Journal of Integrative Agriculture
Advance Online Publication 2015
Doi: 10.1016/S2095-3119(15)61246-1
Response of root morphology, physiology and endogenous hormones in maize (Zea
mays L.) to potassium deficiency
1*
1,2*
ZHAO Xin-hua , YU Hai-qiu1*, WEN Jing , WANG Xiao-guang1*, DU Qi1, WANG Jing, WANG Qiao1
1
College of Agronomy, Shenyang Agricultural University, Shenyang 110866, P.R.China
2
Agro-Biotechnology Research Institute, Jilin Academy of Agricultural Sciences, Changchun 130124, P.R.China
Abstract Potassium (K) deficiency is one of the major abiotic stresses which has drastically influenced maize
growth and yield around the world. However, the physiological mechanism of K deficiency tolerance is not yet
fully understood. In present study, the differences of root morphology, physiology and endogenous hormones
were investigated using two typical inbred lines under high K (+K) and low K (-K) treatments. The results
indicated that the root length, volume and surface area of 90-21-3 were significantly higher than those of D937
under –K treatment on different growing stages. It is worth noted that the lateral roots of 90-21-3 were
dramatically higher than that of D937 on tasseling and flowering stage under –K treatment. Meanwhile, the
value of superoxide dismutase (SOD) and oxidizing force of 90-21-3 was apparently higher than that of D937,
whereas malondialdehyde (MDA) content of D937 was obviously increased. Compared with +K treatment, the
indole-3-acetic acid (IAA) content of 90-21-3 was largely increased under –K treatment, whereas it was sharply
decreased in D937. On the contrary, abscisic acid (ABA) content of 90-21-3 was slightly increased, but that of
D937 was significantly increased. The Zeatin Riboside (ZR) content of 90-21-3 was significantly decreased,
while that of D937 was relatively increased. These results indicated that the endogenous hormones were
stimulated in 90-21-3 to adjust lateral root development and to maintain the physiology function thereby
alleviating K deficiency.
Key words: potassium deficiency, maize, root morphology, physiological variation, endogenous hormone1
1. Introduction
Potassium (K) as one of essential macronutrients has various functions on crop development, growth and yield
formation process, serving as a cofactor for more than 40 enzymes in different metabolic pathways, regulation of
cell osmotic pressure and stomatal movements (Clarkson and Hanson 1980; Marschner 2011). Soil nutrient
deficiency has directly endangered the world food security in developing and underdeveloped countries (Tan et al.
2005). Large farmlands of the world were reported to be deficient in K including India, China, Southern
Australia, etc. (Rengel and Damon 2008; Römheld and Kirkby 2010; Jin 2012). In addition, lower fertilizer K
application in many cases of unbalanced fertilization has led to declined soil fertility (Vitousek et al. 2009; Zörb et
al. 2014). Maize (Zea mays L.) is one of the most important cereal crops world-wide as well as an important
source of feed, fibre and biofuel. Therefore, improving the efficiency of K acquisition ability and utilization is
important for Maize production.
The ability of plant to absorb nutrients and water from soil is related to their capacity to develop an abundant
root system, working as intermediary between plant and soil. Not only can plant root system regulate morphology
and architecture to adapt to soil condition, but also significantly adjust their metabolism in order to more
Correspondence YU Haiqiu, Mobile: 13674201361, Tel: +86-024-88487135, E-mail: [email protected],
* indicates the authors who contributed equally to this study
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effectively acquire the nutrients (López-Bucio et al. 2003; Schachtman and Shin 2007; Zhang et al. 2010).
Localized application of appropriate nitrogen (N) plus phosphorus (P) fertilization significantly improved maize
root growth and nutrient uptake at seedling stage by intensifying root proliferation and rhizosphere acidification
on calcareous soil (Jing et al. 2012; Ma et al. 2014). However, excessive application of N fertilizer also
inhibited root morphological development during intensive maize production (Tian et al. 2008). Conversely,
nutrient starvation could affect shoot and root growth. Under K or Mg deficiency, sucrose export in phloem
exudate was remarkably decreased in leaves and much lower proportions of photosynthates were distributed to the
roots, thus changing metabolite concentrations in bean plant organs (Cakmak et al. 1994a, b). The rice root
growth and root-shoot ratio were decreased due to soluble sugar declining in roots under K deficiency stress (Cai
et al. 2012). Moreover, Wissuwa et al. (2005) indicated that plant could preferential allocated the acquired
nutrient to root thus improving nutrient absorption to the point that nutrients do not impose any longer such
limitation. K deficiency has a depressive effect on primary root growth in Arabidopsis, whereas it appeared
stimulated lateral root initiation and development (Armengaud et al. 2004; Gruber et al. 2013; Kellermeier et al.
2013). Comprehending the adaptations of root systems to nutrient deficiency has been pointed out regard as the
key issue in modern agriculture (Herder et al. 2010).
Plant root development is adjusted by the integration of endogenous factors, such as phytohormones, with
integrate environmental stimuli, such as soil nutrients (Osmont et al. 2007; Petricka et al. 2012). Particularly,
phytohormones play an important role in plant development and in acclimating response to abiotic stress
(Franco-Zorrilla et al. 2004; Gao et al. 2014). Some reports have indicated that P starvation lead to a variation in
ethylene and auxin responsiveness in the root (Borch et al. 1999; López-Bucio et al. 2002). In addition, auxin
(IAA), cytokinin (CK), abscisic acid (ABA) and ethylene have been considered to play a role in plant response to
K deficiency in Arabidopsis (Jung et al. 2009; Nam et al. 2012; Shin 2014). Chen et al. (2015) reported that
WOX11 gene was controlling root and shoot phenotypes in the OsHAK 16p: WOX 11 transgenic lines improved
expression of several RR genes encoding type-A cytokinin-responsive regulators, PIN genes was encoding auxin
transporters and Aux/IAA genes in rice root under K-deficiency condition. Moreover, the increased ethylene
crosstalks with ABA, IAA and CK to adapt flooding stress condition, especially aerenchyma cells were improved
by those kind of interactions under waterlogging stress (Shimamura et al. 2014; Kim et al. 2015). However,
roles of phytohormone in K deficiency are still unclear in maize at present time.
In the present study, two representative maize inbred lines, screened in our previous research (Cao et al.
2007), were carried out to investigate relationship among root growth, physiological traits and endogenous
hormones. The objections are to identify variations of root structure under K deficiency stress, to explore
interactions between enzyme and root, and to expound the effects of phytohormones on maize plant growth.
2. Results
2.1 Root morphological variations
The root morphological variations were showed on Fig. 1 on different growing stages under different K treatments.
Under –K treatment, the total root length of 90-21-3 were decreased before booting stage, whereas it was gently
increased 274 and 347 cm on shooting stage and on tasseling and flowering stage (Fig. 1-A), respectively. By
contrast, the total length of D937 were slightly decreased compared with +K treatment during the whole growing
period. Under -K treatment, the total root lengths of 90-21-3 were significantly longer than D937 during the
whole growing period.
Under +K treatment, there was no significant difference on primary and lateral roots between 90-21-3 and
D937 (Fig. 1-B). However, under –K treatment, the primary and lateral roots of 90-21-3 were significantly more
than those of D937. It is noteworthy that an abundant of lateral roots in 90-21-3 were developed on tasseling and
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flowering stage under –K treatment, which was significantly more than D937.
Root volume of two inbred lines showed different variation trend under low potassium stress (Fig. 1-C). On
seedling and shooting stages, root volumes of two inbred lines under –K treatment were less than those under +K
treatment. The root volume of 90-21-3 were gently increased under –K treatment on booting, tasselling and
flowering stages. On the contrary, the root volumes of D937 were significantly decreased during the whole
growing period, especially on tasselling and flowering stage. The root volume of 90-21-3 was 16.57 cm3 under
–K treatment on booting stage, which was 1.50 times of D937. However, on tasselling and flowering stage, the
root volume of 90-21-3 was 38.8 cm3, which was significantly higher than D937.
Root surface area also displayed a similar variation trend with root volume (Fig. 1-D). There is no
significant different difference observed in the root surfaces of 90-21-3 under –K and +K treatments. However,
compared with +K treatment, the root surface area of D937 was largely decreased under –K treatment. It is very
obvious that the root surface area of D937 was significantly decreased from 1431 to 822 cm2 on tasseling and
flowering stage. The root surface areas of 90-21-3 were 1388.4 and 2074.0 cm2 under –K treatment on booting,
tasseling and flowering stages, which were significantly higher than those of D937, 2.65 and 2.53 times,
respectively.
The root average diameter only on shooting stage of 90-21-3 was significantly higher than D937 under +K
treatment (Fig. 1-E). However, there is no significantly different under –K treatment. It is showed that the root
average diameter of 90-21-3 was slightly decreased on different growing stages under –K treatment, whereas
those of D937 were appreciably increased on shooting stage and on tasseling and flowering stage.
Overall, the root length, volume, surface area of 90-21-3 were relatively increased under –K treatment
especially on tasseling and flowering stage, which were significantly less than 90-21-3. Additionally, the rate of
lateral root of 90-21-3 was significantly higher than D937.
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Fig. 1 Root morphological variations in two inbred lines under different potassium treatments.
A, C, D and E showed the variations
of root length, root volume, root surface area and average diameter on different growing periods, respectively.
proportion of primary root and lateral root on tasseling and flowering stage.
significant different at the P<0.05 level.
B showed the
a, b and c on line charts and histograms means the
The same as below.
2.2 Physiological variations
The contents of MDA and SOD were showed on Fig. 2 (A, B). Although there was slightly different between
two inbred lines for the content of MDA under +K treatment on tasseling and flowering stage, the content of MDA
in D937 was significantly increased from 0.91 to 1.94 nmol g-1 under –K treatment (Fig. 2-A), and it was only
increased from 0.46 to 0.56 nmol g-1 in 90-21-3. Compared with +K treatment, the activity of SOD in 90-21-3
(84.7 u g-1) was relatively higher under –K treatment (Fig. 2-B), whereas it was decreased from 76.3 to 68.5 u g-1
in D937. It is meaningful that the activity of SOD in 90-21-3 was largely higher than that in D937 under –K
treatment.
The oxidizing force and reducing ability of root were shown in Fig. 2 (C, D) under different K treatments.
Under –K treatment, oxidizing force of 90-21-3 and D937 roots were significantly increased compared with +K
treatment (Fig. 2-C). Oxidizing force of 90-21-3 root was significantly higher than that of D937 under –K
treatment. Contrarily, the reducing ability of two inbred line roots were significantly decreased under –K
treatment (Fig. 2-D). The variation of reducing ability of D937 root was larger than that of 90-21-3 root.
In brief, under –K treatment, the SOD activities and oxidizing ability of 90-21-3 were largely increased,
while the MDA content was slightly increased. The reducing ability of 90-21-3 and D937, however, were
sharply dropped to similar level.
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Fig. 2 Physiological variations in two maize inbred lines under different potassium treatments. A, B, C and D showed the difference
of MDA, SOD, Oxidizing ability and Reducing ability in two inbred lines on tasseling and flowering stage, respectively.
2.3 Root endogenous hormone variations
The variations of endogenous hormone in root were shown on tasseling and flowering stage under +K and –K
treatments (Fig. 3). Under +K treatment, the IAA content of 90-21-3 (1.49 μg g-1) was slightly higher than
D937 (1.31μg g-1) (Fig. 3-A), whereas the IAA content of 90-21-3 was significantly higher than D937 under –K
treatment. Meanwhile, the ABA content of 90-21-3 under –K treatment only little higher than that under +K
treatment, whereas the ABA content of D937 under –K treatment was significantly increased compared with +K
treatment. Under +K treatment, the GA3 content of D937 was largely higher than 90-21-3 (Fig. 3-C). The GA3
content of 90-21-3 under –K treatment was increased from 6.28 to 6.57μg g-1 compared with +K treatment,
whereas the GA3 content of D937 was decreased 7.63 to 7.05μg g-1 under –K treatment. The ZR content of
90-21-3 under –K treatment was significantly decreased from 21.55 to 14.67μg g-1 compared with +K treatment,
whereas the ZR content of D937 under –K treatment was largely increased from 20.21 to 23.49μg g-1 compared
with +K treatment. In short, IAA content of 90-21-3 was dramatically higher than D937 under –K treatment, but
ABA and ZR contents of D937 were significantly higher than 90-21-3.
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The endogenous hormone variations in two inbred line under different potassium treatments.
A, B, C and D showed the
difference of IAA, ABA, GA3 and ZR in two inbred lines, respectively.
3. Discussion
3.1 Root morphology response to K deficiency
Significant difference capacities of absorbing nutrients from soil were demonstrated in plants by modifying root
development, architecture and exudation. Under nutrient deficiency stress, one of the most significant variations
in root architecture is that abundant root hairs were formed as a result of induction of some epidermal cell files
(López-Bucio et al. 2003). Under P deficiency condition, Arabidopsis root hairs became longer and denser
contributing to the vast increased epidermal cells that differentiate into trichoblasts (Bates and Lynch 1996; Ma et
al. 2001). In maize, when the growth of nodal root, the main component of mature roots system, was inhibited
by P deficiency, the maintenance of its formation could result in an increased proportion of lateral root
development in the adventitious root system (Zhu and Lynch 2004; Bayuelo-Jiménez et al. 2011;
Bayuelo-Jiménez and Ochoa-Cadavid 2014). Meanwhile, Johnson et al. (1996) discovered that the proteoid
roots, a clusters of short lateral roots, were developed under low-P stress in root systems of white lupin to
specialize P acquirability. Homologous, N deficiency stimulated maize roots to develop longer axial roots,
including primary roots, seminal roots, and nodal roots, which was conducive to explore a wider rhizosphere soil
and suffice the spatial N availability (Tian et al. 2008). In the present study, the root length, root volume, root
surface area of inbred line D937 sensitive to K deficiency were declined through the whole growing period under
–K treatment, especially on tasselling and flowering stage. However, it was gently increased on booting stage
and on tasselling and flowering stage in K deficiency tolerance of 90-21-3. Under –K treatment, the root length,
root volume, root surface area of 90-21-3 were attractively higher than D937. The root average diameters of
90-21-3 were slightly decreased under –K treatment, but those of D937 were relatively increased on shooting
stage and on tasseling and flowering stage. Nevertheless, on tasselling and flowering stage, the ratio of lateral
roots of 90-21-3 was significantly higher than that in D937. These results indicated that the root morphology
modifying was particularly attributed to promotion of lateral root, which enable root to absorb adequate nutrients
from soil and maintain plant growth to adapt effectively to the K deficiency environment.
Inbred lines 90-21-3 tolerance to K deficiency with increased elongation of lateral root development under –K
treatment had superior ability to absorb K and to maintain crop development.
3.2 Root physiology variations under K deficiency
In plants, potassium deficiency in soil causes marginal chlorosis, roots had an increased susceptibility to
root-rotting fungi and inducing early senescence (Taize and Zeiger 2010). Deficiency in macronutrients
including K resulted in oxidative stress, as evidenced by accumulation of reactive oxygen species (ROS) and
membrane lipid peroxidation (Shin and Schachtman 2004; Miao et al. 2010). It was well known that the SOD
activity was one vital part of plant inner protective enzyme system and played an important role in plant senescing
and stresses (Mittler 2002). Tewari et al. (2007) reported that K deficiency lead to increasing SOD activity in
maize leaves. Moreover, Miao et al. (2010) indicated that a significant increase in MDA concentrations in
soybean leaves when grown in K-deficient medium, while the activities of SOD, POD and CAT were increased
substantially. In present study, root MDA content, which was the production of membrane lipid peroxidation, in
D937 was obviously higher than that in 90-21-3 on tasselling and flowering stage under –K treatment, whereas it
was at the similar level under +K treatment. Contrarily, variations of antioxidant enzymes showed that the
activity of SOD in root of 90-21-3 was obviously higher than that in root of D937 under –K treatment.
Furthermore, under –K treatment, oxidizing force of 90-21-3 root were significantly enhanced on different
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growing periods, whereas the reducing ability of root were significantly decreased. These results indicated that
the inbred line 90-21-3 tolerance to K deficiency alleviated K deprivation-induced oxidative damage to root
development by modulating antioxidant enzyme activity.
3.3 Endogenous hormones variations under K deficiency
Plant hormones were important endogenous factors for the integration of different environmental stimuli,
including temperature fluctuations, availability of nutrients, water accommodation, and stimulated to regulate
plant development for stress adaptation (Vanstraelen and Benková 2012). IAA as a major of auxin in higher
plants has been demonstrated to promote roots development by exogenous application, whereas it also could stunt
the root development by applying IAA transport inhibitors, or at excess levels (Blakely et al. 1988; Philippar et al.
2003; Ivanchenko et al. 2008). Certifiable evidence demonstrated that auxins were an important signal
controlling the development of lateral roots under phosphorus deficiency (Niu et al. 2013). ABA was negative
regulator of lateral root emergence and could function as a growth inhibitor in certain cells in the presence of
severe drought stress (Cheng et al. 2002; De Smet et al. 2006). Lateral root formation was an important
organogenetic process that determined the establishment of root architecture in higher plants, which contribute to
water-use efficiency and improve the absorbance of micro- and macro-nutrients from the soil (Fukaki et al. 2007).
Several previous studies had showed that N deficiency increased carbon partitioning to roots and shifted the root
system architectural balance, accelerating root growth, developing fewer, longer axial roots with longer and
denser lateral roots (Eghball and Maranville 1993; Gaudin et al. 2011; Yu et al. 2014). Many physiological and
genetic studies had certified that IAA played an important role in Lateral root initiation and primordium
development (Casimiro et al. 2003; Fukaki et al. 2007). In recent years, it had become apparent that hormonal
pathways had interoperable function by a complicated network and respond to determine the final outcome of the
individual hormone actions (Vanstraelen and Benková2012; Hofmann 2015). Yang et al. (2002) demonstrated
that both ABA and cytokinins were concerned with controlling rice senescence under water stress, and the
elevated ABA level enhanced the carbon remobilization in rice plants subjected to water stress. Kim et al. (2015)
conducted that the ethylene production ratio was significantly higher in tolerant line than susceptible line under
waterlogging stress condition in soybean, whereas ABA contents were dramatically lower in plant. In the present
study, the IAA content of 90-21-3 root was significantly higher than that of D937 root under –K treatment,
whereas they were at similar level under +K treatment. To the contrary, the ABA content of D937 root was
significantly increased under –K treatment, while it was slightly increased in 90-21-3 root. Furthermore, the
GA3 content of 90-21-3 was slightly increased under –K treatment, but that of D937 root was relatively decreased.
Under –K treatment, the ZR content of 90-21-3 was significantly decreased, whereas it was slightly increased in
D937 root. In addition, the lateral root rate of 90-21-3 was in significantly increased under –K treatment on
tasselling and flowering stage. These results indicated that the maize inbred line, 90-21-3, tolerance to low
potassium could maintain the content of ABA in a relatively low level as far as possible, in order to reduce the
degree of senescence at late growth stage and delay the senescence procedure. Meanwhile, the IAA content of
root was increased resulting in stimulating root development especially the lateral root adjustment.
4. Conclusion
The present study demonstrated that maize inbred line 90-21-3 tolerance to K deficiency could adjust root
morphological variations, maintain the physiological variations and regulate the endogenous hormone content to
alleviate K deficiency in maize. These results highlight that the interaction of IAA and ABA content in root
stimulated root development to absorb adequate K nutrient from soil and maintained plant growth under lower K
level. These results will certainly have important practical meaning in agronomic production to improve nutrient
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efficiency under nutrient-deficient conditions.
5. Materials and methods
Two maize inbred lines, 90-21-3 tolerance to potassium deficiency and D937 sensitive to potassium deficiency,
were measured in the long-term K fertilizer experimental pool of Shenyang Agricultural University. The pool
soil was collected from natural potassium deficiency field in Manduhu village (41°32′ N, 122°43′ E), Shenyang
City, Liaoning province, China. The experimental soil was classified as sandy with lack of potassium,
un-application K fertilizer for five years. The initial plough layer (20cm) nutrient elements were 50.4 mg kg-1
available K, organism 12.9 g kg-1, alkali-hydrolysable N 108.5 mg kg-1, available P 16.1 mg kg-1.
5.1 Experimental design
Low potassium (-K) and high potassium (+K) treatments were implemented on 5th May 2014. In the –K
treatment, potash fertilizer was not applied in the test pool, while sulfuric acid potassium fertilizer of 150 kg ha-1
was applied in the +K treatment to adjust the available K content to 130 mg kg-1 (normal conduction). In
addition, 420 kg ha-1 urea and 210 kg ha-1 (NH4)2HPO4 were applied as seed manure, and 315 kg ha-1 urea was
added in booting stage. Four rows per inbred line spacing 0.50 m, length with 3.5 m, were arranged on each
experiment plots. Hill spacing was 0.30 m. Randomized block design with three replications was used.
Roots for morphology trait measurement were sampled on seedling stage, shooting stage, booting stage, tasseling
and flowering stage, while the samples collected on tasseling and flowering stage were frozen in liquid N and
stored at -80℃ for physiological variation and endogenous hormone assay.
5.2 Measuring methods of root morphology and physiology traits
Root morphological traits, including root length, volume, surface area and average diameter, were measured with
WinRHIZO Program (Regent Instruments Inc., Canada). Lipid peroxidation was determined by measuring the
Malondialdehyde (MDA) content in thiobarbituric acid method as described by Tewari et al. (2007). Superoxide
dismutase (SOD) activity was measured by the photochemical photo reduction method with Nitro-blue
tetrazolium chloride (NBT) as described by Miao et al. ( 2010).
5.3 Measuring methods of endogenous hormones
The measurement of Indole-3-acetic acid (IAA), gibberellins (GA3), abscisic acid (ABA) and Zeatin Riboside (ZR)
was performed by high performance liquid chromatography (HPLC) as described by Han et al. (2011) and Crozier
and Moritz (1999). The phytohormone standard samples of GA3, IAA, and ABA were purchased from the Sigma
Chemical Co. (St. Louis. MO, USA). The other relevant reagents were either chromatographically or
analytically pure.
About 1.0g (freeze-dried) root sample was ground in ice-bath and cold homogenized, and its hormone was
extracted with 8 times 80% methanol at 1.0℃ for 24 h in dark. Next day, the extract liquor was centrifuged at
16000g for 10min at 4℃. The extracted supernatants were draw and added polyvinylpolypyrolidone (PVPP),
then shake and centrifuged at 16000g for 10 min at 4℃. Then the filer residue was extracted with little 80%
methanol in order to minimize the loss of phytohormones, and the filtrate was collected together finally.
The filtrate was filtrated with C18 cartridges. The effluent was blow dry and dissolved with 1 mL
phosphate buffer which pH is 3.0, and then extracted three times with an equal volume ethyl acetate. Upper
layer was the ethyl acetate phase, lower was the aqueous phase. After the aqueous phase was blow dry,
dissolved with 1ml phosphate buffer (pH=8.0), and extracted three times with 85% N-butanol saturated with water,
pooled the aqueous phase, blow dry, dissolved with 1 mL methanol, through 0.45 um organic membrane, than
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could determine ZR content. Ethyl acetate phase in upper layer lowed dry, dissolved with 1 mL methanol,
through 0.45um organic membrane than could determine the content of IAA, ABA and GA3.
An aliquot (10 uL) of the methanol preparation was injected into an Agilent 1100 HPLC, which was
equipped with a DAD detector and a Dimas Bor C18 column (5 um, 250×4.6 mm). The methanol preparation
was eluted through C18 column with methanol: 0.1% acetic acid (v:v=55:45) forming its mobile phase. The flow
rate and temperature were set at 0.8 mL min-1 and 35℃, respectively.
The standard samples of GA3, IAA, ABA and ZR were used to determine the plant hormones under the
selected conditions. Precise 9mg IAA, GA3, ABA and ZR were each dissolved into 10 mL volumetric flask with
chromatogram methanol as mother liquor (concentration of 900 mg L-1). Then the mother liquor was diluted into
6 different concentration gradients: 90, 30, 9, 3, 0.9, and 0.3 to determine the sample in the conditions of selected
chromatography in turn.
The determination of the three hormones of the maize root samples was repeated three times. The means
of hormones were the averages of their corresponding three determination replications, were compared by Duncan
test at P=0.01 using SPSS.
5.4 Statistical analysis
A two-way analysis of variance (ANOVA) (repeated) procedure was calculated by SPSS19.0 Statistics software to
detect the significance of the interaction between different genotypes and K treatments. Different letters on line
charts and histograms indicated that means were statistically different at the P<0.05 level.
Acknowledgements
The work was financially supported by the Program for Liaoning Excellent Talents in University (LR2013032),
the National Natural Science Foundation of China (31301259, 31101106) and the Tianzhu Mountian Scholars
Support Plan of Shenyang Agricultural University, China.
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