1

Chloroplast-generated ROS dominates NaCl-induced K+ efflux in wheat leaf mesophyll 1

2

Honghong Wu, Lana Shabala, Meixue Zhou, Sergey Shabala* 3

4

School of Land and Food, University of Tasmania, Private Bag 54, Hobart, Tas 7001, 5

Australia 6

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*Corresponding author 8

Phone +613 62267539; Fax + 613 62262642; E-mail: Sergey.Shabala@utas.edu.au 9

10

The present paper is an addendum to the paper: Wu H, Shabala L, Zhou M, Shabala S. 11

Durum and Bread wheat differ in their ability to retain potassium in leaf mesophyll: 12

implications for salinity stress tolerance. Plant Cell Physiol 2014; 55 (10): 1749–1762. 13

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2

Abstract 15

Mesophyll K+ retention ability has been recently reported as an important component of 16

salinity stress tolerance in wheat. In order to investigate the role of ROS in regulating NaCl-17

induced K+ efflux in wheat leaf mesophyll, a series of pharmacological experiments was 18

conducted using MV (methyl viologen, superoxide radical inducer), DPI (an inhibitor of 19

NADPH oxidase), H2O2 (to mimic apoplastic ROS), and EGCG ((−)-Epigallocatechin 20

gallate, ROS scavenger). Mesophyll pre-treatment with 10 μM MV resulted in a significantly 21

higher NaCl-induced K+ efflux in leaf mesophyll, while 50 μM EGCG pre-treatment 22

alleviated K+ leakage under salt stress. No significant change in NaCl-induced K

+ efflux in 23

leaf mesophyll was found in specimens pre-treated by H2O2 and DPI, compared with the 24

control. The highest NaCl-induced H+ efflux in leaf mesophyll was also found in samples 25

pre-treated with MV, suggesting a futile cycle between increased H+-ATPase activity 26

required and ROS-induced K+ leak. Overall, it is suggested that, under saline stress, K

+ efflux 27

from wheat mesophyll is mediated predominantly by non-selective cation channels (NSCC) 28

regulated by ROS produced in chloroplasts, at least in bread wheat. 29

30

Keywords 31

Chloroplast, K+ channels, K

+ retention, leaf mesophyll, ROS 32

33

3

Potassium is a critical nutrient in plant’s life. It has a central role in the maintenance of key 34

metabolic processes, and its deficiency in plants can result in significant suppression of their 35

photosynthetic ability.1-3

To cope with low availability of K+ in soil (0.1-1mM)

4, a 36

sophisticated K+ uptake system was evolved in plants to reach a high concentration of 37

cytosolic K+ (100 mM)

4,5 in plant cells. Besides root K

+ uptake, efficient K

+ retention was 38

also believed to be involved in this process, since up to 80% K+ in shoot may be recirculated 39

back to the root.6 Under stress conditions such as salinity, K

+ efflux is thermodynamically 40

favoured.7 Over the last decade, the importance of K

+ retention in the cytosol has emerged as 41

an additional component in plant salinity tolerance mechanisms, both in root8-12

and leaf13-15

42

tissues in various species. 43

Earlier experiments have suggested that NaCl-induced K+ efflux in leaf mesophyll is 44

mediated mainly by two types of plasma membrane channels: KOR channels (K+ outward 45

rectifying channel) and NSCC (non-selective cation) channels.16

KOR channels are always 46

gated by membrane depolarization4, 17

, and their activity is increased in the presence of ROS 47

(i.e. OH•)

18. In their turn, some of NSCC can also be activated by various ROS species such 48

as H2O2 or OH•, as revealed in direct patch -clamp experiments.

19-21 Given the prominent role 49

ROS production plays in salt-stress responses22, 23

, it is therefore important to understand 50

which of the channels plays a major role in cytosolic K+ retention under stress conditions. In 51

our seminal paper14

, we showed that NSCC but not KOR channels dominated NaCl-induced 52

K+ efflux from wheat leaf mesophyll. We also found no significant effects of DPI (an 53

inhibitor of NADPH oxidase) pre-treatment on NaCl-induced K+ efflux from leaf mesophyll, 54

suggesting that ROS produced by NADPH oxidase was not the main source in regulation of 55

the NaCl-induced K+ efflux in wheat leaf mesophyll. The aim of current work was to reveal 56

the identity and source of ROS signals mediating K+ efflux from leaf mesophyll under saline 57

conditions. 58

4

Many sources of intracellular ROS production were characterized in plants. This includes 59

ROS production by the plasma membrane located NADPH oxidase system, peroxisomes, 60

chloroplast, mitochondria, vacuole, and apoplast.24, 25

While in animal systems mitochondria 61

are the main sources of ROS production, chloroplasts and peroxisomes are the largest ROS 62

producers in green plant tissues, producing 20-fold more ROS than mitochondria under the 63

light.26

With this notion, internal H2O2 concentration in leaf mitochondria remained 64

unchanged under salt stress in both salt-tolerant and salt-sensitive pea varieties, compared 65

with the control.27

Importantly, chloroplasts not only provide cell with the energy but also 66

represent a sensor of environmental information, and chloroplast redox signals help to 67

acclimate the organism to environmental stresses.28

68

In the present paper, NaCl-induced K+ efflux in leaf mesophyll was investigated in 69

samples pre-treated with methyl viologen (MV, a redox-active compound that generates 70

superoxide anions in chloroplasts29

), H2O2 (mimicking apoplastic ROS), and EGCG ((−)-71

Epigallocatechin gallate, a ROS scavenger30, 31

), by using MIFE technique (Fig. 1; see also 72

Wu et al. 2014 for details14

). MV and H2O2 are widely used in plant research for oxidative 73

stress. EGCG is a well-known ROS scavenger that protects lipids and membrane proteins 74

from oxidative damage.32, 33

It is widely used in animal research34, 35, 36, 37

, and was also 75

applied to plant species by several authors.38, 39

As showed in Fig. 2, specimens pre-treated 76

with MV showed significantly higher NaCl-induced peak K+ efflux from leaf mesophyll as 77

compared with non-treated control. The opposite trend was observed in EGCG pre-treated 78

samples, where significantly lower NaCl-induced peak K+ efflux was detected (Fig. 2). No 79

significant difference in NaCl-induced K+ efflux in leaf mesophyll was found between 80

control and H2O2 pre-treated samples (Fig. 2a, b). It is suggested that compared with apoplast 81

and plasma membrane-located NADPH oxidase, the ROS generated by chloroplasts play the 82

main role in regulating NaCl-induced K+ efflux in leaf mesophyll, at least in wheat. 83

5

Consistent with K+

data, the highest NaCl-induced H+ efflux in leaf mesophyll was found in 84

specimens pre-treated with MV, showing over 10-fold higher H+ efflux than in the control 85

(Fig. 3). These results suggest that, in an effort to retain high cytosolic K+ levels under MV-86

induced K+ leak, plants are pumping out more H

+ to maintain membrane potential to take up 87

external K+ via inward-rectifying (KIR) channels. Yet, being unable to control MV-induced 88

K+ leak via NSCC this pumping might result in a futile cycle, potentially leading to a 89

substantial waste of the ATP pool. Further studies are required to directly prove this model 90

and understand a causal link between mitochondria-produced superoxide anions and NSCC 91

activation in leaf mesophyll, in order to confer salinity stress tolerance in plants, that is 92

directly proportional to plant’s ability to retain K+ in leaf mesophyll.

3, 7 93

In conclusion, chloroplast-generated ROS play a main role in regulating NaCl-induced K+ 94

efflux in wheat leaf mesophyll. Both reducing NSCC sensitivity to ROS and alleviating ROS 95

generation in chloroplast may be instrumental in improving mesophyll K+ retention ability in 96

wheat. However, the identity of specific NSCC channels has to be revealed in order to control 97

NaCl-induced K+ efflux. Additionally, pyramiding the ROS regulated mesophyll K

+ retention 98

trait with other important traits (e.g. Na+ exclusion) might be a promising way to improve 99

salinity tolerance in wheat. Furthermore, as ROS play a dual role in plant salt tolerance, 100

understanding the network between K+ transport and the dynamic processes of the generation 101

and scavenging of different ROS species in plant cell under salt stress would benefit the 102

deciphering of the complexity of plant salt tolerance mechanisms as well as promote the 103

program of breeding salt tolerant crop varieties. 104

105

Conflict of interest statement 106

The authors declare that the research was conducted in the absence of any commercial or 107

financial relationships that could be construed as a potential conflict of interest. 108

6

109

Acknowledgements 110

This work was supported by the Grain Research and Development Corporation grants to SS 111

and MZ and by the Australian Research Council Discovery grants to SS. 112

113

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Figure legends 222

Fig. 1 – Logistics of ion flux measurements using the MIFE technique. (A), Measuring 223

chamber, leaf segments holder, and the plastic stopper used for immobilisation; (B), leaf 224

segments immobilized in the measuring chamber; (C), the measuring chamber with 225

immobilized specimen assembled on the cartridge of hydraulic manipulator; (D), the tips of 226

K+ and H

+ ion selective microelectrodes are aligned next to the surface of exposed leaf 227

mesophyll. See also Wu et al. 2014 for details14

. 228

Fig. 2 – Kinetics of NaCl-induced K+ efflux in leaf mesophyll (seven to 10 days old leaves of 229

bread wheat seedlings used, cultivar Janz) pre-treated with different chemicals (A), peak K+ 230

11

efflux values from mesophyll samples exposed to 1 mM H2O2, 20 μM DPI, 10 μM MV, and 231

50 μM EGCG pre-treatments (1h) (B). Mean ± SE (n = 5 - 10). Different lower case letters 232

represent significant difference (P < 0.05; one way ANOVA based on Duncan’s multiple 233

range test, SPSS 20.0). 234

Fig. 3 − Kinetics of NaCl-induced H+ efflux in leaf mesophyll (seven to 10 days old leaves of 235

bread wheat seedlings used, cultivar Janz) pre-treated with different chemicals (A), peak H+ 236

efflux values from mesophyll samples exposed to 1 mM H2O2, 20 μM DPI, 10 μM MV, and 237

50 μM EGCG pre-treatments (1h) (B). Mean ± SE (n = 5 - 10). Different lower case letters 238

represent significant difference (P < 0.05; one way ANOVA based on Duncan’s multiple 239

range test, SPSS 20.0). 240

241

Fig. 1

A

B

A

B

C

D

-3000

-2700

-2400

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0Control H2O2 DPI MV EGCG

Peak K

+ f

lux,

nm

olm

-2s

-1

B

a

ab

c

b b

Fig. 2

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-2100

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-900

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0

300

0 2 4 6 8 10 12 14 16

Control

H2O2

DPI

EGCG

MV

Net

K+ flu

x,

nm

olm

-2s

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A

100 mM NaCl

Fig. 3

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-1400

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0

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0 2 4 6 8 10 12 14 16

Control

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Net

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x,

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olm

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A 100 mM NaCl

-1600

-1400

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0Control H2O2 DPI MV EGCG

Peak H

+ f

lux,

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a

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a a