Molecular Plant Advance Access published November 27, 2013
3
Nitric oxide is required for homeostasis of oxygen and reactive oxygen species in barley roots under aerobic conditions
4
Kapuganti J. Gupta1, Kim H. Hebelstrup2, Nicholas J. Kruger1, R. George Ratcliffe*1
1 2
5 6
1
7
OX1 3RB, UK
8
2
9
4200 Slagelse, Denmark
Department of Plant Sciences, University of Oxford, South Parks Road, Oxford,
Downloaded from http://mplant.oxfordjournals.org/ at Ondokuz Mayis University on May 2, 2014
Department of Molecular Biology and Genetics, Aarhus University, Forsøgsvej 1,
10 11 12 13
*To whom correspondence should be addressed:
14
Email: [email protected]
15
Tel +44 (0)1865 275000
16
Fax +44 (0)1865 275074
17 18
Running title: NO and oxygen homeostasis in aerobic barley roots
19 20
Article type: Research letter to the editor
21
Number of words: 1542
22
Number of references: 12
23
1
24
Dear Editor,
25
Oxygen, the terminal electron acceptor for mitochondrial electron transport, is vital
27
for plants because of its role in the production of ATP by oxidative phosphorylation.
28
While photosynthetic oxygen production contributes to the oxygen supply in leaves,
29
reducing the risk of oxygen limitation of mitochondrial metabolism under most
30
conditions, root tissues often suffer oxygen deprivation during normal development
31
due to the lack of an endogenous supply and isolation from atmospheric oxygen.
32
Since changes in oxygen concentration have multiple effects on metabolism and
33
energy production (Geigenberger, 2003), tight control of oxygen consumption and
34
homeostasis is likely to be particularly important in underground tissues such as
35
roots.
36 37
Nitric oxide (NO) is involved in many plant processes (Mur et al., 2013) and under
38
hypoxia there is good evidence that nitric oxide (NO) contributes to the recycling of
39
NADH (Stoimenova et al., 2007), the synthesis of ATP (Stoimenova et al., 2007) and
40
the regulation of oxygen consumption (Borisjuk et al., 2007). The involvement of NO
41
in the metabolic response to low oxygen is consistent with increased NO production
42
during oxygen deprivation (Borisjuk et al., 2007), but the extent to which NO might
43
also play a role in the energy metabolism of roots under normal aerobic conditions is
44
unknown. Mitochondria, whose functions are central to aerobic metabolism, are the
45
major source of NO in plants, and potential targets for NO include cytochrome c
46
oxidase in the mitochondrial electron transport chain (Gupta et al., 2011). Thus NO
47
could influence oxygen consumption under normal aerobic conditions in roots, and it
48
is this specific function that is assessed here.
49 50
The role of NO in oxygen homeostasis in normoxic roots was investigated using
51
barley plants over-expressing non-symbiotic haemoglobin 1 (Hb+) under the control
52
of the ubiquitin-2 promoter from Zea mays. The plants were engineered by
53
Agrobacterium-mediated transformation, after cloning the cDNA of the barley class 1
54
non-symbiotic haemoglobin 1 (accession number: U94968) into the vector pUCE-
55
UBI-USER-NOS, and over-expression of non-symbiotic haemoglobin in the
56
transgenic lines was confirmed by qRT-PCR and western blotting (Hebelstrup et al.,
57
2013). Non-symbiotic class 1 haemoglobins have NO dioxygenase activity and so 2
Downloaded from http://mplant.oxfordjournals.org/ at Ondokuz Mayis University on May 2, 2014
26
provide a scavenging mechanism for NO in conjunction with methaemoglobin
59
reductase (Igamberdiev et al., 2006). In order to confirm that over-expression of non-
60
symbiotic haemoglobin affected NO levels, NO was first measured using
61
diaminofluorescein diacetate (DAF-2DA). This cell-permeant dye is cleaved
62
intracellularly to diaminofluorescein which can then diffuse to the site of NO
63
production, where it reacts with NO in the presence of oxygen to yield highly
64
fluorescent triazolofluorescein. Root slices were incubated with DAF-2DA and the
65
measured fluorescence showed that NO levels were higher in wild type (WT) roots
66
than in Hb+ roots (Fig 1A), suggesting that over-expression of non-symbiotic
67
haemogobin leads to scavenging of NO. In order to confirm that the observed
68
fluorescence was related to NO, root slices were incubated with 200 µM of the
69
known NO scavenger cPTIO 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-
70
oxyl-3-oxide), which when used in low concentrations, oxidizes NO to nitrite.
71
Reduced fluorescence in the presence of cPTIO (Fig 1A) showed that the observed
72
DAF fluorescence was associated with NO.
73 74
Given the potential unreliability of NO measurements (Gupta and Igamberdiev,
75
2013), further support for the difference between the WT and Hb+ roots was
76
obtained using two other methods. First, NO production was assayed by gas phase
77
chemiluminescence, yielding initial rates of 2.28 ± 0.129 nmol g FW -1 h-1 for WT
78
roots and 1.11 ± 0.097 nmol g FW -1 h-1 for Hb+ roots (Fig. 1B). Secondly, a gas
79
phase Griess reagent assay also showed that the NO level was higher in the WT
80
roots than the Hb+ roots (Supplementary Figure 1). Thus all three NO assays
81
showed that over-expression of non-symbiotic haemoglobin reduced the NO level in
82
normoxic roots.
83 84
Since NO is a potent inhibitor of cytochrome c oxidase (complex IV, COX) in the
85
mitochondrial electron transport chain, increased levels would be expected to
86
decrease the respiration rate. The respiratory rates measured using a Clark type
87
oxygen electrode were 13 ± 1.28 nmol-1 mg FW -1 min-1 for Hb+ roots and 9.3 ± 1.2
88
nmol-1 mg FW -1 min-1 in WT roots, indicating that the scavenging of NO in the Hb+
89
roots led to an increase in respiration (Fig. 1C). The effect of the observed increase
90
in respiration at low levels of NO on the internal oxygen concentration was
91
investigated non-invasively using an oxygen-sensitive fluorescent foil. Roots excised 3
Downloaded from http://mplant.oxfordjournals.org/ at Ondokuz Mayis University on May 2, 2014
58
from 3-week-old plants were transferred to hydroponic medium, and then 1-2 cm root
93
slices were cut and placed on a slide containing 500 µl of nutrient solution. A 1-2 cm
94
segment of oxygen sensor foil was placed on the root and used to monitor the
95
internal oxygen distribution using a ViSisens microscope (PreSens, Regensburg,
96
Germany). The sensor foil was calibrated by placing a drop of sodium dithionite (100
97
mg ml-1) in the middle of the sensor. After scanning, oxygen concentrations were
98
derived from a calibration function based on exposure to known oxygen
99
concentrations (i.e. 0% and 100%). The Stern–Vollmer plot (incorporated into the
100
software) leads to a linear relation which was used for calculating the oxygen
101
concentration in the tissue (Tschiersch et al., 2012). The results showed that the
102
internal oxygen concentration in WT root segments (216 ± 13 µM) was significantly
103
greater than in Hb+ root segments (155 ± 10 µM) (Fig 1D). Thus the lower NO level
104
in plants over-expressing non-symbiotic haemoglobin 1 led to a drop in the local
105
internal oxygen concentration as a result of the increase in respiration.
106 107
Maintaining an appropriate rate of carbohydrate oxidation is also important for the
108
plant and this can be achieved by control of respiration. In order to determine
109
whether NO plays a role in the regulation of carbohydrate oxidation via its effect on
110
respiration, roots were supplied with positionally labelled [14C]glucose (at C1, C2,
111
C3,4 and C6) and the release of
112
capture respired CO2. Significantly, Hb+ roots released almost twice as much
113
as those of WT plants (Fig 1E), suggesting that control of respiration is relaxed in the
114
presence of lower concentrations of NO, leading to increased consumption of
115
storage carbohydrates.
14
CO2 was monitored for 6 h using alkaline traps to 14
CO2
116 117
Hypoxia is associated with increased production of reactive oxygen species (ROS)
118
(Vergara et al. 2012) raising the possibility that the lower internal oxygen level in the
119
Hb+ roots could influence the production of ROS. Accordingly, ROS production was
120
assessed by monitoring the intracellular cleavage of 2',7'-dichlorodihydrofluorescin
121
diacetate (H2DCFDA) to dichlorofluorescin (H2DCF) and its subsequent oxidation to
122
2',7'-dichlorofluorescein
123
measurements showed that the ROS levels were indeed higher in the Hb+ roots (Fig
124
1F).
(DCF)
which
was
quantified
fluorimetrically.
The
125 4
Downloaded from http://mplant.oxfordjournals.org/ at Ondokuz Mayis University on May 2, 2014
92
Under aerobic conditions, over-expression of non-symbiotic haemoglobin 1 in barley
127
roots decreased the NO level, increased respiration and carbohydrate oxidation, and
128
reduced the internal oxygen level. These observations reveal an intricate interplay
129
between NO and oxygen metabolism, and they suggest that NO has a significant
130
role in the reciprocal regulation of respiration and internal oxygen availability. It was
131
already known that NO is important in regulating hypoxic metabolism, and the
132
observations reported here extend this role to the regulation of oxygen consumption
133
under normoxia. Moreover the decrease in oxygen level in Hb+ roots was associated
134
with an increase in ROS, suggesting that NO could also be indirectly important in
135
determining the balance between ROS production and scavenging. Regulation of
136
oxygen concentration by NO could act as a first line of defence against ROS
137
production, running in parallel with the effect of NO on the induction of alternative
138
oxidase activity (Huang et al., 2002) and the direct scavenging of ROS by NO
139
(Beligni and Lamattina 1999). Since over-expression of haemoglobin results in
140
increased ROS production, delay in germination and reduced growth rate and yield
141
under normoxia (Hebelstrup et al., 2013; Supplementary Figures 2, 3), it seems likely
142
that plants only induce haemoglobin production under specific conditions, such as
143
hypoxia, where NO scavenging via the NO dioxygenase activity of the non-symbiotic
144
haemoglobin would have a beneficial effect on the energy state of the tissue.
145 146
In conclusion, the present study provides strong evidence that NO modulates
147
respiration, internal oxygen, carbohydrate consumption and ROS levels in roots
148
under normoxia. A decrease in NO, caused by over-expression of non-symbiotic
149
haemoglobin 1, was accompanied by a drop in internal oxygen, an increase in
150
glucose consumption and elevation of ROS. Thus NO is important for maintaining
151
steady state oxygen concentrations and for keeping ROS at low levels in barley roots
152
under normal aerobic conditions. It remains to be seen whether over-expression of
153
non-symbiotic haemoglobin 1, and the resulting decrease in NO, has other
154
consequences for the response of roots to abiotic stress under aerobic conditions.
155 156
Funding
157
5
Downloaded from http://mplant.oxfordjournals.org/ at Ondokuz Mayis University on May 2, 2014
126
158
This work was supported by a Marie Curie Intra-European Fellowship for Career
159
Development within the 7th European Community Framework Programme (K.J.G &
160
R.G.R).
161 162
Acknowledgments
163 164
K.J.G. thanks PreSens, Gmbh (Regensberg, Germany) for providing access to a
165
VisiSens device as a part of the VisiSens Competition. We thank Abir Igamberdiev
166
(Memorial University of Newfoundland) for comments on the manuscript.
168
Supplementary data
169 170
Supplementary Figure 1. Gas phase Griess reagent assay for NO.
171 172
Supplementary Figure 2. Comparison of the growth phenotype of WT and Hb+
173
barley plants.
174 175
Supplementary Figure 3. Comparison of the root growth phenotype of 16-day-old
176
wild type (WT) and haemoglobin over-expressing (Hb+) barley plants grown in
177
hydroponic culture.
178 179
References
180 181
Beligni, M.V., and Lamattina, L. (1999). Nitric oxide counteracts cytotoxic
182
processes mediated by reactive oxygen species in plant tissues. Planta. 208, 337-
183
344.
184 185
Borisjuk, L., Macherel, D., Benamar, A., Wobus, U., and Rolletschek, H. (2007).
186
Low oxygen sensing and balancing in plant seeds: a role for nitric oxide. New Phytol.
187
176, 813–823.
188 189
Geigenberger, P. (2003). Response of plant metabolism to too little oxygen. Curr.
190
Opin. Plant Biol. 6, 247–256.
191 6
Downloaded from http://mplant.oxfordjournals.org/ at Ondokuz Mayis University on May 2, 2014
167
192
Gupta, K.J., and Igamberdiev, A.U., (2013). Recommendations of using at least
193
two different methods for measuring NO. Front. Plant Sci. 4:58; doi:
194
10.3389/fpls.2013.00058
195 196
Gupta, K.J., Igamberdiev, A.U., Manjunatha, G., Segu, S., Moran, J.F.,
197
Neelawarne, B., Bauwe, H., and Kaiser, W.M. (2011). The emerging roles of nitric
198
oxide (NO) in plant mitochondria. Plant Science. 181, 520-526.
199
Hebelstrup, K. H., Shah, J.K., Simpson, C., Schjoerring, J.K., Mandon, J.,
201
Cristescu, S.M., Harren, F.J.M., Christiansen, M.W., Mur, L.A.J., and
202
Igamberdiev, A.U. (2013). An assessment of the biotechnological use of
203
hemoglobin modulation in cereals. Physiol. Plant. doi: 10.1111/ppl.12115
Downloaded from http://mplant.oxfordjournals.org/ at Ondokuz Mayis University on May 2, 2014
200
204 205
Huang, X., von Rad, U., and Durner, J. (2002). Nitric oxide induces transcriptional
206
activation of the nitric oxide-tolerant alternative oxidase in Arabidopsis suspension
207
cells. Planta. 215, 914–923.
208 209
Igamberdiev, A.U., Bykova, N.V., and Hill, R.D. (2006). Scavenging of nitric oxide
210
by barley hemoglobin is facilitated by a monodehydroascorbate reductase-mediated
211
ascorbate reduction of methemoglobin. Planta. 223, 1033-1040.
212 213
Mur, L.A.J., Mandon, J., Persijn, S., Cristescu, S.M., Moshkov, I.E., Novikova,
214
G.V., Hall, M.A., Harren, F.J.M., Hebelstrup, K.H., and Gupta, K.J. (2013). Nitric
215
oxide in plants: an assessment of the current state of knowledge. AoB Plants. 5,
216
pls052; doi: 10.1093/aobpla/pls052
217 218
Stoimenova, M., Igamberdiev, A.U., Gupta, K.J., and Hill, R.D. (2007). Nitrite-
219
driven anaerobic ATP synthesis in barley and rice root mitochondria. Planta. 226,
220
465–474.
221 222
Tschiersch, H., Liebsch, G., Borisjuk, L., Stangelmayer, A., and Rolletschek, H.
223
(2012). An imaging method for oxygen distribution, respiration and photosynthesis at
224
a microscopic level of resolution. New Phytol. 196, 926-936.
225 7
226
Vergara, R., Parada, F., Rubio, S., and Pérez, F.J. (2012). Hypoxia induces H2O2
227
production and activates antioxidant defence system in grapevine buds through
228
mediation of H2O2 and ethylene. J. Exp. Bot. 63, 4123-4131.
229 230
Figure Legend
231
Figure 1. Scavenging of NO by non-symbiotic haemoglobin 1 leads to increased
233
respiration, decreased internal oxygen, increased ROS production and increased
234
glucose consumption in barley roots. Data are presented as the mean ± SD of the
235
specified number of measurements (n). WT and Hb+ plants were compared using
236
Student’s T-test assuming unequal variance; * indicates a comparison for which P
3
Nitric oxide is required for homeostasis of oxygen and reactive oxygen species in barley roots under aerobic conditions
4
Kapuganti J. Gupta1, Kim H. Hebelstrup2, Nicholas J. Kruger1, R. George Ratcliffe*1
1 2
5 6
1
7
OX1 3RB, UK
8
2
9
4200 Slagelse, Denmark
Department of Plant Sciences, University of Oxford, South Parks Road, Oxford,
Downloaded from http://mplant.oxfordjournals.org/ at Ondokuz Mayis University on May 2, 2014
Department of Molecular Biology and Genetics, Aarhus University, Forsøgsvej 1,
10 11 12 13
*To whom correspondence should be addressed:
14
Email: [email protected]
15
Tel +44 (0)1865 275000
16
Fax +44 (0)1865 275074
17 18
Running title: NO and oxygen homeostasis in aerobic barley roots
19 20
Article type: Research letter to the editor
21
Number of words: 1542
22
Number of references: 12
23
1
24
Dear Editor,
25
Oxygen, the terminal electron acceptor for mitochondrial electron transport, is vital
27
for plants because of its role in the production of ATP by oxidative phosphorylation.
28
While photosynthetic oxygen production contributes to the oxygen supply in leaves,
29
reducing the risk of oxygen limitation of mitochondrial metabolism under most
30
conditions, root tissues often suffer oxygen deprivation during normal development
31
due to the lack of an endogenous supply and isolation from atmospheric oxygen.
32
Since changes in oxygen concentration have multiple effects on metabolism and
33
energy production (Geigenberger, 2003), tight control of oxygen consumption and
34
homeostasis is likely to be particularly important in underground tissues such as
35
roots.
36 37
Nitric oxide (NO) is involved in many plant processes (Mur et al., 2013) and under
38
hypoxia there is good evidence that nitric oxide (NO) contributes to the recycling of
39
NADH (Stoimenova et al., 2007), the synthesis of ATP (Stoimenova et al., 2007) and
40
the regulation of oxygen consumption (Borisjuk et al., 2007). The involvement of NO
41
in the metabolic response to low oxygen is consistent with increased NO production
42
during oxygen deprivation (Borisjuk et al., 2007), but the extent to which NO might
43
also play a role in the energy metabolism of roots under normal aerobic conditions is
44
unknown. Mitochondria, whose functions are central to aerobic metabolism, are the
45
major source of NO in plants, and potential targets for NO include cytochrome c
46
oxidase in the mitochondrial electron transport chain (Gupta et al., 2011). Thus NO
47
could influence oxygen consumption under normal aerobic conditions in roots, and it
48
is this specific function that is assessed here.
49 50
The role of NO in oxygen homeostasis in normoxic roots was investigated using
51
barley plants over-expressing non-symbiotic haemoglobin 1 (Hb+) under the control
52
of the ubiquitin-2 promoter from Zea mays. The plants were engineered by
53
Agrobacterium-mediated transformation, after cloning the cDNA of the barley class 1
54
non-symbiotic haemoglobin 1 (accession number: U94968) into the vector pUCE-
55
UBI-USER-NOS, and over-expression of non-symbiotic haemoglobin in the
56
transgenic lines was confirmed by qRT-PCR and western blotting (Hebelstrup et al.,
57
2013). Non-symbiotic class 1 haemoglobins have NO dioxygenase activity and so 2
Downloaded from http://mplant.oxfordjournals.org/ at Ondokuz Mayis University on May 2, 2014
26
provide a scavenging mechanism for NO in conjunction with methaemoglobin
59
reductase (Igamberdiev et al., 2006). In order to confirm that over-expression of non-
60
symbiotic haemoglobin affected NO levels, NO was first measured using
61
diaminofluorescein diacetate (DAF-2DA). This cell-permeant dye is cleaved
62
intracellularly to diaminofluorescein which can then diffuse to the site of NO
63
production, where it reacts with NO in the presence of oxygen to yield highly
64
fluorescent triazolofluorescein. Root slices were incubated with DAF-2DA and the
65
measured fluorescence showed that NO levels were higher in wild type (WT) roots
66
than in Hb+ roots (Fig 1A), suggesting that over-expression of non-symbiotic
67
haemogobin leads to scavenging of NO. In order to confirm that the observed
68
fluorescence was related to NO, root slices were incubated with 200 µM of the
69
known NO scavenger cPTIO 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-
70
oxyl-3-oxide), which when used in low concentrations, oxidizes NO to nitrite.
71
Reduced fluorescence in the presence of cPTIO (Fig 1A) showed that the observed
72
DAF fluorescence was associated with NO.
73 74
Given the potential unreliability of NO measurements (Gupta and Igamberdiev,
75
2013), further support for the difference between the WT and Hb+ roots was
76
obtained using two other methods. First, NO production was assayed by gas phase
77
chemiluminescence, yielding initial rates of 2.28 ± 0.129 nmol g FW -1 h-1 for WT
78
roots and 1.11 ± 0.097 nmol g FW -1 h-1 for Hb+ roots (Fig. 1B). Secondly, a gas
79
phase Griess reagent assay also showed that the NO level was higher in the WT
80
roots than the Hb+ roots (Supplementary Figure 1). Thus all three NO assays
81
showed that over-expression of non-symbiotic haemoglobin reduced the NO level in
82
normoxic roots.
83 84
Since NO is a potent inhibitor of cytochrome c oxidase (complex IV, COX) in the
85
mitochondrial electron transport chain, increased levels would be expected to
86
decrease the respiration rate. The respiratory rates measured using a Clark type
87
oxygen electrode were 13 ± 1.28 nmol-1 mg FW -1 min-1 for Hb+ roots and 9.3 ± 1.2
88
nmol-1 mg FW -1 min-1 in WT roots, indicating that the scavenging of NO in the Hb+
89
roots led to an increase in respiration (Fig. 1C). The effect of the observed increase
90
in respiration at low levels of NO on the internal oxygen concentration was
91
investigated non-invasively using an oxygen-sensitive fluorescent foil. Roots excised 3
Downloaded from http://mplant.oxfordjournals.org/ at Ondokuz Mayis University on May 2, 2014
58
from 3-week-old plants were transferred to hydroponic medium, and then 1-2 cm root
93
slices were cut and placed on a slide containing 500 µl of nutrient solution. A 1-2 cm
94
segment of oxygen sensor foil was placed on the root and used to monitor the
95
internal oxygen distribution using a ViSisens microscope (PreSens, Regensburg,
96
Germany). The sensor foil was calibrated by placing a drop of sodium dithionite (100
97
mg ml-1) in the middle of the sensor. After scanning, oxygen concentrations were
98
derived from a calibration function based on exposure to known oxygen
99
concentrations (i.e. 0% and 100%). The Stern–Vollmer plot (incorporated into the
100
software) leads to a linear relation which was used for calculating the oxygen
101
concentration in the tissue (Tschiersch et al., 2012). The results showed that the
102
internal oxygen concentration in WT root segments (216 ± 13 µM) was significantly
103
greater than in Hb+ root segments (155 ± 10 µM) (Fig 1D). Thus the lower NO level
104
in plants over-expressing non-symbiotic haemoglobin 1 led to a drop in the local
105
internal oxygen concentration as a result of the increase in respiration.
106 107
Maintaining an appropriate rate of carbohydrate oxidation is also important for the
108
plant and this can be achieved by control of respiration. In order to determine
109
whether NO plays a role in the regulation of carbohydrate oxidation via its effect on
110
respiration, roots were supplied with positionally labelled [14C]glucose (at C1, C2,
111
C3,4 and C6) and the release of
112
capture respired CO2. Significantly, Hb+ roots released almost twice as much
113
as those of WT plants (Fig 1E), suggesting that control of respiration is relaxed in the
114
presence of lower concentrations of NO, leading to increased consumption of
115
storage carbohydrates.
14
CO2 was monitored for 6 h using alkaline traps to 14
CO2
116 117
Hypoxia is associated with increased production of reactive oxygen species (ROS)
118
(Vergara et al. 2012) raising the possibility that the lower internal oxygen level in the
119
Hb+ roots could influence the production of ROS. Accordingly, ROS production was
120
assessed by monitoring the intracellular cleavage of 2',7'-dichlorodihydrofluorescin
121
diacetate (H2DCFDA) to dichlorofluorescin (H2DCF) and its subsequent oxidation to
122
2',7'-dichlorofluorescein
123
measurements showed that the ROS levels were indeed higher in the Hb+ roots (Fig
124
1F).
(DCF)
which
was
quantified
fluorimetrically.
The
125 4
Downloaded from http://mplant.oxfordjournals.org/ at Ondokuz Mayis University on May 2, 2014
92
Under aerobic conditions, over-expression of non-symbiotic haemoglobin 1 in barley
127
roots decreased the NO level, increased respiration and carbohydrate oxidation, and
128
reduced the internal oxygen level. These observations reveal an intricate interplay
129
between NO and oxygen metabolism, and they suggest that NO has a significant
130
role in the reciprocal regulation of respiration and internal oxygen availability. It was
131
already known that NO is important in regulating hypoxic metabolism, and the
132
observations reported here extend this role to the regulation of oxygen consumption
133
under normoxia. Moreover the decrease in oxygen level in Hb+ roots was associated
134
with an increase in ROS, suggesting that NO could also be indirectly important in
135
determining the balance between ROS production and scavenging. Regulation of
136
oxygen concentration by NO could act as a first line of defence against ROS
137
production, running in parallel with the effect of NO on the induction of alternative
138
oxidase activity (Huang et al., 2002) and the direct scavenging of ROS by NO
139
(Beligni and Lamattina 1999). Since over-expression of haemoglobin results in
140
increased ROS production, delay in germination and reduced growth rate and yield
141
under normoxia (Hebelstrup et al., 2013; Supplementary Figures 2, 3), it seems likely
142
that plants only induce haemoglobin production under specific conditions, such as
143
hypoxia, where NO scavenging via the NO dioxygenase activity of the non-symbiotic
144
haemoglobin would have a beneficial effect on the energy state of the tissue.
145 146
In conclusion, the present study provides strong evidence that NO modulates
147
respiration, internal oxygen, carbohydrate consumption and ROS levels in roots
148
under normoxia. A decrease in NO, caused by over-expression of non-symbiotic
149
haemoglobin 1, was accompanied by a drop in internal oxygen, an increase in
150
glucose consumption and elevation of ROS. Thus NO is important for maintaining
151
steady state oxygen concentrations and for keeping ROS at low levels in barley roots
152
under normal aerobic conditions. It remains to be seen whether over-expression of
153
non-symbiotic haemoglobin 1, and the resulting decrease in NO, has other
154
consequences for the response of roots to abiotic stress under aerobic conditions.
155 156
Funding
157
5
Downloaded from http://mplant.oxfordjournals.org/ at Ondokuz Mayis University on May 2, 2014
126
158
This work was supported by a Marie Curie Intra-European Fellowship for Career
159
Development within the 7th European Community Framework Programme (K.J.G &
160
R.G.R).
161 162
Acknowledgments
163 164
K.J.G. thanks PreSens, Gmbh (Regensberg, Germany) for providing access to a
165
VisiSens device as a part of the VisiSens Competition. We thank Abir Igamberdiev
166
(Memorial University of Newfoundland) for comments on the manuscript.
168
Supplementary data
169 170
Supplementary Figure 1. Gas phase Griess reagent assay for NO.
171 172
Supplementary Figure 2. Comparison of the growth phenotype of WT and Hb+
173
barley plants.
174 175
Supplementary Figure 3. Comparison of the root growth phenotype of 16-day-old
176
wild type (WT) and haemoglobin over-expressing (Hb+) barley plants grown in
177
hydroponic culture.
178 179
References
180 181
Beligni, M.V., and Lamattina, L. (1999). Nitric oxide counteracts cytotoxic
182
processes mediated by reactive oxygen species in plant tissues. Planta. 208, 337-
183
344.
184 185
Borisjuk, L., Macherel, D., Benamar, A., Wobus, U., and Rolletschek, H. (2007).
186
Low oxygen sensing and balancing in plant seeds: a role for nitric oxide. New Phytol.
187
176, 813–823.
188 189
Geigenberger, P. (2003). Response of plant metabolism to too little oxygen. Curr.
190
Opin. Plant Biol. 6, 247–256.
191 6
Downloaded from http://mplant.oxfordjournals.org/ at Ondokuz Mayis University on May 2, 2014
167
192
Gupta, K.J., and Igamberdiev, A.U., (2013). Recommendations of using at least
193
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Figure Legend
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Figure 1. Scavenging of NO by non-symbiotic haemoglobin 1 leads to increased
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respiration, decreased internal oxygen, increased ROS production and increased
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glucose consumption in barley roots. Data are presented as the mean ± SD of the
235
specified number of measurements (n). WT and Hb+ plants were compared using
236
Student’s T-test assuming unequal variance; * indicates a comparison for which P
