Introduction
Nitrogen
(N) is very important for the growth and development of crops (Jones et al.
2005). As the expanding of the population, the demand for food continues to
increase. In order to improve crop yields, large amounts of nitrogen
fertilizer, particularly nitrate fertilizer, are widely used (Bian et al. 2020). Excessive utilization of nitrogen
fertilizer reduced the efficiency of nitrogen fertilizer, led to environmental
contamination, accelerated soil acidification, caused the accumulation of nitrate
in plants and reduced the quality of crops (Popa et al. 2021). Temme et al. (2011) thought nitrate was one of the
most important factors influencing the quality of crops.
Vegetables are among the most important sources of food which provides essential
vitamins, minerals
and secondary metabolites for human
beings. Meanwhile, vegetable is also one kind of crops
that are particularly easy to accumulate nitrate. More than 80% of human
nitrate comes from vegetables (Velzen et al.
2008). Vegetables with high nitrate content was a threat to human health, because
it may lead to gastric cancer and methemoglobinemia (Song et al. 2015). The
nitrate problem in vegetables has gradually attracted the attention of
scientists and farmers (Bian et al. 2018). The European Union has limited the maximum nitrate
content of fresh spinach to 2000 mg/kg (Irigoyen et al. 2006).
Nitrate is absorbed by the roots,
but only a small part of it is assimilated in the roots, the rest was
assimilated in the leaves or other organs by nitrate reductase (NR) and other
metabolic enzymes (Lam et al. 1996). So, the final nitrate
content in plants depends on the absorption, transportation and metabolism of nitrate. Studies showed that the absorption, assimilation and transportation of nitrate in plants
were affected by both internal (plant itself) and external factors
(environmental factors). The content of nitrate varied among vegetables, growth
stages and plant tissues. Escobar-Gutierrez et al. (2002)
demonstrated that nitrate content varied greatly among varieties. Nitrate
content of white leaf lettuce was significantly higher than that of common
variety (Reinink et al. 1994).
The nitrate content of dark and wrinkled spinach was significantly higher than
that of light and smooth spinach (Barker et al. 1974). Transport capacity of plants to nitrate was
affected by the concentration of external nitrate. When NO3-
was added into the culture medium, the activity of high-affinity transport system
of barley was significantly increased (Aslam
et
al. 1992). The induced high-affinity transport system also
had stronger nitrate induction effect (Yaeesh et al. 1990) and the absorption capacity of nitrate nitrogen
in plants was inhibited by NH4+ (Aslam et al. 1994). How to reduce nitrate content in plants has become a concern of
everyone. It was well known that nitrification was one of a cause for nitrate accumulation.
So, nitrification inhibitors such as dicyandiamide (DCD) have been applied to
regulate on nitrate accumulation in plants. Irigoyen et al. (2006)
showed that DCD reduced the nitrate content of spinach by 18˗˗61%
under Mediterranean growing conditions. Elrys et al. (2021) concluded that DCD can limit nitrate
content of potatoes to acceptable levels.
The radish (Raphanus sativus L.) belongs to
the Brassicaceae family, which is a kind
of vegetable that is easy to accumulate nitrate. In this study, seedlings of
hydroponic cherry radish were used as the materials to study the effects of DCD
on physiological changes and nitrate accumulation, hope to lay a foundation for
study of the nitrate regulation in cherry radish in future.
Materials
and Methods
Plant material
Seeds of cherry radish (Raphanus
sativus L. var.radculus pers) namely Tianqian
(produced by Meifa seed Co. Ltd, China) were selected and soaked with water for 4 h, then sown in the plugs (54 cm×28
cm) with mixtures of peat and vermiculite (3:1 w/w). Seedlings were cultured at
20±1oC chamber with carefully management until grew to three
leaves.
Selected healthy and
uniform size seedlings with three leaves, cleaned the roots with deionized water, then cultured in the boxes filled
with Hoagland nutrient solution. After two-day culture, replaced the nutrient
solution with new ones containing different concentration of DCD (0, 3 and 6%).
Leaves were harvested for assessment of the following indexes at the 1st,
3rd, 5th, 7th and 9th day of
treatment. All experiments were randomized block design with 3 replicates.
Pigment analysis
The contents
of chorophyll-a (Chl-a), Chl-b, Chl-a+b, and carotenoid
(Car) were determined according to the method of Wang et al. (2008). A 1.0 g of leaf tissue was
homogenized with 80% acetone,
centrifuged at 12,000×g for 8 min. The supernatants were used for determination
at 662, 645 and 440 nm with a UV-5200 spectrophotometer (Shanghai Metash Instruments Co. Ltd, Shanghai, China).
Anthocyanin content
assay
The content of
anthocyanin was assayed with the method of Wang et al. (2019) with some modifications. A 1.0 g of leaf tissue
was incubated in 20 mL acidified methanol (with 1% HCl) at room temperature for 12 h. The extract
was use for the measurement of anthocyanin content at 530 and 600 nm with a
UV-5200 spectrophotometer.
Measurement of
nitrate content
Nitrate
content was determined via the method of salicylic acid (SA) according to
Machado et al. (2021) with some modifications as following steps. A 1.0 g of leaf tissue was homogenized with 15 mL ddH2O,
incubated at 900C for 30 min, cooled at room temperature for 30 min,
centrifuged at 12,000×g for 15 min, transferred the supernatant to a 25 mL
volumetric flask, and the volume was fixed to scale with ddH2O. Took
the extract (0.1 mL) to a new tube, added 0.4 mL solution of 5% SA-H2SO4,
mixed carefully, cooled at room temperature for 20 min, added 9.5 mL NaOH (8%),
cooled at room temperature again for 30 min. The OD of the reaction solution
was measured at 410 nm.
Measurement of
NR activity
NR activity
was assayed with the method of Zhang et al. (2021). A 0.5 g
of leaf tissue was homogenized with 5 mL buffer (100 mM Tris/HCl pH 8.0,
1 mM EDTA, 10 mM cysteine), then centrifuged at 12,000 g for 10
min. The supernatant was used for NR determination.
Stomatal density
Stomatal
density was determined according to Kumar et al. (2021). A
thin layer of transparent nail polish was applied on detached leaves, peeled
off after being dried according to the method of Kusumi
(2017), then observed stoma at 40× magnification with an Olympus imaging
system.
Statistical analysis
Data were subject to analysis of variance (ANOVA) and analyzed using
SPSS19.0. Differences between the means were separated by Duncan’s Multiple
Range Test (P <0.05).
Results
Effects
of DCD on pigment content in cherry radish leaves
Chl-a content: Chl-a content showed an upward trend during the
period of treatment (Fig. 1). In the first three days, there was little
difference in Chl-a content among three treatments,
and the differences appeared from the fifth day. On the fifth day, the Chl-a content of 6% DCD was the highest, then followed by
the control, and 3% DCD was the lowest. From the 7th day, Chl-a content of both 3 and 6% DCD were significantly (P
<0.05) higher than that of the control. On the 7th and 9th
day of treatment, Chl-a content of 3 and 6% DCD were
1.23, 1.15 and 1.35, 1.18 times higher than the control, respectively. In
conclusion, DCD increased the content of Chl-a in
cherry radish leaves, but this effect was related to the treatment time, that
was to say the effect was shown only in the late stage of the DCD treatment.
Chl-b
content: The change of Chl-b
content was similar to that of Chl-a under DCD
treatment. The difference was not significant (P >0.05) at the early stage
of treatment (1–5 d), but it became significantly (P <0.05) obvious later (7–9
d) %20209-214_files/image002.png)
Fig. 1: Chl-a content in leaves of cherry radish
Note: In this and subsequent figures, 0% : control; 3%: 3% DCD; 6%: 6%
DCD. Data are the mean ± SD of three repeats. Different letters are
significantly different at P < 0.05, the same as below
%20209-214_files/image004.png)
Fig. 2: Chl-b content in leaves of hydroponic cherry
radish
%20209-214_files/image006.png)
Fig. 3: Chl-a+b content in leaves of hydroponic
cherry radish
(Fig. 2). From the 7th
day of the treatment, the contents of Chl-b of both 3
and 6% DCD were all lower than those of the control, which indicated that DCD
treatment reduced the content of Chl-b at this
period.
Chl-a+b content: It can be seen that with the treatment of the time prolong, the content of
Chl-a+b increased (Fig. 3). It also showed that from
the 1st to 3rd day, the contents of Chl-a+b
in three treatments were almost the same without any differences (Fig. 3). On
the 5th and 9th day, the differences appeared, the Chl-a+b content of 6% DCD treatment was 1.13 times and 1.09
times of the control, respectively. But on the 7th day, the
difference disappeared. It suggested that DCD treatment increased the content
of Chl-a+b of radish only on certain periods, but
this need to be studied further.
Car content: During the treatment period, the content of Car
increased in all three treatments (Fig. 4). The Fig. 4 also showed that the Car
content of 6% DCD was significantly higher than that of control from the 5th
to 9th day and the Car content of 3% DCD was also significantly
higher than that of control from the7th to 9th day (Fig.
4) (P <0.05). On the 7th and 9th day, Car content of 3
and 6% DCD was 1.12, 1.29 times and 1.14, 1.26 times of the control,
respectively. In conclusion, DCD treatment increased the Car content of cherry
radish and the effect under 6% DCD was better.
%20209-214_files/image008.png)
Fig. 4: Car content in leaves of hydroponic cherry radish
%20209-214_files/image010.png)
Fig. 5: Anthocyanin content in leaves of hydroponic
cherry radish
Anthocyanin content: With the extension of treatment time, anthocyanin
content increased in all three treatments (Fig. 5). From the 5th day
onwards, the anthocyanin content of 3 and 6% DCD was significantly higher than
that of the control (Fig. 5) (P <0.05). On the 5th, 7th
and 9th day, the anthocyanin content of 3% DCD was 1.21, 1.20, 1.33
times of the control (0%), respectively. For the 6% DCD group, it was 1.23,
1.17, 1.34 times of the control, respectively. However, there was little
difference between 3 and 6% DCD treatment. It concluded that DCD promoted
anthocyanin accumulation in cherry radish leaves.
Effects of DCD on nitrate accumulation in leaves
During the whole treatment time,
the nitrate content of leaves in all treatments increased (Fig. 6). Among the
three treatments, the nitrate content of 3% DCD was the lowest. There was no
significant difference in nitrate content between 3% DCD and the control from
the first day to the fifth day (P > 0.05). From the 7th day to 9th
day, nitrate content of 3% DCD was significantly lower than the control (P
<0.05). Therefore, 3% DCD treatment inhibited the accumulation of nitrate in
leaves. There was no significant difference in nitrate content between 6% DCD
and the control from the first day to the fifth day (P >0.05). But on the 7th
day, the nitrate content of 6% DCD was significantly higher than that of the
control (P <0.05). It can also be seen from Fig. 6 that the curve of the 6%
DCD was always above the control, which suggested that this treatment promoted
nitrate accumulation in leaves of cherry radish.
On the 5th, 7th and 9th day of the
treatment, the nitrate content of 3% DCD was 0.89, 0.84 and 0.79 times, while
the 6% DCD was 1.06, 1.14, 1.16 times of the control, respectively. In
conclusion, 3% DCD has an inhibitory effect on nitrate accumulation in cherry
radish leaves, while 6% DCD did an opposite effect.
DCD treatment also affected NR activity of radish leaves (Fig. 7). During
the period of treatment, NR activity showed an upward trend, which was similar
to the change of nitrate content (Fig. 6). Compared with the control, the NR
activity of 3% DCD showed no significant difference in the first three days (P
>0.05), but it was higher than that of the control from the 5th
to 9th day. There was no significant difference in the NR activity
between 6% DCD treatment and the control in the first 5 days (P >0.05). But
from the 7th to 9th day, NR activity of 6% DCD was lower
than the control. On the 5th, 7th and 9th day
of treatment, the NR activity of 3% DCD was 1.05, 1.07, 1.06 times of the
control, while it was 0.96, 0.85, 0.83 times of the control under 6% DCD,
respectively. In conclusion, 3% DCD treatment increased NR activity in cherry
radish leaves, but 6% DCD treatment inhibited it, which suggested that the
influence of DCD on NR activity may be related to DCD concentrations. The
results further showed that there was a negative correlation between nitrate
content and NR activity in cherry radish leaves under DCD treatment.
%20209-214_files/image012.png)
Fig. 6: Nitrate content in leaves of hydroponic cherry radish
%20209-214_files/image014.png)
Fig. 7: NR activity in leaves of hydroponic cherry radish
%20209-214_files/image016.png)
Fig. 8: Stomatal density in leaves of hydroponic
cherry radish
Effects of DCD on stomatal density in leaves of
cherry radish
Stomatal density in all treatments
increased all the time (Fig. 8). Difference was not significant between the 3%
DCD treatment and the control (P>0.05). However, 6% DCD influenced stomatal
density obviously. From the 5th to 9th day, the stomatal
density in 6% DCD treatment was significantly lower than both control and 3%
DCD (P<0.05). These results indicated that 6% DCD treatment had a certain
inhibitory effect on the increase of stomatal density, while 3% DCD did not.
Discussion
Fertilizer is very important for crops. However,
unreasonable use of fertilizers can cause nitrate accumulations in plants,
which was harmful to human beings (Popa et al. 2021). Reducing nitrate content in vegetables has
become a concern of everyone, and various measures are being explored.
Management of the nitrogen fertilizer was a common measure for reducing the
nitrate accumulation. Liu and Yang (2012) verified that reducing nitrate concentration in nutrient solution
significantly reduced nitrate content of lettuce. However, reducing inputs of
nitrogen fertilizer also affected crop growth and development, thus led to
yield loss. Application of nitrifying inhibitor such as DCD to regulate nitrate was
seemed to be a suitable measure. Yu et al. (2006) showed
that adding 10–20% DCD reduced the nitrate content in leaves and stems of
radish by 29.9–34.6% and 11.7–32.2%, respectively. The results of this experiment agreed with the above. Since
NR is one of important enzymes in the process of nitrogen metabolism, it is
also a rate limiting enzyme in the process of nitrogen uptake, assimilation and
translocation. The activity of NR determines the rate of nitrate assimilation
into organic nitrogen compounds; therefore, the decrease of nitrate
accumulation in plants by DCD may be related to NR activity.
It was thought that a higher NR activity promoted the
reduction of nitrate, thus reduced nitrate accumulation levels. For example,
low nitrate content of pakchoi, its amount of NR
activity and gene expression was higher (Luo et al. 2006). The
nitrate content of potato transformed by exogenous NR gene was also
significantly reduced (Djennane et al. 2002). It seemed that NR activity was negatively
correlated with nitrate content, which was similar to the results of this experiment.
However, the
results of Blom-Zandstra and Alberr (1986)
suggested that nitrate content was positively correlated with NR activity. Analysis showed that the relationship between nitrate content and NR activity may be associated with many factors, such as plant tissue, culture method,
growth environment, etc. Nitrate is absorbed by roots, most of it is
transported to other tissue and organs including leaves to assimilate under NR
and other enzymes, which may be affected by many factors. Liu et al. (2006)
believed that the influence on nitrate content might not be the absolute value
of NR, but the expression degree of potential NR activity. The higher the potential NR activity, the more
nitrate is reduced and less accumulated in plant tissue (Liu et al. 2006). Nitrate
content may also be related to the ratio of endogenous/exogenous NR activity
(Huang et al. 2012).
Chl-a and Chl-b
were similar in structure except that the side chain at C-7 was a methyl group
in Chl-a, while it was a formaldehyde group in Chl-b. Biosynthesis of Chl-b
started and ended with Chl-a, which was considered to
be a “Chl cycle”. Biosynthesis of Chl-a
need only one step while Chl-b need two steps. Plants
adjusted the Chl-a/b ratio according to different
conditions (Wang et al. 2008). Many studies found that as long as the Chl-b content decreased, there must be an increase in Chl-a content (Akoyunoglou and Akoyunoglou 1985; Tanaka et al. 1991). The results
of this study was consistent with the results above, which suggested that DCD
affected the Chl metabolism of cherry radish.
Perhaps, there were some action sites for DCD in the Chl
metabolism. Anyway, future research needs to be identified and studied.
Conclusion
DCD increased
the content of Chl-a, Car and anthocyanin of cherry
radish. 3% DCD also deduced the nitrate accumulation. Data indicated that DCD
not only affected the photosynthesis, but also improved the color and reduced
nitrate accumulation of cherry radish. It is well-known that Chl is an important pigment for photosynthesis, Car and
anthocyanin are important color pigments and nitrate is harmful for human
beings. So, application of a certain amount of DCD in cherry radish production
may be an approval method to improve its appearance and safety quality.
Acknowledgement
This work was
supported by the National Natural Science Foundation of China (31471867).
Author
Contributions
WF planned the
experiments, LG and ZX interpreted the results. WF and LG made the write up and
JY statistically analyzed the data and made illustrations.
Conflict of
Interest
All authors
declare no conflict of interest.
Data
Availability
Data presented
in this study will be available on a fair request to the corresponding author.
Ethics
Approval
Not applicable
in this paper.
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