in photosynthetic and physiological adjustment

to potassium deciency in cotton (Gossypium hirsutum)
Ning Wang a, Hanbai Hua a, A. Egrinya Eneji b, Zhaohu Li a, Liusheng Duan a, Xiaoli Tian a,
a
State Key Laboratory of Plant Physiology and Biochemistry, Key Laboratory of Crop Cultivation and Farming System, Center of Crop Chemical Control,
China Agricultural University, Beijing 100193, China
b
Department of Soil Science, Faculty of Agriculture, Forestry and Wildlife Resources Management, University of Calabar, Nigeria

a r t i c l e

i n f o

Article history:
Received 29 October 2011
Received in revised form 13 January 2012
Accepted 7 February 2012
Available online 15 February 2012
Keywords:
Cotton
Potassium deciency
Carbon metabolism
Nitrogen metabolism
Genotypic variation

a b s t r a c t
A hydroponic culture experiment was conducted to determine genotypic variation in photosynthetic rate
and the associated physiological changes in response to potassium (K) deciency in cotton (Gossypium
hirsutum L.) seedlings with contrasting two cotton cultivars in K efciency. The K-efcient Liaomian18
produced 66.7% more biomass than the K-inefcient NuCOTN99B under K deciency, despite their similar
biomass under K sufciency. Compared with NuCOTN99B, Liaomian18 showed 19.4% higher net photosynthetic rate (Pn, per unit leaf area) under K decient solutions and this was associated with higher photochemical efciency and faster export of soluble sugars from the phloem. The lower net Pn of
NuCOTN99B was attributed to higher capacity for nitrate assimilation and lower export of soluble sugars.
Furthermore, NuCOTN99B showed 38.4% greater ETR/Pn than Liaomian18 under K deciency, indicating
that more electrons were driven to other sinks. Higher superoxide dismutase (SOD) and lower catalase
(CAT) and ascorbate peroxidase (APX) activities resulted in higher levels of reactive oxygen species
(ROS; e.g. O2and H2O2) in NuCOTN99B relative to Liaomian18. Thus, the K inefciency of NuCOTN99B,
indicated by lower biomass and net Pn under K deciency, was associated with excessively high nitrogen
assimilation, lower export of carbon assimilates, and greater ROS accumulation in the leaf.
Crown Copyright 2012 Published by Elsevier B.V. All rights reserved.

1. Introduction
Potassium (K) is one of the macronutrients essential for plant
growth and development, and plays an important role in a wide
range of physiological processes, such as maintenance of electrical
potential gradients across cell membranes, turgor generation,
maintenance of anioncation balances, activation of numerous enzymes, protein synthesis, and maintenance of photosynthesis and
related processes [1]. However, compared with nitrogen (N), K is
less applied worldwide. The N:K application ratio declined from
1:0.74 in the 1960s to 1:0.27 in the early 2000s [2], thus causing
soil K mining. For example, China had a negative K balance (K
application with potash fertilizers minus K removal by crops) of
about 60 kg ha1 y1 in the late 1990s with an ongoing downward trend [3]. The lack of K application and soil K mining will
cause unavoidable K deciency in plants.
Cotton (Gossypium hirsutum L.) has a high requirement for K and
shows a greater response to K fertilizer than does corn or soybean
[4]. Widespread K deciency in cotton crops has occurred in many
Corresponding author. Address: Department of Agronomy, College of Agronomy
and Biotechnology, China Agricultural University, No. 2 Yuanmingyuan West Road,
Haidian, Beijing 100193, China. Tel.: +86 10 62732567; fax: +86 10 62731569.
E-mail address: [email protected] (X. Tian).

countries [5,6], because of the negative K balance in the soil, adoption of modern cultivars characterized by faster fruit set and greater boll load [7], and popularization of transgenic Bt (Bacillus
thuringiensis Berliner) cotton [6], which is more susceptible to K
deciency [8,9].
Plants have developed a wide range of adaptive or resistance
mechanisms to maintain productivity under a variety of environmental stress conditions, including K deciency. Genotypes showing high K efciency usually have greater uptake or utilization
efciency [10,11]. Our previous work has indicated great genotypic
differences in K efciency among cotton genotypes [8,12]. For
example, the larger root system and greater internal K utilization
efciency in Liaomian18 compared with NuCOTN99B resulted in
greater K efciency [12]. We demonstrated that the greater internal K utilization efciency of Liaomian18 was independent of certain biophysical functions of K; for example, the osmotic potential
and relative water content in leaves of Liaomian18 were similar to
those of NuCOTN99B under K deciency [12]. Consequently, it was
necessary to study the biochemical and physiological functions of
K (e.g., the activation of enzymes and related processes, and maintenance of photosynthesis), which would be mainly responsible for
the genotypic variations in K utilization efciency between these
two cultivars.

1011-1344/$ - see front matter Crown Copyright 2012 Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.jphotobiol.2012.02.002

N. Wang et al. / Journal of Photochemistry and Photobiology B: Biology 110 (2012) 18

The objectives of the present study were to (i) investigate the

effect of K on photosynthesis and related physiological processes
such as chlorophyll (Chl) uorescence, nitrogen metabolism and
reactive oxygen species (ROS) accumulation in cotton seedlings
and (ii) examine differences in the responses to K supply between
a K-efcient cultivar, Liaomian18 and a K-inefcient cultivar,
NuCOTN99B. The data obtained will reveal the mechanisms underlying genotypic variations in internal K utilization efciency, and
provide a framework to identify breeding targets for improving
the K use efciency of cotton.

2. Materials and methods

2.1. Plant material
A K-efcient cotton cultivar, Liaomian18 (non-Bt cotton) developed by the Liaoning Cash Crops Research Institute, and one
K-inefcient cotton cultivar NuCOTN99B (Bt cotton) developed by
Monsanto Company and distributed by the Hebei Jidai Cotton Seed
Company in China, were used in the present study. The former
cultivar produced the same biomass as the latter under K sufciency (2.5 mM), but produced much higher biomass than the
latter under K deciency (0.03 mM) [12].
2.2. Growth conditions
Hydroponic culture experiments were conducted in a growth
chamber with a 14 h photoperiod at 28/20 C (day/night) temperatures, 7080% relative humidity, and 450 lmol m2 s1 photosynthetically active radiation. Seeds were surface-sterilized with 9%
H2O2 for 30 min and germinated in a K-free sand medium. After
emergence (4 d after germination), uniform seedlings were cultured hydroponically by transferring to 35 cm  27 cm  12 cm
plastic pots that contained 2.2 L half-strength modied Hoaglands
solution containing 2.5 mM Ca (NO3)2, 1 mM MgSO4, 0.5 mM
(NH4)H2PO4, 0.2 lM CuSO4, 1 lM ZnSO4, 0.1 mM Fe Na EDTA,
20 lM H3BO3, 5 pM (NH4)6Mo7O24 and 1 lM MnSO4. The concentration of K in the form of potassium sulfate (K2SO4) in solutions
was 0.03 mM for the K-decient treatment and 2.5 mM for the
K-sufcient treatment. Each pot contained six seedlings. All
solutions were changed twice weekly; deionized water was added
daily to replace the water lost by evapotranspiration. The solution
pH was maintained at 6.5 by addition of concentrated NaOH solution and the solution was continuously aerated with an air pump
to provide O2 and achieve nutrient homogeneity. A completely
randomized design was used with four replications (pots).
2.3. Carbon assimilation, chlorophyll a uorescence and carbohydrate
concentration
At the ve-leaf stage (26 d after transfer [DAT] to nutrient solution), the gas exchange, including stomatal conductance (gs), of the
youngest fully-expanded main-stem leaf (the 4th leaf from apex)
was measured three times per leaf between 9:00 and 11:30 with
a Li-6400 (Li-COR, Lincoln, USA) at 27 C, 60% relative humidity,
500 lmol mol1 CO2 concentration and 1000 lmol m2 s1 quantum ux. The value of photorespiration (PR) was determined as
the difference in photosynthesis rate between low and normal oxygen content in the air [13]. During the same period, Chla uorescence of the same leaf was determined three times per leaf with
a modulated uorometer (PAM 2100, Walz, Effeltrich, Germany)
following the method of Schreiber et al. [14]. The electron transport rate (ETR) was calculated with the following equation:

ETR f  I  aleaf  F 0m  F 0s =F 0m 

where f is the fraction of absorbed quanta used by photosystem II

(PSII), typically assumed to be 0.5, I is the incident photon ux density (mmol m2 s1), and aleaf is the leaf absorbance assumed to be
0.84 [15], and F 0m and F 0s represent the maximum and steady-state
Chla uorescence in light-adapted leaves.
After measurement of Chla uorescence and gas exchange, the
youngest fully-expanded leaves were sampled and separated into
two parts. One part was preserved at 80 C for the determination
of total Chl content, Rubisco (EC 4.1.1.39, IRA) activity and carbohydrate content, and the other portion was oven-dried at 80 C
for measurement of biomass and analysis of K with an atomic
absorption spectrophotometer (SpectAA-50/55, Varian, Australia)
[8]. Specic leaf area was calculated by dividing the leaf area by
the dry weight of the same leaf. Concentrations of Chl were determined in 80% acetone extract [16]. IRA initial activity was assayed
according to modied method of Cheng and Fuchigami [17], 50 lL
of sample extract was added to 900 lL of assay solution, immediately followed by 50 lL of 10 mM RuBP. The assay solution consisted of 100 mM Hepes-KOH (pH 8.0), 25 mM KHCO3, 20 mM
MgCl2, 3.5 mM ATP, 5 mM phosphocretaine, 5 units NAD-glyceraldehyde-3-phosphate dehydrogenase (NAD-GAPDH, EC1.2.1.12),
5 units 3-phosphoglyceric phospokinase (PCK, EC 2.7.2.3), 17.5
units creatine phosphokinase (EC2.7.3.2), and 0.25 mM NADH.
The enzyme reaction was measured as the decrease in absorbance
at 340 nm for 1 min. Starch, soluble sugars, sucrose and reducing
sugars were analyzed as described in Cross et al. [18].
2.4. Nitrogen assimilation and recycling
Leaf samples were prepared using the same method as for
determination of IRA. Nitrate reductase (EC 1.7.99.4, NR) activity
and glutamine synthetase (EC 6.3.1.2, GS) activity were determined
according to Ding et al. [19]. The concentration of soluble protein
was determined with Coomassie brilliant blue G-250 [20], using
bovine serum albumin (BSA) as a standard. Protease activity and
peptidase activity were assayed as described in Setlow [21], and
total free amino acid was measured using spectrophotometric
analyses as described by Cross et al. [18].
2.5. Phloem export of soluble sugars and free amino acids
Phloem exudate was collected from detached youngest fullyexpanded main-leaf leaves using the EDTA-promoted technique
[22]. The cut ends of petioles were placed in 8 mL of 20 mM EDTA
solution (pH 6). In order to avoid contamination with xylem exudates, the EDTA solution was discarded after 15 min in the dark,
then the leaves were rinsed, transferred to fresh 8 mL EDTA
solution, and kept in the dark for 5 h to avoid transpiration. The
exudation solutions were stored at 20 C for measurement of
soluble sugar and free amino acid contents [18].
2.6. Reactive oxygen species metabolism
Leaf samples were prepared using the same method as for
determination of IRA. Leaf tissues (0.5 g) were homogenized in
1 mL of 50 mM potassium phosphate (pH 7.0) and 1% (v/v) Triton
X-100. The homogenate was centrifuged at 12,000g for 20 min at
4 C and the supernatant was immediately used for enzyme assays.
Superoxide dismutase (SOD; EC 1.15.1.1) activity was determined
following the method of Stewart and Bewley [23]. Catalase (CAT;
EC 1.11.1.6) and ascorbate peroxidase (APX; EC 1.11.1.11) activities
were measured according to Patra et al. [24] and Nakano and Asada
[25], respectively. Malondialdehyde (MDA) content was determined with thiobarbituric acid as described in Dhindsa et al.
[26]. Superoxide anion (O
2 ) production was measured by monitoring nitrate formation from hydroxylamine in the presence of O
2

N. Wang et al. / Journal of Photochemistry and Photobiology B: Biology 110 (2012) 18

[27]. The H2O2 content was assayed according to Mukherjee and

Choudhuri [28].
2.7. Statistical analysis
The experiment was repeated three times with results showing
similar trends; hence the pooled data are presented here. Statistical analysis was performed with SPSS 10.0 (SPSS Inc. Chicago, USA)
using one-way analysis of variance, and treatment means were
compared using Duncans multiple range test (p < 0.05). All data
were tested for normality and homogeneity; non-normal data
were transformed with an appropriate transformation method
(e.g., log 10, log(1+x) or LN).
3. Results
3.1. Biomass production and partitioning
There were no signicant differences in biomass production and
its partitioning to root, stem and leaf between Liaomian18 and NuCOTN99B receiving sufcient K supply (Fig. 1). Potassium deciency severely impeded seedling growth; the K-inefcient
cultivar, NuCOTN99B was more severely affected than the K-efcient cultivar, Liaomian18. For example, biomass production under
K deciency was only 22.9% of that in the K-sufcient treatment for
NuCOTN99B but 60.0% for Liaomian18 (Fig. 1A). Biomass partitioning to the stem (Liaomian18) or the root (NuCOTN99B) was decreased by K deciency in favor of the leaf (Fig. 1B).
3.2. Carbon assimilation
Under K sufciency, the youngest fully-expanded leaf of two
cultivars showed the same specic leaf area (SLA); the Chl content
and stomatal conductance (gs) of NuCOTN99B were higher than
those of Liaomian18, but total photosynthesis (Pn), both per unit
area and per unit fresh weight (FW), did not differ signicantly between the cultivars, possibly because of similarities in their intercellular CO2 concentrations (Ci) and IRA (Table 1). The net Pn of
NuCOTN99B tended to be lower than that of Liaomian18, owing
to its greater photorespiration (PR; Table 1).
Potassium deciency caused a severe decrease in SLA, Pn and
associated parameters (except PR of Liaomian18 and Ci; Table 1).
Averaged for cultivars, the SLA, total and net Pn (per unit FW),
Chl, gs, and IRA decreased by 62.1, 22.1, 22.5, 56.7, 82.8 and
78.2%, respectively. The SLA, Chl and Pn in NuCOTN99B were more
affected than in Liaomian18 (Table 1), although both cultivars had
the same leaf K concentration (about 0.3%) under K deciency. In
addition, K deciency caused a 1.2-fold increase in PR per unit

Table 1
Effect of potassium (K) on concentrations of total chlorophyll (Chl), total photosynthesis rate (Pn), net photosynthesis rate (nPn), specic leaf area (SLA), stomatal
conductance (gs), intercellular CO2 concentration (Ci), Rubisco activity (IRA), and
photorespiration (PR) in the youngest fully-expanded leaf of cotton seedlings at the
ve-leaf stage (26 d after transfer to hydroponic culture). Seedlings were grown in
either K-decient (0.03 mM; K1) or K-sufcient (2.5 mM; K2) solutions. Values are
the means of four replications. Means within the same row followed by the same
letter are not signicantly different (P 6 0.05) according to Duncans multiple range
test.
NuCOTN99B

Parameters

Liaomian18
K1

K2

K1

K2

Pn (lmol CO2 m2 s1)

PR (lmol CO2 m2 s1)
nPn (lmol CO2 m2 s1)
Pn (lmol CO2 g1 FW s1)
PR (lmol CO2 g1 FW s1)
nPn (lmol CO2 g1 FW s1)
SLA (cm2 g1 DW)
Chl (mg g1 FW)
gs (lmol H2O m2 s1)
Ci (lmol CO2 mol1)
IRA (lmol g1 FW min1)

8.65b
3.33a
5.32b
36.24b
15.66a
26.55b
83.8b
2.69c
32.3c
390.8ab
0.228b

10.48a
1.84b
8.63a
41.18a
7.24c
33.92a
169.7a
5.33b
175.2b
327.4b
1.260a

5.75c
1.28c
4.46c
28.13c
6.28 c
21.81c
42.5c
2.22d
38.4c
421.3a
0.269b

10.83a
3.43a
7.40a
41.55a
13.16b
28.40a
160.3a
6.13a
241.3a
325.3b
1.056a

FW in Liaomian18 compared to K sufciency, but caused a 1.1-fold

decrease in NuCOTN99B (Table 1).
3.3. Chlorophyll uorescence
There were no differences between Liaomian18 and NuCOTN99B supplied with sufcient K in terms of maximum quanH
tum yield of PSII (Fv/Fm), actual quantum yield of PSII ( PSII),
non-photochemical quenching (qN), photochemical quenching
(qP) and ETR in the youngest fully-expanded leaf (Table 2). PotasH
sium deciency signicantly suppressed Fv/Fm, PSII, qP and ETR,
indicating destruction of the PSII reaction center and decreased
photosynthesis occurred. Similar to Chl and Pn, Chl uorescence
was more strongly affected in NuCOTN99B than in Liaomian18.
The ETR/Pn (per unit area) ratio was increased greater in
NuCOTN99B than in Liaomian18 under K deciency (Table 2).
Non-photochemical quenching was depressed by K deciency in
both cultivars (Table 2).
3.4. Leaf carbohydrate content and soluble sugar export in phloem
There were no signicant differences in the contents of sucrose,
reducing sugars, and soluble sugars in the youngest fully-expanded
leaves of Liaomian18 and NuCOTN99B under K sufciency. However, the starch concentration in the leaf of Liaomian18 was higher

Fig. 1. Effect of potassium (K) supply on dry matter (a) and its partitioning to leaf, stem and root (b) of cotton seedlings at the ve-leaf stage (26 d after transfer to hydroponic
culture). Seedlings were grown in either K-decient (0.03 mM) or K-sufcient (2.5 mM) solutions. The columns represent the mean of four replications. Columns with the
same letter are not signicantly different (P 6 0.05) according to Duncans multiple range test.

N. Wang et al. / Journal of Photochemistry and Photobiology B: Biology 110 (2012) 18

Table 2
Effect of potassium (K) on maximal efciency of PSII photochemistry (Fv/Fm), nonphotochemical quenching coefcient (qN), photochemical quenching (qP), photoH
chemical quantum yield of photosystem ( PSII), electron transport rate (ETR) and
ETR/Pn in the youngest fully-expanded leaf of cotton seedlings at the ve-leaf stage
(26 d after transfer to hydroponic culture). Seedlings were grown in either K-decient
(0.03 mM; K1) or K-sufcient (2.5 mM; K2) solutions. Values are means of four
replications. Means within the same row followed by the same letter are not
signicantly different (P 6 0.05) according to Duncans multiple range test.
Trait

Fv/Fm
qN
qP
H
PSII
ETR (lmol m2 s1)
ETR/Pn

Table 4
Effect of potassium (K) on phloem export of soluble sugar from the youngest fullyexpanded leaf of cotton seedlings at the ve-leaf stage (26 d after transfer to
hydroponic culture). Phloem exudates were collected from detached leaves using the
EDTA-promoted technique. Seedlings were grown in either K-decient (0.03 mM; K1)
or K-sufcient (2.5 mM; K2) solutions. Values are means of four replications. Means
within the same row followed by the same letter are not signicantly different
(P 6 0.05) according to Duncans multiple range test.
Trait

NuCOTN99B

Liaomian18
K1

K2

K1

K2

0.738b
0.137b
0.882b
0.673b
127.2b
14.71b

0.791a
0.256a
0.942a
0.731a
138.2a
13.21c

0.669c
0.147b
0.829c
0.618c
116.9c
20.36a

0.801a
0.286a
0.947a
0.726a
137.3a
12.68c

Liaomian18

NuCOTN99B

K1

K2

K1

K2

39.7c

78.8b

15.1d

92.7a

37.3c

55.5a

33.3d

49.7b

Export per leaf

(lg h1 leaf1)
Export per unit FW 2
(lg g1 FW h1)
Phloem:leaf ratio 3
(100 lg g1 FW h1 lg g1 FW)

0.341b

0.867a

0.268c

0.867a

Exported soluble sugar per hour per leaf.

Exported soluble sugar per hour per unit leaf fresh weight.
Ratio of exported soluble sugar content in the phloem exudates to that in leaf
per unit leaf fresh weight.
2
3

than that of NuCOTN99B (Table 3). Potassium deciency dramatically enhanced the accumulation of sucrose, reducing sugars and
soluble sugars in leaf, especially for NuCOTN99B (Table 3). The
starch content under K deciency decreased substantially in Liaomian18, but increased signicantly in NuCOTN99B (Table 3).
On the basis of a unit leaf (the youngest fully-expanded), NuCOTN99B showed higher phloem export of soluble sugars under
K sufciency than Liaomian18. However, the reverse was observed
on the basis of leaf FW, i.e. Liaomian18 exceeded NuCOTN99B
(Table 4). Under K deciency, the export of soluble sugars was reduced considerably in both cultivars, but the actual level of export
was higher in Liaomian18 than that in NuCOTN99B, regardless of
the unit of measurement (Table 4). When the export capacity of
a leaf was evaluated based on the ratio of soluble sugars in the
phloem to soluble sugars in the leaf (phloem:leaf ratio) per unit
FW, the export was also more strongly impaired by K deciency
in NuCOTN99B than in Liaomian18 (Table 4).

3.5. Nitrogen assimilation and recycling

B

Under K sufciency, NuCOTN99 showed 77.3% higher NR and

78.5% higher GS activities in the youngest fully-expanded leaf compared with Liaomian18 (Table 5). The concentration of soluble protein in the leaf of NuCOTN99B was similar to that of Liaomian18
(Table 5). In contrast to NR and GS, protease activity of NuCOTN99B
was 72.2% lower than that of Liaomian18, and no difference in peptidase activity was observed (Table 5). The AA content in the leaf of
NuCOTN99B was 118.9% higher than that of Liaomian18, which
perhaps resulted from the higher PR in NuCOTN99B (Table 1) and
the imbalance between nitrate assimilation and protein synthesis.
Potassium deciency substantially depressed nitrogen assimilation, characterized by much lower NR activity and lower protein
content, but greatly enhanced protein degradation, characterized

Table 3
Effect of potassium (K) on concentrations of starch, soluble sugar, sucrose and
reducing sugars in the youngest fully-expanded leaf of cotton seedlings at the veleaf stage (26 d after transfer to hydroponic culture). Seedlings were grown in either
K-decient (0.03 mM; K1) or K-sufcient (2.5 mM; K2) solutions. Values are means of
four replications. Means within the same row followed by the same letter are not
signicantly different (P 6 0.05) according to Duncans multiple range test.
Trait

Starch (mg g1 FW)

Soluble sugar (mg g1 FW)
Sucrose (mg g1 FW)
Reducing sugars (mg g1 FW)

Table 5
Effect of potassium (K) on the activities of nitrate reductase (NR), glutamine
synthetase (GS), protease and peptidase, and the concentrations of soluble protein
and free amino acids (AA) in the youngest fully-expanded leaf of cotton seedlings at
the ve-leaf stage (26 d after transfer to hydroponic culture). Seedlings were grown in
either K-decient (0.03 mM; K1) or K-sufcient (2.5 mM; K2) solutions. Values are
means of four replications. Means within the same row followed by the same letter
are not signicantly different (P 6 0.05) according to Duncans multiple range test.
NuCOTN99B

Trait

Liaomian18
K1

K2

K1

K2

NR (lg g1 FW h1)

GS (lmol g1 FW h1)
Protease (lg g1 FW h1)
Peptidase (lg g1 FW h1)
Soluble protein (mg g1 FW)
AA (mg g1 FW)

9.85d
413.4a
298.8a
14.96a
12.06c
1.607b

26.08b
212.3b
206.7b
2.70b
19.48a
0.443d

16.91c
411.0a
118.7c
13.15a
14.89b
2.498a

46.25a
407.6a
57.3d
1.64b
19.79a
0.970c

by much higher protease and peptidase activities and higher AA

content (Table 5). The activity of GS did not decline under K deciency. Compared with the K-efcient cultivar, Liaomian18, the
K-inefcient cultivar, NuCOTN99B showed greater capacity for
nitrogen assimilation and lower capacity for protein degradation
under K deciency, as indicated by higher NR activity, higher soluble protein content, and lower protease activity (Table 5). In addition, the AA content in the leaf of NuCOTN99B was 55.4% higher
than that of Liaomian18.
3.6. Amino acid export in phloem
In accordance with the higher AA content in the leaf of NuCOTN99B, this cultivar exported more phloem AA under K sufciency, either on the basis of a unit leaf or the leaf FW (Table 6).
However, the phloem:leaf ratio per unit FW indicated that the
capacity for AA export in the leaf of NuCOTN99B was lower than
that of Liaomian18 (Table 6). Under K deciency, amino acid export
decreased dramatically, regardless of the unit of measurement (except the value per unit FW in NuCOTN99B), and NuCOTN99B
showed greater export capability on the basis of leaf FW or
phloem:leaf ratio (Table 6).

NuCOTN99B

Liaomian18
K1

K2

K1

K2

8.83c
10.93b
2.90b
7.60b

11.78a
6.40c
2.74d
2.88c

10.84b
12.41a
4.00a
9.05a

9.54c
5.73c
2.45d
2.86c

3.7. Reactive oxygen species metabolism

Under K sufciency, no differences were found for O
2 and H2O2
contents in the youngest fully-expanded leaves of Liaomian18
and NuCOTN99B (Table 7). Consequently, the MDA content was

N. Wang et al. / Journal of Photochemistry and Photobiology B: Biology 110 (2012) 18

Table 6
Effect of potassium (K) on phloem export of free amino acids from the youngest fully-expanded leaf of cotton seedlings at the ve-leaf stage (26 d after transfer to hydroponic
culture). Phloem exudates were collected from detached leaves using the EDTA-promoted technique. Seedlings were grown in either K-decient (0.03 mM; K1) or K-sufcient
(2.5 mM; K2) solutions. Values are means of four replications. Means within the same row followed by the same letter are not signicantly different (P 6 0.05) according to
Duncans multiple range test.

1
2
3

NuCOTN99B

Trait

Liaomian18
K1

K2

K1

K2

Export per leaf1 (lg h1 leaf1)

Export per unit FW2 (lg g1 FW h1)
Phloem:leaf ratio3 (100 lg g1 FW h1 lg g1 FW)

0.250c
0.235d
0.014d

0.497b
0.351c
0.079a

0.259c
0.572a
0.022c

0.925a
0.496b
0.059b

Exported free amino acids per hour per leaf.

Exported free amino acids per hour per unit leaf fresh weight.
Ratio of exported free amino acid content in phloem exudates to that in the leaf per unit leaf fresh weight.

Table 7
Effect of potassium (K) on concentrations of O
2 , H2O2 and malondialdehyde (MDA),
and activity of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase
(APX) in the youngest fully-expanded leaf of cotton seedlings at the ve-leaf stage
(26 d after transfer to hydroponic culture). Seedlings were grown in either K-decient
(0.03 mM; K1) or K-sufcient (2.5 mM; K2) solutions. Values are means of four
replications. Means within the same row followed by the same letter are not
signicantly different (P 6 0.05) according to Duncans multiple range test.
NuCOTN99B

Trait

Liaomian18
K1

K2

K1

K2

1
O
FW min1)
2 (nmol g
H2O2 (mmol g1 FW)
MDA (lmol g1 FW)
SOD (U g1 FW)
APX (mmol g1 FW min1)
CAT (U g1 FW min1)

4.59b
15.77b
4.34b
724.8b
3.98a
27.35a

3.91b
14.43b
2.94c
647.4b
2.38c
15.92b

10.19a
19.82a
5.03a
1090.8a
2.87b
16.33b

4.39b
14.45b
3.45c
760.0b
1.56d
9.55c

statistically similar in the two cultivars. Potassium deciency reB

sulted in accumulation of O
2 and H2O2 in the leaf of NuCOTN99 ,
but not in that of Liaomian18. However, the MDA content in the
leaf of Liaomian18 was enhanced by K deciency, albeit to a lower
extent than that of NuCOTN99B (Table 7), possibly because of increased levels of other undetermined ROS, such as singlet oxygen
(1O2) in the leaf.
Several antioxidative enzymes play important roles in O
2 and
H2O2 removal. Liaomian18 showed similar SOD activity, but higher
CAT and APX activities, under K sufciency compared with
NuCOTN99B (Table 7). In response to K deciency, both cultivars
signicantly increased SOD, CAT and APX activities (except SOD
in Liaomian18; Table 7). With regard to the differences between
the two cultivars, Liaomian18 had lower SOD activity, but greater
CAT and APX activities than NuCOTN99B under K deciency
(Table 7).

4. Discussion
4.1. K deciency inhibits CO2 and nitrate assimilation, restricts
assimilate transport, and impairs ROS balance in cotton seedlings
In K-decient plants, the loss of K+ by guard cells results in decreased gs and, thereby, depresses photosynthesis [29]. Also, nonstomatal reductions in photosynthesis of higher plants exposed to
K-decient environments are well known. For example, K deciency
has been shown to destroy Chl ultrastructure [30,31] and reduce Chl
content [32], thus disrupting the photochemical reactions of photosynthesis, such as Hill reaction activity [33], and production rate of
ATP and NADP [34]. The biochemical reactions of photosynthesis,
such as Rubisco biosynthesis [35] and IRA are also impaired by
K deciency [36]. Furthermore, decreased photosynthesis in
K-decient plants is associated with restricted carbohydrate

translocation [30,37], and the accumulated sucrose can affect gene

expression related to photosynthesis [38].
The study of Bednarz et al. [39] on cotton indicated that during
mild K deciency, decreased gs is the rst response to result in decreased photosynthesis and, as K deciency becomes more acute,
biochemical factors also contribute. In the present study, cotton
seedlings were grown in severely K-decient solution (0.03 mM),
in which both gs and IAR (Table 1) declined dramatically compared
with plants grown in K-sufcient solution (2.5 mM). However,
because of the increase in Ci (albeit not signicant in the leaf of
Liaomian18; Table 1), it appears that non-stomatal factors play a
more important role in decreasing photosynthesis in cotton seedlings under severe K deciency.
Nitrate reductase is the key enzyme that catalyzes the ratelimiting step in the nitrate-assimilation pathway [40]. Glutamine
synthetase functions as the major assimilatory enzyme for NH
4
incorporation into amino acids [41]. As earlier reported by other
authors [1,42], nitrate assimilation and protein synthesis were
strongly impeded by K deciency. We found that mean NR activity
and protein content in the leaf of K-decient seedlings of the two
cultivars declined by about 60% and 31.5%, respectively, whereas
the content of amino acids increased 190.8% compared with K-sufcient seedlings (Table 5). The activity of GS did not decline under
K deciency, probably because its substrate, ammonia, is supplied
from multiple pathways in addition to nitrate assimilation, such as
PR and protein breakdown [43].
Increased concentrations of soluble sugars in leaves of K-decient bean [32], cotton [30,37] and soybean [44] were considered
to be related to restricted photosynthate transport from the source
to sink organs [32]. Gerardeaux et al. [31] demonstrated that even
before leaf photosynthesis was affected by K deciency, assimilate
export already declined. In the present study, we calculated the
transport of soluble sugars using a different basis of measurement.
Firstly, calculation on the basis of leaf FW as used by Cakmak et al.
[32] makes sense when the export of assimilates among different K
levels is compared. Secondly, comparison on the basis of a unit leaf
is meaningful to investigate the supply capacity of a whole source
leaf and therefore the available photosynthate obtained by sink organs. Lastly, the ratio of soluble sugars in phloem exudates to that
in the leaf (phloem:leaf) per unit leaf FW indicates the proportion
of total potential assimilate that are exported. The results of the
present study indicated that K deciency restricted assimilate export less (33% averaged across the two cultivars) when evaluated
on the basis of leaf FW, but more when evaluated on the basis of
a unit leaf (68% averaged across the two cultivars) due to the smaller leaves of K-decient plants. Similarly, export based on the
phloem:leaf ratio was reduced by 65% under K deciency (Table
4). These results indicate that the application of different methods
of calculation are useful in describing overall assimilate export.
Translocation of AA via the sieve tubes requires mass ow of sucrose driven by active sucrose transport [45]. Cakmak et al. [32]

N. Wang et al. / Journal of Photochemistry and Photobiology B: Biology 110 (2012) 18

found that the export of AA from a source leaf was decreased in Kdecient bean plants, which occurred simultaneously with the
reduction in sucrose export. In the present study, amino acid transport from the source leaf, averaged across the two cultivars, declined only 4.6% under K deciency when expressed on the basis
of leaf FW. However, it decreased by 64 and 71% when expressed
on the basis of a unit leaf and phloem:leaf ratio (Table 6), which
were similar to that of soluble sugars.
Chloroplasts are the main organelles that produce ROS, such as
O2, H2O2 and 1O2 during photosynthesis [46]. Under normal conditions, up to 20% of the total photosynthetic electron ux is distributed to form ROS [47]. However, under K deciency,
utilization of absorbed light energy during CO2 xation is limited,
thus the electron ux to O2 is intensied, resulting to an accumulation of large quantities of ROS in chloroplasts [48]. These ndings
are consistent with those obtained for NuCOTN99B in the present
study (Table 7). Although SOD (catalyzing the dismutation of O
2
into O2 and H2O2), CAT (catalyzing the decomposition of H2O2 to
water and O2) and APX (utilizing reductant in the form of ascorbate
to detoxify H2O2) activities increased under K deciency (Table 7),
ROS were not scavenged completely and thus led to membrane
damage characterized by MDA accumulation (Table 7) and Chl degradation characterized by lower Chl content (Table 1).
4.2. Why is Liaomian18 K efcient and NuCOTN99B K inefcient?
The K-efcient cultivar, Liaomian18 showed greater capacity for
carbon assimilation under K deciency compared with the K-inefcient NuCOTN99B, distinctly associated with the higher photochemical efciency (Table 2), faster utilization of carbon
assimilation (e.g. less soluble carbohydrates and starch in leaf; Table 3) and faster export of soluble sugars (Table 4). In addition, we
assumed that the lower Pn in the leaf of NuCOTN99B exposed to K
deciency was related to greater capacity for nitrate assimilation
(Table 5). Because carbon and nitrogen metabolism must share organic carbon and energy supplied directly from photosynthetic
electron transport and CO2 xation, up to 55% of net plant carbon
is committed to nitrogen assimilation and metabolism in some tissues [49].
Chlorophyll uorescence can be used to characterize energy utilization during photosynthetic electron transport [14]. In the present study, ETR/Pn in the leaf of NuCOTN99B was signicantly higher
than that of Liaomian18 under K deciency (Table 2), suggesting
that more electrons were driven to other sinks including oxidation
of molecular O2 at the expense of reduced CO2 assimilation. Moreover, a marked decline in CO2 assimilation accompanied by a small
decrease in PSII photochemical activity under abiotic stress will
result in excessive excitation energy, since absorbed light energy
exceeds the capacity of chloroplasts to use it in CO2 xation. Net
H
Pn per unit area and PSII in the leaf of the K-efcient Liaomian18
declined 17.4% and 7.9%, respectively, under K deciency. However, in the leaf of the K-inefcient NuCOTN99B these two traits
decreased 46.9% and 14.9%, respectively, indicating more severe
imbalance between absorbed light energy and utilization for
carbon assimilation in NuCOTN99B.
Plants have developed several mechanisms to cope with excessive excitation energy during photosynthesis, such as thermal dissipation characterized by qN [50], and PR [51] that functions as a
safety valve by preventing excess NADPH and ATP from reacting
with oxygen and producing ROS. No difference was observed in
qN between the two cultivars under either K sufciency or deciency, suggesting that thermal dissipation was not related to
genotypic variation in the present study. Nevertheless, a strong difference in PR existed between Liaomian18 and NuCOTN99B. For
example, Liaomian18 showed lower PR under K sufciency. When

grown in K-decient solution, PR in the leaf of Liaomian18 showed

a 1.2-fold increase, but that of NuCOTN99B showed a 1.1-fold decrease, and the actual PR value of Liaomian18 was about 1.5-fold
greater than that of NuCOTN99B (Table 1), indicating the former
could eliminate excessive excitation energy more effectively. This
kind of genotypic variation in PR under K deciency is similar to
differences among species reported in Yamada et al. [52] where
K deciency did not affect PR in wheat and soybean, but caused
it to increase in sunower. The increased PR of Liaomian18 under
K deciency also provided an explanation, at least partly, for the
44.8% higher GS activity in response to K deciency, because the
magnitude of the ammonium ux through the PR pathway was
estimated to exceed that produced from nitrate reduction by veto ten-fold in the leaves of C3 plants [53].
With respect to antioxidant enzymes, NuCOTN99B exposed to K
deciency showed higher SOD activity but lower APX and CAT
activity relative to Liaomian18. Accompanied with the tendency
to produce more excessive excitation energy and lower capability
of PR protection in NuCOTN99B under K deciency (see above), this
imbalance between SOD and CAT and APX resulted in greater accuB
mulation of O
2 and H2O2 in leaf of NuCOTN99 compared with
B
Liaomian18 (Table 7). Thus NuCOTN99 suffered more severe oxidation degradation of Chl and membranes, as well as photoinhibition compared with Liaomian18, as indicated by its lower Chl
content (Table 1), greater MDA content (Table 7), and lower Fv/
Fm, respectively (Table 2).
Amino acids in the leaf originate from several pathways, such as
NO
3 assimilation, PR, and the hydrolysis of proteins and organic N
[54]. In the present study, although both PR (Table 1) and protease
activity (Table 5) in the leaf of NuCOTN99B (Bt cotton) were less
than 50% of those of Liaomian18 (non-Bt cotton) under K deciency, NuCOTN99B leaves accumulated 55.4% higher AA, which
explicitly resulted from the 71.7% higher NR activity. In agreement
with our results, Chen et al. [55] reported that Bt cotton cultivars
showed higher AA content, higher NR activity, and lower protease
activity in the leaf than the parental lines. Although it remains unclear why Bt cotton genotypes accumulate greater quantities of
amino acids, we speculate it might be associated with Bt toxin
biosynthesis or other processes related to Bt gene expression and
toxin function.
5. Conclusions
The K-inefcient cotton cultivar, NuCOTN99B (Bt cotton)
showed similar biomass production and partitioning as the K-efcient cultivar, Liaomian18 (non-Bt cotton) under K sufciency.
However, NuCOTN99B was affected much more by K deciency
than Liaomian18 in terms of biomass and photosynthesis. Potassium deciency inhibited CO2 and nitrate assimilation, restricted
assimilates transport, and impaired the ROS balance in cotton
seedlings. Factors contributing to the lower photosynthesis rate
of NuCOTN99B relative to Liaomian18 include: (1) higher excess
excitation energy, such as greater ETR/Pn; (2) inefcient photoprotection, such as lower PR; (3) imbalance between SOD and CAT and
APX activities; (4) higher ROS (O
2 and H2O2) accumulation and
therefore more severe photoinhibition (e.g. lower Fv/Fm); (5) greater capacity for nitrate assimilation; and (6) decreased export of soluble sugars.
These results shed light on the mechanisms underlying genotypic variations in internal K utilization efciency in cotton. Future
studies in this eld need to address the issues of biochemical reaction related to photosynthesis such as Rubisco biosynthesis, dark
respiration consumption, interaction between carbon and nitrogen
metabolism, and effects of Bt gene introduction on carbon and
nitrogen metabolism in cotton.

N. Wang et al. / Journal of Photochemistry and Photobiology B: Biology 110 (2012) 18

Abbreviations
AA
APX
CAT
Chl
Chla
Ci
ETR
Fv/Fm
gs
GS
IRA
MDA
NR
nPn
Pn
PR
qN
qP
H
PSII
ROS
SLA
SOD

free amino acids

ascorbate peroxidase
catalase
chlorophyll
chlorophyll a
intercellular CO2 concentration
electron transport rate
maximal efciency of PSII photochemistry
stomatal conductance
glutamine synthetase
initial Rubisco activity
malondialdehyde
nitrate reductase
net photosynthesis rate
total photosynthesis rate
photorespiration
non-photochemical quenching coefcient
photochemical quenching
photochemical quantum yield of photosystem
reactive oxygen species
specic leaf area
superoxide dismutase

Acknowledgments
This research was supported by the National Natural Science
Foundation of China (Nos. 30571118 and 30971708). The authors
thank Liaoning Cash Crops Research Institute, Liaoyang, Liaoning,
China, and Hebei Jidai Cotton Seed Company Ltd., Shijiazhuang, Hebei, China, for providing cotton seeds.
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