Selenium Inhibits Root Elongation by Repressing the Generation of Endogenous Hydrogen Sulfide in Brassica rapa
et al. (2014) Selenium Inhibits Root Elongation by Repressing the Generation of Endogenous Hydrogen
Sulfide in Brassica rapa. PLoS ONE 9(10): e110904. doi:10.1371/journal.pone.0110904
Selenium Inhibits Root Elongation by Repressing the Generation of Endogenous Hydrogen Sulfide in Brassica rapa
Yi Chen 0
Hai-Zhen Mo 0
Mei-Yu Zheng 0
Ming Xian 0
Zhong-Qiang Qi 0
You-Qin Li 0
Liang-Bin Hu 0
Jian Chen 0
Li-Fei Yang 0
Anil Kumar, University of Missouri-Kansas City, United States of America
0 1 College of Horticulture, Nanjing Agricultural University , Nanjing , China , 2 Institute of Food Quality and Safety, Jiangsu Academy of Agricultural Sciences , Nanjing , China , 3 Department of Food Science, Henan Institute of Science and Technology , Xinxiang, Henan Province, China, 4 Lishui Plant Science Base , Jiangsu Academy of Agricultural Sciences , Nanjing , China , 5 Department of Chemistry, Washington State University , Pullman, Washington , United States of America
Selenium (Se) has been becoming an emerging pollutant causing severe phytotoxicity, which the biochemical mechanism is rarely known. Although hydrogen sulfide (H2S) has been suggested as an important exogenous regulator modulating plant physiological adaptions in response to heavy metal stress, whether and how the endogenous H2S regulates Se-induce phytotoxicity remains unclear. In this work, a self-developed specific fluorescent probe (WSP-1) was applied to track endogenous H2S in situ in the roots of Brassica rapa under Se(IV) stress. Se(IV)-induced root growth stunt was closely correlated with the inhibition of endogenous H2S generation in root tips. Se(IV) stress dampened the expression of most LCD and DCD homologues in the roots of B. rapa. By using various specific fluorescent probes for bio-imaging root tips in situ, we found that the increase in endogenous H2S by the application of H2S donor NaHS could significantly alleviate Se(IV)induced reactive oxygen species (ROS) over-accumulation, oxidative impairment, and cell death in root tips, which further resulted in the recovery of root growth under Se(IV) stress. However, dampening the endogenous H2S could block the alleviated effect of NaHS on Se(IV)-induced phytotoxicity. Finally, the increase in endogenous H2S resulted in the enhancement of glutathione (GSH) in Se(IV)-treated roots, which may share the similar molecular mechanism for the dominant role of H2S in removing ROS by activating GSH biosynthesis in mammals. Altogether, these data provide the first direct evidences confirming the pivotal role of endogenous H2S in modulating Se(IV)-induced phytotoxicity in roots.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its
Supporting Information files.
Funding: This study was supported by the National Natural Science Foundation of China (31101537, http://www.nsfc.gov.cn/). The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
Selenium (Se) contamination is a global environmental safety
issue because Se is becoming an emerging health hazards due to
the dramatic increase in Se concentration in the environment
[1,2]. The rapid development of metal industry promotes the
biogeochemical cycle of Se, which results in the remarkably
anthropogenic release of Se into soil [1,3]. Se is an essential
micronutrient for plants because Se-containing proteins play vital
roles in regulating plant growth and plant adaption to the
environment [4–6]. Additionally, human prefer to consume
Serich foods because Se appears to have an critical role in
strengthening the immune system in human body [7,8]. Thus,
the importance of Se for both human and plants has driven the
long-term application of Se fertilizers in farm work, which is
another important factor contributing to the increasing
anthropogenic release of Se into the agricultural environment [9,10].
Se with low dose often acts as a protector helping plants against
various environmental stimuli , but the great concern has been
raised about the possible adverse effects of the excessive Se in
plants. Treatment with Se (8–16 ppm) significantly inhibits the
growth of barley shoot . Se at the concentration of 4–6 ppm
show remarkable inhibitory effect on the growth of both shoot and
root in bean seedlings . By using image analysis of roots, the
root development of lettuce and ryegrass can be completely
inhibited by Se as low as 1 ppm . The mechanism of
Seinduced phytotoxicity is rarely reported because of the limited
studies about the adverse effects of Se on plants. Several studies
suggest that excessive Se can trigger oxidative stress in plants by
inducing the production of reactive oxygen species (ROS) and the
subsequent lipid peroxidation, which may contribute to
Seinduced phytotoxicity [12,13,15]. A recent study indicated that
Se-induced growth stunt of root was closely associated with the
disturbance of plant hormones and endogenous nitric oxide (NO)
in Arabidopsis , but the biochemical mechanisms for
Seinduced phytotoxicity are still elusive.
Hydrogen sulfide (H2S), the third gasotransmitter generated
endogenously in mammals after NO and carbon monoxide (CO),
has been highly appreciated for its clinical relevance [17–20]. In
plants, H2S is produced from cysteine desulfuration catalyzed by
L-cysteine desulfhydrase (LCD, EC18.104.22.168) and D-cysteine
desulfhydrase (DCD, EC22.214.171.124), both of which belonging to pyridoxal
59-phosphate (PLP)-dependent protein family . Both genes
(LCD and DCD) have been characterized in Arabidopsis [22–24].
Recently, H2S has been drawing increasing attention in plants
because it shows great potential in the regulation of multiple
physiological processes in plants, but the detailed studies in the
biological role of H2S in plants are still very limited as compared to
those in mammals [25,26]. The exogenous application of NaHS, a
H2S donor, can alleviate the phytotoxicity induced by various
metal species, such as copper (Cu) , chromium (Cr) ,
boron (B) , lead (Pd) [30,31], aluminum (Al) [32–34], and
cadmium (Cd) [35–37]. All of these reports suggest that H2S may
be an important player regulating plant response to heavy metal
stress. Nevertheless, the specific role of endogenous H2S in
modulating the phytotoxicity induced by heavy metals (including
Se) is largely unknown because of the lack of the data of tracking
endogenous H2S in situ in plants. Our recent study demonstrate
that Washington Stat Probe 1 (WSP-1) is a very useful fluorescent
probe for selectively capturing and tracking H2S in vivo in plant
root, which provides a powerful tool for identifying the role of
endogenous H2S as a true cellular signaling molecule in regulating
plant physiology [38,39].
In this work, we investigated whether and how endogenous H2S
responds to Se-induced toxicity in the roots of Brassica rapa. The
effect of Se stress on the generation of endogenous H2S was
studied in vivo by using fluorescent microscopy. To get deeper
insights into the role of H2S in Se-induced toxicity, the
involvement of the endogenous H2S in root elongation, cell death,
and oxidative injury was investigated further by pharmacological
experiments. These results were able to help our understanding for
the role of H2S in plants under Se stress, which could extend our
knowledge of H2S in plants and Se-induced phytotoxicity.
Materials and Methods
Plant culture and chemicals
Seeds of B. rapa (LvLing) seeds were surface-sterilized with 1%
NaClO for 10 min followed by washing with distilled water. Seeds
were germinated for 1 day in the dark on the floating plastic nets.
Then the selected identical seedlings with radicles 0.5 cm were
transferred to another Petri dish containing various treatment
solutions in a chamber with a photosynthetic active radiation of
200 mmol/m2/s, a photoperiod of 12 h, and the temperature at
Seedling roots were exposed to Na2SeO3 (sodium selenite,
Se(IV)) with different concentrations (0.03–0.46 mM) for various
treatment time (0–72 h). The 0–2.0 mM of NaHS (sodium
hydrosulphide) was applied as H2S donor. PAG
(DL-propargylglicine) (0.05–0.2 mM) and HT (hypotaurine) (0.1–0.4 mM) are H2S
biosynthesis inhibitor and H2S scavenger, respectively. The
treatment solution is composed of different chemicals as
mentioned above according to the experimental design. After
treatments, the roots were washed with distilled water for
physiological, histochemical, and biochemical analysis.
The intracellular H2S was visualized using specific fluorescent
[39-methoxy-3-oxo-3H-spiro[isobenzofuran-1,99xanthen]-69-yl 2-(pyridin-2-yldisulfanyl)benzoate] in situ
according to our previous method . The roots of seedlings after
treatments were incubated at 20 mM Hepes-NaOH (pH 7.5)
buffer solution containing 20 mM of WSP-1 at 25uC for 40 min.
Then the roots were washed with distilled water three times and
were visualized immediately by a fluorescence microscope with a
465/515 nm and an excitation/emission filter set (ECLIPSE,
TE2000-S, Nikon). The relative fluorescent density of the
fluorescent images was analyzed using Image-Pro Plus 6.0 (Media
Intracellular ROS was visualized using specific fluorescent
probe DCFH-DA (29,79-dichlorofluorescein diacetate) in situ
described by Foreman et al. . The roots of seedlings were
incubated in 10 mM of DCFH-DA at 25uC for 10 min. Then the
roots were rinsed with distilled water for three times followed by
the visualization (excitation 488 nm and emission 525 nm) with a
fluorescence microscope (ECLIPSE, TE2000-S, Nikon). The
relative fluorescent density of the fluorescent images was analyzed
using Image-Pro Plus 6.0 (Media Cybernetics, Inc.).
Intracellular superoxide radical was visualized using specific
fluorescent probe DHE (dihydroethidium) in situ described by
Yamamoto et al . The roots of seedlings after treatment were
incubated in 15 mM of DHE at 25uC for 15 min. Then the roots
were rinsed with distilled water for three times and were visualized
(excitation 535 nm and emission 610 nm) by a fluorescence
microscope (ECLIPSE, TE2000-S, Nikon). The relative
fluorescent density of the fluorescent images was analysed using
ImagePro Plus 6.0 (Media Cybernetics, Inc.).
Histochemical detection of cell death was performed by using
propidium iodide (PI) in situ as described by Kellermeier et al
. The roots of seedlings after treatment were incubated in
20 mM of PI solution for 20 min. Then the roots were rinsed with
distilled water for three times and were visualized (excitation
535 nm and emission 615 nm) by a fluorescence microscope
(ECLIPSE, TE2000-S, Nikon). The relative fluorescent density of
the fluorescent images was analyzed using Image-Pro Plus 6.0
(Media Cybernetics, Inc.).
Histochemical detection of glutathione (GSH) was performed by
using specific molecular probe monochlorobimane in situ as
described by Liso et al . The endogenous GSH in root was
visualized after conjugation with monochlorobimane to give
fluorescent GS-bimane adduct. The roots of seedlings after
treatment were incubated in 100 mM of monochlorobimane
solution for 30 min. Then the roots were rinsed with distilled
water for three times and were visualized (excitation 390 nm and
emission 478 nm) by a fluorescence microscope (ECLIPSE,
TE2000-S, Nikon). The relative fluorescent density of the
fluorescent images was analyzed using Image-Pro Plus 6.0 (Media
Figure 2. Effect of Se(IV) on the endogenous H2S in the root tips of B. rapa. The roots of seedlings were exposed to 0, 0.03, 0.06, 0.12, 0.23,
and 0.46 mM of Se(IV) solution for 48 h. Afterwards, the roots were loaded with WSP-1 for fluorescent imaging (A) and the calculation of relative
fluorescent density (B). (C–D) The image and density of WSP-1 fluorescence were obtained when the roots of seedlings were exposed to 0.06 mM of
Se(IV) solution for 0, 3, 6, 12, and 24 h, respectively. (E) The correlation analysis between WSP-1 fluorescent density and root elongation under Se(IV)
treatment with concentration at 0, 0.03, 0.06, 0.12, 0.23, and 0.46 mM. Asterisk indicates that mean values of three replicates are significantly different
between the treatments of Se(IV) and the control group (CK) (P,0.05).
Histochemical detection of lipid peroxidation was achieved by
using Schiff’s regent as described by Wang and Yang . The
roots of seedlings after treatment were incubated in Schiff’s regent
for 20 min. Then the stained roots were rinsed with a solution
containing 0.5% (w/v) K2S2O5 (prepared in 0.05 M of HCl) until
the root colour became light red. After that, the roots were
photographed using a digital camera.
Histochemical detection of loss of plasma membrane integrity
was performed by using Evans blue as described by Yamamoto
et al. . The roots of seedlings after treatment were incubated in
5 ml of 0.025% Evans blue solutions (w/v) for 20 min. After that,
the roots were rinsed with distilled water for three times followed
by photographed using a digital camera.
Screening and analysis of LCD and DCD from the genome
of B. rapa
The sequences of AtLCD (AT5G28030) and AtDCDs
(AT3G26115 and AT2G48420) from Arabidopsis were used as
baits for BLAST research in the genome of B. rapa from BRAD
(http://brassicadb.org/brad/index.php). The obtained sequences
were retrieved and analyzed. The phylogenetic trees were
constructed using the maximum likelihood method in MEGA
5.2. The multialignment of amino acid sequences was performed with ClustalX 2.0 and DNAMAN 5.2.2. Protein structure prediction was performed on SMART (http://smart.emblheidelberg.de/).
The DNA sequences with the length of 2 kb were retrieved
from the upstream of LCDs and DCDs in B. rapa for promoter
analysis. The sequence between the start of target gene and the
end of its upstream gene was obtained for promoter analysis if the
length of this sequence was less than 2 kb. The cis-elements in the
retrieved promoter regions were analyzed using online tool
Analysis of transcripts
Total RNA was extracted from root tissues using Trizol
(Invitrogen) according to the manufacturer’s instructions. Reverse
transcription was performed at 42uC in 25 ml reaction mixture
including 3 mg of RNA, 0.5 mg of oligo (dT) primers, 12.5 nmol of
dNTPs, 20 units of RANase inhibitor and 200 units of M-MLV.
The first cDNA was used as a template for polymerase chain
amplification and to analyse the transcripts of genes by using
realtime quantitative reverse transcription-polymerase chain reaction
(qRT-PCR) (Applied Biosystems 7500 Fast Real-Time PCR
System, LifeTechnologies). The primers designed for the
amplification of the genes are listed in Table S1.
Measurement of Se concentration in roots
About 0.2 g of dried root sample was mixed with 10 mL of
HNO3:HClO4 (4:1, v/v) in a test tube and covered with Parafilm
for 24 h. Then the samples were digested. The digested solution
was analyzed for Se concentration by using hydride generation
atomic fluorescence spectrometry (AFS-230a, Beijing Wantuo).
The calibration was performed by using standard Se solution with
concentration of 10–80 mg/L .
Each result was presented as the mean 6 standard deviation
(SD) of at least three replicated measurements. The significant
differences between treatments were statistically evaluated by SD
and one-way analysis of variance (ANOVA) using SPSS 2.0. The
data between two specific different treatments were compared
statistically by ANOVA, followed by F-test if the ANOVA result is
significant at P,0.05. For multiple comparison analysis, least
significant difference test (LSD) was performed on all data
following ANOVA tests to test for significant (P,0.05) differences
among different treatments.
Se(IV) treatment inhibited root elongation of B. rapa
Treatment with Se(IV) significantly inhibited root elongation in
both dose- and time-dependent manners. The roots of B. rapa
were exposed to 0–0.46 mM of Se(IV) for up to 72 h. Compared
to the control group, root elongation decreased by 25%, 63%,
77%, 88%, and 93% at 0.03, 0.06, 0.12, 0.23, and 0.46 mM of
Se(IV) levels, respectively (Figure 1A). In a time-course
experiment, exposure of 0.06 mM of Se(IV) showed significantly
inhibitory effect on root elongation. Compared to the control
group, root elongation began to decreased after treatment with
0.06 mM of Se(IV) for 24 h, and continued to decrease up to 72 h
Se(IV) treatment inhibited the generation of endogenous
H2S in root tips
Root tip is the main expansion zone for root elongation . In
order to test the effect of Se(IV) stress on endogenous H2S in root
tips, we performed in situ detection of endogenous H2S generation
by using specific fluorescent probe WSP-1. Compared to the
control, the decreased WSP-1 fluorescent density was observed in
root tips in the presence of Se(IV) in a dose-dependent manner
(Figure 2A and B). In a time-course experiment, the endogenous
H2S indicated by WSP-1 fluorescence maintained stable up to
24 h in control groups. However, WSP-1 fluorescence began to
decrease significantly after treatment with 0.06 mM of Se(IV) for
6 h (Figure 2C and D). The correlation analysis suggested that the
changes of endogenous H2S level occurred in parallel with the
changes of root elongation under Se(IV) stress. Initially, WSP-1
fluorescent density decreased slowly with the light decrease in root
elongation, followed by a quick decrease with the dramatic
inhibition of root elongation induced by Se(IV) at high
concentrations (Figure 2E). These results suggested that the generation of
endogenous H2S decreased significantly in root tips upon Se(IV)
Se(IV) stress differentially regulated the expression of LCD
and DCD in roots
In order to understand how Se(IV) stress impacted the
generation of endogenous H2S, we further investigated the effect
of Se(IV) stress on the expression of LCD and DCD in the roots of
B. rapa. According to BLAST search, sequence identity, and
phylogenetic analysis, we obtained two DCD homologues
(Bra025184 and Bra018726) and ten LCD homologues
(Bra020605, Bra001131, Bra014529, Bra004781, Bra006115,
Bra037682, Bra039708, Bra009985, Bra036910, and Bra006114)
from B. rapa (Figure S1). All of the retrieved LCDs and DCDs
have typical PLP domains (Figure S2). LCD has been well studied
in Arabidopsis and Brassica napus [23,47]. The multialignment of
deduced amino acid sequences revealed that the obtained LCDs
from B. rapa had many typically structural features of plant LCDs,
such as PLP-binding sites, the substrate binding site, and the SAT
protein-interaction site (Figure S3) .
The expression levels of LCDs and DCDs under 0.06 mM of
Se(IV) treatment were tested by using qRT-PCR (Figure 3). The
results suggested that Se(IV) stress showed extensively inhibitory
effect on the expression of both LCDs and DCDs (Figure 3). Two
DCDs in roots were down-regulated upon Se(IV) stress (Figure 3).
Figure 6. Effect of NaHS, PAG, and HT on the endogenous H2S level in root tips with or without Se(IV) stress. In the presence of
0.06 mM of Se(IV) or not, the roots were treated with water, 0.5 mM of NaHS, 100 mM of PAG, and 200 mM of HT alone or their combinations. After
various treatments for 3 h, the roots were loaded with WSP-1 for fluorescent imaging (A) and the calculation of relative fluorescent density (B). The
mean values of three replicates followed by different letters indicate significance of difference between the treatments (P,0.05, ANOVA, LSD).
Compared to the control, Se(IV) treatment decreased the
expression of most LCDs. Among them, Bra001131, Bra020605,
and Bra039708 showed relatively more decreased transcription as
compared to their controls, respectively (Figure 3). These results
suggested that Se(IV)-induced inhibition of endogenous H2S might
resulted from the down-regulation of LCDs and DCDs in the roots
of B. rapa.
Analysis of nitric oxide-, auxin-, and metal-responsive
cis-elements in the promoter region of LCDs and DCDs
According to the identification of NO-responsive cis-element
(NRE) from higher plants [48,49], several NREs (e.g. ACGT Box,
MYCL, and W-BOX) could be found in the promoter region of all
the LCDs and DCDs obtained from B. rapa (Table S2). In
addition, the auxin-responsive cis-element (ARE) could be found
in most LCDs and DCDs except for Bra018726 (Table S2).
Application of H2S donor NaHS alleviated Se(IV)-induced
root inhibition by enhancing endogenous H2S level
To obtain more evidence for the involvement of H2S in the
regulation of root elongation under Se(IV) stress, the H2S donor
NaHS was added to the treatment solution. A preliminary
experiment with NaHS at 0.06–2.0 mM was carried out to
determine the point where NaHS showed the most significant
effect. Treatment with NaHS at 0.5 mM had the greatest effect on
the alleviation of Se(IV)-induced inhibition of root elongation
(Figure 4A). The root elongation increased by 90% in seedlings
treated with 0.5 mM NaHS+0.06 mM Se(IV) as compared to
0.06 mM Se(IV) treatment alone (Figure 4A). In a time-course
experiment, Se(IV)-induced reduction in root elongation was
significantly recovered when roots were incubated in the treatment
solution containing both Se(IV) and 0.5 mM of NaHS (Figure 4B),
which may resulted from the enhancement of endogenous H2S
To verify the alleviated effect of NaHS on root elongation under
Se(IV), we tested the root elongation treated with PAG
(endogenous H2S biosynthesis inhibitor) and HT (H2S scavenger),
respectively. Compared to the control, PAG and HT resulted in
the significant decreases in root elongation (Figure 5A),
respectively, suggesting that the endogenous H2S is essential for root
elongation. Furthermore, the addition of PAG or HT could
partially blocked the alleviated effect of NaHS on Se(IV)-induced
root inhibition (Figure 5B), which may resulted from the decrease
in endogenous H2S level.
Subsequently, we test the effect of NaHS application on the
endogenous H2S level in roots under Se(IV) stress. In Se(IV)-free
roots, NaHS could enhance the level of endogenous H2S while
both PAG and HT were able to decrease endogenous H2S level
(Figure 6). Additionally, the addition of NaHS could recover the
decrease in endogenous H2S level in Se(IV)-treated roots.
However, in both Se(IV)-free and Se(IV)-treated roots, the
enhancement of endogenous H2S level by NaHS supplement
could be blocked by the addition of PAG and HT, respectively
(Figure 6). All of these results suggested that the enhancement of
endogenous H2S could alleviate Se(IV)-induced inhibition in root
Treatment with NaHS attenuated Se(IV)-induced ROS
generation, cell death, and oxidative injury in roots
Compared to the control, treatment with 0.06 mM of Se(IV)
resulted in the over-generation of total endogenous ROS in root
Figure 7. Effect of NaHS on endogenous ROS, super oxide radical, and cell death in root tips under Se(IV) stress. The roots were
exposed to water, 0.06 mM of Se(IV), 0.06 mM of Se(IV) and 0.5 mM of NaHS, and 0.5 mM of NaHS for 3 h. Then roots were loaded with DCFH-DA (A),
DHE (C), and PI (E) for fluorescent imaging, respectively. The fluorescent density of DCF (B), DHE (D), and PI (F) was estimated, respectively. The mean
values of three replicates followed by different letters indicate significance of difference between the treatments (P,0.05, ANOVA, LSD).
tips indicated by staining with specific fluorescent probe
DCFHDA. However, the addition of NaHS significantly decreased the
accumulation of total ROS induced by Se(IV) (Figure 7A and B).
Superoxide radical, one of the most important ROS, was detected
with specific fluorescent probe DHE. The addition of NaHS
significantly inhibited the increase in superoxide radical level in
root tips under Se(IV) stress (Figure 7C and D).
Cell death in root tips were fluorescently detected with PI. The
application of NaHS was able to significantly alleviate
Se(IV)induced cell death in root tips (Figure 7E and F). Because the
overgeneration of ROS is closely related to the oxidative injury to plant
cells, we further determined the peroxidation of membrane lipids
and the loss of membrane integrity by using histochemical staining
with Shiff’s regent and Evans blue, respectively . Compared to
the control, the roots treated with Se(IV) showed more extensive
staining. However, the roots treated with Se(IV)+NaHS had only
light staining as compared to Se(IV) treatment alone (Figure 8).
These results indicated that the enhancement of endogenous H2S
by applying NaHS could alleviate Se(IV)-induced cell injury in
Treatment with NaHS enhanced endogenous GSH level
in Se(IV)-treated roots
By using specific molecular probe for detecting endogenous
GSH in situ, treatment with 0.06 mM of Se(IV) significantly
decreased the endogenous GSH level in root as compared to the
control. However, the addition of NaHS could remarkabley
enhance the GSH level (Figure 9A). The relative GS-bimane
fluorescent density indicated that treatment with Se(IV)+NaHS
increased the endogenous GSH by 82.6% as compared to Se(IV)
treatment alone (Figure 9B).
The concentration of Se in roots were not affected
effectively by the application of NaHS
In order to test the effect of NaHS treatment on the uptake of Se
by the roots, the Se concentration in roots were measured and
compared between Se(IV) treatment and Se(IV)+NaHS treatment.
In a time-course experiment, treatment with 0.6 mM of Se(IV)
resulted in the continuous increase in concentration of Se in roots
as compared to the control groups (Figure S4). However, the
addition of 0.5 mM of NaHS didn’t affect the concentration of Se
in roots significantly as compared to the treatment of Se(IV) alone
In comparison with other heavy metals (e.g. Cd, Zn, Al, Pb, and
Hg), whose phytotoxicity have been well documented [50–53], the
biochemical mechanisms for plant responses to Se are rarely
known. It has been demonstrated that the exogenous application
of H2S can modify plant physiology in response to heavy metal
stress . However, whether and how the endogenous H2S
influences plant growth under heavy metal stress remains unclear.
In the present study, by using in situ fluorescent tracking of
endogenous H2S, we found that the inhibition of endogenous H2S
generation underlay Se(IV)-induced inhibition of root elongation
in B. rapa, which could be supported by four lines of evidence.
First, Se(IV)-induced inhibition of root elongation was closely
correlated with the decrease in endogenous H2S in root tips.
Second, Se(IV)-induced inhibition of endogenous H2S generation
may result from the down-regulation of LCDs and DCDs. Third,
the application of H2S donor NaHS could enhance endogenous
H2S level in root tips, which further resulted in the recovery of
root elongation under Se(IV) stress. The decrease in endogenous
H2S level by the addition of PAG and HT could block the
recoverable effect of NaHS on root elongation under Se(IV) stress.
Fourth, the enhancement of endogenous H2S by NaHS resulted in
the alleviation of Se(IV)-induced ROS accumulation, cell death,
and oxidative injury in root tips.
It has been reported that H2S is required for the organogenesis
of lateral root and adventitious root [38,54–56]. In the present
study, decreasing the endogenous H2S level using PAG or HT
could inhibit the root elongation of B. rapa (Figure 5A, suggesting
that endogenous H2S is indispensable for root elongation.
Se(IV)induced inhibition of endogenous H2S generation in root tips may
probably contributed to the depression of root elongation
(Figure 1 and 2). Several LCDs and DCDs responsible for the
endogenous generation of H2S have been identified from plants
[23,24,47], but the regulation of the expression of these genes by
environmental stimuli is rarely reported. In this study, we detected
the genome-wide expression pattern of LCDs and DCDs in B. rapa
under Se(IV) stress. We found an effective inhibitory action of
Se(IV) to the expression of LCDs and DCDs in the roots of B. rapa
(Figure 3), which may contributed to the significant decrease in
endogenous H2S level (Figure 2). The similarly depressed mode of
LCD expression was also observed in B. napus under Cd stress
. Interestingly, we also found some typical NO- or
auxinresponsive elements in the promoter region of LCDs (Table S2),
which may explain the previous observation that exogenous NO
and auxin could stimulate the expression of LCD in B. napus .
Supported by genetic evidences it was proposed that the inhibition
of endogenous NO and auxin contributed to Se(IV)-induced root
growth inhibition in Arabidopsis . Thus, it can an indication
that NO and auxin may act upstream of H2S by manipulating the
expression of LCDs and DCDs during Se(IV)-induced inhibition of
root growth. In addition, the transcriptional regulation of LCDs
and DCDs in B. rapa by Se(IV) may also results from the presence
of MREs in their promoter region.
ROS has been suggested as the main inducer of plant cell death
. In the present study, Se(IV)-induced over-accumulation of
ROS may contribute to the cell death in root tips, which was also
accompanied with the decrease in endogenous H2S (Figure 2 and
7). The enhancement of endogenous H2S by supplement with
NaHS could reverse the inducible effect of Se(IV) on ROS
accumulation, cell death, and oxidative injury (Figure 7),
suggesting that H2S has an important role in the plant protection from
Se(IV) stress by scavenging the over-accumulated of ROS. In
mammals, the effective stimulation of GSH biosynthesis induced
by low level of H2S contribute to the suppression of oxidative stress
more efficiently than the scavenging of ROS by H2S itself .
Cystine is indispensable for the biosynthesis of glutathione. H2S
can enhance the activity of cysteine/glutamate antiporter, leading
to the increase in the transport of cystine into cells . Cystine is
subsequently reduced to cysteine in cells and incorporated into
glutathione. Additionally, H2S can directly interact with
glutamylcysteine synthase, a limiting enzyme for glutathione
biosynthesis, thereby increasing the production of glutathione [59,60].
Our present results demonstrated that the increase in endogenous
H2S by applying NaHS significantly enhance GSH level in roots
under Se(IV) stress (Figure 9). Glutathione plays important role in
protecting plants from metal toxicity by scavenging ROS or
chelating metals . Plants share similar mechanism with
mammals for glutathione biosynthesis . Therefore, in
Se(IV)treated plants, whether H2S depressed the generation of ROS
through the similar mechanism mentioned above remains to be
The biology of H2S in mammals has been significantly
advanced, but evaluating the role of endogenous H2S in plants
is just beginning. By using in situ fluorescent detection of
endogenous H2S in plant, we provide the direct evidence that
Se(IV) stress can inhibits the generation of endogenous H2S in the
roots of B. rapa. The enhancement of endogenous H2S can
alleviate Se(IV)-induced root inhibition by depressing ROS
generation and decreasing cell death. These data support the fact
that Se(IV) induces phytotoxicity by hijacking the generation of
endogenous H2S in B. rapa. Despite of the observation in this
study, H2S-mediated signaling components upon Se stress is still
elusive. Thus, a more precise understanding of this question will
accelerate the investigation on the mechanism of Se-induced
phytotoxicity, which in turn will help the improvement of crop
production in Se-polluted environment.
Figure S1 The phylogenetic relationship of LCDs and
DCDs in B. rapa with their related member in higher
plants. NCBI accession numbers are NP_974843.1 for
Arabidopsis thaliana LCD (AtLCD), AFS17242.1 for Brassica napus
LCD (BnLCD), NP_175275.3 for Arabidopsis thaliana DCD1
(AtDCD1), NP_974363.1 for Arabidopsis thaliana DCD2
(AtDCD2), NP_001234368.1 for Solanum lycopersicum DCD
(SlDCD), XP_007037066.1 for Theobroma cacao DCD1
(TcDCD1), XP_007037067.1 for Theobroma cacao DCD2
Figure S2 The location of PLP-dependent domain in
LCDs and DCDs from B. rapa. The protein structure of two
LCDs and two DCDs were analyzed by online tool SMART. The
typical PLP-dependent domains were indicated as orange box. Bar
indicated 100 amino acids (aa).
Figure S3 Alignment of the predicted amino acid
sequences of LCDs in A. thaliana, B. napus, and B.
rapa. Dark shading with white letters and gray shading with black
letters reveal 100% and 75% sequence similarity, respectively.
Database accession numbers are the same as described in Figure
S1. The PLP-binding sites are shown by red box. The substrate
binding site is indicated by blue box. The SAT protein-interaction
site is indicated by red box.
Figure S4 The concentration of Se in the roots of B. rapa
exposed to Se(IV) or Se(IV)+NaHS. The roots were exposed
to Se(IV) (0.06 mM) or Se(IV) (0.06 mM)+NaHS (0.5 mM) for 6,
12, 24, 48, 72 h, respectively. The roots were harvested at each
point of treatment time for Se analysis. Each value was presented
as the mean of three replicates with SD.
Conceived and designed the experiments: JC L-FY YC. Performed the
experiments: YC H-ZM M-YZ Z-QQ. Analyzed the data: JC YC Y-QL.
Contributed reagents/materials/analysis tools: MX L-BH. Wrote the
paper: JC YC L-BH.
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