Phosphorus deficiency restricts plant growth but induces pigment formation in the flower stalk of Chinese kale

Abstract

The effect of phosphorus (P) nutrition on plant growth and pigment formation in the flower stalk was studied under hydroponic conditions for 2 Chinese kale (Brassica alboglabra Bailey) cultivars: ‘Jianyexia’ (green flower stalk) and ‘Hongjiao’ (mauve flower stalk). Three different P treatments were used: 30 (normal-P), 7.5 (low-P), and 0 mg·L−1 (P-deficient). The results showed that the biomass, yield, plant height, stem diameter, and leaf number of Chinese kale were significantly reduced in the low-P and P-deficient treatments compared to the normal-P treatment. The chlorophyll content in the flower stalk epidermis was not affected by different P levels in ‘Jianyexia’, but was significantly reduced by the P-deficient treatment in ‘Hongjiao’. Decreased P levels caused the flavonoid, soluble phenol, and anthocyanin content of the flower stalks to gradually increase in both Chinese kale cultivars. The pH value of the flower stalk epidermis gradually decreased with the declining P levels, and was significantly different among the 3 treatments. As the P levels declined, phenylalanine ammonia-lyase (PAL) and chalcone isomerase (CHI) activities in the flower stalk epidermis gradually increased, and were significantly different among the 3 treatments. P nutrition may control the synthesis of anthocyanins in the flower stalk by regulating the epidermal pH value, and the activities of PAL and CHI.

Full text

Phosphorus Deficiency Restricts Plant Growth but Induces Pigment
Formation in the Flower Stalk of Chinese Kale
Riy uan Chen1,2
ŏ
, Shiwei Song2
ŏ
, X iuchun Li2, Houcheng Liu2, and Danfeng Huang1*
1College of Agriculture and Biology, Shanghai Jiaotong University, Shanghai 201101, P R China
2College of Horticulture, South China Agricultural University, Guangzhou 510642, P R China
*Corresponding author: hdf@sjtu.edu.cn
ಳThese authors are contributed equally to this work.
Received February 11, 2013 / Revised April 26, 2013 / Accepted May 3, 2013
GKorean Society for Horticultural Science and Springer 2013
Abstract. The effect of phosphorus (P) nutrition on plant growth and pigment formation in the flower stalk was studied
under hydroponic conditions for 2 Chinese kale (Brassica alboglabra Bailey) cultivars: ‘Jianyexia’ (green flower stalk)
and ‘Hongjiao’ (mauve flower stalk). Three different P treatments were used: 30 (normal-P), 7.5 (low-P), and 0 mg·L-1
(P-deficient). The results showed that the biomass, yield, plant height, stem diameter, and leaf number of Chinese kale
were significantly reduced in the low-P and P-deficient treatments compared to the normal-P treatment. The chlorophyll
content in the flower stalk epidermis was not affected by different P levels in ‘Jianyexia’, but was significantly
reduced by the P-deficient treatment in ‘Hongjiao’. Decreased P levels caused the flavonoid, soluble phenol, and
anthocyanin content of the flower stalks to gradually increase in both Chinese kale cultivars. The pH value of the
flower stalk epidermis gradually decreased with the declining P levels, and was significantly different among the 3
treatments. As the P levels declined, phenylalanine ammonia-lyase (PAL) and chalcone isomerase (CHI) activities in
the flower stalk epidermis gradually increased, and were significantly different among the 3 treatments. P nutrition may
control the synthesis of anthocyanins in the flower stalk by regulating the epidermal pH value, and the activities of
PAL and CHI.
Additional key words: anthocyanins, chalcone isomerase, phenylalanine ammonia-lyase
Hort. Environ. Biotechnol. 54(3):243-248. 2013.
DOI 10.1007/s13580-013-0018-x
Research Report
Introduction
Phosphorus (P) is one of the most important nutrients for
plant growth and development. P plays an essential role in
numerous biological functions. For example, P serves as a
structural element in phospholipids and nucleic acids, con-
tributes to energy metabolism, and is involved in the regulation
of enzymatic activities and signal transduction cascades
(Raghothama, 1999; Rausch and Bucher, 2002). Furthermore,
previous studies had indicated that P deficiency restricts the
plant growth and development (Akhtar et al., 2007; Li et al.,
2006; Zhou et al., 2009).
Stress caused by P deficiency may induce the synthesis of
numerous secondary metabolites, particularly flavonoids in
plant. For instance, the anthocyanin content of Arabidopsis
thaliana increases when P was deficient, regardless of whether
it is a wild-type, a P-deficient mutant, or a low P-insensitive
mutant (Sánchez-Calderón et al., 2006; Zakhleniuk et al.,
2001). Generally, anthocyanin content increases and plant
growth decreases under N and P deficiency, when compared
to plants with complete nutrient access (Hodges and Nozzolillo,
1996; Rajendran et al., 1992). Furthermore, P deficiency was
shown to result in an evident increase of anthocyanin content
in tomatoes, independent of varieties and cultivation conditions
(Ulrychová and Sosnová, 1970). The biosynthesis of anthocyanin
was influenced by pH in plant cell (Cabrita et al., 2000), in
addition to the activities of various enzymes (Martin et al.,
1991). However, the mechanism of anthocyanin formation
under P deficiency requires further study.
Chinese kale (Brassica alboglabra Bailey) is an important
vegetable in South China, with a large growing area and
marketable supply in this region. The flower stalk of Chinese
kale is the edible organ, which is crisp and full of nutrients,
e.g. Vitamin C, glucosinolates and minerals. There are 2
types of Chinese kale, which are differentiated according to
flower stalk color, i.e., green and mauve. Flower stalk color
of Chinese kale is determined mainly by the anthocyanin
and chlorophyll contents in its epidermis. Flower stalk color

Riyuan Chen, Shiwei Song, Xiuchun Li, Houcheng Liu, Danfeng Huang
244
was also affected by the presence of other pigments, such as
flavonoids and carotenoids (Liu et al., 2004). The flower
stalk color of Chinese kale may change from green to mauve
when P is deficient. This color change seriously affects the
visual quality of the flower stalks in the green-type Chinese
kale.
In this study, the aforementioned 2 types of Chinese kale
were grown under hydroponic conditions, to study the effect
of P nutrition on plant growth and pigment formation in
flower stalk epidermis. The aim is to clarify how P nutrition
regulates the synthesis of pigment, especially anthocyanins,
in flower stalk.
Materials and methods
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The study was conducted in a plastic greenhouse located
at the experimental vegetable farm of the South China
Agricultural University. Two Chinese kale cultivars ‘Jianyexia’
with green flower stalks and ‘Hongjiao’ with mauve flower
stalks, were seeded in plastic plug trays with 72 cells. In
total, 15 seedlings with 3 true leaves were transplanted in a
plastic box (61 cm length × 42 cm width × 8 cm height)
filled with 15 L nutrient solution. The nutrient solution
consisted of : N, 200; P, 30; K, 240; Ca, 130; Mg, 48; Fe, 2;
B, 0.5; Mn, 0.5; Zn, 0.05; Cu, 0.02; and Mo, 0.02 mgL-1.
The nutrient solution was aerated for 15 minutes every hour
via an automatically controlled pump, and was completely
refreshed every 9 days.
Three different P treatments were used: 30 (normal-P),
7.5 (low-P), and 0 mg·L-1 (P-deficient). There were 4 replicates
of each treatment (one plastic box represented one replicate)
and for each cultivar, with a randomized block arrangement.
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Chinese kale plants were sampled once the mature stage
was reached to measure their growth parameters. Three
representative plants from each replicate were selected at
random. ‘Jianyexia’, an early maturing cultivar, was sampled
earlier than ‘Hongjiao’, a medium maturing cultivar. Plant
height and stem diameter (in the middle of 5th and 6th nodes
of the flower stalk) were measured, respectively. Each plant
was divided into root, rootstock, and flower stalk respectively,
and fresh weight and dry weight (after drying at 70°C to
constant weight) were obtained.
The epidermis of the flower stalk was used to measure the
pigment content. Approximately 0.5 g fresh ground epidermis
was extracted in 80% acetone, and the chlorophyll and
carotenoid content were assayed according to Lichtenthaler
(1987). The contents of soluble phenols, flavonoids, and
anthocyanins were analyzed according to Pirie and Mullins
(1976). 0.5 g sample of flower stalk epidermis was extracted
with methanol-HCl (0.1% HCl v/v) for 3 h. The extracts
were measured at 530 and 600 nm using a spectrophotometer
(Thermo Spectronic Helios Ȗ, Madison, USA), and anthocyanin
concentration was calculated as A530-A600. After 50 ȝL
extract was added to 4.5 mL methanol-HCl (0.1% HCl v/v),
soluble phenols were measured at 280 nm. Flavonoid levels
were measured at 325 nm after 1 mL extract was added to 4
mL methanol-HCl (0.1% HCl v/v). The pH value was measured
after 2.0 g of flower stalk epidermis was homogenized with
10 mL double-distilled water.
Enzyme activity related to anthocyanin biosynthesis was
measured using the epidermis sample of plant flower stalk.
A total of 0.5 g sample was added to 5 mL of extract solution
consisted of 0.05 M Na2HPO4 (pH 7.0), 0.05 M ascorbic
acid, and 0.018 M mercaptoethanol, and then homogenized
at 4°C, and centrifuged at 15000 rpm for 20 minutes. The
supernatant was a crude enzyme extraction solution, which
was used to determine the activity of phenylalanine ammonia-
lyase (PAL) and chalcone isomerase (CHI). PAL activity
was measured according to the method of McCallum and
Walker (1990), and CHI activity was determined according
to the method of Boland and Wong (1975).
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All data were statistically analyzed by ANOVA, using the
SPSS software package (version 16.0 for Windows). Two-way
ANOVA was used to evaluate the effect of P levels and
cultivars, and their interactions were evaluated using
Duncan’s multiple range tests.
Results
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Growth characteristics (e.g., plant height, stem diameter,
and leaf number) of the 2 Chinese kale cultivars were
significantly affected by different P levels (Table 1). All
indicators showed the same tendency in both cultivars: plant
height, stem diameter and leaf number were not significantly
different between normal P and low-P treatments, but
significantly lower and less in the P-deficient treatment than
other 2 treatments.
P levels had a significant effect on growth of both Chinese
kale cultivars. Shoot fresh weight gradually decreased with
the decreasing P levels, with a significant difference being
recorded among all 3 treatments. Compared with the normal-P
treatment, the shoot fresh weight of low-P and P-deficient
treatments was reduced by 23.2% and 58.9% in ‘Jianyexia’,
respectively, and by 29.0% and 84.7% in ‘Hongjiao’, re-
spectively. A similar trend was observed for the effect of P
levels on root fresh weight.

Hort. Environ. Biotechnol. 54(3):243-248. 2013. 245
Table 1. The effect of P levels on the growth of Chinese kale.
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The root/shoot ratio of both Chinese kale cultivars was
significantly higher in the P-deficient treatment than the
normal-P and low-P treatments; however, the difference
between the latter two was not significant. This result
indicated that P deficiency promotes limited biomass production
to be preferentially allocated to the roots. The root/shoot
ratio of ‘Hongjiao’ was higher than ‘Jianyexia’ at the same
P level.
The yield (weight of flower stalks) gradually decreased
with decreasing P levels in both cultivars. Compared to the
normal-P treatment, the yield with low-P and P-deficient
treatments was reduced by 22.0% and 65.4% in ‘Jianyexia’,
respectively, and by 18.8% and 80.4% in ‘Hongjiao’,
respectively. There was a significant difference among the 3
treatments.
Two-way analyses (P levels and cultivars) were conducted
for the growth parameters of Chinese kale, with the results
showing that both factors significantly affect plant growth.
Shoot and root fresh weight, along with the root/shoot ratio,
had significant interaction effects by P levels and cultivars.
However, yield, plant height, stem diameter, and leaf number
had no interaction effect.
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The chlorophyll and carotenoid contents in the flower
stalk epidermis were not significantly affected by P levels
(Table 2), with the exception that the P-deficient treatment
significantly reduced the chlorophyll content in ‘Hongjiao’.
The ratio of chlorophyll a/b gradually decreased with decreasing
P levels, and was significantly lower in the P-deficient
treatment than the normal-P treatment. This result indicated
that although there was no decrease in the total chlorophyll
content of the flower stalk epidermis, chlorophyll a content
was lower in the P-deficient treatment.
The contents of soluble phenols, flavonoids, and antho-
cyanins in the flower stalk epidermis generally increased
with decreasing P levels. The pigment content was significantly
higher in the P-deficient treatment than the normal-P treatment.

Riyuan Chen, Shiwei Song, Xiuchun Li, Houcheng Liu, Danfeng Huang
246
Fig. 1. The effect of P level on the pH of the flower stalk epidermis
in Chinese kale. The vertical bars represent the standard error
of measurements (n = 4) and the values followed by different
letter are significantly different at P < 0.05 (Duncan’s method).
Fig. 2. The effect of P level on the activity of 2 enzymes related to anthocyanin biosynthesis in Chinese kale. The vertical bars represent
the standard error of measurements (n = 4) and the values followed by different letter are significantly different at P < 0.05 (Duncan’s
method).
Anthocyanin content was 4.5 and 1.34 times higher in the
P-deficient treatment than the normal P treatment for ‘Jianyexia’
and ‘Hongjiao’, respectively.
Two-way analyses (P levels and cultivars) were performed
for the pigment parameters of Chinese kale. The P levels
significantly affected pigment content, except for carotenoids.
Pigment content was significantly different between the 2
Chinese kale cultivars. For instance, the contents of chlorophyll,
carotenoid, and soluble phenol were significantly higher in
‘Jianyexia’ compared to ‘Hongjiao’. In comparison, the
anthocyanin content was significantly higher in ‘Hongjiao’
than ‘Jianyexia’. This result was consistent with the color
performance of the 2 cultivars. Chlorophyll and carotenoid
content showed no interaction effect with P level and
cultivar, while the other 4 parameters had a significant
interaction effect.
S+ RI )ORZHU 6WDON (SLGHUPLV
The pH value of the flower stalk epidermis for both
cultivars ranged from 5.76 to 6.41 (Fig. 1). The pH value
gradually decreased with decreasing P levels, and a significant
difference was obtained among the 3 treatments.
$FWLYLW\ RI $QWKRF\DQLQ %LRV\QWKHVLV (Q]\PHV
The activities of pigment metabolism enzymes were
markedly affected by the P levels (Fig. 2). PAL activity in
‘Jianyexia’ was the highest in the P-deficient treatment,
followed by the low-P treatment and the normal-P treatment.
The difference among the 3 treatments was significant. In
‘Hongjiao’, the PAL activity was significantly higher in the
P-deficient treatment than the normal P and low P treatments.
There was no significant difference between the latter 2
treatments. The CHI activity was the highest in the P-deficient
treatment, followed by the low-P and normal-P treatments.
The difference among the 3 treatments was significant.
Discussion
Low-P and P-deficient treatments significantly reduced
the biomass of the 2 Chinese kale cultivars, with this being
more severe in the P-deficient treatment. Growth inhibition
was greater in the P-deficient treatment for ‘Hongjiao’
compared to ‘Jianyexia’. Similarly, P deficiency decreased
plant growth in Glycine max (Tsvetkova and Georgiev,
2003), Arabidopsis thaliana (Jain et al., 2005; Li et al.,
2006) and Brassica (Akhtar et al., 2007). Many studies had
demonstrated that this decrease was caused by the leaf
photosynthetic rate and carbon metabolism of plants being
inhibited (Li et al., 2006; Zhou et al., 2009).
P deficiency may lead to the accumulation of secondary

Hort. Environ. Biotechnol. 54(3):243-248. 2013. 247
metabolites, such as flavonoids (Plaxton and Carswell, 1999)
and anthocyanins (Yamakawa et al., 1983). In this study,
P-deficiency induced anthocyanin and flavonoid accumulation
in the flower stalk epidermis, in parallel to reducing chloro-
phyll content. Thus, the green epidermis of ‘Jianyexia’ became
mauve, whereas the mauve color ‘Hongjiao’ became deep
purple.
Anthocyanin is characteristically involved in ionization,
with color expression being highly influenced by its structure
and pH (Cabrita et al., 2000). Low-P and P-deficient treatments
caused a decrease in flower stalk pH, while increasing
anthocyanin content. This result indicated that Chinese kale
could promote the synthesis of anthocyanins by reducing
epidermal pH. A previous study showed that acidic conditions
promote the formation of anthocyanins in apple (Saure, 1990).
The formation of anthocyanin is regulated by structural
and regulatory genes. For example, structural genes encode
dozens of enzymes from phenylalanine (Phe) to form antho-
cyanins (Martin et al., 1991). PAL is a key enzyme in
anthocyanin synthesis via the phenylpropanoid pathway.
CHI is an enzyme associated with flavonoid synthesis (Heller
et al., 1985), and catalyzes the formation of primary flavonoids
(2s-flavanone). Recent study on anthocyanin biosynthesis
focused on these enzymes’ activities and their encoding
gene expression.
During apple ripening, ethylene initiates rapid anthocyanin
accumulation by increasing the amount of the rate-limiting
enzyme, PAL, in the skin (Faragher and Brohier, 1984). When
day/night temperatures were low, the total anthocyanin and
polyphenol content in lettuce were positively correlated with
the activities of PAL (Boo et al., 2011). Moreover, the active
expression of a PAL gene (PAL6) in tomato was compatible
with flavonoid synthesis (Løvdal et al., 2010). In the process
that causes an apple to turn red, increased CHI activity was
associated with increased anthocyanin content (Lister et al.,
1996). Furthermore, the simultaneous gene expressions of
chalcone synthase (CHS) and CHI in flax resulted in a
significant increase in the levels of flavones and anthocyanins
(Lorenc-Kukuáa et al., 2007).
Methyl jasmonate/sucrose treatment stimulated PAL and
CHS gene expression, which triggered the accumulation of
both anthocyanins and piceids in grapevine cells (Belhadj et
al., 2008). In the current study, the P-deficient experiment
significantly improved PAL and CHI activity in Chinese
kale, causing anthocyanins to accumulate in the flower stalk
epidermis. Thus it could be deduced that under P-deficient
conditions, Chinese kale might promote anthocyanin synthesis
by enhancing PAL and CHI activity, which in turn caused
the flower stalk color to change.
Ac know ledg ment: This study was financially supported
by China Agriculture Research System (CARS-25-C-04).
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