Planta DOI 10.1007/s00425-015-2316-2

ORIGINAL ARTICLE

Dissecting blue light signal transduction pathway in leaf epidermis using a pharmacological approach Branka D. Zˇivanovic´1,2 Sergey N. Shabala1

•

Lana I. Shabala1 • Theo J. M. Elzenga3

•

Received: 1 February 2015 / Accepted: 28 April 2015 Ó Springer-Verlag Berlin Heidelberg 2015

Abstract Main conclusion Blue light signalling pathway in broad bean leaf epidermal cells includes key membrane transporters: plasma- and endomembrane channels and pumps of H1, Ca21 and K1 ions, and plasma membrane redox system. Blue light signalling pathway in epidermal tissue isolated from the abaxial side of fully developed Vicia faba leaves was dissected by measuring the effect of inhibitors of second messengers on net K?, Ca2? and H? fluxes using non-invasive ion-selective microelectrodes (the MIFE system). Switching the blue light on–off caused transient changes of the ion fluxes. The effects of seven groups of inhibitors were tested in this study: CaM antagonists, ATPase inhibitors, Ca2? anatagonists or chelators, agents affecting IP3 formation, redox system inhibitors, inhibitors of endomembrane Ca2? transport systems and an inhibitor of plasma membrane Ca2?-permeable channels. Most of the inhibitors had a significant effect on steady-state (basal) net fluxes, as well as on the magnitude of the transient ion flux responses to blue light fluctuations. The data presented in this study suggest that redox signalling and, specifically, plasma membrane NADPH oxidase and coupled Ca2? and K? fluxes play an essential role in blue light signal transduction.

Keywords Broad bean
Inhibitors
Ion fluxes
Microelectrodes Abbreviations BL Blue light CaM Calmodulin CRY Cryptochrome DPI Diphenyleneiodonium ErB Erythrocin B EY Eosin yellow HCFIII Hexacyanoferrate (III) MIFE Microelectrode ion flux estimation NM Neomicyn PHOT Phototropin RR Ruthenium red TG Thapsigargin TFP Trifluoperazine TMB-8 8-(N,N-diethylamino) octyl-3,4,5trimethoxybenzoate W7 N-(6-aminohexyl)-5-chloro-1naphthalenesulfonamide

Introduction & Branka D. Zˇivanovic´ [email protected] 1

School of Land and Food, University of Tasmania, Private Bag 54, Hobart, TAS 7001, Australia

2

Institute for Multidisciplinary Research, University of Belgrade, Kneza Visˇeslava 1, Belgrade, Serbia

3

Ecophysiology of Plants, University of Groningen, Nijenborgh 7, Groningen, The Netherlands

Higher plants measure quantity, quality, direction and periodicity of incident light via a collection of specific photoreceptors, belonging to several classes of photoreceptors that perceive different wavelengths of light (Gyula et al. 2003; Jenkins 2009). Two of these—phototropins and cryptochromes—detect incident light from the UV-A/blue region of the spectrum (Cashmore et al. 1999; Kimura and Kagawa 2006) and are classified as blue light (BL)

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photoreceptors. Blue light responses are not restricted to a single cell type in plants. For instance, blue light controls extension growth of epidermal cells in leaf and stem, stomatal aperture in guard cells and chloroplast position in mesophyll cells. Both the expression of BL photoreceptors and their mode of signalling appear to be highly tissue specific. While in hypocotyls BL inhibits cell expansion growth (Cosgrove 1994), this process is enhanced by BL in leaf epidermis (Staal et al. 1994). The major difference comes from signal transduction pathways to downstream targets. It is believed that BL perception in hypocotyls is mediated by CRY1 photoreceptors (Ahmad and Cashmore 1993). These photoreceptors subsequently activate Clchannels, resulting in plasma membrane depolarization (Spalding and Cosgrove 1992). This depolarization activates a certain class of outward-rectifying K? channels causing massive K? efflux. The water efflux follows suit, and the cell volume decreases. The process is rather different in epidermis, where BL perception increases the rate of H? efflux (Staal et al. 1994), resulting in acidification of the apoplast, acid-induced cell wall loosening and solute accumulation for turgor maintenance (Stiles and Van Volkenburgh 2002). Despite the broad spectrum of physiological responses, it appears that the downstream targets of BL signalling are limited to a set of key transport proteins mediating movements of osmotically active inorganic ions (mainly K? and Cl-) across the plasma membrane (Spalding 2000). However, despite many years of research, signal transduction pathways between BL photoreceptors and plasma membrane effectors (downstream targets) have largely remained elusive. Numerous second messengers were suggested including various kinases and phospholipases (Shimazaki et al. 1992; Kaufman 1993). BL-induced increase in cytosolic free Ca2? is also widely reported (Kinoshita et al. 1995; Baum et al. 1999).Voltage gating via regulation of the plasma membrane H?-ATPase (Takemiya et al. 2006) and involvement of a redox system (Long and Jenkins 1998; Baum et al. 1999) are also widely discussed, although many details remain unclear. One of the major hurdles in studying signal transduction pathways from BL receptors to membrane effectors is the lack of convenient tools allowing in planta study of activity of specific plasma membrane transporters, which have sufficient temporal resolution to study the rapid kinetics of BL responses and are capable of doing this under physiologically relevant conditions. The MIFE technique for noninvasive microelectrode ion flux measurements (Shabala et al. 1997, 2012) is one of these tools. The technique allows in situ measurements of net ion fluxes across a membrane with high spatial (\2 lm) and temporal (5 s) resolution (Shabala et al. 2012). Earlier, we have successfully applied the MIFE technique to study ion fluxes

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associated with phototropism (Babourina et al. 2002) and the localization of BL receptors (Babourina et al. 2003) in plants, the association between light-induced transient ion fluxes and leaf growth and photosynthesis (Shabala and Newman 1999; Zˇivanovic´ et al. 2005; Shabala and Hariadi 2005), and their spectral and dose dependency (Zˇivanovic´ et al. 2007). In the current study, we use the MIFE technique in combination with a pharmacological approach to dissect BL-induced signal transduction pathways in broad bean leaf epidermis. Data presented in the current work suggests that redox signalling and, specifically, plasma membrane NADPH oxidase, and coupled Ca2? and K? fluxes, play an essential role in BL signal transduction.

Materials and methods Plant material Broad bean (Vicia faba L. cv Oswald; Hollander Imports, Hobart, Australia) plants were grown from seeds in standard potting mix (70 % composted pine bark, 20 % coarse sand and 10 % sphagnum peat, pH 6.0) supplemented with fertilizer mix (1.8 kg m-3 Limil, 1.8 kg m-3 dolomite, 6.0 kg m-3 Osmocote Plus and 0.5 kg m-3 ferrous sulphate) in 2 L pots in temperature-controlled glasshouse facilities at the University of Tasmania (26/19 °C mean day/night temperatures; 16-h day length; 55–70 % relative humidity; natural sunlight) as previously described (Shabala 2000; Percey et al. 2014). Four seeds per pot were sown and thinned to two per pot when the plants reached the two-leaf stage. Plants were watered daily with tap water and used for measurements after 2–3 weeks. Broad bean epidermal strips for MIFE measurements The newest fully developed leaves were excised from 15to 20-day-old broad bean plants. The abaxial epidermis was peeled off by means of fine forceps (Eye-Instruments, Albert Heiss H3376, Tuttlingen, Germany). Isolated epidermal strips were left floating on aerated experimental solution (0.1 mM KCl, 0.1 mM NaCl, 0.1 mM MgCl2, 0.05 mM CaCl2, 0.05 mM NH4Cl, 10 mM sucrose; pH approximately 5.3, non-buffered) for 1 h before starting the measurements to avoid wounding effects and to stabilize ion fluxes. The isolated epidermal strips were rolled around a glass holder with the mesophyll-facing side on the outside, exposed to the experimental solution. The epidermal strip attached to its glass holder was placed in the measuring chamber filled with measuring solution containing 0.1 mM KCl, 0.1 mM CaCl2 and 10 mM sucrose, pH 5.3 (non-buffered). The positions of

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Ion flux measurements Net fluxes of K?, Ca2? and H? were measured using the MIFE technique (University of Tasmania, Hobart, Australia) for non-invasive microelectrode ion flux measurements. All details of fabrication and calibration of ionselective microelectrodes are available in our previous publications (Shabala and Newman 1999; Shabala and Shabala 2002; Babourina et al. 2003). In brief, glass microelectrodes were pulled from non-filamentous glass capillaries (GC150-10, Clark Electrochemical instruments, Pangbourne, UK), salinized with tributylchlorosilane (Fluka Chemicals 90796), backfilled with solutions

(a)

-6

H+

5.751

-7

-8 5.743

-9

5.739

5.735

-10

(b)

pH units

5.747

Net fluxes (nmol m-2 s-1)

the measuring chamber and the microelectrodes were adjusted under dim green microscope light. The electrodes were placed 40 lm above the epidermal tissue surface with their tips aligned and separated by 3–4 lm. During measurements, electrodes were moved by a computer-controlled stepper motor at a frequency of 0.1 Hz in a squarewave manner between 40 and 60 lm from the tissue surface. Net ion fluxes were calculated from recorded potential differences at these positions, assuming cylindrical diffusion geometry (Shabala et al. 1997; Shabala and Newman 1999; Newman 2001). The epidermal strips were illuminated with blue light (30 lmol m-2 s-1) from a 150 W quartz halogen lamp (Phillips, Type 6423) with a flexible light guide (KL 1500 LCD, Schott, Mainz, Germany) and blue light filter (BG37, 481 nm, Schott). Light intensity was measured by a Li-Cor quantum photometer (LI-250 Light meter, Lincoln, NE, USA).The epidermal strips were exposed to several light/dark cycles (5/5 min light/dark) until they obtained stable light-induced transient changes in ion fluxes before the addition of an inhibitor (Fig. 1). The choice of the 5/5 min cycle was driven by the fact that this period was close to the frequency of the natural (self-sustained) oscillations in membrane-transport activity in plant cells (see Shabala et al. 2006 for comprehensive review and modelling). As a result, although the entire transient response takes typically between 30 and 40 min (Shabala and Newman 1999), the strongest response (the peak value) is achieved at around 5 min. Hence, using 5/5 min cycle was the most optimal, as it came at a resonant frequency and allowed us to maximize the magnitude of response (hence, the resolution of the method). The inhibitor of appropriate concentration was added to the experimental chamber during a dark cycle, and light/dark transients of ion fluxes in the presence of the inhibitor were continuously recorded for several cycles. For clarity reasons, the spurious peaks in the recording during the addition of the inhibitor are omitted from the analysis (Fig. 2).

0

5

10

15

20

25

15

20

25

15

20

25

15

Ca2+ 10

5

0

-5

-10 0

(c)

5

10

-5

K+ -15

-25

-35

-45 0

5

10

Time (min)

Fig. 1 Representative tracings of BL-induced transient changes in net fluxes of H? (a), Ca2? (b) and K? (c) ions (in nmol m-2 s-1) (open circle) and pH units (filled circle) recorded from broad bean (Vicia faba L.) leaf epidermis in the absence of inhibitors. Epidermal tissue was exposed to several light-on/off (L/D, 5 min/5 min) cycles before the onset of inhibitory treatment. White and black bars indicate 5 min period of light and dark, respectively

(15 mM NaCl, 40 mM KH2PO4, adjusted to pH 6.0 by NaOH for H?; 0.5 mM CaCl2 for Ca2?), and filled with appropriate ion-selective cocktail (Fluka: K?: 60031; Ca2?: 21048; H?: 95297). Electrodes were then calibrated in a set of pH, K? or Ca2? standards and used for measurements. Inhibitors The effects of seven groups of the inhibitors were tested in this study (Table 1) [(1) CaM (calmodulin) antagonists: W7, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide; and TFP, trifluoperazine; (2) inhibitors of P2B-type ATPase: EY, eosin yellow; and ErB, erythrocin B; (3) Ca2? antagonists or

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Net H+ flux (nmol m-2 s-1)

20 µM DPI 6

amplitudes of the light-induced transients recorded from different epidermal strips were averaged before and after treatment. The effect of pharmacological treatment was expressed as the difference in net basal fluxes and lightinduced amplitudes before (control) and after treatment. Data are presented as mean ± SE and tested for significance by using Student’s t test. Mean values of basal fluxes were simply tested for difference between control and treated samples, while the magnitude of the flux response to the light before and after treatment was presented as a relative value (% of control) which was also tested for significant difference.

BL on

4 2 0 -2

BL off -4

6.05

pH units

5.95 5.85

Results

5.75

Blue light-induced transients of the ion fluxes 5.65 10

20

30

40

50

60

70

80

90

Time (min)

Fig. 2 Experimental protocol illustrated by a typical example of recording of net H? flux and pH from broad bean leaf epidermis. Leaf epidermis was immobilized in the measuring chamber and exposed to a rhythmical blue light treatment (30 lmol m-2 s-1; ON/OFF 5 min/ 5 min cycle). After five or six cycles (50–60 min later), a metabolic inhibitor was added to the bath solution (20 lM DPI in this particular example) and fluxes were recorded for another 60–80 min. Black and white bars indicate 5 min period of dark and light, respectively

chelators: TMB-8, 8-(N,N-diethylamino) octyl-3,4,5trimethoxybenzoate; and EGTA, ethylene glycol tetraacetic acid; (4) agents affecting IP3 formation: NM, neomycin; and LiCl; (5) redox system inhibitors: DPI, diphenyleneiodonium; and HCFIII, hexacyanoferrate (III); (6) inhibitors of endomembrane Ca2? transport systems: TG, thapsigargin; and RR, ruthenium red; and (7) inhibitor of plasma membrane Ca2?-permeable channels: LaCl3]. The stock solutions of the inhibitors were prepared in distilled water, except DPI and TG which were dissolved in DMSO and ethanol, respectively. Potential effects of all inhibitors on ion-selective microelectrode characteristics and pH of the experimental solution were tested separately. Ion-selective microelectrodes were calibrated in the experimental solution first and then in the presence of the inhibitor after the experiment. Data processing and analysis Initial calculations of ion fluxes were performed after the measurements and data were processed in the spreadsheet software Excel (Microsoft). The ion fluxes obtained from individual epidermal strips were averaged. There were two groups of ion flux values analysed, i.e. steady-state (net basal fluxes) values and the amplitudes of the blue lightinduced transients of ion fluxes. Basal fluxes and the

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Net basal H? ion fluxes recorded from different leaf samples were rather heterogeneous showing either net uptake (influx) or efflux, while for Ca2? and K? ions influx and efflux were observed in most cases, respectively (Figs. 3, 5, 6, 7, 8, 9). The blue light induced transient changes of H?, Ca2? and K? ions close (25 lm) to the surface of the epidermal strips. BL caused bimodal changes (efflux-influx) of H? fluxes, while Ca2? and K? fluxes displayed a transient influx upon BL illumination (Fig. 1). Switching the light off caused a change in all ion fluxes opposite of the transients that were induced by light-on. After obtaining stable BL-induced transients in ion fluxes, the inhibitors were added to the experimental solution during a dark period and the ion fluxes were recorded for at least three more BL-on/-off cycles. Such experimental protocol for the application of all inhibitors used in this study is illustrated in Fig. 2 by showing an example of one treatment with 20 lm DPI. Most of the inhibitors induced a significant effect on steady-state (basal) net fluxes (Fig. 3), as well as on the magnitude of ion flux responses to blue light-on/light-off changes (Fig. 4). Effect of CaM antagonists The application of CaM antagonists (W7 and TFP) aimed at testing the involvement of CaM in BL-induced activation of proton pump and coupled K? and Ca2? ion channels, which is expected for the leaf epidermis upon BL illumination. Moreover, the inhibition of CaM by these CaM antagonists was previously reported to stimulate proton pumping outside of the guard cell (Shimazaki et al. 1992). It was expected that CaM antagonists-induced increase H? pumping outside of the epidermis should stimulate K? and Ca2? uptake. Indeed, both 200 lM W7 and 100 lM TFP caused a significant (P \ 0.05) shift towards

Planta Table 1 Details on metabolic inhibitors (CaM antagonists, inhibitors of P2B type ATPase, Ca2? antagonists, Ca2? chelators, agents affecting IP3 formation, redox inhibitors, inhibitors of endomembrane Ca2?transport systems) and channel blockers used in this study Abbreviation

Full name

Mode of action

Concentration

References

W7

N-(6-aminohexyl)-5-chloro-1naphthalenesulfonamide

CaM antagonist

200 lM

Li et al. (2004)

TFP

Trifluoperazine

CaM antagonist

100 lM

Liu et al. (2005)

EY

Eosin yellow

Inhibitor of P2B type ATPase

0.5 lM

De Michelis et al. (1993)

ErB

Erythrocin B

Inhibitor of P2B type ATPase

4 lM

Cocucci and Marre´ (1986)

TMB-8

8-(N,N-diethylamino)octyl-3,4,5trimethoxybenzoate

Intracellular Ca2? antagonist

50 lM

Mayer et al. (1997), Schumaker and Sze (1987), Alexandre et al. (1990)

EGTA NM

Ethylene glycol tetraacetic acid Neomycin

Ca2? chelator Decreases formation of IP3

2 mM 10 lM

Vitrac et al. (2000) Mayer et al. (1997), Toyoda et al. (1992)

LiCl

LiCl

Increases IP3 levels

5 mM

Monreal et al. (2007)

DPI

Diphenyleneiodonium

NADPH oxidase inhibitor

20 lM

HCFIII

Hexacyanoferrate (III)

Electron acceptor

0.5 mM

Khokon et al. (2011), Morre´ (2002) Grabov and Bo¨ttger (1994)

TG

Thapsigargin

Endomembrane Ca2? ATPase inhibitor

5 lM

Pang et al. (2007), Ordenes et al. (2002)

RR

Ruthenium red

Endomembrane Ca2? channel inhibitor

1 lM

Muir et al. (1997)

La3?

LaCl3

Plasma membrane Ca2? channel blocker

500 lM

Huang et al. (1994)

increased net basal Ca2? and K? uptake (Fig. 3b, c). Basal H? fluxes were also significantly affected and shifted towards a net H? efflux (Fig. 3a). The addition of CaM antagonists to the bath also potentiated the magnitude of BLinduced ion flux responses significantly (Fig. 4), with three- to fivefold higher responses compared to pre-treated tissue (Fig. 5). Adding TFP to the bath proved to have a major detrimental effect on H?-specific electrodes. H? flux data in response to TFP are therefore unavailable in Figs. 3 and 4.

Ca2? flux responses (Figs. 4b, 6), while at the same time induced a doubling of the K? flux (Figs. 4c, 6). These data argue in favour of the proposed model given in Fig. 10. No significant effects of either 0.5 lM EY or 4 lM ErB on the basal net Ca2? fluxes were found (Fig. 3b), while net K? efflux was slightly enhanced by ErB (Fig. 3c). Also, no clear trends were noticed for basal H? fluxes and BL-induced H? flux responses (Figs. 3a, 4a, 6).

Effect of inhibitors of P2B-type ATPase

To explore the involvement of calcium in blue light signalling in the epidermis, Ca2? chelators and antagonists were employed in this study. It was expected that decrease in intracellular Ca2? concentration would promote proton pumping outside the cell and activate KIR/KOR channels due to voltage change on plasma membrane in the presence of these inhibitors. Intracellular Ca2? antagonist 50 lM TMB and Ca2? chelator 2 mM EGTA both resulted in increased net basal H? efflux (Fig. 3a) and reduced net Ca2? uptake (Fig. 3b), which is in the line with our expectation. Significant (P \ 0.05) effects on net K? basal fluxes were also detected (Fig. 3c). When epidermal strips were subjected to cyclic BL fluctuation, pre-treatment with TMB and EGTA significantly reduced the magnitude of Ca2? flux responses, but led to up to threefold increase in

The inhibitors of P2B-type ATPase (EY and ErB) have been previously shown to affect Ca2? pumping out of the plant cells (Romani et al. 2004; Beffagna et al. 2005; Nemchinov et al. 2008), so we tested their potential effect on broad bean epidermal tissue. We supposed that the inhibition of Ca2? ATPase by P2B type ATPase inhibitors could result in accumulation of positive ions in the cell (depolarization of the membrane potential), which should stimulate opening of K? channels (KOR) enabling the efflux of K? ions from the cell and subsequently return membrane potential to hyperpolarized level. When epidermal strips were subjected to cyclic BL fluctuation, pre-treatment with these compounds indeed induced a twofold (P \ 0.05) reduction in magnitude of

Effect of Ca21 antagonists and chelators

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(a) 350

*

RR

TG

HCFIII

La3+

Fig. 3 Steady-state (basal) net fluxes (in nmol m-2 s-1) of H? (a), Ca2? (b) and K? (c) ions measured 60 min after the addition of a pharmacological agents (for details, see Table 1). Positive and negative values of ion fluxes correspond to influx and efflux, respectively. Mean ± SE (n = 5–12). Control bars refer to the basal flux values before addition of a pharmacological agent. Treatments labelled with asterisks are significantly different from controls at P \ 0.05

the magnitude of H? and K? flux responses (Fig. 4a, c, 7). Thus, data presented in this study confirmed the involvement of intracellular Ca2? concentration in BL signal transduction pathway. Effect of agents affecting IP3 formation It has been previously reported that the enzymes and metabolites of the phosphoinositide cycle are present in plants (Stevenson et al. 2000). Plant vacuoles were suggested as a major IP3-regulated intracellular Ca2? store. In this study we tested the effects of the two compounds affecting IP3 formation: neomycin (NM) and LiCl, to explore

RR

TG

DPI

NM

TMB

EGTA

EY

ErB

W7

TFP

LiCl

NM

EGTA

EY

ErB

TFP

TMB

*

* DPI

* NM

TMB

EY

ErB

-100

EGTA

*

LiCl

-50 TFP

RR

TG

HCFIII

NM

* * W7

*

W7

*

0

0

Control

*

100

*

123

K+ flux

*

200

K+ flux

*

* TG

DPI

300

50

* * *

HCFIII

500 400

100

*

(c)

200

*

* * * *

600

(c) 150

* EGTA

*

* LiCl

EGTA

TMB

EY

ErB

W7

TFP

Control

NM

*

-50

* *

La3+

*

RR

0

*

HCFIII

* *

TG

*

Ca2+ flux

TMB

Ca2+ flux

100

* *

EY

*

150

700 600 500 400 300 200 100 0

ErB

200

(b)

TFP

(b)

- 50

W7

La3+

TG

RR

DPI

HCFIII

NM

LiCl

TMB

EGTA

ErB

EY

W7

TFP

Control

-20

Magnitude of response (% control)

*

50

* *

50

-15

HCFIII

*

*

150

*

DPI

*

*

DPI

* *

LiCl

-5 -10

Net basal fluxes (nmol m-2 s-1)

*

250

0

La3+

*

5

H+ flux

*

La3+

10

RR

flux

450

LiCl

H+

15

La3+

(a)

Fig. 4 Effect of metabolic inhibitors (for details, see Table 1) on the magnitude of flux responses (% control) for H? (a), Ca2? (b) and K? (c) measured in response to blue light on/off cycles. Mean ± SE (n = 8–25 values from at least four biological specimens). Treatments labelled with asterisks are significantly different from controls at P \ 0.05

the existence of IP3-gated endomembrane Ca2? channels. Neomycin (NM) decreases IP3 formation via inhibition of enzyme phospholipase C (Mayer et al. 1997), while LiCl increases cytosolic IP3 levels (Monreal et al. 2007). NM (10 lM) caused non-significant shift in basal H? towards net influx and non-significant effect on K? fluxes, while the Ca2? net efflux was increased significantly (P \ 0.05) (Fig. 3). Moreover, the use of NM reduced the magnitude of BL-induced Ca2? flux responses to about 50 % of that in control (Fig. 4b), while the effects on H? and K? fluxes were not significant (Fig. 4a, c). Opposite effects were observed for 5 mM LiCl. In the latter case, responses of both Ca2? and K? fluxes to BL were substantially potentiated (about twofold; Figs. 4b, c, 8), and basal H? flux was shifted to towards net efflux (Fig. 3a). Although the effects of NM and LiCl on Ca2? ions were observed in this work, our data did not show clearly the existence of IP3 gated Ca2? channel responsible for regulation of intracellular Ca2? concentration.

Planta Before treatment

After treatment

-2

0

-3

4 3

-8

H+

2

-4

1

-16

-5

Net ion fluxes (nmol m-2 s-1)

Fig. 5 Effect of CaM antagonist W7 on blue lightinduced ion flux responses measured from broad bean epidermis. Typical flux recordings are shown for each ion (H?, Ca2? and K?) before and after 200 lM W7 adding to the bath solution. Different scales for Y axis are given for fluxes recorded ‘‘before’’ and ‘‘after treatment’’ to make visible kinetics of control fluxes of all ions measured. The right column shows mean peak magnitudes (in nmol m-2 s-1) before (control; C) and after treatment (T). Mean ± SE (n = 16)

0

-6

C

-24 0

5

10

15

20

0

15

550

10

450

5

350

5

10

15

T

20 30 20

0

Ca2+ 10

250 0

5

10

15

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20

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5

10

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20

C

-10

500

-15

300

60

-20

100

40

-25

-100

-30

-300

T

80

K+

20

0

5

10

15

20

0 C 0

5

10

15

T

20

Time (min)

Before treatment

3

After treatment

3

1.5 1

2

2

1

1

H+

0.5 0 C

0

Net ion fluxes (nmol m-2 s-1)

Fig. 6 Effect of Erythrosin B (an inhibitor of P2B-type Ca2?ATPase) on blue light-induced ion flux responses measured from broad bean epidermis. Typical flux recordings are shown for each ion (H?, Ca2? and K?) before and after 4 lM ErB adding to the bath solution. The right column shows the mean peak magnitudes (in nmol m-2 s-1) before (control; C) and after treatment (T). Mean ± SE (n = 20)

T

0 0

5

10

15

20

0

5

10

15

20

45

35

6

40

30

4

35

25

2

30

20

25

15 0

5

10

15

0 C 0

20

30

0

10

-20

-10

-40

-30

-60

Ca2+

5

10

15

40 30 20

K+

10 0 C

-50 0

5

10

15

20

T

20

T

-80 0

5

10

15

20

Time (min)

Effect of redox system inhibitors Reduction of impermeable artificial electron acceptors, such as hexacyanoferrate III (HCFIII), has been reported on several intact plants since the early 1980s (Lu¨thje et al. 1997; Be´rczi and Mu`ller 2000). Depolarization of the

plasma membrane and increase in apoplastic acidification were also observed upon application of redox inhibitors. The application of the two redox system inhibitors on broad epidermis in this study, 20 lM DPI and 0.5 mM HCFIII, did reduce the net basal Ca2? uptake and decreased basal K? leak (Fig. 3b, c), but significantly

123

Planta Before treatment

0.1

Net ion fluxes (nmol m-2 s-1)

Fig. 7 Effect of EGTA (a known Ca2? chelator) on blue light-induced ion flux responses measured from broad bean epidermis. Typical flux recordings are shown for each ion (H?, Ca2?, and K?) before and after 2 mM EGTA adding to the bath solution. Different scale for Y axis is given for H? fluxes recorded ‘‘before’’ and ‘‘after treatment’’. The right column shows mean peak magnitudes (in nmol m-2 s-1) before (control; C) and after treatment (T). Mean ± SE (n = 13)

After treatment -5

0

-6

-0.1

-7

6 5 4 3 2 1 0

H+

-8

-0.2 0

5

10

15

C 0

20

10

10

5

5

5

10

15

T

20 5 4

Ca2+

3 2

0

1

0

0 C -5

T

-5 0

5

10

15

20

0

0

-70

-20

-90

5

10

15

20 40 30

K+

20 -40

10

-110

0 -60 0

5

10

15

20 -130

C 0

5

10

15

T

20

Time (min)

Before treatment

-3

After treatment -3

-5

4 3

-5

H+

2 -7

-7

-9

-9

1 0

Net ion fluxes (nmol m-2 s-1)

Fig. 8 Effect of LiCl (a known IP3 signalling inhibitor) on blue light-induced ion flux responses measured from broad bean epidermis. Typical flux recordings are shown for each ion (H?, Ca2?, and K?) before and after adding 5 mM LiCl to the bath solution. The right column shows the mean peak magnitudes (in nmol m-2 s-1) before (control; C) and after treatment (T). Mean ± SE (n = 20)

0

5

10

15

20

C 0

5

10

15

29

14

8

22

7

6

15

0

4

8

-7

2

1 5

10

15

0

20

5

10

15

C

20

-30

-30

-40

-40

30

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-50

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-60

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-70

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increased H? efflux (Fig. 3a), which is in the line with previously reported data (Menckhoff and Lu¨thje 2004). The magnitude of BL-induced oscillations in net ion fluxes was significantly (P \ 0.05) inhibited for all ions (Fig. 4)

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in epidermal strips pre-treated with these agents, resulting in a reduction ranging from three to fivefold (Fig. 9). These data confirmed the involvement of the redox systems in BL signalling of broad bean epidermis.

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Effect of inhibitors of endomembrane Ca21 transport systems

as previously reported on Arabidopsis (Wang et al. 2004). La3? has been also shown to block both inward-rectifying (KIR) (Tester and MacRobbie 1990) and outward-rectifying (KOR) (Ketchum and Poole 1991) K? channels. The application of 500 lM La3? on epidermis has significantly reduced net basal Ca2? uptake and increased basal H? efflux (Fig. 3b, a), while the basal K? efflux was reduced (Fig. 3c). A significant (3 to 4-fold) reduction in the magnitude of BL-induced oscillations in net fluxes of Ca2? and K? ions and increased BL-induced oscillations in net H? fluxes were observed in epidermal strips pre-treated with La3? (Fig. 4). Thus, the results obtained in the present work with La3? are consistent with previously reported data (Tester and MacRobbie 1990; Ketchum and Poole 1991; Wang et al. 2004).

Endomembrane Ca2? transporters regulate cytosolic Ca2? concentration either passively by Ca2? channels enabling export Ca2? from internal stores to cytosol or actively by Ca2? efflux mechanisms (Ca2? ATPase) which rapidly remove the excess of Ca2? from cytosol by sequestering it into internal stores. The effects of the two inhibitors (thapsigargin, TG, and ruthenium red, RR) of the endomembrane Ca2? transporters, mostly used in animal systems (Thastrup et al. 1990; Bae et al. 2003), were also tested on plants (Pottosin et al. 1999; Chang et al. 2001; Ordenes et al. 2002; Urbina et al. 2006). In this study, TG (5 lM) and RR (1 lM) significantly reduced basal Ca2? uptake, but had no impact on basal K? fluxes (Fig. 3b, c). The net H? efflux was stimulated by both compounds (Fig. 3a). Both TG and RR reduced (about twofold; P \ 0.05) the magnitude of BLinduced net Ca2? flux oscillations (Fig. 4b), but had no significant effect on K? flux (Fig. 4c) and H? flux (Fig. 4a) responses to BL. These results indicate that endomembrane Ca2? channel (inhibited by RR) and Ca2? ATPase (inhibited by TG) might be included in the model on BL signalling in the broad bean epidermis (Fig. 10).

Discussion Ion transporters involved in BL signalling in bean epidermis Since the guard cells form only a small fraction of overall membrane area in leaf epidermis, and the BL-induced H? flux was stimulated by calmodulin antagonists (Fig. 4) which is opposite to reported responses from V. faba guard cells (Shimazaki et al. 1992), it is likely that the fluxes measured in this work are predominantly associated with the basement cells and, given the leaf age, could be part of the BL-related growth response. As growth of the epidermal

Effect of plasma membrane Ca21-permeable channels block La3? ion was added in the form of LaCl3 solution to inhibit Ca2?-permeable channel activity at the plasma membrane,

After treatment

Before treatment 3

3

3

1

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-1

-1

-3

-3

H+

1 0

Net ion fluxes (nmol m-2 s-1)

Fig. 9 Effect of diphenyleneiodonium (DPI; a known NADPH oxidase inhibitor) on blue light-induced ion flux responses measured from broad bean epidermis. Typical flux recordings are shown for each ion (H?, Ca2?, and K?) before and after adding 20 lM to the bath solution. The right column shows the mean peak magnitudes (in nmol m-2 s-1) before (control; C) and after treatment (T). Mean ± SE (n = 15)

0

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K+

H2O2

O 2-

DPI, HCF

NSCC

e-

O2

Ca2+

Voltage

HACC H+

Ca2+

NADPH

La3+

La3+

ATP

Y

TMB-8, EGTA

EY, ErB

Ca2+

LiCl

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ATP

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CaM

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W7,TFP

IP3

?

ATP

TG

NM

BL Ca2+

RR

KIR

K+

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Ca2+

Fig. 10 Tentative model for blue light signalling in broad bean epidermal cells. HACC hyperpolarization-activated Ca2?-permeable channel, KIR inward-rectifying hyperpolarization-activated K? channel, KOR outward-rectifying depolarization-activated K? channel, NSCC non-selective cation channel, R receptor; at least two unidentified signalling molecules are postulated (X and Y): one

mediates transduction of BL stimulus from receptor to IP3 (factor X in the model), while another (factor Y in the model) acts as a negative feedback regulator between CaM and H?-ATPase. The relationship between IP3 and endomembrane Ca2?-permeable channel was not confirmed pharmacologically in this model (indicated by question mark). See text for more details and other abbreviations

basement cells is mainly the result of cell expansion, it is essential that, firstly, both the vacuole and the cytoplasm need to accumulate osmolytes to allow water uptake leading to volume increase and, secondly, that the pH in the cell wall becomes or remains low, to increase the plasticity and allow cell increase. For both of them H?-pumping ATPase needs to be activated and the driving force created for the uptake of K? (or other small solutes) and the acidification of the cell wall. In our working hypothesis, BL activates the H?-pumping ATPase through a signalling cascade that involves an increase in the cytoplasmic calcium concentration (released from internal stores or coming from outside the cell), which in turn activates CaM/CDPK, leading to changes in the activity of K? influx. Since the Ca2?/CaM signal transduction is a central pathway in signal transduction of many external factors, other elements (redox reactions) might directly or indirectly interfere with the coupling between BL perception and ATPase and K? channel activation. The tentative BL signal transduction pathways and key membrane transporters involved in BL signalling are depicted in Fig. 10. The latter include four channels and two pumps: K?-permeable inward (KIR) and outward (KOR) rectifying channels and non-selective cation channels (NSCC); Ca2? transporting hyperpolarization-activated cannel (HACC) and P2B-type Ca2?-ATPase; and H?-translocation ATPase. In addition, endomembrane

Ca2? channel and P2A-type Ca2?-ATPase are included in the model as a part of the endomembrane systems. The reasons for this are given below.

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H1-ATPase In our working hypothesis, activation of the proton pump is essential to acidify the cell wall to increase extensibility and generate the driving force for K? influx to maintain an osmotic difference between cytoplasm and apoplast and thus maintain turgor. Modulation of BL has resulted in significant fluctuation in net H? fluxes (all figures). The fact that in most cases background H? was outwardly directed (net efflux; Fig. 3) and that BL-induced changes in H? fluxes were suppressed by vanadate (Shabala and Hariadi 2005; Percey et al. 2014) explicitly suggests involvement of plasma membrane H?-ATPase. BL-induced activation of H?-ATPase has been reported before, both for stomata guard cells (Schwartz et al. 1991; Taylor and Assmann 2001) and leaf epidermis (Staal et al. 1994). K1 transport systems For the inward-directed K? fluxes, several candidates are included in our model. TEA-sensitive K? channels play a key role in BL-induced cell expansion growth (Stiles and

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van Volkenburgh 2002). KIR channels are activated by plasma membrane hyperpolarization caused by BL-induced activation of H?-ATPase and are responsible for solute accumulation for turgor maintenance, while KOR channels are activated by depolarization to drive K? efflux (Stiles and van Volkenburgh 2002). In our case, increased magnitude of BL-induced response in H? flux was often accompanied by the concurrent increase in the magnitude of K? flux response (e.g. for W7; TMB; EGTA; Fig. 4a, c) suggesting that this was the case for broad bean as well. Addition of depolarization-activated KOR channels is justified by the fact that net K? efflux was measured for some treatments (Fig. 3c). In addition, BL-induced K? flux responses were strongly suppressed by La3?, a known blocker of K?-permeable NSCC (Figs. 3c, 4c). Hence, the latter are also considered in the model.

work also confirmed La3? inhibitory effect on K? channels (Figs. 3c, 4c). At the same time, Ca2? flux responses were also sensitive to ErB and EY (Fig. 4b), two inhibitors of P2B-type Ca2?-ATPase located at the plasma membrane (Axelsen and Palmgren 2001). Thus, the latter is also added to the model (Fig. 10). The two known major types of Ca2? efflux systems, Ca2?-pumps and Ca2?/H? antiporters, are essential for restoring the basal levels of cytosolic free calcium at normal physiological condition (Ca2?-ATPase) or under extreme stress (Ca2?/H? antiporters), intracellular calcium concentration ([Ca2?]cyt) (Sze et al. 2000; White and Broadley 2003) and, together with Ca2? influx channels are responsible for stimulus-induced calcium ‘‘signatures’’ in plant cells (Bose et al. 2011) required for growth and development. Endomembrane systems

Ca21 transport systems The cellular signal transduction system responsible for the coupling of excitation of the BL photoreceptor and the activation of H?-ATPase is hypothesized to be the Ca2?/ CaM system. Any treatment that will increase the cytoplasmic Ca2? concentration (release form internal stores or entry from the apoplast) is assumed to activate the H?ATPase, while inhibition of CaM is expected to reduce the H? efflux. Ca2? plays an important role in plant growth, development, and signal transduction. Ca2? uptake into cytosol is a thermodynamically passive process mediated by a range of Ca2?-permeable channels, while the removal of Ca2? from the cytosol and return to endomembrane compartments is against an electrochemical potential gradient for Ca2? and is therefore an energy-dependent process driven by ATP-dependent Ca2? pumps (reviewed in Medvedev 2005). Furthermore, plants have two types of Ca2? efflux transporters, ATP-dependent Ca2? pumps (Sze et al. 2000; Boursiac and Harper 2007) and Ca2?/H? antiporters (exchangers) that utilize the proton motive force (Shigaki and Hirschi 2006). Ca2?-permeable plasma membrane channels can be separated into voltage-gated (e.g. HACC, or hyperpolarization-activated Ca2? channels; Miedema et al. 2001 and references therein) and weakly voltage dependent (NSCC, or non-selective cation channels; Demidchik and Maathuis 2007). Both of them are known to be sensitive to trivalent cations such as La3? and Gd3? (Demidchik et al. 2002; Hetherington and Brownlee 2004) and in our case BL-induced fluctuations of Ca2? flux were significantly reduced by La3? (Fig. 4b), suggesting the involvement of such NSCC and/or HACC (Fig. 10). It cannot be ignored that La3? apart from inhibiting Ca2? channels (Wang et al. 2004) has been reported to block both KIR and KOR K? channels (Tester and MacRobbie 1990; Ketchum and Poole 1991). The results shown in this

Net ion flux response were significantly modulated by RR, a known inhibitor of endomembrane Ca2? channels, as well as by TG, an inhibitor of P2A-type Ca2?-ATPase (endomembrane-located) suggesting involvement of both these transport systems in cytosolic Ca2? signalling and homeostasis (Figs. 3b, 4b, 10). BL signalling to downstream targets Regulation of H?-ATPase The mechanism by which the perception of BL is transduced into the pump activation is far from being clear (Assmann et al. 1985). It was suggested for guard cells that BL receptor PHOT1 contains in addition to the lightsensing LOV domain(s) a serine/threonine protein kinase domain which enables activation of PHOT1 by autophosphorylation upon BL illumination, resulting in the binding of 14-3-3 proteins to the H?-ATPase. Thus, in guard cells BL activates the H?-ATPase via phosphorylation of the C terminus with subsequent binding of 14-3-3 protein to the H?-ATPase (Palmgren 2001; Kinoshita et al. 2003). By contrast, in leaf pulvina BL perception by phototropin results in dephosphorylation of the phosphorylated Thr in the C terminus of H?-ATPase and the subsequent dissociation of 14-3-3 protein (Gaxiola et al. 2007). The mechanism of activation (phosphorylation)/deactivation (dephosphorylation) of H?-ATPase is not clear. Our data reported here suggests that CaM may be an important component of the signal transduction pathway from BL receptor to H?ATPase. Indeed, several-fold increase in BL-induced ion flux fluctuations were observed in epidermal strips treated with two CaM antagonists, W7 and TFP (Figs. 4, 5). CaM antagonists increased H? efflux (stimulate H? pumping outside) and increased Ca2? and K? influxes (Fig. 3).

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Thus, it can be assumed that BL-increased H? pump activity has hyperpolarized plasma membrane leading to opening of the HACC channel and KIR channels to uptake Ca2? and K? ions from apoplast to cytosol, respectively (Fig. 10). Moreover, increased [Ca2?]cyt should activate CaM protein to indirectly inhibit (via some unknown Y component) H? pump, which is antagonized by CaM inhibitors (Fig. 10). This suggests that CaM acts either as a negative feedback regulator of 14-3-3 protein binding to H?-ATPase, or the observed regulation is indirect and may be related to altered ionic conditions in cell cytosol. It is known that K? may act as an intrinsic uncoupler of the proton pump by binding to its site involving Asp-617 in the cytoplasmic domain and inducing dephosphorylation of the reaction cycle intermediate by a mechanism involving Glu184 in the conserved TGES motif of the pump phosphatase domain (Buch-Pedersen et al. 2006). H?-ATPase is also known to be sensitive to [Ca2?]cyt (Kinoshita et al. 1995). Thus, stomatal opening is promoted by activation of H?ATPase due to lowered [Ca2?]cyt, whereas the increase in [Ca2?]cyt reduces the activity of H?-ATPase leading to stomatal closure. We show here that activation of increased cytosolic Ca2? inhibits modulation of BL-induced Ca2? flux responses by TFP or W7 may have a domino effect on intracellular K? and Ca2? concentrations and, this, indirectly regulate H?-ATPase activity. BL-induced [Ca2?]cyt elevation BL-induced [Ca2?]cyt elevation has always been considered to be central to BL signalling, both in guard cells (Shimazaki et al. 1992) and leaf epidermis (Elzenga et al. 1997). Early pharmacological studies have suggested that CaM- and Ca2?/CaM-dependent myosin light chain kinases (MLCK) are the components of the signal transduction pathway in BL-dependent proton pumping in guard cells (Shimazaki et al. 1992). In this study, the evidence for [Ca2?]cyt involvement is from the observed modulation of BL responses by EGTA (Ca2? chelator), W7 and TFP (CaM antagonists) (Fig. 4). Intracellular Ca2? storage (and, hence, endomembrane Ca2? transport systems) appears to be central to BL signal transduction. Baum et al. (1999) have suggested that Ca2? may regulate the activity of the nuclear pore controlling movement of constitutively photomorphogenic repressor protein 1 (COP1) between the cytosol and the nucleus. Photo 2 was implicated in Ca2? release from intracellular stores in Arabidopsis leaves (Baum et al. 1999; Babourina et al. 2002; Harada et al. 2003). One of the possible candidates for the pathway for Ca2? release from the intracellular stores may be IP3-gated Ca2?-permeable channels (Allen et al. 1995). At the same time, some other researchers have questioned the existence

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of such ligand-gated channels’ endomembranes (reviewed by Pottosin and Scho¨nknecht 2007). In our case, BL-induced flux responses were modulated by LiCl and NM (Fig. 4b, 8), two chemicals known to modify cytosolic IP3 levels (Monreal et al. 2007), and also by RR (Fig. 4), a known blocker of IP3-gated Ca2? channels (Pineros and Tester 1997). However, the question of whether IP3 may directly modulate such endomembrane Ca2? channels, or this process is mediated by some other additional signal component, needs to be resolved in direct patch-clamp experiments. This is reflected by putting a question mark between IP3 and Ca2? channels in our model (Fig. 10). Endomembrane Ca2?-ATPases are also involved in signal transduction pathway, as BL-induced responses were modulated by TG, an inhibitor of P2A-type ATPase Ca2?ATPase (endomembrane-located) (Figs. 4b, 10). Redox signalling component Redox reactions have long been suggested as the early steps in BL signalling (Long and Jenkins 1998; Cashmore et al. 1999). Baum et al. (1999) provided evidence that BL signal transduction involves changes in redox process; according to their model, the LOV domains of NPH1 detect BL modification in its redox state and activate protein kinase. Alternatively, redox changes may modify PM potential stimulating Ca2? uptake via voltage-gated channels (Baum et al. 1999). UV-B-induced stimulation of NADPH oxidase activity has been reported elsewhere (Jenkins 2009 and references within). Also, BL-stimulated medium acidification by guard cells was found to be vanadate insensitive and interpreted as evidence for the operation of proton-extruding redox system (Raghavendra 1990), although this view was challenged later by Taylor and Assmann (2001) who showed that BL-stimulated H? currents in guard cell protoplasts were not affected by either NADH or NADPH added to pipette. Our data also provide strong evidence that redox signalling and, specifically, plasma membrane NADPH oxidase, play essential role in BL signal transduction. Both DPI (NADPH oxidase inhibitor) and HCFIII (electron acceptor) resulted in significant reduction of the magnitude of BL-induced Ca2? and K? flux oscillations (Figs. 4b, c, 9). This can be explained by the presence of positive feedback between NADPH oxidase and Ca2? uptake via HACC mediated by apoplastic H2O2 production, and by concurrent stimulation of ROS-induced K? leak via NSCC, respectively (Fig. 10). Both effects have been previously reported in direct patch-clamp experiments on Arabidopsis roots (e.g. Demidchik et al. 2002). It remains to be shown that the same channels are present in the bean epidermis. In this study we used pharmacological approach to dissect blue light signal transduction pathway in the leaf

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epidermal tissue of the broad bean plant. Taking into account the fact that the specificity of inhibitors is not absolute, we proposed a tentative model for blue light signalling in broad bean epidermal cells. Data presented suggest the involvement of various plasma membrane/endomembrane ion channels and pumps in blue light signalling, i.e. plasma membrane K? channels (KIR, KOR), Ca2? channel (HACC), non-selective cation channels (NSCC), endomembrane Ca2? channel, plasma membrane pumps as P-type H?-ATPase, Ca2?-ATPase, endomembrane Ca2?-ATPase, and plasma membrane redox system (NADPH oxidase). It is also reasonable to include in the model blue light photoreceptors such as cryptochromes and phototropins. Additional electrophysiological and biochemical experimentation are required to confirm the involvement of all these components given in the hypothetical model of blue light signalling in broad bean epidermal tissue. Author contribution statement SS and BZˇ designed the experiments and wrote the manuscript. BZˇ performed MIFE measurements and analysed data. LS contributed to methodological aspects of this study and critically assessed the data. TE critically read the manuscript and contributed to data interpretation. Acknowledgments This work was supported by the Australian Research Council Discovery grant to Prof Sergey Shabala and partly by Grant No. 173040 from the Serbian Ministry of Education and Science. Conflict of interest of interest.

The authors declare that they have no conflict

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