- Email: [email protected]
Hydrogen sulfide interacts with calcium signaling to enhance the chromium tolerance in Setaria italica
Accepted Manuscript Title: Hydrogen sulfide interacts with calcium signaling to enhance the chromium tolerance in Setaria italica Author: Huihui Fang Tao Jing Zhiqiang Liu Liping Zhang Zhuping Jin Yanxi Pei PII: DOI: Reference:
S0143-4160(14)00159-6 http://dx.doi.org/doi:10.1016/j.ceca.2014.10.004 YCECA 1612
To appear in:
Cell Calcium
Received date: Revised date: Accepted date:
25-6-2014 1-9-2014 14-10-2014
Please cite this article as: H. Fang, T. Jing, Z. Liu, L. Zhang, Z. Jin, Y. Pei, Hydrogen sulfide interacts with calcium signaling to enhance the chromium tolerance in Setaria italica, Cell Calcium (2014), http://dx.doi.org/10.1016/j.ceca.2014.10.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Graphical Abstract
ip t
1
4
The summary of the crosstalk between H2S and Ca2+ signaling in foxtail millet responding to Cr6+ stress.
us
3
cr
2
This study exhibited that H2S interacts with Ca2+ signaling to enhance the Cr6+
6
tolerance in foxtail millet by activation of the HM chelators and regulation of the
7
antioxidant system. H2S dependent manner is a necessary way in which Ca2+ activated
8
the synthesis of MT and PC by up-regulating the expressions of MT3A and PCS.
9
H2S dependent pathway is a component of the Ca2+ activating antioxidant system and
13
M
d
te
11 12
H2S partially contributes Ca2+-activating antioxidant system.
Ac ce p
10
an
5
1
Page 1 of 34
13
Highlight
14
signaling.
H2S-dependent pathway partially contributes Ca2+-activating antioxidant system.
us
19
cr
H2S-dependent manner.
17 18
Ca2+ activates the HM chelators synthesis-related genes in a
ip t
15 16
H2S enhances the Cr6+ tolerance in millet by interacting with Ca2+
an
20
Ac ce p
te
d
M
21
2
Page 2 of 34
22
Hydrogen sulfide interacts with calcium signaling to enhance the chromium tolerance in Setaria italica
23
Huihui Fanga#, Tao Jingb#, Zhiqiang Liua, Liping Zhanga, Zhuping Jina & Yanxi Peia*
21
a
School of Life Science, Shanxi University, Taiyuan 030006, China
25
b
Department of Biological Sciences, University of Alabama, Tuscaloosa, Alabama, 35487, USA
26
#
These authors contributed equally to this study.
27
* Corresponding author, Email: [email protected]
ip t
24
The running title
30
H2S participates in Ca2+ induced Cr6+ tolerance
31 32
Abstract
us
29
cr
28
The oscillation of intracellular calcium (Ca2+) concentration is a primary event in
34
numerous biological processes in plants, including stress response. Hydrogen sulfide
35
(H2S), an emerging gasotransmitter, was found to have positive effects in plants
36
responding to chromium (Cr6+) stress through interacting with Ca2+ signaling. While
37
Ca2+ resemblances H2S in mediating biotic and abiotic stresses, crosstalk between the
38
two pathways remains unclear. In this study, Ca2+ signaling interacted with H2S to
39
produce a complex physiological response, which enhanced the Cr6+ tolerance in
40
foxtail millet (Setaria italica). Results indicate that Cr6+ stress activated endogenous
41
H2S synthesis as well as Ca2+ signaling. Moreover, toxic symptoms caused by Cr6+
42
stress were strongly moderated by 50 μM H2S and 20 mM Ca2+. Conversely,
48
Ac ce p
te
d
M
an
33
49
antioxidant system.
43 44 45 46 47
50 51
treatments with H2S synthesis inhibitor and Ca2+ chelators prior to Cr6+-exposure aggravated these toxic symptoms. Interestingly, Ca2+ upregulated expression of two important factors in metal metabolism, MT3A and PCS, which participated in the biosynthesis of heavy metal chelators, in a H2S-dependent manner to cope with Cr6+ stress. These findings also suggest that the H2S dependent pathway is a component of the Ca2+ activating antioxidant system and H2S partially contributes Ca2+-activating Key words: hydrogen sulfide, calcium signaling, chromium stress, Setaria italica
52 53 54
1. Introduction Hydrogen sulfide (H2S), which was often considered to be a poisonous gas, has
3
Page 3 of 34
been verified as the third gasotransmitter, the other two being nitric oxide (NO) and
56
carbon monoxide (CO) [1-3]. In mammals, these gases have been shown to play
57
essential roles in various physiological processes [2,3]. In the plant kingdom, NO and
58
CO are known to regulate physiological processes and to defend against abiotic and
59
biotic stresses [4,5]. As early as 1978, Wilson found that leaves of some plants could
60
release H2S [6], which subsequently motivated researchers to investigate the
61
physiological functions of H2S in the growth and development of plants [7-9].
62
Resulting studies revealed that H2S, as a gasotransmitter, is of great importance in
63
helping plants respond to abiotic and biotic stresses, including heat, drought, salinity,
64
nonionic osmotic, and heavy metals (HM) stresses [7,10-19]. Endogenous H2S
65
metabolic enzymes in plants have also been widely investigated [20]. Several studies
66
have shown that the activation of desulfhydrases (CDes) plays a central role in H2S
67
generation. Currently, two specific desulfhydrases, L-cysteine desulfhydrase (LCD)
68
and D-cysteine desulfhydrase (DCD), have been reported to be the most unambiguous
69
CDes, which generate endogenous H2S [20,21]. Other enzymes, such as DCD2, NIFs,
70
OAS-TL, and DES1, have been reported to generate H2S to a lesser degree [21]. Recently, studies have focused on the crosstalk between H2S and other molecules
72
involved in plant growth and development, resulting in some notable progress. For
73
instance, H2S interacting with abscisic acid (ABA) enhanced drought tolerance [14],
74
while its interaction with NO induced stomatal closure and enhanced stress response
75
[10,11]. Additionally, H2S was found to regulate the effects of H2O2 [22], and to
76
82 83
mediated by the concerted action of Ca2+ channels, pumps and carriers located in
84
plasma membranes and vacuole membranes. These proteins include Ca2+ cyclic
85
nucleotide-gated channels (CNGC), two-pore channels (TPC) and the ATP binding
86
cassette (ABC) protein MRP5, which are responsible for increasing the [Ca2+]cyt, as
87
well as the high affinity P-type Ca2+-ATPase (ACA) and the moderate affinity
88
Ca2+-H+ cation antiporter (CAX), which can lead to the decrease in [Ca2+]cyt [25].
77 78 79 80 81
te
d
71
Ac ce p
M
an
us
cr
ip t
55
modulate glutathione homeostasis and heme oxygenase-1 expression to delay GA-triggered programmed cell death [23]. Ca2+, a universal second messenger, is regarded as a core transducer and
regulator in many adaptive processes in plants. A transient elevation in cytosolic calcium concentration ([Ca2+]cyt) is an early event in a large array of biological processes and various stresses, which then delivers this signal to cells and subsequently activates adaptive responses [24,25]. The rapid increase in [Ca2+]cyt is
4
Page 4 of 34
Next, [Ca2+]cyt oscillation is perceived by other Ca2+ binding proteins, such as
90
calcium-dependent protein kinase (CDPK), calmodulin (CaM) and calcineurin B-like
91
protein (CBL), which combine with Ca2+ to interact with other downstream proteins.
92
Ca2+ binding proteins, known as “Ca2+ sensors”, can perceive the rapid increase in
93
[Ca2+]cyt and transmit this specific signal to help plants make an adaptive response
94
[25,26]. Some papers also reported that Ca2+ could compete with HM for channels,
95
enhancing HM tolerance [27].
ip t
89
Chromium (Cr), considered to be the second-most common HM, is often present
97
in the form of Cr3+ and Cr6+ [28]. Both forms are becoming an increasingly serious
98
environmental pollution, creating enduring problems because of the difficulty in
99
degrading and removing them from the environment. Moreover, the negative effects
100
of their toxicity remain in the environment for a very long time [28,29]. Therefore, it
101
is worthwhile and necessary to investigate the detoxification of Cr6+ in plants. With millions of years of evolution at hand, plants have evolved a series of smart
103
and complex strategies to quickly respond and adapt to the toxic effects of HM,
104
including controlling the uptake of HM or excluding the toxic ions to reduce the
105
accumulation of metals [29]. Compartmentalizing HM within vacuoles or activating
106
metal ion chelators, such as metallothioneins (MT) and phytochelatins (PC), to chelate
107
the HM and reduce the toxicity of metal ions are also plant strategies to minimize the
108
toxic effects of HM [29-31]. In addition, plants can alleviate oxidative damage
109
derived from HM stress by activating antioxidant systems, which consist of
110
116 117
system is also due to its ability to regenerate the AsA in AsA-GSH pathway [33].
118
It is noteworthy that H2S can alleviate toxic symptoms caused by HM stress [7,16-19]. However, the molecular mechanism by which H2S works is still unclear and other signaling molecules involved in this protective process remain to be identified. Given that Ca2+ functions in plants responding to stresses have similarities to H2S mediated stress responses [25-27], we investigated crosstalk between H2S and Ca2+ in terms of enhancing Cr6+ tolerance, employing foxtail millet (Setaria italica) as
111 112 113 114 115
119 120 121 122 123
te
d
M
102
Ac ce p
an
us
cr
96
antioxidant enzymes, such as superoxide dismutase (SOD), peroxidase (POD), glutathione reductase (GR), and nonenzymatic antioxidant molecules like glutathione (GSH) and ascorbic acid (AsA) [31,32]. A highly reduced state of GSH or AsA acts as a key regulator of antioxidant defenses [33]. When facing oxidative damage, the plant generally maintains the intracellular redox balance through some redox reactions. Therefore, the ratios of GSH/GSSG and AsA/DHA are important factors indicating the cell resistance to oxidative damage. GSH’s role in the plant antioxidative defense
5
Page 5 of 34
125
the experimental material. Foxtail millet has exhibited considerable tolerance to various stresses, including Cr6+. In this study, we explored the mechanism by which foxtail millet responds to
126 6+
stress, focusing on the crosstalk between H2S and Ca2+ signaling in this
127
Cr
128
protective process.
129
2. Materials and methods
130
2.1 Plant growth and treatments
ip t
124
Seeds of Setaria italica ecotype (Jingu-21) were used in this study. For each
132
experiment, seeds were sterilized in 75% (v/v) ethanol for 30 s and in 6% (v/v)
133
sodium hypochlorite solution for 9 min, and then grown on the petri dishes with three
134
layers of gauzes at the bottom and containing 8 mL water. After two days’ cultivation
135
in darkness at 23°C, with 60% relative humidity, these materials were then kept in
136
16/8 h (light/dark), 160 μEm-2 s-1. Additionally, the gauzes should be kept wet.
an
us
cr
131
Ten days later, the water in the petri dishes was replaced with different chemical
138
solutions (20 mM CaCl2, 5 mM EGTA, and/or 1 mM HA), and NaHS fumigation (50
139
μM) was given at the same time. NaHS, acting as an exogenous H2S donor, was used
140
to provide H2S by fumigating the seedlings. All manipulations were done as described
141
in Jin et al. [13]. For this fumigation, plants were kept in their own petri dishes placed
142
in a sealed glass container and then fumigated with 50 μM NaHS for 12 h. After 12
143
hours pretreatment, all the chemical reagents were sucked out from petri dishes then
144
Cr6+ solution (5 mM K2Cr2O7) was added to stress these seedlings. Gene expressions
145
were detected at 12 h time-point of Cr6+ exposure, while the physiological indexes
147 148 149 150 151
d
te
Ac ce p
146
M
137
were analyzed after 24 h Cr6+ exposure. 2.2 Measurement of the width of leaves To indicate the leaves curled degree, the width of foxtail millet cotyledons in
natural state (not stretched) after various treatments were measured. The data represent means ± SE of 30 cotyledons with three independent repeats. 2.3 Extraction of total RNA and qRT-PCR
152
The total RNA was isolated from 0.1 g of foxtail millet leaves using RNA
153
isolation TRizol® Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s
154
instructions. Then the target gene expressions were detected with quantitative
155
real-time PCR (qRT-PCR) as in Shen et al. [15]. Meanwhile, the gene ACTIN was
156
used as the internal control. The primers used for qRT-PCR are listed in Table S1.
157
Annotation
158
(http://foxtailmillet.genomics.org.cn/page/species/index.jsp). Each experiment was
was
performed
in
the
foxtail
millet
database
6
Page 6 of 34
159
repeated independently for three replicates.
160
2.4 Determination of H2S production rate
161
CDes activity was determined by testing the production rate of H2S according to Jin et al. [13].
163
2.5 Physiological indexes assays
ip t
162
The contents of MDA and H2O2, the activities of SOD and POD as well as the
165
content of GSH, GSSG, AsA and DHA were measured according to published
166
methods [13-19,34].
cr
164
MDA content assay
168
The MDA content, an important indicator of the lipid peroxidation level, was
169
determined by Thiobarbituric acid (TBA) reaction [17,19]. 0.15 g of leaves with
170
various treatments was ground in 1.5 mL of 5% Trichloroacetic acid (TCA). The
171
solution was centrifuged at 5000 g for 5 min at 4°C, then 1 mL of supernatant was
172
mixed with 1 mL of 0.67% TBA in a test tube and boiled in water at 95°C for 30 min.
173
The reaction was terminated in an ice bath. The solution was centrifuged at 5000 g for
174
2 min at 4°C and the supernatant was measured at 450 nm, 532 nm and 600 nm,
175
respectively. Finally, MDA content was expressed as nmol g-1 FW (fresh weight). The detection of H2O2
177
Qualitative detection of H2O2 was carried out with 3, 3-diaminobenzidine (DAB)
178
(Sigma, MO, USA) [17]. The leaves of plants with various treatments were soaked in
179
1 mg L-1 DAB in darkness for 1 h, and then transferred into light conditions for 12 h.
180
186 187
buffer and 1 mL of 1 M KI. The reaction ran for 1 h in darkness and the absorbance at
188
390 nm was measured. The content of H2O2 was calculated based on a standard curve
189
of known concentrations of H2O2.
181 182 183 184 185
te
d
176
Ac ce p
M
an
us
167
After rinsing with distilled water three times, the leaves were boiled in 95% ethanol for 10 min to remove the pigments, and then H2O2 production in the form of reddish-brown coloration was visualized. Quantitative detection of H2O2 was carried out with potassium iodide (KI) as
described in a published method [19]. 0.15 g of leaves was ground in 1.5 mL of 5% TCA and the solution was centrifuged at 5000 g for 5 min at 4°C. The reaction mixture consisted of 500 μL of extracted supernatant, 500 μL of potassium phosphate
190
SOD activity assay
191
The activity of SOD was assayed by detecting the inhibition of reduction level of
192
nitro blue tetrazolium (NBT) according to Giannopolitis and Ries (1977) with some
7
Page 7 of 34
modifications [34]. The total enzyme was extracted using 0.05 M phosphate buffer
194
(pH 7.0), and centrifuged at 12000 g for 5 min at 4°C. Then the reaction was
195
performed in a test tube containing 3 mL of the mixture consisting of 50 mM
196
potassium phosphate buffer (pH 7.8), 13 mM methionine, 75 mM NBT, 0.1 mM
197
EDTA, 2 mM riboflavin and 100 μL enzyme solution. Then this mixture was exposed
198
to light with the intensity of 5000 lx for 15 min, and the absorbance was monitored at
199
560 nm. One unit of SOD activity was defined as the amount of enzyme that caused
200
50% inhibition of NBT reduction.
cr
ip t
193
POD activity assay
202
The activity of POD was assayed according to our published methods with some
203
modifications [16,34]. The total enzyme was extracted as described above. 3 mL of
204
the assay mixture for the POD activity included: 0.05 M phosphate buffer (pH 7.0)
205
containing 10 mM guaiacol, 0.1 mM EDTA and 5 mM H2O2. This mixture was
206
incubated for 5 min at 25°C. The 50 μL 20 times-diluted enzyme solution was
207
subsequently added to start this reaction, and the absorbance at 470 nm was
208
immediately monitored.
M
an
us
201
GSH and GSSG content assay
210
GSH and GSSG content was assayed using a GSH and GSSG assay kit S0053
211
(Beyotime Institute of Biotecnology, China) according to the manufacturer’s
212
instructions.
te
AsA and DHA content assay
220
Ac ce p
213
d
209
221
Probes, Eugene, OR, USA). All manipulations were done according to Jin et al. [14].
222
2.7 Statistical analysis
214 215 216 217 218 219
AsA content was assayed using an AsA assay kit A009 (Nanjing Jiancheng
Bioengineering Institute, China) and DHA content was assayed using an DHA assay kit SM102 (Beijing Solarbio Science ＆ Tecnology, China). All operations were performed according to the manufacturer’s instructions. 2.6 Fluo-3/AM loading and detection of cytoplasmic Ca2+ For detecting the [Ca2+]cyt, the lower epidermis peeled off from the leaves were
used to load the Ca2+-sensitive Fluorescent probe 5 mM Fluo-3/AM (Molecular
223
Each experiment was carried out for three independent repetitions. The results
224
were expressed as the means ± SE. Data were analyzed using SPSS (version 17, IBM
225
SPSS, Chicago, IL), and error bars were made according to Tukey’s multiple range
226
test (P < 0.05).
8
Page 8 of 34
227
3. Results
228
3.1 H2S alleviated Cr6+ toxicity in foxtail millet
229
3.1.1 Cr6+ stress damaged the foxtail millet To study the negative influence of Cr6+ stress on foxtail millet, 10-day-old plants
231
were exposed to increasing concentration of Cr6+ (0, 5, 10, 15, 20 mM Cr6+) for 24 h
232
respectively. It is clear to see that Cr6+ treatment led to toxic symptoms of dim and
233
curly leaves in plants (Fig. 1A), and moreover the width of cotyledons in a natural
234
state after Cr6+ treatment was measured to indicate the leaves curled degree (Fig. 1B).
235
Notably, these damaged symptoms were dose-dependently induced.
cr
ip t
230
HM are known to disturb the redox balance and consequently expose plants to
237
oxidative stress. In this study, the content of MDA and H2O2 went up as Cr6+
238
concentration increased from 0 to 20 mM (Fig. 1C and D). 10 mM Cr6+ stress is
239
strong enough to stimulate these toxic symptoms (P < 0.05), so 10 mM Cr6+ was
240
chosen in further experiments.
241
3.1.2 Cr6+ stress stimulated the H2S emission
M
an
us
236
In order to investigate the effect of Cr6+ stress on the H2S-emission system, the
243
expressions of LCD, DCD1, DCD2, DES and the rate of H2S production in plants
244
treated with Cr6+ were measured. It was found that the expressions of H2S-emission
245
related genes LCD, DCD2, DES were markedly increased during the first 12 h of Cr6+
246
exposure following decline at 24 h, while the expression of DCD1 was consistently
247
increased from 0 h to 24 h under Cr6+ stress (Fig. 2A). In spite of this, all the gene
248
expressions were markedly up-regulated at the 12 h time-point of Cr6+ exposure
254
Ac ce p
te
d
242
255
was exposed to Cr6+.
256
3.1.3 H2S enhanced the Cr6+ tolerance
249 250 251 252 253
compared to the control seedlings without stress, so we decided to choose 12 h to detect the associated gene expressions. Additionally, the H2S production rate was dose and time dependently induced by
6+
Cr stress, and this activation was the most significant with 24 h of 10 mM Cr6+ treatment (Fig. 2B).
These results implied that H2S emission system was activated when foxtail millet
257
The results above implied that H2S might be of importance to foxtail millet’s
258
response to Cr6+ stress. In order to confirm the positive effect of H2S, 50 μM of NaHS
259
acting as an exogenous H2S donor, within the physiological range of H2S, was used in
260
subsequent experiments. 1 mM of HA, acting as a H2S synthesis inhibitor, was also
9
Page 9 of 34
261
employed for the reverse verification. As is shown in Fig. 3, 50 μM of H2S pretreatment significantly alleviated the
263
toxic symptoms and restored the dim and curly leaves caused by Cr6+ stress back to a
264
normal level (Fig. 3A and B). Moreover, whether the plants were exposed to Cr6+ or
265
not, H2S fumigation could help maintain the contents of H2O2 (Fig. 3C and D) and
266
MDA (Fig. 8) at the normal level, while the contents of H2O2 (Fig. 3C and D) and
267
MDA (Fig. 8) increased considerably in stressed plants without H2S pretreatment or
268
addition of HA.
269
3.2 Ca2+ signaling involved in foxtail millet responding to Cr6+ stress
270
3.2.1 Cr6+ stress stimulated the Ca2+ signaling
us
cr
ip t
262
The rapid shock in [Ca2+]cyt is an original mediator in a series of stresses in plants.
272
In this study, the foxtail millet leaves with or without 10 mM Cr6+ stress were loaded
273
with Fluo-3/AM to detect the changes of [Ca2+]cyt. Obviously, stronger Ca2+
274
fluorescence was observed in the leaves with Cr6+ stress (Fig. 6C).
an
271
Also, the expression levels of Ca2+ transporter-encoding genes in foxtail millet
276
with increasing time of Cr6+ treatment (0, 1, 3, 6, 12, 24 h) were detected. As is shown
277
in Fig. 4, the TPC1 and MRP5 expressions were time-dependently up-regulated by
278
Cr6+ treatment, and meanwhile the expression of ACA9 decreased when Cr6+ treating
279
time was extended. As we expected, this result was in accordance with the stronger
280
Ca2+ fluorescence in the previous experiment (Fig. 6C).
te
d
M
275
Numerous studies have shown that the rapid increase in [Ca2+]cyt is perceived by
282
some following members known as “Ca2+ sensors”. To explore whether Ca2+ signaling
283 284 285 286 287 288 289
Ac ce p
281
participated in foxtail millet’s response to Cr6+ stress, the CaM, CBL and CDPK, which encode the [Ca2+]cyt direct perceivers, as well as CIPK, the CBL downstream protein-encoding gene, were further investigated. It revealed in Fig. 4 that the expressions of all these Ca2+ signaling related genes were enhanced by Cr6+ stress to various degrees in a time-dependent manner, and this up-regulation was notable at 12 h of Cr6+ stress (Fig. 4).
As is mentioned above, we concluded that the Ca2+ signaling was involved in
290
response against Cr6+ stress in foxtail millet.
291
3.2.2 Ca2+ significantly alleviated the Cr6+ toxicity
292
In order to test the effect of Ca2+ in this process, 20 mM CaCl2 was used as an
293
exogenous Ca2+ donor. The toxic symptoms in leaves of foxtail millet exposed to Cr6+
294
stress were obvious. However, 20 mM Ca2+ pretreatment, to a large extent, alleviated
10
Page 10 of 34
these symptoms, and at the same time rescued the dim and curly leaves (Fig. 5A and
296
B). Interestingly, the Cr6+ tolerance was weakened by addition of Ca2+ chelator EGTA
297
(Fig. 5A and B). Furthermore, Ca2+ pretreatment greatly reduced the accumulation of
298
H2O2 (Fig. 5C and D) and MDA (Fig. 8), whereas their accumulation largely increased
299
in stressed plants with EGTA pretreatment.
300
3.3 H2S interacting with Ca2+ signaling enhanced Cr6+ tolerance
301
3.3.1 The connection of H2S and Ca2+ signaling in foxtail millet responding to
302
Cr6+ stress
cr
ip t
295
In order to investigate the crosstalk between H2S and Ca2+ signaling in the
304
process of foxtail millet responding to Cr6+ stress, we detected the effects of Ca2+ and
305
EGTA on the increased H2S production, as well as the influences of H2S and HA on
306
the rapid shock of [Ca2+]cyt and the up-regulated expressions of Ca2+ signaling
307
associated genes.
an
us
303
When the plants were exposed to Cr6+ stress, Ca2+ pretreatment hugely improved
309
the H2S production rate, and EGTA reduced the increased H2S emission induced by
310
Cr6+ stress (Fig. 6A). As fluorescent staining has shown, the stronger Ca2+
311
fluorescence derived from Cr6+ stress can be moderated by H2S fumigation.
312
Correspondingly, HA pretreatment strengthened the Ca2+ fluorescence to some degree
313
(Fig. 6C).
Simultaneously, the up-regulated expressions of CaM and CBL caused by Cr6+
315
stress can be increased by Ca2+ pretreatment, while this effect of Ca2+ can be strongly
316
322 323
biosynthesis of these chelators.
317 318 319 320 321
te
314
Ac ce p
d
M
308
enhanced by H2S fumigation, but weakened by addition of HA (Fig. 6B). However, these treatments have little influence on the expression of CDPK (Fig. 6B). 3.3.2 H2S participated in Ca2+ up-regulating expressions of HM chelators-related genes
It is widely known that the accumulation of chelators, such as MT and PC, play
an important role in plant defense against HM stresses, we thus explored the crosstalk of H2S and Ca2+ signaling in regulating the expressions of genes involved in the
324
As Fig. 7 showed, during Cr6+-exposure, the expressions of MT3A and PCS were
325
increased significantly. Furthermore, H2S or Ca2+ pretreatment strengthened the
326
expressions of these genes. Additionally, duplicate effects could be observed after H2S
327
and Ca2+ combined treatment. Interestingly, EGTA has no influence on the
328
up-regulation of these genes induced by H2S. However, the up-regulation of these
11
Page 11 of 34
329
gene expressions caused by Ca2+ can be strongly blocked by HA, a H2S synthesis
330
inhibitor.
331
3.3.3 H2S interacting with Ca2+ signaling to alleviate the oxidative damage from
332
Cr6+ stress As is mentioned above, both H2S and Ca2+ can enhance Cr6+ tolerance.
334
Interestingly, H2S and Ca2+ signaling have a potential crosstalk in this process. As is
335
shown in Fig. 8, both H2S and Ca2+ can relieve the accumulation of MDA during Cr6+
336
stress and this protective effect was remarkably observed with H2S and Ca2+
337
combined pretreatment. However, the accumulation of MDA can be strengthened by
338
addition of HA and EGTA, respectively. Further results demonstrated that EGTA can
339
partly weaken the protective role of H2S. Similarly, HA also attenuated the positive
340
role of Ca2+ to some degree (Fig. 8).
us
cr
ip t
333
Plants facing with stress can activate their antioxidant system to defend against
342
the oxidative damages. Hereby, the relationship between H2S and Ca2+ in regulating
343
this system was analyzed.
M
an
341
Under Cr6+-exposure, the SOD and POD activities were elevated markedly to
345
fight against oxidative damage (Fig. 9A and B). Additionally, whether the plants were
346
exposed to Cr6+ stress or not, the SOD and POD activities could be kept at the normal
347
level by H2S fumigation, while their activities increased more significantly in stressed
348
plants with addition of HA (Fig. 9A). On the contrary, Ca2+ pretreatment led to a
349
higher SOD and POD activity in stressed seedlings. Correspondingly, EGTA
350
pretreatment prior to Cr6+-exposure evidently suppressed the SOD and POD activities
352 353 354 355 356
te
Ac ce p
351
d
344
(Fig. 9B). Interestingly, the effect of Ca2+ on these antioxidant enzymes was totally different from that of H2S.
As both H2S and Ca2+ can relieve the oxidative damage caused by Cr6+ stress,
why is their regulation on the activities of these antioxidant enzymes quite different? In order to explore this intriguing problem, the influences of H2S and Ca2+ on the antioxidants were studied.
357
As the results have shown, under Cr6+ stress, the expressions of GSH1 and GR
358
were up-regulated (Fig. 9C and D), the reduced GSH content correspondingly went up
359
(Fig. 10A), and this increase could be markedly strengthened by H2S or Ca2+
360
pretreatment. Furthermore, duplicate effects were observed after the H2S and Ca2+
361
treatment together. Besides, when EGTA was used as Ca2+ chelator, little influence on
362
the up-regulation of GSH1 and GR induced by H2S was observed (Fig. 9C and D).
12
Page 12 of 34
However, the positive effect of H2S on the reduced GSH content was partly restrained
364
by EGTA addition (Fig. 10A). Interestingly, HA, acting as a H2S synthesis inhibitor,
365
could obviously weaken Ca2+-induced GSH1 and GR expressions (Fig. 9C and D) and
366
the GSH content (Fig. 10A). In addition, during Cr6+ exposure, the redox state
367
GSH/GSSG decreased markedly, and this decrease was weakened by H2S and Ca2+
368
pretreatment (Fig. 10C), also this effect of H2S and Ca2+ could be partially depressed
369
by EGTA and HA, respectively (Fig. 10C).
ip t
363
Additionally, the AsA content was simultaneously measured, the result of which
371
indicated that the pattern of Cr6+ stress regulating the content of AsA did not seem to
372
be so consistent with that regulating GSH content. AsA content was depressed
373
markedly by Cr6+-exposure, while H2S or Ca2+ pretreatment led to more AsA
374
accumulation in stressed seedlings, and this accumulation was more obvious by H2S
375
and Ca2+ combined pretreatment (Fig. 10B ). Besides, when EGTA pretreated, the
376
accumulation of AsA induced by H2S was partly inhibited; and HA pretreatment could
377
obviously weaken the positive effect of Ca2+ on the AsA content to some degree (Fig.
378
10B). Simultaneously, Cr6+ stress led to a significant decrease in the ratio of
379
AsA/DHA (even decreased by 66%). H2S or Ca2+ had little positive effect on this
380
decrease derived from Cr6+ stress (Fig. 10D).
381 382
Recently, it has been reported that H2S as a gasotransmitter participates in plant
390
Ac ce p
383
4. Discussion
te
d
M
an
us
cr
370
391
could both dramatically alleviate these negative effects. Thus, we focused on the
392
crosstalk between H2S and Ca2+ signaling in this process.
384 385 386 387 388 389
defense responses to abiotic and biotic stresses [1-3,10-19]. However, the potential molecular mechanism and its signaling pathways remain limited. This study was aimed at exploring the molecular mechanism of the positive
effects of H2S and its signaling pathways on foxtail millet that responds to Cr6+ stress. It has been obviously noticed that Cr6+ stress could lead to toxic symptoms and accumulation of ROS. Meanwhile both the endogenous H2S emission system and Ca2+ signaling were activated during this stress. In addition, exogenous H2S and Ca2+
393
The expressions of the H2S-emission related genes LCD, DCD2 and DES were
394
markedly increased during the first 12 h of Cr6+ exposure, and then decreased; while
395
the DCD1 expression was being increased from 0 h to 24 h under Cr6+ stress (Fig. 2A).
396
This unsynchronized result may be due to the different transcription regulation of
13
Page 13 of 34
these genes. Although the expressions of H2S-generating genes LCD, DCD2 and DES
398
were decreased at 24 h of Cr6+ exposure, we did not find the difference of H2S
399
production rate at 12 h and 24 h of Cr6+ exposure. H2S production rate directly reflects
400
the intensity of enzymatic activity of H2S-generating proteins, and it will take time for
401
transcribed mRNA to be translated into protein following posttranslational
402
modification to have complete activity. It is really the case, and we did observe that
403
H2S production rate is significantly reduced at 36 h and 48 h of Cr6+ exposure (Fig.
404
2B), indicative of a delayed response to the reduced gene expression of LCD, DCD2
405
and DES at 24 h of Cr6+ exposure.
cr
ip t
397
It was found that fumigation with different doses of NaHS (0, 10, 20, 30, 40, 50,
407
60 μM) could enhance Cr6+ tolerance in foxtail millet to different degrees, 50 μM
408
NaHS pretreatment showed significantly protective role (data not shown). Hence, the
409
50 μM NaHS was employed as an exogenous H2S donor, which is within the H2S
410
physiological concentration detected in animals and plants [13,15]. Exogenous donors (50 μM NaHS and 20 mM CaCl2) and endogenous inhibitors
412
(1 mM HA and 5 mM EGTA) of H2S and Ca2+ were used in this study. It is
413
noteworthy that during Cr6+ stress, the H2S emission could be raised by Ca2+ but
414
suppressed by EGTA (Fig. 6A). This may suggest that Cr6+ stress activate the H2S
415
emission system in a Ca2+-dependent manner. Furthermore, H2S could participate in
416
Ca2+ signaling through affecting the [Ca2+]cyt and regulating some downstream
417
molecules (Fig. 6B and C) in the process. All these results implied that H2S and Ca2+
418
424 425
and PC by up-regulating the expressions of MT3A and PCS to defend against this
426
stress. These chelators could chelate the Cr6+ and reduce its toxicity. As is shown in
427
Fig. 7, their expressions could be elevated by H2S or Ca2+, even superimposed effects
428
could be observed after H2S and Ca2+ combined treatment. This activation of the
429
MT3A and PCS expression induced by H2S could not be influenced by EGTA.
430
Surprisingly, the addition of HA, to inhibit the endogenous synthesis of H2S, even
419 420 421 422 423
te
d
M
411
Ac ce p
an
us
406
signaling have a potential connection during foxtail millet responding to this stress. However, despite their complex interaction in this process, some differences in
the mode of their actions were also observed. We then further explained their crosstalk and differences in two aspects: the activation of HM chelators and the regulation of antioxidant system.
HM chelators are extremely important in defending against HM stress [30]. As
we expected, when exposed to Cr6+ stress, foxtail millet activated the synthesis of MT
14
Page 14 of 34
431
completely suppressed this positive role of Ca2+. These results illustrated that Ca2+
432
could enhance the synthesis of MT and PC by up-regulating the expressions of MT3A
433
and PCS in a H2S-dependent manner to deal with the Cr6+ stress, and Ca2+-activated
434
H2S was essential for this adaptive response. It is generally considered that toxicity of HM exposes plants to oxidative damage.
436
Once exposed to Cr6+ stress, plants can strongly activate the antioxidant system. In
437
this paper, the expressions of GSH1 and GR as well as the content of reduced GSH in
438
foxtail millet during Cr6+-exposure could be enormously strengthened by H2S or Ca2+,
439
and their combined pretreatment could even cause duplicate effects (Fig. 9C and D,
440
Fig. 10A). Moreover, we also explored the expression of GSH2 (data not shown). In
441
contrast to GSH1, the expression of GSH2 remained unchanged when faced with Cr6+
442
stress as well as H2S and/or Ca2+pretreatment. GSH1 has been well reported to act as
443
the rate-limiting enzyme in the process of GSH generation [17], so we speculate that
444
H2S and Ca2+ mainly improve the GSH content by increasing GSH1 expression.
an
us
cr
ip t
435
It is well known that AsA is an important antioxidant in plants, and therefore its
446
content was measured. In contrast to the change of GSH content, AsA content was
447
depressed markedly by Cr6+-exposure, while H2S or Ca2+ pretreatment led to more
448
AsA accumulation in stressed seedlings (Fig. 10A). Under Cr6+ stress, the AsA content
449
decreased while the GSH content increased. We thus inferred that the AsA made an
450
earlier contribution than GSH to defending against the oxidative damage. The AsA
451
scavenged ROS in the way that it became an oxidized form DHA, so the ratio
452
AsA/DHA decreased dramatically during Cr6+stress (Fig. 10D). H2S and Ca2+ had no
458
Ac ce p
te
d
M
445
459
speculated that Ca2+ up-regulated the antioxidant molecules in a H2S dependent or
460
independent manner. That is to say, H2S dependent pathway is just an alternative in
461
Ca2+ induced activation of these antioxidant molecules, suppressing this H2S pathway
462
could not block the function of Ca2+ in this process.
453 454 455 456 457
effect on AsA/DHA ratio whereas they could regulate the content of GSH and AsA as well as the GSH/GSSG ratio. Therefore we speculate that H2S and Ca2+ might activate this GSH-AsA pathway and then regulate the plant antioxidative defense system. Meanwhile, GSH could also act as an antioxidant molecule to fight against ROS. It was clear that the positive effect of Ca2+ on the expression of GSH1 and GR as
well as the content of GSH and AsA could be partly weakened by HA. Thus we
463
However, some inconsistencies can be found in the influences of H2S and Ca2+
464
on the activities of antioxidant enzymes. During Cr6+ exposure, Ca2+ could help
15
Page 15 of 34
foxtail millet defend against oxidative damage by increasing the activities of SOD and
466
POD (Fig. 9B), but exogenous H2S pretreatment kept the SOD and POD activities at
467
the normal level (Fig. 9A). These results may indicate that H2S alleviated
468
Cr6+-induced oxidative damage not via regulating the antioxidant enzymes activities
469
directly. Thus it was inferred that H2S might alleviate oxidative damage by regulating
470
the nonenzymatic antioxidant molecules or decline the generation of ROS, such as
471
suppressing the activity of NADPH oxidase, as H2S has been reported to decrease the
472
ROS production by inhibiting the NADPH oxidase 4-related signaling [32].
473
Compared to H2S, Ca2+ could activate the antioxidant system not only by enhancing
474
SOD and POD activities but also by increasing the contents of GSH and AsA to cope
475
with Cr6+-induced oxidative damage. This illustrated that Ca2+ might be more
476
extensive in the exercise of its protective functions compared to H2S. All of these
477
results strongly suggested that there may be a complicated crosstalk between H2S and
478
Ca2+ in this process, but not a simple linear relationship between them. A model based on the results described in this study was proposed to expound
480
the signaling pathways of H2S and Ca2+ in foxtail millet’s response to Cr6+ stress (Fig.
481
11). Cr6+ stress activated the H2S and Ca2+ signaling, and then H2S interacting with
482
Ca2+ signaling participated in complex physiological processes to defend against Cr6+
483
stress. H2S dependent manner is a necessary way in which Ca2+ activated the
484
synthesis of MT and PC by up-regulating the expressions of MT3A and PCS. Whereas
485
in the aspect of regulating antioxidant system, Ca2+ activated this system not only
486 487 488 489 490 491 492
te
d
M
479
Ac ce p
an
us
cr
ip t
465
through the antioxidant enzymes but also via the antioxidant molecules to fight against the Cr6+-induced oxidative damage. H2S alleviated oxidative damage mainly by up-regulating the antioxidant molecules. In other words, H2S dependent pathway is a component of the Ca2+ activating antioxidant system and H2S partially contributes Ca2+-activating antioxidant system. Overall, concerted efforts with Ca2+, H2S promptly enhanced the Cr6+ tolerance
in foxtail millet.
493 494 495 496
Acknowledgement This work was supported by the National Natural Science Foundation of China (31372085 to Yanxi Pei; 31300236 to Zhiqiang Liu; 31400237 to Zhuping Jin).
497
We give sincere appreciation to Dr. Margaret Johnson and Dr. Kim Lackey from
498
Department of Biological Sciences at the University of Alabama for their professional
16
Page 16 of 34
499
and generous help with the language.
Ac ce p
te
d
M
an
us
cr
ip t
500
17
Page 17 of 34
500 501
References
502
1.
gaseous transmitter? The FASEB Journal. 16 (2002) 1792-1798. 2.
blossomed, Physiol Rev. 92 (2012) 791-896.
505
3. 4.
M. Delledonne, NO news is good news for plants, Curr Ppin Plant Biol. 8 (2005) 390-396.
509 510
cr
mice with deletion of cystathionine γ-lyase. Science 322(2008) 587-590.
507 508
G.D. Yang, L.Y. Wu, B. Jiang, H2S as a physiologic vasorelaxant: hypertension in
5.
us
506
R. Wang, Physiological implications of hydrogen sulfide: a whiff exploration that
ip t
503 504
R. Wang, Two’s company, three’s a crowd: can H2S be the third endogenous
W. Xuan, F.Y. Zhu, S. Xu, et al., The heme oxygenase/carbon monoxide system is involved in the auxin-induced cucumber adventitious rooting process, Plant
512
Physiol. 148 (2008) 881-893. 6.
sulfide from plants, Plant Physiology. 61 (1978) 184-189.
514 515
L.G. Wilson, R.A. Bressan, P. Filner, Light-dependent emission of hydrogen
7.
M
513
an
511
H. Zhang, L.Y. Hu, K.D. Hu, Y.D. He, S.H. Wang, J.P. Luo, Hydrogen sulfide promotes wheat seed germination and alleviates oxidative damage against copper
517
stress, J Integr Plant Biol. 50 (2008) 1518-1529. 8.
H. Zhang, S.L. Hu, Z.J. Zhang, et al., Hydrogen sulfide acts as a regulator of
te
518
d
516
flower senescence in plants, Postharvest Biol Tech. 60 (2011) 251-257. J. Chen, F.H. Wu, W.H. Wang, et al., Hydrogen sulfide enhances photosynthesis
520
9.
521
Ac ce p
519
522 523 524 525 526 527
through promoting chloroplast biogenesis, photosynthetic enzyme expression, and thiol redox modification in Spinacia oleracea seedlings, J Exp Bot. 62 (2011) 4481-4493.
10. Z.G. Li, S.Z. Yang, W.B. Long, G.X. Yang, Z.Z. Shen, Hydrogen sulfide may be a novel downstream signal molecule in nitric oxide-induced heat tolerance of maize (Zea mays L.) seedlings, Plant cell Environ. 36 (2013) 1564-1572.
11. Y.Q. Wang, L. Li, W.T. Cui, S. Xu, W.B. Shen, R. Wang, Hydrogen sulfide
528
enhances alfalfa (Medicago sativa) tolerance against salinity during seed
529
germination by nitric oxide pathway, Plant Soil. 351 (2012) 107-119.
530
12. A. Christou, G.A. Manganaris, I. Papadopoulos, V. Fotopoulos, Hydrogen sulfide
531
induces systemic tolerance to salinity and non-ionic osmotic stress in strawberry
532
plants through modification of reactive species biosynthesis and transcriptional
533
regulation of multiple defence pathways, J Exp Bot. 64 (2013) 1953-1966.
18
Page 18 of 34
13. Z.P. Jin, J.J. Shen, Z.J. Qiao, et al., Hydrogen sulfide improves drought resistance
535
in Arabidopsis thaliana, Biochem Biophys Res Commu. 414 (2011) 481-486.
536
14. Z.P. Jin, S.W. Xue, Y.N. Luo, et al., Hydrogen sulfide interacting with abscisic
537
acid in stomatal regulation responses to drought stress in Arabidopsis, Plant
538
Physiol Biochem. 62 (2013) 41-46.
ip t
534
15. J.J. Shen, T.J. Xing, H.H. Yuan, et al., Hydrogen Sulfide improves drought
540
tolerance in Arabidopsis thaliana by MicroRNA Expressions, PLOS One. 8
541
(2013) e77047.
543
16. J.J. Shen, Z.J. Qiao, T.J. Xing, et al., Cadmium toxicity is alleviated by AtLCD and AtDCD in Escherichia coli, J Appl Microbiol. 113 (2012) 1130-1138.
us
542
cr
539
17. Y.W. Li, Z.H. Gong, Y. Mu, et al., An Arabidopsis mutant atcsr-2 exhibits high
545
cadmium stress sensitivity involved in the restriction of H2S emission, J Zhejiang
546
Univ-Sci B. 13 (2012) 1006-1014.
an
544
18. M. Dawood, F.B. Cao, M.M. Jahangir, G.P. Zhang, F.B. Wu, Alleviation of
548
aluminum toxicity by hydrogen sulfide is related to elevated ATPase, and
549
suppressed aluminum uptake and oxidative stress in barley, J Hazard Mater.
550
209-210 (2012) 121-128.
M
547
19. B.L. Wang, L. Shi, Y.X. Li, W.H. Zhang, Boron toxicity is alleviated by hydrogen
552
sulfide in cucumber (Cucumis sativus L.) seedlings, Planta. 231 (2010)
553
1301-1309.
554
20. A. Riemenschneider, R. Wegele, A. Schmidt, J. Papenbrock, Isolation and
555
Ac ce p
te
d
551
556 557 558 559 560 561
characterization of a D-cysteine desulfhydrase protein from Arabidopsis thaliana, FEBS J. 272 (2005) 1291-1304.
21. A. Riemenschneider, K. Riedel, R. Hoefgen, J. Papenbrock, H. Hesse, Impact of reduced O-acetylserine (thiol) lyase isoform contents on potato plant metabolism, Plant Phys. 137 (2005) 892-900.
22. Y.G. Luo, X.A. Liu, Q.S. Zheng, et al., Hydrogen sulfide prevents hypoxia-induced apoptosis via inhibition of an H2O2-activated calcium signaling
562
pathway in mouse hippocampal neurons, Biochem Biophys Res Commun. 425
563
(2012) 473–477.
564
23. Y.J. Xie, C. Zhang, D.W. Lai, et al., Hydrogen sulfide delays GA-triggered
565
programmed cell death in wheat aleurone layers by the modulation of glutathione
566
homeostasis and heme oxygenase-1 expression, J Plant Physiol. 171 (2013)
567
53-62.
19
Page 19 of 34
568
24. F.W. Leng, On Ca2+ signalling research, Sci China Life Sci. 55 (2012) 744-746.
569
25. J. Kudla, O. Batistič, K. Hashimoto, Calcium signals: the lead currency of plant
570
information processing, Plant Cell. 22 (2010) 541-563. 26. H.C. Wu, D.L. Luo, F. Vignols, T.L. Jinn, Heat shock-induced biphasic Ca2+
572
signature and OsCaM1-1 nuclear localization mediate downstream signalling in
573
acquisition of thermotolerance in rice (Oryza sativa L.), Plant Cell Environ. 35
574
(2012) 1543-1557.
578 579 580 581 582 583 584
cr
28. A.K. Shanker, C. Cervantes, H. Loza-Tavera, S. Avudainayagam, Chromium
us
577
boil. 53 (2002) 247-273. toxicity in plants, Environ Int. 31 (2005) 739-753.
29. J.L. Hall, Cellular mechanisms for heavy metal detoxification and tolerance, J Exp Bot. 53 (2002) 1-11.
an
576
27. J.K. Zhu, Salt and drought stress signal transduction in plants, Annu Rev plant
30. C. Cobbett, P. Goldsbrough, Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis, Annu Rev Plant Biol. 53 (2002) 159-182.
M
575
ip t
571
31. C.S. Seth, T. Remans, E. Keunen, et al., Phytoextraction of toxic metals: a central role for glutathione, Plant Cell Environ. 35 (2012) 334-346. 32. L.L. Pan, X.H. Liu, Y.Q. Shen, et al., Inhibition of NADPH oxidase 4-related
586
signaling by sodium hydrosulfide attenuates myocardial fibrotic response, Int J
587
Cardiol. 168 (2013) 3770-3778.
588
33. N.A. Anjum, I. Ahmad, I. Mohmood, et al., Modulation of glutathione and its
589
Ac ce p
te
d
585
590 591 592 593 594
related enzymes in plants' responses to toxic metals and metalloids-A review, Environmental and Experimental Botany. 75 (2012) 307-324.
34. C.N. Giannopolitis, S.K. Ries, Superoxide dismutases I. Occurrence in higher plants, Plant physiology. 59 (1977) 309-314.
20
Page 20 of 34
594
Fig. 1 The negative influences of Cr6+ stress on foxtail millet.
596
(A) Phenotype of seedling leaves. (B) The width of leaves in natural state. (C) & (D)
597
Accumulations of MDA and H2O2.
598
10-day-old seedlings were exposed to different concentrations of Cr6+ (0, 5, 10, 15, 20
599
mM) for 24 h. Data are means ± SE of three independent repeats.
600
Fig. 2 The responses of the H2S-emission system to Cr6+ stress.
601
(A) The expressions of H2S synthase-encoding gene LCD, DCD1, DCD2 and DES in
602
foxtail millet at different times of Cr6+ exposure. Seedlings treated with 10 mM Cr6+ at
603
different periods of time (0, 1, 3, 6, 12, 24 h) were used to analyze the gene
604
expressions;
605
(B) I: The H2S production rate of foxtail millet with different concentrations (0, 5, 10,
606
15, 20 mM) of Cr6+ stress for 24 h; II: The H2S production rate of foxtail millet with
607
10 mM Cr6+ stress for different treating times (0, 12, 24, 36, 48 h); Data are means ±
608
SE of three independent repeats. The different letters labeled in this figure show
609
significant differences of H2S production rate (P < 0.05).
610
Fig. 3 Effects of 50 μM H2S on the foxtail millet.
611
(A) Phenotype of leaves. (B) The width of leaves in natural state. (C) & (D) The
cr
us
an
M
d
te
accumulation of H2O2.
613
10-day-old seedlings were pretreated with 50 μM H2S (NaHS as exogenous H2S
614
donor) or 1 mM HA for 12 h, and then exposed to 10 mM Cr6+ for 24 h, with
615
621
Ac ce p
612
ip t
595
622
Fig. 4 The expressions of Ca2+ transporters (TPC1, MRP5, ACA9) and “Ca2+ sensors”
623
(CaM, CBL, CDPK, CIPK1, CIPK2) related genes induced by Cr6+ stress.
624
The mRNAs were isolated from 10-day-old seedlings at different time points (0, 1, 3,
625
6, 12, 24 h) of 10 mM Cr6+ treatment. Data are means ± SE of three independent
626
repeats
616 617 618 619 620
respective controls.
Control: pretreated with H2O, no stressed. Cr6+: pretreated with H2O, stressed with 10 mM Cr6+. H2S: pretreated with 50 μM H2S, no stressed. H2S+Cr6+: pretreated with 50 μM H2S, stressed with 10 mM Cr6+. HA+Cr6+: pretreated with 1 mM HA, stressed with 10 mM Cr6+.
Data are means ± SE of three independent repeats. Bars with different letters are significantly different (P < 0.05).
21
Page 21 of 34
Fig. 5 Effects of 20 mM Ca2+ on the foxtail millet.
628
(A) Phenotype of leaves. (B) The width of leaves in natural state. (C) & (D) H2O2
629
accumulation.
630
10-day-old seedlings were pretreated with 20 mM Ca2+ or 5 mM EGTA for 12 h, and
631
then exposed to 10 mM Cr6+ stress for 24 h, with respective controls.
632
Control: pretreated with H2O, no stressed. Cr6+: pretreated with H2O, stressed with 10
633
mM Cr6+. Ca2+: pretreated with 20 mM Ca2+, no stressed. Ca2++Cr6+: pretreated with
634
20 mM Ca2+, stressed with 10 mM Cr6+. EGTA+Cr6+: pretreated with 5 mM EGTA,
635
stressed with 10 mM Cr6+.
636
Data are means ± SE of three independent repeats. Bars with different letters are
637
significantly different (P < 0.05).
638
Fig. 6 The connection of H2S and Ca2+ in Cr6+ stressed foxtail millet.
639
(A) The effects of 20 mM Ca2+ or 5 mM EGTA on the H2S production rate in foxtail
640
millet stressed with 10 mM Cr6+ for 24 h.
641
(B) The influences of 50 μM H2S (NaHS as exogenous H2S donor) or 1 mM HA on
642
the expressions of Ca2+ sensors-related genes CaM, CBL and CDPK in foxtail millet
643
stressed with 10 mM Cr6+ for 12 h.
644
(C) The influences of 50 μM H2S (NaHS as exogenous H2S donor) or 1 mM HA on
645
the [Ca2+]cyt in stressed foxtail millet observed under fluorescence microscopy at 488
646
nm (400×). 10-day-old seedlings with various pretreatments were exposed to 10 mM
647
Cr6+ for 24 h.
648
654
Ac ce p
655
with different letters are significantly different (P < 0.05).
656
Fig. 8 Influences of H2S and Ca2+ on the content of MDA in foxtail millet with or
657
without Cr6+ stress.
658
10-day-old seedlings with different pretreatments (50 μM H2S or 20 mM Ca2+ or
659
1mM HA or 5 mM EGTA or some combined pretreatments) for 12 h, were then
660
treated with (black columns) or without (white columns) 10 mM Cr6+ for 24 h. Data
649 650 651 652 653
te
d
M
an
us
cr
ip t
627
Data are means ± SE of three independent repeats. Bars with different letters are significantly different (P < 0.05).
Fig. 7 Effects of H2S and Ca2+ on the expressions of PCS1 and MT3 in foxtail millet. 10-day-old seedlings with different pretreatments (50 μM H2S or 20 mM Ca2+ or 1 mM HA or 5 mM EGTA or some combined pretreatments) for 12 h were then treated with (black columns) or without (white columns) 10 mM Cr6+ for 12 h. Data are means ± SE of three independent repeats, error bars indicate error standard and bars
22
Page 22 of 34
are means ± SE of three repeats, error bars indicate error standard and bars with
662
different letters are significantly different (P < 0.05).
663
Fig. 9 Effects of H2S and Ca2+ on the antioxidant system in foxtail millet.
664
(A) Effects of 50 μM H2S (NaHS as exogenous H2S donor) and 1 mM HA on the
665
activities of SOD and POD. (B) Effects of 20 mM Ca2+ and 5mM EGTA on the
666
activities of SOD and POD. (C) & (D) Effects of H2S and Ca2+ on the expressions of
667
GSH1 and GR.
668
10-day-old seedlings with different pretreatments (50 μM H2S or 20 mM Ca2+ or
669
1mM HA or 5 mM EGTA or some combined pretreatments) for 12 h, were then
670
treated with or without 10 mM Cr6+ for 12 h to detect the genes expressions, and for
671
24 h to detect the activities of SOD and POD.
672
Data are means ± SE of three independent repeats, error bars indicate error standard
673
and bars with different letters are significantly different (P < 0.05).
674
Fig. 10 Effects of H2S and Ca2+ on the contents of GSH and AsA as well as the ratio
675
of GSH/GSSG and AsA/DHA in foxtail millet.
676
(A) Effects of 50 μM H2S (NaHS as exogenous H2S donor) and 20 mM Ca2+ on the
677
content of GSH.
678
(B) Effects of 50 μM H2S (NaHS as exogenous H2S donor) and 20 mM Ca2+ on the
679
content of AsA.
680
(C) Effects of 50 μM H2S (NaHS as exogenous H2S donor) and 20 mM Ca2+ on the
681
ratio of GSH/GSSG.
682
(D) Effects of 50 μM H2S (NaHS as exogenous H2S donor) and 20 mM Ca2+ on the
688
Ac ce p
te
d
M
an
us
cr
ip t
661
689
Fig. 11 The summary of the crosstalk between H2S and Ca2+ signaling in foxtail millet
690
responding to Cr6+ stress. Arrows indicate enhanced expressions and hyphens indicate
691
suppressed expression.
683 684 685 686 687
ratio of AsA/DHA.
10-day-old seedlings with different pretreatments (50 μM H2S or 20 mM Ca2+ or 1mM HA or 5 mM EGTA or some combined pretreatments) for 12 h, were then treated with or without 10 mM Cr6+ for 24 h to detect the contents of GSH and AsA as well as their oxidized forms GSSG and AsA contents then calculate the GSH/GSSG and AsA/DHA ratios.
23
Page 23 of 34
Ac ce p
te
d
M
an
us
cr
ip t
Figure.1
Page 24 of 34
Ac
ce
pt
ed
M
an
us
cr
i
Figure.2
Page 25 of 34
Ac ce p
te
d
M
an
us
cr
ip t
Figure.3
Page 26 of 34
Ac ce p
te
d
M
an
us
cr
ip t
Figure.4
Page 27 of 34
Ac ce p
te
d
M
an
us
cr
ip t
Figure.5
Page 28 of 34
Ac
ce
pt
ed
M
an
us
cr
i
Figure.6
Page 29 of 34
Ac
ce
pt
ed
M
an
us
cr
i
Figure.7
Page 30 of 34
Ac
ce
pt
ed
M
an
us
cr
i
Figure.8
Page 31 of 34
Ac ce p
te
d
M
an
us
cr
ip t
Figure.9
Page 32 of 34
Ac
ce
pt
ed
M
an
us
cr
i
Figure.10
Page 33 of 34
Ac
ce
pt
ed
M
an
us
cr
i
Figure.11
Page 34 of 34
S0143-4160(14)00159-6 http://dx.doi.org/doi:10.1016/j.ceca.2014.10.004 YCECA 1612
To appear in:
Cell Calcium
Received date: Revised date: Accepted date:
25-6-2014 1-9-2014 14-10-2014
Please cite this article as: H. Fang, T. Jing, Z. Liu, L. Zhang, Z. Jin, Y. Pei, Hydrogen sulfide interacts with calcium signaling to enhance the chromium tolerance in Setaria italica, Cell Calcium (2014), http://dx.doi.org/10.1016/j.ceca.2014.10.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Graphical Abstract
ip t
1
4
The summary of the crosstalk between H2S and Ca2+ signaling in foxtail millet responding to Cr6+ stress.
us
3
cr
2
This study exhibited that H2S interacts with Ca2+ signaling to enhance the Cr6+
6
tolerance in foxtail millet by activation of the HM chelators and regulation of the
7
antioxidant system. H2S dependent manner is a necessary way in which Ca2+ activated
8
the synthesis of MT and PC by up-regulating the expressions of MT3A and PCS.
9
H2S dependent pathway is a component of the Ca2+ activating antioxidant system and
13
M
d
te
11 12
H2S partially contributes Ca2+-activating antioxidant system.
Ac ce p
10
an
5
1
Page 1 of 34
13
Highlight
14
signaling.
H2S-dependent pathway partially contributes Ca2+-activating antioxidant system.
us
19
cr
H2S-dependent manner.
17 18
Ca2+ activates the HM chelators synthesis-related genes in a
ip t
15 16
H2S enhances the Cr6+ tolerance in millet by interacting with Ca2+
an
20
Ac ce p
te
d
M
21
2
Page 2 of 34
22
Hydrogen sulfide interacts with calcium signaling to enhance the chromium tolerance in Setaria italica
23
Huihui Fanga#, Tao Jingb#, Zhiqiang Liua, Liping Zhanga, Zhuping Jina & Yanxi Peia*
21
a
School of Life Science, Shanxi University, Taiyuan 030006, China
25
b
Department of Biological Sciences, University of Alabama, Tuscaloosa, Alabama, 35487, USA
26
#
These authors contributed equally to this study.
27
* Corresponding author, Email: [email protected]
ip t
24
The running title
30
H2S participates in Ca2+ induced Cr6+ tolerance
31 32
Abstract
us
29
cr
28
The oscillation of intracellular calcium (Ca2+) concentration is a primary event in
34
numerous biological processes in plants, including stress response. Hydrogen sulfide
35
(H2S), an emerging gasotransmitter, was found to have positive effects in plants
36
responding to chromium (Cr6+) stress through interacting with Ca2+ signaling. While
37
Ca2+ resemblances H2S in mediating biotic and abiotic stresses, crosstalk between the
38
two pathways remains unclear. In this study, Ca2+ signaling interacted with H2S to
39
produce a complex physiological response, which enhanced the Cr6+ tolerance in
40
foxtail millet (Setaria italica). Results indicate that Cr6+ stress activated endogenous
41
H2S synthesis as well as Ca2+ signaling. Moreover, toxic symptoms caused by Cr6+
42
stress were strongly moderated by 50 μM H2S and 20 mM Ca2+. Conversely,
48
Ac ce p
te
d
M
an
33
49
antioxidant system.
43 44 45 46 47
50 51
treatments with H2S synthesis inhibitor and Ca2+ chelators prior to Cr6+-exposure aggravated these toxic symptoms. Interestingly, Ca2+ upregulated expression of two important factors in metal metabolism, MT3A and PCS, which participated in the biosynthesis of heavy metal chelators, in a H2S-dependent manner to cope with Cr6+ stress. These findings also suggest that the H2S dependent pathway is a component of the Ca2+ activating antioxidant system and H2S partially contributes Ca2+-activating Key words: hydrogen sulfide, calcium signaling, chromium stress, Setaria italica
52 53 54
1. Introduction Hydrogen sulfide (H2S), which was often considered to be a poisonous gas, has
3
Page 3 of 34
been verified as the third gasotransmitter, the other two being nitric oxide (NO) and
56
carbon monoxide (CO) [1-3]. In mammals, these gases have been shown to play
57
essential roles in various physiological processes [2,3]. In the plant kingdom, NO and
58
CO are known to regulate physiological processes and to defend against abiotic and
59
biotic stresses [4,5]. As early as 1978, Wilson found that leaves of some plants could
60
release H2S [6], which subsequently motivated researchers to investigate the
61
physiological functions of H2S in the growth and development of plants [7-9].
62
Resulting studies revealed that H2S, as a gasotransmitter, is of great importance in
63
helping plants respond to abiotic and biotic stresses, including heat, drought, salinity,
64
nonionic osmotic, and heavy metals (HM) stresses [7,10-19]. Endogenous H2S
65
metabolic enzymes in plants have also been widely investigated [20]. Several studies
66
have shown that the activation of desulfhydrases (CDes) plays a central role in H2S
67
generation. Currently, two specific desulfhydrases, L-cysteine desulfhydrase (LCD)
68
and D-cysteine desulfhydrase (DCD), have been reported to be the most unambiguous
69
CDes, which generate endogenous H2S [20,21]. Other enzymes, such as DCD2, NIFs,
70
OAS-TL, and DES1, have been reported to generate H2S to a lesser degree [21]. Recently, studies have focused on the crosstalk between H2S and other molecules
72
involved in plant growth and development, resulting in some notable progress. For
73
instance, H2S interacting with abscisic acid (ABA) enhanced drought tolerance [14],
74
while its interaction with NO induced stomatal closure and enhanced stress response
75
[10,11]. Additionally, H2S was found to regulate the effects of H2O2 [22], and to
76
82 83
mediated by the concerted action of Ca2+ channels, pumps and carriers located in
84
plasma membranes and vacuole membranes. These proteins include Ca2+ cyclic
85
nucleotide-gated channels (CNGC), two-pore channels (TPC) and the ATP binding
86
cassette (ABC) protein MRP5, which are responsible for increasing the [Ca2+]cyt, as
87
well as the high affinity P-type Ca2+-ATPase (ACA) and the moderate affinity
88
Ca2+-H+ cation antiporter (CAX), which can lead to the decrease in [Ca2+]cyt [25].
77 78 79 80 81
te
d
71
Ac ce p
M
an
us
cr
ip t
55
modulate glutathione homeostasis and heme oxygenase-1 expression to delay GA-triggered programmed cell death [23]. Ca2+, a universal second messenger, is regarded as a core transducer and
regulator in many adaptive processes in plants. A transient elevation in cytosolic calcium concentration ([Ca2+]cyt) is an early event in a large array of biological processes and various stresses, which then delivers this signal to cells and subsequently activates adaptive responses [24,25]. The rapid increase in [Ca2+]cyt is
4
Page 4 of 34
Next, [Ca2+]cyt oscillation is perceived by other Ca2+ binding proteins, such as
90
calcium-dependent protein kinase (CDPK), calmodulin (CaM) and calcineurin B-like
91
protein (CBL), which combine with Ca2+ to interact with other downstream proteins.
92
Ca2+ binding proteins, known as “Ca2+ sensors”, can perceive the rapid increase in
93
[Ca2+]cyt and transmit this specific signal to help plants make an adaptive response
94
[25,26]. Some papers also reported that Ca2+ could compete with HM for channels,
95
enhancing HM tolerance [27].
ip t
89
Chromium (Cr), considered to be the second-most common HM, is often present
97
in the form of Cr3+ and Cr6+ [28]. Both forms are becoming an increasingly serious
98
environmental pollution, creating enduring problems because of the difficulty in
99
degrading and removing them from the environment. Moreover, the negative effects
100
of their toxicity remain in the environment for a very long time [28,29]. Therefore, it
101
is worthwhile and necessary to investigate the detoxification of Cr6+ in plants. With millions of years of evolution at hand, plants have evolved a series of smart
103
and complex strategies to quickly respond and adapt to the toxic effects of HM,
104
including controlling the uptake of HM or excluding the toxic ions to reduce the
105
accumulation of metals [29]. Compartmentalizing HM within vacuoles or activating
106
metal ion chelators, such as metallothioneins (MT) and phytochelatins (PC), to chelate
107
the HM and reduce the toxicity of metal ions are also plant strategies to minimize the
108
toxic effects of HM [29-31]. In addition, plants can alleviate oxidative damage
109
derived from HM stress by activating antioxidant systems, which consist of
110
116 117
system is also due to its ability to regenerate the AsA in AsA-GSH pathway [33].
118
It is noteworthy that H2S can alleviate toxic symptoms caused by HM stress [7,16-19]. However, the molecular mechanism by which H2S works is still unclear and other signaling molecules involved in this protective process remain to be identified. Given that Ca2+ functions in plants responding to stresses have similarities to H2S mediated stress responses [25-27], we investigated crosstalk between H2S and Ca2+ in terms of enhancing Cr6+ tolerance, employing foxtail millet (Setaria italica) as
111 112 113 114 115
119 120 121 122 123
te
d
M
102
Ac ce p
an
us
cr
96
antioxidant enzymes, such as superoxide dismutase (SOD), peroxidase (POD), glutathione reductase (GR), and nonenzymatic antioxidant molecules like glutathione (GSH) and ascorbic acid (AsA) [31,32]. A highly reduced state of GSH or AsA acts as a key regulator of antioxidant defenses [33]. When facing oxidative damage, the plant generally maintains the intracellular redox balance through some redox reactions. Therefore, the ratios of GSH/GSSG and AsA/DHA are important factors indicating the cell resistance to oxidative damage. GSH’s role in the plant antioxidative defense
5
Page 5 of 34
125
the experimental material. Foxtail millet has exhibited considerable tolerance to various stresses, including Cr6+. In this study, we explored the mechanism by which foxtail millet responds to
126 6+
stress, focusing on the crosstalk between H2S and Ca2+ signaling in this
127
Cr
128
protective process.
129
2. Materials and methods
130
2.1 Plant growth and treatments
ip t
124
Seeds of Setaria italica ecotype (Jingu-21) were used in this study. For each
132
experiment, seeds were sterilized in 75% (v/v) ethanol for 30 s and in 6% (v/v)
133
sodium hypochlorite solution for 9 min, and then grown on the petri dishes with three
134
layers of gauzes at the bottom and containing 8 mL water. After two days’ cultivation
135
in darkness at 23°C, with 60% relative humidity, these materials were then kept in
136
16/8 h (light/dark), 160 μEm-2 s-1. Additionally, the gauzes should be kept wet.
an
us
cr
131
Ten days later, the water in the petri dishes was replaced with different chemical
138
solutions (20 mM CaCl2, 5 mM EGTA, and/or 1 mM HA), and NaHS fumigation (50
139
μM) was given at the same time. NaHS, acting as an exogenous H2S donor, was used
140
to provide H2S by fumigating the seedlings. All manipulations were done as described
141
in Jin et al. [13]. For this fumigation, plants were kept in their own petri dishes placed
142
in a sealed glass container and then fumigated with 50 μM NaHS for 12 h. After 12
143
hours pretreatment, all the chemical reagents were sucked out from petri dishes then
144
Cr6+ solution (5 mM K2Cr2O7) was added to stress these seedlings. Gene expressions
145
were detected at 12 h time-point of Cr6+ exposure, while the physiological indexes
147 148 149 150 151
d
te
Ac ce p
146
M
137
were analyzed after 24 h Cr6+ exposure. 2.2 Measurement of the width of leaves To indicate the leaves curled degree, the width of foxtail millet cotyledons in
natural state (not stretched) after various treatments were measured. The data represent means ± SE of 30 cotyledons with three independent repeats. 2.3 Extraction of total RNA and qRT-PCR
152
The total RNA was isolated from 0.1 g of foxtail millet leaves using RNA
153
isolation TRizol® Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s
154
instructions. Then the target gene expressions were detected with quantitative
155
real-time PCR (qRT-PCR) as in Shen et al. [15]. Meanwhile, the gene ACTIN was
156
used as the internal control. The primers used for qRT-PCR are listed in Table S1.
157
Annotation
158
(http://foxtailmillet.genomics.org.cn/page/species/index.jsp). Each experiment was
was
performed
in
the
foxtail
millet
database
6
Page 6 of 34
159
repeated independently for three replicates.
160
2.4 Determination of H2S production rate
161
CDes activity was determined by testing the production rate of H2S according to Jin et al. [13].
163
2.5 Physiological indexes assays
ip t
162
The contents of MDA and H2O2, the activities of SOD and POD as well as the
165
content of GSH, GSSG, AsA and DHA were measured according to published
166
methods [13-19,34].
cr
164
MDA content assay
168
The MDA content, an important indicator of the lipid peroxidation level, was
169
determined by Thiobarbituric acid (TBA) reaction [17,19]. 0.15 g of leaves with
170
various treatments was ground in 1.5 mL of 5% Trichloroacetic acid (TCA). The
171
solution was centrifuged at 5000 g for 5 min at 4°C, then 1 mL of supernatant was
172
mixed with 1 mL of 0.67% TBA in a test tube and boiled in water at 95°C for 30 min.
173
The reaction was terminated in an ice bath. The solution was centrifuged at 5000 g for
174
2 min at 4°C and the supernatant was measured at 450 nm, 532 nm and 600 nm,
175
respectively. Finally, MDA content was expressed as nmol g-1 FW (fresh weight). The detection of H2O2
177
Qualitative detection of H2O2 was carried out with 3, 3-diaminobenzidine (DAB)
178
(Sigma, MO, USA) [17]. The leaves of plants with various treatments were soaked in
179
1 mg L-1 DAB in darkness for 1 h, and then transferred into light conditions for 12 h.
180
186 187
buffer and 1 mL of 1 M KI. The reaction ran for 1 h in darkness and the absorbance at
188
390 nm was measured. The content of H2O2 was calculated based on a standard curve
189
of known concentrations of H2O2.
181 182 183 184 185
te
d
176
Ac ce p
M
an
us
167
After rinsing with distilled water three times, the leaves were boiled in 95% ethanol for 10 min to remove the pigments, and then H2O2 production in the form of reddish-brown coloration was visualized. Quantitative detection of H2O2 was carried out with potassium iodide (KI) as
described in a published method [19]. 0.15 g of leaves was ground in 1.5 mL of 5% TCA and the solution was centrifuged at 5000 g for 5 min at 4°C. The reaction mixture consisted of 500 μL of extracted supernatant, 500 μL of potassium phosphate
190
SOD activity assay
191
The activity of SOD was assayed by detecting the inhibition of reduction level of
192
nitro blue tetrazolium (NBT) according to Giannopolitis and Ries (1977) with some
7
Page 7 of 34
modifications [34]. The total enzyme was extracted using 0.05 M phosphate buffer
194
(pH 7.0), and centrifuged at 12000 g for 5 min at 4°C. Then the reaction was
195
performed in a test tube containing 3 mL of the mixture consisting of 50 mM
196
potassium phosphate buffer (pH 7.8), 13 mM methionine, 75 mM NBT, 0.1 mM
197
EDTA, 2 mM riboflavin and 100 μL enzyme solution. Then this mixture was exposed
198
to light with the intensity of 5000 lx for 15 min, and the absorbance was monitored at
199
560 nm. One unit of SOD activity was defined as the amount of enzyme that caused
200
50% inhibition of NBT reduction.
cr
ip t
193
POD activity assay
202
The activity of POD was assayed according to our published methods with some
203
modifications [16,34]. The total enzyme was extracted as described above. 3 mL of
204
the assay mixture for the POD activity included: 0.05 M phosphate buffer (pH 7.0)
205
containing 10 mM guaiacol, 0.1 mM EDTA and 5 mM H2O2. This mixture was
206
incubated for 5 min at 25°C. The 50 μL 20 times-diluted enzyme solution was
207
subsequently added to start this reaction, and the absorbance at 470 nm was
208
immediately monitored.
M
an
us
201
GSH and GSSG content assay
210
GSH and GSSG content was assayed using a GSH and GSSG assay kit S0053
211
(Beyotime Institute of Biotecnology, China) according to the manufacturer’s
212
instructions.
te
AsA and DHA content assay
220
Ac ce p
213
d
209
221
Probes, Eugene, OR, USA). All manipulations were done according to Jin et al. [14].
222
2.7 Statistical analysis
214 215 216 217 218 219
AsA content was assayed using an AsA assay kit A009 (Nanjing Jiancheng
Bioengineering Institute, China) and DHA content was assayed using an DHA assay kit SM102 (Beijing Solarbio Science ＆ Tecnology, China). All operations were performed according to the manufacturer’s instructions. 2.6 Fluo-3/AM loading and detection of cytoplasmic Ca2+ For detecting the [Ca2+]cyt, the lower epidermis peeled off from the leaves were
used to load the Ca2+-sensitive Fluorescent probe 5 mM Fluo-3/AM (Molecular
223
Each experiment was carried out for three independent repetitions. The results
224
were expressed as the means ± SE. Data were analyzed using SPSS (version 17, IBM
225
SPSS, Chicago, IL), and error bars were made according to Tukey’s multiple range
226
test (P < 0.05).
8
Page 8 of 34
227
3. Results
228
3.1 H2S alleviated Cr6+ toxicity in foxtail millet
229
3.1.1 Cr6+ stress damaged the foxtail millet To study the negative influence of Cr6+ stress on foxtail millet, 10-day-old plants
231
were exposed to increasing concentration of Cr6+ (0, 5, 10, 15, 20 mM Cr6+) for 24 h
232
respectively. It is clear to see that Cr6+ treatment led to toxic symptoms of dim and
233
curly leaves in plants (Fig. 1A), and moreover the width of cotyledons in a natural
234
state after Cr6+ treatment was measured to indicate the leaves curled degree (Fig. 1B).
235
Notably, these damaged symptoms were dose-dependently induced.
cr
ip t
230
HM are known to disturb the redox balance and consequently expose plants to
237
oxidative stress. In this study, the content of MDA and H2O2 went up as Cr6+
238
concentration increased from 0 to 20 mM (Fig. 1C and D). 10 mM Cr6+ stress is
239
strong enough to stimulate these toxic symptoms (P < 0.05), so 10 mM Cr6+ was
240
chosen in further experiments.
241
3.1.2 Cr6+ stress stimulated the H2S emission
M
an
us
236
In order to investigate the effect of Cr6+ stress on the H2S-emission system, the
243
expressions of LCD, DCD1, DCD2, DES and the rate of H2S production in plants
244
treated with Cr6+ were measured. It was found that the expressions of H2S-emission
245
related genes LCD, DCD2, DES were markedly increased during the first 12 h of Cr6+
246
exposure following decline at 24 h, while the expression of DCD1 was consistently
247
increased from 0 h to 24 h under Cr6+ stress (Fig. 2A). In spite of this, all the gene
248
expressions were markedly up-regulated at the 12 h time-point of Cr6+ exposure
254
Ac ce p
te
d
242
255
was exposed to Cr6+.
256
3.1.3 H2S enhanced the Cr6+ tolerance
249 250 251 252 253
compared to the control seedlings without stress, so we decided to choose 12 h to detect the associated gene expressions. Additionally, the H2S production rate was dose and time dependently induced by
6+
Cr stress, and this activation was the most significant with 24 h of 10 mM Cr6+ treatment (Fig. 2B).
These results implied that H2S emission system was activated when foxtail millet
257
The results above implied that H2S might be of importance to foxtail millet’s
258
response to Cr6+ stress. In order to confirm the positive effect of H2S, 50 μM of NaHS
259
acting as an exogenous H2S donor, within the physiological range of H2S, was used in
260
subsequent experiments. 1 mM of HA, acting as a H2S synthesis inhibitor, was also
9
Page 9 of 34
261
employed for the reverse verification. As is shown in Fig. 3, 50 μM of H2S pretreatment significantly alleviated the
263
toxic symptoms and restored the dim and curly leaves caused by Cr6+ stress back to a
264
normal level (Fig. 3A and B). Moreover, whether the plants were exposed to Cr6+ or
265
not, H2S fumigation could help maintain the contents of H2O2 (Fig. 3C and D) and
266
MDA (Fig. 8) at the normal level, while the contents of H2O2 (Fig. 3C and D) and
267
MDA (Fig. 8) increased considerably in stressed plants without H2S pretreatment or
268
addition of HA.
269
3.2 Ca2+ signaling involved in foxtail millet responding to Cr6+ stress
270
3.2.1 Cr6+ stress stimulated the Ca2+ signaling
us
cr
ip t
262
The rapid shock in [Ca2+]cyt is an original mediator in a series of stresses in plants.
272
In this study, the foxtail millet leaves with or without 10 mM Cr6+ stress were loaded
273
with Fluo-3/AM to detect the changes of [Ca2+]cyt. Obviously, stronger Ca2+
274
fluorescence was observed in the leaves with Cr6+ stress (Fig. 6C).
an
271
Also, the expression levels of Ca2+ transporter-encoding genes in foxtail millet
276
with increasing time of Cr6+ treatment (0, 1, 3, 6, 12, 24 h) were detected. As is shown
277
in Fig. 4, the TPC1 and MRP5 expressions were time-dependently up-regulated by
278
Cr6+ treatment, and meanwhile the expression of ACA9 decreased when Cr6+ treating
279
time was extended. As we expected, this result was in accordance with the stronger
280
Ca2+ fluorescence in the previous experiment (Fig. 6C).
te
d
M
275
Numerous studies have shown that the rapid increase in [Ca2+]cyt is perceived by
282
some following members known as “Ca2+ sensors”. To explore whether Ca2+ signaling
283 284 285 286 287 288 289
Ac ce p
281
participated in foxtail millet’s response to Cr6+ stress, the CaM, CBL and CDPK, which encode the [Ca2+]cyt direct perceivers, as well as CIPK, the CBL downstream protein-encoding gene, were further investigated. It revealed in Fig. 4 that the expressions of all these Ca2+ signaling related genes were enhanced by Cr6+ stress to various degrees in a time-dependent manner, and this up-regulation was notable at 12 h of Cr6+ stress (Fig. 4).
As is mentioned above, we concluded that the Ca2+ signaling was involved in
290
response against Cr6+ stress in foxtail millet.
291
3.2.2 Ca2+ significantly alleviated the Cr6+ toxicity
292
In order to test the effect of Ca2+ in this process, 20 mM CaCl2 was used as an
293
exogenous Ca2+ donor. The toxic symptoms in leaves of foxtail millet exposed to Cr6+
294
stress were obvious. However, 20 mM Ca2+ pretreatment, to a large extent, alleviated
10
Page 10 of 34
these symptoms, and at the same time rescued the dim and curly leaves (Fig. 5A and
296
B). Interestingly, the Cr6+ tolerance was weakened by addition of Ca2+ chelator EGTA
297
(Fig. 5A and B). Furthermore, Ca2+ pretreatment greatly reduced the accumulation of
298
H2O2 (Fig. 5C and D) and MDA (Fig. 8), whereas their accumulation largely increased
299
in stressed plants with EGTA pretreatment.
300
3.3 H2S interacting with Ca2+ signaling enhanced Cr6+ tolerance
301
3.3.1 The connection of H2S and Ca2+ signaling in foxtail millet responding to
302
Cr6+ stress
cr
ip t
295
In order to investigate the crosstalk between H2S and Ca2+ signaling in the
304
process of foxtail millet responding to Cr6+ stress, we detected the effects of Ca2+ and
305
EGTA on the increased H2S production, as well as the influences of H2S and HA on
306
the rapid shock of [Ca2+]cyt and the up-regulated expressions of Ca2+ signaling
307
associated genes.
an
us
303
When the plants were exposed to Cr6+ stress, Ca2+ pretreatment hugely improved
309
the H2S production rate, and EGTA reduced the increased H2S emission induced by
310
Cr6+ stress (Fig. 6A). As fluorescent staining has shown, the stronger Ca2+
311
fluorescence derived from Cr6+ stress can be moderated by H2S fumigation.
312
Correspondingly, HA pretreatment strengthened the Ca2+ fluorescence to some degree
313
(Fig. 6C).
Simultaneously, the up-regulated expressions of CaM and CBL caused by Cr6+
315
stress can be increased by Ca2+ pretreatment, while this effect of Ca2+ can be strongly
316
322 323
biosynthesis of these chelators.
317 318 319 320 321
te
314
Ac ce p
d
M
308
enhanced by H2S fumigation, but weakened by addition of HA (Fig. 6B). However, these treatments have little influence on the expression of CDPK (Fig. 6B). 3.3.2 H2S participated in Ca2+ up-regulating expressions of HM chelators-related genes
It is widely known that the accumulation of chelators, such as MT and PC, play
an important role in plant defense against HM stresses, we thus explored the crosstalk of H2S and Ca2+ signaling in regulating the expressions of genes involved in the
324
As Fig. 7 showed, during Cr6+-exposure, the expressions of MT3A and PCS were
325
increased significantly. Furthermore, H2S or Ca2+ pretreatment strengthened the
326
expressions of these genes. Additionally, duplicate effects could be observed after H2S
327
and Ca2+ combined treatment. Interestingly, EGTA has no influence on the
328
up-regulation of these genes induced by H2S. However, the up-regulation of these
11
Page 11 of 34
329
gene expressions caused by Ca2+ can be strongly blocked by HA, a H2S synthesis
330
inhibitor.
331
3.3.3 H2S interacting with Ca2+ signaling to alleviate the oxidative damage from
332
Cr6+ stress As is mentioned above, both H2S and Ca2+ can enhance Cr6+ tolerance.
334
Interestingly, H2S and Ca2+ signaling have a potential crosstalk in this process. As is
335
shown in Fig. 8, both H2S and Ca2+ can relieve the accumulation of MDA during Cr6+
336
stress and this protective effect was remarkably observed with H2S and Ca2+
337
combined pretreatment. However, the accumulation of MDA can be strengthened by
338
addition of HA and EGTA, respectively. Further results demonstrated that EGTA can
339
partly weaken the protective role of H2S. Similarly, HA also attenuated the positive
340
role of Ca2+ to some degree (Fig. 8).
us
cr
ip t
333
Plants facing with stress can activate their antioxidant system to defend against
342
the oxidative damages. Hereby, the relationship between H2S and Ca2+ in regulating
343
this system was analyzed.
M
an
341
Under Cr6+-exposure, the SOD and POD activities were elevated markedly to
345
fight against oxidative damage (Fig. 9A and B). Additionally, whether the plants were
346
exposed to Cr6+ stress or not, the SOD and POD activities could be kept at the normal
347
level by H2S fumigation, while their activities increased more significantly in stressed
348
plants with addition of HA (Fig. 9A). On the contrary, Ca2+ pretreatment led to a
349
higher SOD and POD activity in stressed seedlings. Correspondingly, EGTA
350
pretreatment prior to Cr6+-exposure evidently suppressed the SOD and POD activities
352 353 354 355 356
te
Ac ce p
351
d
344
(Fig. 9B). Interestingly, the effect of Ca2+ on these antioxidant enzymes was totally different from that of H2S.
As both H2S and Ca2+ can relieve the oxidative damage caused by Cr6+ stress,
why is their regulation on the activities of these antioxidant enzymes quite different? In order to explore this intriguing problem, the influences of H2S and Ca2+ on the antioxidants were studied.
357
As the results have shown, under Cr6+ stress, the expressions of GSH1 and GR
358
were up-regulated (Fig. 9C and D), the reduced GSH content correspondingly went up
359
(Fig. 10A), and this increase could be markedly strengthened by H2S or Ca2+
360
pretreatment. Furthermore, duplicate effects were observed after the H2S and Ca2+
361
treatment together. Besides, when EGTA was used as Ca2+ chelator, little influence on
362
the up-regulation of GSH1 and GR induced by H2S was observed (Fig. 9C and D).
12
Page 12 of 34
However, the positive effect of H2S on the reduced GSH content was partly restrained
364
by EGTA addition (Fig. 10A). Interestingly, HA, acting as a H2S synthesis inhibitor,
365
could obviously weaken Ca2+-induced GSH1 and GR expressions (Fig. 9C and D) and
366
the GSH content (Fig. 10A). In addition, during Cr6+ exposure, the redox state
367
GSH/GSSG decreased markedly, and this decrease was weakened by H2S and Ca2+
368
pretreatment (Fig. 10C), also this effect of H2S and Ca2+ could be partially depressed
369
by EGTA and HA, respectively (Fig. 10C).
ip t
363
Additionally, the AsA content was simultaneously measured, the result of which
371
indicated that the pattern of Cr6+ stress regulating the content of AsA did not seem to
372
be so consistent with that regulating GSH content. AsA content was depressed
373
markedly by Cr6+-exposure, while H2S or Ca2+ pretreatment led to more AsA
374
accumulation in stressed seedlings, and this accumulation was more obvious by H2S
375
and Ca2+ combined pretreatment (Fig. 10B ). Besides, when EGTA pretreated, the
376
accumulation of AsA induced by H2S was partly inhibited; and HA pretreatment could
377
obviously weaken the positive effect of Ca2+ on the AsA content to some degree (Fig.
378
10B). Simultaneously, Cr6+ stress led to a significant decrease in the ratio of
379
AsA/DHA (even decreased by 66%). H2S or Ca2+ had little positive effect on this
380
decrease derived from Cr6+ stress (Fig. 10D).
381 382
Recently, it has been reported that H2S as a gasotransmitter participates in plant
390
Ac ce p
383
4. Discussion
te
d
M
an
us
cr
370
391
could both dramatically alleviate these negative effects. Thus, we focused on the
392
crosstalk between H2S and Ca2+ signaling in this process.
384 385 386 387 388 389
defense responses to abiotic and biotic stresses [1-3,10-19]. However, the potential molecular mechanism and its signaling pathways remain limited. This study was aimed at exploring the molecular mechanism of the positive
effects of H2S and its signaling pathways on foxtail millet that responds to Cr6+ stress. It has been obviously noticed that Cr6+ stress could lead to toxic symptoms and accumulation of ROS. Meanwhile both the endogenous H2S emission system and Ca2+ signaling were activated during this stress. In addition, exogenous H2S and Ca2+
393
The expressions of the H2S-emission related genes LCD, DCD2 and DES were
394
markedly increased during the first 12 h of Cr6+ exposure, and then decreased; while
395
the DCD1 expression was being increased from 0 h to 24 h under Cr6+ stress (Fig. 2A).
396
This unsynchronized result may be due to the different transcription regulation of
13
Page 13 of 34
these genes. Although the expressions of H2S-generating genes LCD, DCD2 and DES
398
were decreased at 24 h of Cr6+ exposure, we did not find the difference of H2S
399
production rate at 12 h and 24 h of Cr6+ exposure. H2S production rate directly reflects
400
the intensity of enzymatic activity of H2S-generating proteins, and it will take time for
401
transcribed mRNA to be translated into protein following posttranslational
402
modification to have complete activity. It is really the case, and we did observe that
403
H2S production rate is significantly reduced at 36 h and 48 h of Cr6+ exposure (Fig.
404
2B), indicative of a delayed response to the reduced gene expression of LCD, DCD2
405
and DES at 24 h of Cr6+ exposure.
cr
ip t
397
It was found that fumigation with different doses of NaHS (0, 10, 20, 30, 40, 50,
407
60 μM) could enhance Cr6+ tolerance in foxtail millet to different degrees, 50 μM
408
NaHS pretreatment showed significantly protective role (data not shown). Hence, the
409
50 μM NaHS was employed as an exogenous H2S donor, which is within the H2S
410
physiological concentration detected in animals and plants [13,15]. Exogenous donors (50 μM NaHS and 20 mM CaCl2) and endogenous inhibitors
412
(1 mM HA and 5 mM EGTA) of H2S and Ca2+ were used in this study. It is
413
noteworthy that during Cr6+ stress, the H2S emission could be raised by Ca2+ but
414
suppressed by EGTA (Fig. 6A). This may suggest that Cr6+ stress activate the H2S
415
emission system in a Ca2+-dependent manner. Furthermore, H2S could participate in
416
Ca2+ signaling through affecting the [Ca2+]cyt and regulating some downstream
417
molecules (Fig. 6B and C) in the process. All these results implied that H2S and Ca2+
418
424 425
and PC by up-regulating the expressions of MT3A and PCS to defend against this
426
stress. These chelators could chelate the Cr6+ and reduce its toxicity. As is shown in
427
Fig. 7, their expressions could be elevated by H2S or Ca2+, even superimposed effects
428
could be observed after H2S and Ca2+ combined treatment. This activation of the
429
MT3A and PCS expression induced by H2S could not be influenced by EGTA.
430
Surprisingly, the addition of HA, to inhibit the endogenous synthesis of H2S, even
419 420 421 422 423
te
d
M
411
Ac ce p
an
us
406
signaling have a potential connection during foxtail millet responding to this stress. However, despite their complex interaction in this process, some differences in
the mode of their actions were also observed. We then further explained their crosstalk and differences in two aspects: the activation of HM chelators and the regulation of antioxidant system.
HM chelators are extremely important in defending against HM stress [30]. As
we expected, when exposed to Cr6+ stress, foxtail millet activated the synthesis of MT
14
Page 14 of 34
431
completely suppressed this positive role of Ca2+. These results illustrated that Ca2+
432
could enhance the synthesis of MT and PC by up-regulating the expressions of MT3A
433
and PCS in a H2S-dependent manner to deal with the Cr6+ stress, and Ca2+-activated
434
H2S was essential for this adaptive response. It is generally considered that toxicity of HM exposes plants to oxidative damage.
436
Once exposed to Cr6+ stress, plants can strongly activate the antioxidant system. In
437
this paper, the expressions of GSH1 and GR as well as the content of reduced GSH in
438
foxtail millet during Cr6+-exposure could be enormously strengthened by H2S or Ca2+,
439
and their combined pretreatment could even cause duplicate effects (Fig. 9C and D,
440
Fig. 10A). Moreover, we also explored the expression of GSH2 (data not shown). In
441
contrast to GSH1, the expression of GSH2 remained unchanged when faced with Cr6+
442
stress as well as H2S and/or Ca2+pretreatment. GSH1 has been well reported to act as
443
the rate-limiting enzyme in the process of GSH generation [17], so we speculate that
444
H2S and Ca2+ mainly improve the GSH content by increasing GSH1 expression.
an
us
cr
ip t
435
It is well known that AsA is an important antioxidant in plants, and therefore its
446
content was measured. In contrast to the change of GSH content, AsA content was
447
depressed markedly by Cr6+-exposure, while H2S or Ca2+ pretreatment led to more
448
AsA accumulation in stressed seedlings (Fig. 10A). Under Cr6+ stress, the AsA content
449
decreased while the GSH content increased. We thus inferred that the AsA made an
450
earlier contribution than GSH to defending against the oxidative damage. The AsA
451
scavenged ROS in the way that it became an oxidized form DHA, so the ratio
452
AsA/DHA decreased dramatically during Cr6+stress (Fig. 10D). H2S and Ca2+ had no
458
Ac ce p
te
d
M
445
459
speculated that Ca2+ up-regulated the antioxidant molecules in a H2S dependent or
460
independent manner. That is to say, H2S dependent pathway is just an alternative in
461
Ca2+ induced activation of these antioxidant molecules, suppressing this H2S pathway
462
could not block the function of Ca2+ in this process.
453 454 455 456 457
effect on AsA/DHA ratio whereas they could regulate the content of GSH and AsA as well as the GSH/GSSG ratio. Therefore we speculate that H2S and Ca2+ might activate this GSH-AsA pathway and then regulate the plant antioxidative defense system. Meanwhile, GSH could also act as an antioxidant molecule to fight against ROS. It was clear that the positive effect of Ca2+ on the expression of GSH1 and GR as
well as the content of GSH and AsA could be partly weakened by HA. Thus we
463
However, some inconsistencies can be found in the influences of H2S and Ca2+
464
on the activities of antioxidant enzymes. During Cr6+ exposure, Ca2+ could help
15
Page 15 of 34
foxtail millet defend against oxidative damage by increasing the activities of SOD and
466
POD (Fig. 9B), but exogenous H2S pretreatment kept the SOD and POD activities at
467
the normal level (Fig. 9A). These results may indicate that H2S alleviated
468
Cr6+-induced oxidative damage not via regulating the antioxidant enzymes activities
469
directly. Thus it was inferred that H2S might alleviate oxidative damage by regulating
470
the nonenzymatic antioxidant molecules or decline the generation of ROS, such as
471
suppressing the activity of NADPH oxidase, as H2S has been reported to decrease the
472
ROS production by inhibiting the NADPH oxidase 4-related signaling [32].
473
Compared to H2S, Ca2+ could activate the antioxidant system not only by enhancing
474
SOD and POD activities but also by increasing the contents of GSH and AsA to cope
475
with Cr6+-induced oxidative damage. This illustrated that Ca2+ might be more
476
extensive in the exercise of its protective functions compared to H2S. All of these
477
results strongly suggested that there may be a complicated crosstalk between H2S and
478
Ca2+ in this process, but not a simple linear relationship between them. A model based on the results described in this study was proposed to expound
480
the signaling pathways of H2S and Ca2+ in foxtail millet’s response to Cr6+ stress (Fig.
481
11). Cr6+ stress activated the H2S and Ca2+ signaling, and then H2S interacting with
482
Ca2+ signaling participated in complex physiological processes to defend against Cr6+
483
stress. H2S dependent manner is a necessary way in which Ca2+ activated the
484
synthesis of MT and PC by up-regulating the expressions of MT3A and PCS. Whereas
485
in the aspect of regulating antioxidant system, Ca2+ activated this system not only
486 487 488 489 490 491 492
te
d
M
479
Ac ce p
an
us
cr
ip t
465
through the antioxidant enzymes but also via the antioxidant molecules to fight against the Cr6+-induced oxidative damage. H2S alleviated oxidative damage mainly by up-regulating the antioxidant molecules. In other words, H2S dependent pathway is a component of the Ca2+ activating antioxidant system and H2S partially contributes Ca2+-activating antioxidant system. Overall, concerted efforts with Ca2+, H2S promptly enhanced the Cr6+ tolerance
in foxtail millet.
493 494 495 496
Acknowledgement This work was supported by the National Natural Science Foundation of China (31372085 to Yanxi Pei; 31300236 to Zhiqiang Liu; 31400237 to Zhuping Jin).
497
We give sincere appreciation to Dr. Margaret Johnson and Dr. Kim Lackey from
498
Department of Biological Sciences at the University of Alabama for their professional
16
Page 16 of 34
499
and generous help with the language.
Ac ce p
te
d
M
an
us
cr
ip t
500
17
Page 17 of 34
500 501
References
502
1.
gaseous transmitter? The FASEB Journal. 16 (2002) 1792-1798. 2.
blossomed, Physiol Rev. 92 (2012) 791-896.
505
3. 4.
M. Delledonne, NO news is good news for plants, Curr Ppin Plant Biol. 8 (2005) 390-396.
509 510
cr
mice with deletion of cystathionine γ-lyase. Science 322(2008) 587-590.
507 508
G.D. Yang, L.Y. Wu, B. Jiang, H2S as a physiologic vasorelaxant: hypertension in
5.
us
506
R. Wang, Physiological implications of hydrogen sulfide: a whiff exploration that
ip t
503 504
R. Wang, Two’s company, three’s a crowd: can H2S be the third endogenous
W. Xuan, F.Y. Zhu, S. Xu, et al., The heme oxygenase/carbon monoxide system is involved in the auxin-induced cucumber adventitious rooting process, Plant
512
Physiol. 148 (2008) 881-893. 6.
sulfide from plants, Plant Physiology. 61 (1978) 184-189.
514 515
L.G. Wilson, R.A. Bressan, P. Filner, Light-dependent emission of hydrogen
7.
M
513
an
511
H. Zhang, L.Y. Hu, K.D. Hu, Y.D. He, S.H. Wang, J.P. Luo, Hydrogen sulfide promotes wheat seed germination and alleviates oxidative damage against copper
517
stress, J Integr Plant Biol. 50 (2008) 1518-1529. 8.
H. Zhang, S.L. Hu, Z.J. Zhang, et al., Hydrogen sulfide acts as a regulator of
te
518
d
516
flower senescence in plants, Postharvest Biol Tech. 60 (2011) 251-257. J. Chen, F.H. Wu, W.H. Wang, et al., Hydrogen sulfide enhances photosynthesis
520
9.
521
Ac ce p
519
522 523 524 525 526 527
through promoting chloroplast biogenesis, photosynthetic enzyme expression, and thiol redox modification in Spinacia oleracea seedlings, J Exp Bot. 62 (2011) 4481-4493.
10. Z.G. Li, S.Z. Yang, W.B. Long, G.X. Yang, Z.Z. Shen, Hydrogen sulfide may be a novel downstream signal molecule in nitric oxide-induced heat tolerance of maize (Zea mays L.) seedlings, Plant cell Environ. 36 (2013) 1564-1572.
11. Y.Q. Wang, L. Li, W.T. Cui, S. Xu, W.B. Shen, R. Wang, Hydrogen sulfide
528
enhances alfalfa (Medicago sativa) tolerance against salinity during seed
529
germination by nitric oxide pathway, Plant Soil. 351 (2012) 107-119.
530
12. A. Christou, G.A. Manganaris, I. Papadopoulos, V. Fotopoulos, Hydrogen sulfide
531
induces systemic tolerance to salinity and non-ionic osmotic stress in strawberry
532
plants through modification of reactive species biosynthesis and transcriptional
533
regulation of multiple defence pathways, J Exp Bot. 64 (2013) 1953-1966.
18
Page 18 of 34
13. Z.P. Jin, J.J. Shen, Z.J. Qiao, et al., Hydrogen sulfide improves drought resistance
535
in Arabidopsis thaliana, Biochem Biophys Res Commu. 414 (2011) 481-486.
536
14. Z.P. Jin, S.W. Xue, Y.N. Luo, et al., Hydrogen sulfide interacting with abscisic
537
acid in stomatal regulation responses to drought stress in Arabidopsis, Plant
538
Physiol Biochem. 62 (2013) 41-46.
ip t
534
15. J.J. Shen, T.J. Xing, H.H. Yuan, et al., Hydrogen Sulfide improves drought
540
tolerance in Arabidopsis thaliana by MicroRNA Expressions, PLOS One. 8
541
(2013) e77047.
543
16. J.J. Shen, Z.J. Qiao, T.J. Xing, et al., Cadmium toxicity is alleviated by AtLCD and AtDCD in Escherichia coli, J Appl Microbiol. 113 (2012) 1130-1138.
us
542
cr
539
17. Y.W. Li, Z.H. Gong, Y. Mu, et al., An Arabidopsis mutant atcsr-2 exhibits high
545
cadmium stress sensitivity involved in the restriction of H2S emission, J Zhejiang
546
Univ-Sci B. 13 (2012) 1006-1014.
an
544
18. M. Dawood, F.B. Cao, M.M. Jahangir, G.P. Zhang, F.B. Wu, Alleviation of
548
aluminum toxicity by hydrogen sulfide is related to elevated ATPase, and
549
suppressed aluminum uptake and oxidative stress in barley, J Hazard Mater.
550
209-210 (2012) 121-128.
M
547
19. B.L. Wang, L. Shi, Y.X. Li, W.H. Zhang, Boron toxicity is alleviated by hydrogen
552
sulfide in cucumber (Cucumis sativus L.) seedlings, Planta. 231 (2010)
553
1301-1309.
554
20. A. Riemenschneider, R. Wegele, A. Schmidt, J. Papenbrock, Isolation and
555
Ac ce p
te
d
551
556 557 558 559 560 561
characterization of a D-cysteine desulfhydrase protein from Arabidopsis thaliana, FEBS J. 272 (2005) 1291-1304.
21. A. Riemenschneider, K. Riedel, R. Hoefgen, J. Papenbrock, H. Hesse, Impact of reduced O-acetylserine (thiol) lyase isoform contents on potato plant metabolism, Plant Phys. 137 (2005) 892-900.
22. Y.G. Luo, X.A. Liu, Q.S. Zheng, et al., Hydrogen sulfide prevents hypoxia-induced apoptosis via inhibition of an H2O2-activated calcium signaling
562
pathway in mouse hippocampal neurons, Biochem Biophys Res Commun. 425
563
(2012) 473–477.
564
23. Y.J. Xie, C. Zhang, D.W. Lai, et al., Hydrogen sulfide delays GA-triggered
565
programmed cell death in wheat aleurone layers by the modulation of glutathione
566
homeostasis and heme oxygenase-1 expression, J Plant Physiol. 171 (2013)
567
53-62.
19
Page 19 of 34
568
24. F.W. Leng, On Ca2+ signalling research, Sci China Life Sci. 55 (2012) 744-746.
569
25. J. Kudla, O. Batistič, K. Hashimoto, Calcium signals: the lead currency of plant
570
information processing, Plant Cell. 22 (2010) 541-563. 26. H.C. Wu, D.L. Luo, F. Vignols, T.L. Jinn, Heat shock-induced biphasic Ca2+
572
signature and OsCaM1-1 nuclear localization mediate downstream signalling in
573
acquisition of thermotolerance in rice (Oryza sativa L.), Plant Cell Environ. 35
574
(2012) 1543-1557.
578 579 580 581 582 583 584
cr
28. A.K. Shanker, C. Cervantes, H. Loza-Tavera, S. Avudainayagam, Chromium
us
577
boil. 53 (2002) 247-273. toxicity in plants, Environ Int. 31 (2005) 739-753.
29. J.L. Hall, Cellular mechanisms for heavy metal detoxification and tolerance, J Exp Bot. 53 (2002) 1-11.
an
576
27. J.K. Zhu, Salt and drought stress signal transduction in plants, Annu Rev plant
30. C. Cobbett, P. Goldsbrough, Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis, Annu Rev Plant Biol. 53 (2002) 159-182.
M
575
ip t
571
31. C.S. Seth, T. Remans, E. Keunen, et al., Phytoextraction of toxic metals: a central role for glutathione, Plant Cell Environ. 35 (2012) 334-346. 32. L.L. Pan, X.H. Liu, Y.Q. Shen, et al., Inhibition of NADPH oxidase 4-related
586
signaling by sodium hydrosulfide attenuates myocardial fibrotic response, Int J
587
Cardiol. 168 (2013) 3770-3778.
588
33. N.A. Anjum, I. Ahmad, I. Mohmood, et al., Modulation of glutathione and its
589
Ac ce p
te
d
585
590 591 592 593 594
related enzymes in plants' responses to toxic metals and metalloids-A review, Environmental and Experimental Botany. 75 (2012) 307-324.
34. C.N. Giannopolitis, S.K. Ries, Superoxide dismutases I. Occurrence in higher plants, Plant physiology. 59 (1977) 309-314.
20
Page 20 of 34
594
Fig. 1 The negative influences of Cr6+ stress on foxtail millet.
596
(A) Phenotype of seedling leaves. (B) The width of leaves in natural state. (C) & (D)
597
Accumulations of MDA and H2O2.
598
10-day-old seedlings were exposed to different concentrations of Cr6+ (0, 5, 10, 15, 20
599
mM) for 24 h. Data are means ± SE of three independent repeats.
600
Fig. 2 The responses of the H2S-emission system to Cr6+ stress.
601
(A) The expressions of H2S synthase-encoding gene LCD, DCD1, DCD2 and DES in
602
foxtail millet at different times of Cr6+ exposure. Seedlings treated with 10 mM Cr6+ at
603
different periods of time (0, 1, 3, 6, 12, 24 h) were used to analyze the gene
604
expressions;
605
(B) I: The H2S production rate of foxtail millet with different concentrations (0, 5, 10,
606
15, 20 mM) of Cr6+ stress for 24 h; II: The H2S production rate of foxtail millet with
607
10 mM Cr6+ stress for different treating times (0, 12, 24, 36, 48 h); Data are means ±
608
SE of three independent repeats. The different letters labeled in this figure show
609
significant differences of H2S production rate (P < 0.05).
610
Fig. 3 Effects of 50 μM H2S on the foxtail millet.
611
(A) Phenotype of leaves. (B) The width of leaves in natural state. (C) & (D) The
cr
us
an
M
d
te
accumulation of H2O2.
613
10-day-old seedlings were pretreated with 50 μM H2S (NaHS as exogenous H2S
614
donor) or 1 mM HA for 12 h, and then exposed to 10 mM Cr6+ for 24 h, with
615
621
Ac ce p
612
ip t
595
622
Fig. 4 The expressions of Ca2+ transporters (TPC1, MRP5, ACA9) and “Ca2+ sensors”
623
(CaM, CBL, CDPK, CIPK1, CIPK2) related genes induced by Cr6+ stress.
624
The mRNAs were isolated from 10-day-old seedlings at different time points (0, 1, 3,
625
6, 12, 24 h) of 10 mM Cr6+ treatment. Data are means ± SE of three independent
626
repeats
616 617 618 619 620
respective controls.
Control: pretreated with H2O, no stressed. Cr6+: pretreated with H2O, stressed with 10 mM Cr6+. H2S: pretreated with 50 μM H2S, no stressed. H2S+Cr6+: pretreated with 50 μM H2S, stressed with 10 mM Cr6+. HA+Cr6+: pretreated with 1 mM HA, stressed with 10 mM Cr6+.
Data are means ± SE of three independent repeats. Bars with different letters are significantly different (P < 0.05).
21
Page 21 of 34
Fig. 5 Effects of 20 mM Ca2+ on the foxtail millet.
628
(A) Phenotype of leaves. (B) The width of leaves in natural state. (C) & (D) H2O2
629
accumulation.
630
10-day-old seedlings were pretreated with 20 mM Ca2+ or 5 mM EGTA for 12 h, and
631
then exposed to 10 mM Cr6+ stress for 24 h, with respective controls.
632
Control: pretreated with H2O, no stressed. Cr6+: pretreated with H2O, stressed with 10
633
mM Cr6+. Ca2+: pretreated with 20 mM Ca2+, no stressed. Ca2++Cr6+: pretreated with
634
20 mM Ca2+, stressed with 10 mM Cr6+. EGTA+Cr6+: pretreated with 5 mM EGTA,
635
stressed with 10 mM Cr6+.
636
Data are means ± SE of three independent repeats. Bars with different letters are
637
significantly different (P < 0.05).
638
Fig. 6 The connection of H2S and Ca2+ in Cr6+ stressed foxtail millet.
639
(A) The effects of 20 mM Ca2+ or 5 mM EGTA on the H2S production rate in foxtail
640
millet stressed with 10 mM Cr6+ for 24 h.
641
(B) The influences of 50 μM H2S (NaHS as exogenous H2S donor) or 1 mM HA on
642
the expressions of Ca2+ sensors-related genes CaM, CBL and CDPK in foxtail millet
643
stressed with 10 mM Cr6+ for 12 h.
644
(C) The influences of 50 μM H2S (NaHS as exogenous H2S donor) or 1 mM HA on
645
the [Ca2+]cyt in stressed foxtail millet observed under fluorescence microscopy at 488
646
nm (400×). 10-day-old seedlings with various pretreatments were exposed to 10 mM
647
Cr6+ for 24 h.
648
654
Ac ce p
655
with different letters are significantly different (P < 0.05).
656
Fig. 8 Influences of H2S and Ca2+ on the content of MDA in foxtail millet with or
657
without Cr6+ stress.
658
10-day-old seedlings with different pretreatments (50 μM H2S or 20 mM Ca2+ or
659
1mM HA or 5 mM EGTA or some combined pretreatments) for 12 h, were then
660
treated with (black columns) or without (white columns) 10 mM Cr6+ for 24 h. Data
649 650 651 652 653
te
d
M
an
us
cr
ip t
627
Data are means ± SE of three independent repeats. Bars with different letters are significantly different (P < 0.05).
Fig. 7 Effects of H2S and Ca2+ on the expressions of PCS1 and MT3 in foxtail millet. 10-day-old seedlings with different pretreatments (50 μM H2S or 20 mM Ca2+ or 1 mM HA or 5 mM EGTA or some combined pretreatments) for 12 h were then treated with (black columns) or without (white columns) 10 mM Cr6+ for 12 h. Data are means ± SE of three independent repeats, error bars indicate error standard and bars
22
Page 22 of 34
are means ± SE of three repeats, error bars indicate error standard and bars with
662
different letters are significantly different (P < 0.05).
663
Fig. 9 Effects of H2S and Ca2+ on the antioxidant system in foxtail millet.
664
(A) Effects of 50 μM H2S (NaHS as exogenous H2S donor) and 1 mM HA on the
665
activities of SOD and POD. (B) Effects of 20 mM Ca2+ and 5mM EGTA on the
666
activities of SOD and POD. (C) & (D) Effects of H2S and Ca2+ on the expressions of
667
GSH1 and GR.
668
10-day-old seedlings with different pretreatments (50 μM H2S or 20 mM Ca2+ or
669
1mM HA or 5 mM EGTA or some combined pretreatments) for 12 h, were then
670
treated with or without 10 mM Cr6+ for 12 h to detect the genes expressions, and for
671
24 h to detect the activities of SOD and POD.
672
Data are means ± SE of three independent repeats, error bars indicate error standard
673
and bars with different letters are significantly different (P < 0.05).
674
Fig. 10 Effects of H2S and Ca2+ on the contents of GSH and AsA as well as the ratio
675
of GSH/GSSG and AsA/DHA in foxtail millet.
676
(A) Effects of 50 μM H2S (NaHS as exogenous H2S donor) and 20 mM Ca2+ on the
677
content of GSH.
678
(B) Effects of 50 μM H2S (NaHS as exogenous H2S donor) and 20 mM Ca2+ on the
679
content of AsA.
680
(C) Effects of 50 μM H2S (NaHS as exogenous H2S donor) and 20 mM Ca2+ on the
681
ratio of GSH/GSSG.
682
(D) Effects of 50 μM H2S (NaHS as exogenous H2S donor) and 20 mM Ca2+ on the
688
Ac ce p
te
d
M
an
us
cr
ip t
661
689
Fig. 11 The summary of the crosstalk between H2S and Ca2+ signaling in foxtail millet
690
responding to Cr6+ stress. Arrows indicate enhanced expressions and hyphens indicate
691
suppressed expression.
683 684 685 686 687
ratio of AsA/DHA.
10-day-old seedlings with different pretreatments (50 μM H2S or 20 mM Ca2+ or 1mM HA or 5 mM EGTA or some combined pretreatments) for 12 h, were then treated with or without 10 mM Cr6+ for 24 h to detect the contents of GSH and AsA as well as their oxidized forms GSSG and AsA contents then calculate the GSH/GSSG and AsA/DHA ratios.
23
Page 23 of 34
Ac ce p
te
d
M
an
us
cr
ip t
Figure.1
Page 24 of 34
Ac
ce
pt
ed
M
an
us
cr
i
Figure.2
Page 25 of 34
Ac ce p
te
d
M
an
us
cr
ip t
Figure.3
Page 26 of 34
Ac ce p
te
d
M
an
us
cr
ip t
Figure.4
Page 27 of 34
Ac ce p
te
d
M
an
us
cr
ip t
Figure.5
Page 28 of 34
Ac
ce
pt
ed
M
an
us
cr
i
Figure.6
Page 29 of 34
Ac
ce
pt
ed
M
an
us
cr
i
Figure.7
Page 30 of 34
Ac
ce
pt
ed
M
an
us
cr
i
Figure.8
Page 31 of 34
Ac ce p
te
d
M
an
us
cr
ip t
Figure.9
Page 32 of 34
Ac
ce
pt
ed
M
an
us
cr
i
Figure.10
Page 33 of 34
Ac
ce
pt
ed
M
an
us
cr
i
Figure.11
Page 34 of 34