Fast, transient and specific intracellular ROS changes in living root hair cells responding to Nod...

Fast, transient and specific intracellular ROS changesin living root hair cells responding to Nod factors (NFs)

Luis Cardenas*, Adan Martınez, Federico Sanchez and Carmen Quinto

Departamento de Biologıa Molecular de Plantas, Instituto de Biotecnologıa, Universidad Nacional Autonoma de Mexico,

UNAM, Apartado Postal 510-3, Cuernavaca, Morelos 62271, Mexico

Received 11 April 2008; revised 23 June 2008; accepted 21 July 2008; published online 27 August 2008.*For correspondence (fax 52 + 777 3136600; e-mail [email protected]).

Summary

The role of reactive oxygen species (ROS) in root-nodule development and metabolism has been extensively

studied. However, there is limited evidence showing ROS changes during the earliest stages of the interaction

between legumes and rhizobia. Herein, using ratio-imaging analysis, increasing and transient ROS levels were

detected at the tips of actively growing root hair cells within seconds after addition of Nod factors (NFs). This

transient response (which lasted up to 3 min) was Nod-factor-specific, as chitin oligomers (pentamers) failed

to induce a similar response. When chitosan, a fungal elicitor, or ATP was used instead, a sustained increasing

signal was observed. As ROS levels are transiently elevated after the perception of NFs, we propose that this

ROS response is characteristic of the symbiotic interaction. Furthermore, we discuss the remarkable spatial

and temporal coincidences between ROS and transiently increased calcium levels observed in root hair cells

immediately after the detection of NFs.

Keywords: reactive oxygen species, polar growth, root hairs, Nod factors, nodulation.

Introduction

Legumes can acquire nitrogen through a symbiotic inter-

action with rhizobial bacteria. This process involves a

molecular dialogue between the two partners, in which

legume roots exude flavonoids that induce the expression of

bacterial nodulation genes encoding proteins involved in the

synthesis and secretion of specific lipochitooligosacchar-

ides: the so-called Nod factors (NFs). These NFs signal back

to the plant root, and trigger several responses such as ion

changes (K+, Cl), Ca2+and H+), cytoplasmic alkalinization,

calcium oscillations and gene expression that lead to bac-

terial invasion and nodule formation (Cardenas et al., 2000;

Oldroyd and Downie, 2004).

In plants, reactive oxygen species (ROS) modulate

numerous biological processes such as growth, cell cycle,

programmed cell death, plant defense, hormone signaling,

biotic and abiotic stress responses and development. ROS

have also been found to play a key role in regulating polar

growth in root hair cells, fucus zygotes and pollen tubes

through their ability to regulate calcium channels, which are

involved in maintaining the apical calcium gradient (Coelho

et al., 2008; Foreman et al., 2003; Jones et al., 2007; Potocky

et al., 2007). On the other hand, ATP has also emerged as an

important modulator of NADPH oxidases in plant cells (Kim

et al., 2006; Song et al., 2006). In fact, extracellular ATP has

been found to modulate intracellular calcium and the levels

of ROS in root hair cells (Jeter et al., 2004; Kim et al., 2006;

Song et al., 2006).

Whereas compelling evidence indicates that NFs are

signal molecules secreted by bacteria that induce various

plant cellular responses, it remains unknown whether such

rhizobial signal molecules may also elicit pathogen-like

responses. It has been proposed that plant pathogenesis

and symbiosis are variations on a common theme (Baron

and Zambryski, 1995). In this regard, previous results

indicate that rhizobia might be initially recognized as

intruders that somehow evade or overcome the plant

defense response. Indeed, several plant defense responses

are induced during root nodule ontogeny (Gamas et al.,

1998; Parniske et al., 1990; Vasse et al., 1993). In alfalfa, not

all infection threads culminate in successful infection; many

abort even compatible symbiotic interactions because of the

hypersensitive response triggered in the root cortex (Vasse

802 ª 2008 Universidad Nacional Autonoma de MexicoJournal compilation ª 2008 Blackwell Publishing Ltd

The Plant Journal (2008) 56, 802–813 doi: 10.1111/j.1365-313X.2008.03644.x

et al., 1993). In peas, the plant defense response has been

proven to be far more critical for arresting the advancement

of rhizobial infection during incompatible interactions

(Perotto et al., 1994). Wisniewski et al. (2000) proposed that

diamine oxidase (DAO) could be a potential source for

hydrogen peroxide (H2O2), which might be used by cell wall

peroxidase (POD) for hardening the glycoprotein matrix

(MGP). Together, these data indicate the importance of ROS

metabolism during symbiotic interactions (Wisniewski

et al., 2000).

Additional evidence for the role of ROS signaling during

rhizobia–legume interactions has been described in Medi-

cago truncatula, where a peroxidase gene (rip1) is induced in

the presence of NFs from Sinorhizobium meliloti (Cook

et al., 1995). This peroxidase has a cis domain that is ROS

sensitive (Ramu et al., 2002). It is interesting that rip1

transcription and ROS production co-localize in the same

root cells (Ramu et al., 2002). In fact, exogenous H2O2 can

induce rip1 expression, suggesting that cis elements (OCS

and OBP) in the rip1 gene are required for induction after

ROS production. Furthermore, it has been demonstrated

that ROS and ethylene are part of the NFs-induced signal

cascade involved in nodule development in Sesbania

rostrata (D’Haeze et al., 2003).

During the early stages of M. truncatula–S. meliloti sym-

biosis, the oxidation of nitroblue tetrazolium (NBT) was

detected within the infection threads, indicating that O2) is

produced during this process (Santos et al., 2001). Further-

more, it is well known that there is an accumulation of H2O2

during the infection process and during nodule senescence

(Rubio et al., 2004). Accordingly, it is plausible that rhizobia

should have a mechanism to respond against ROS accumu-

lation in plants. In this regard, bacterial ROS scavenging

enzymes such as superoxide dismutase, glutathione-

S-transferase and catalase seem to play a protective role in

the legume–rhizobia interaction (Jamet et al., 2003; Ramu

et al., 2002; Santos et al., 2000; Sigaud et al., 1999). The

increased activity of these ROS scavenging enzymes in

S. meliloti appears to be essential for the regulation of

intracellular ROS production during the establishment and

maintenance of symbiosis with the plant host (Jamet et al.,

2003).

Paradoxically, decreased ROS production in response to

treatment with specific NFs has been also reported in

M. truncatula (Lohar et al., 2007; Shaw and Long, 2003b).

However, these results were obtained several minutes after

the addition of NF, and ROS levels were measured not from

a single cell, but from whole sectioned roots. These

apparently contradictory results prompted us to analyze

ROS responses at the subcellular level using single living

root hair cells from the responsive root region. In addition,

improved cell imaging methodology was used to measure

ROS levels within seconds after the addition of NFs. Herein,

we used a semi-quantitative ratio-imaging approach to

show that intracellular ROS levels transiently increased in

single living root hair cells within a few seconds after

treatment with NFs. This response was specific for NFs, and

was different from that induced by chitosan, a fungal elicitor,

ATP or chitin pentamers that constitute the non-active

backbone of the NFs.

Results

Growing root hairs exhibit a tip-localized ROS distribution

Single growing root hair cells were loaded with different

concentrations of an ROS-sensitive fluorescent dye (CM-

H2DCFDA) in order to achieve optimal loading conditions

that did not alter growth and root hair morphology. Root hair

cells imaged under these conditions presented a tip-loca-

lized ROS signal. Figure 1 (inset) shows the co-localization of

differential interference contrast (DIC), and the fluorescent

image indicates where the ROS distribution extends to in the

subapical region. The location of this signal is similar to that

previously described in root hairs from Arabidopsis thaliana

(Foreman et al., 2003), and hence we considered this to be a

typical ROS distribution for an actively growing root hair

cell. Furthermore, time-lapse imaging microscopy demon-

strated that ROS levels in growing root hair cells remained

unaltered in time, with only discrete fluctuations (Figure 1).

In contrast, when root hair cells entered the mature stage,

the ROS levels were notably decreased and were completely

absent in non-growing cells (data not shown).

NFs induce a rapid and specific transient increase

in intracellular ROS levels

Actively growing Phaseolus vulgaris root hair cells from root

zone II (Heidstra et al., 1994) were analyzed after loading the

roots with the ROS-sensitive dye, under conditions pre-

viously described. After root hair cells showed a typical ROS

distribution (Figure 2a, left-hand image), they were treated

with Rhizobium etli NFs (10)9M). Rapid intracellular ROS

elevation at the apical region of the cell was observed; ROS

levels started to increase 15 s after the addition of NFs to the

growth medium, and reached a maximum level after 1 min

(Figure 2a, middle image; Figure S1). Thereafter, the signal

decreased to basal levels (Figure 2a, right-hand image).

Similar results were obtained when 15 independent root hair

cells were analyzed (Figure 2b). ROS elevation occurred

transiently within the first 3 min, and subsequently declined

to basal intracellular levels that were finally attained

approximately 4 min after treatment with NFs (Figure 2b).

The ROS signal decreased to below the basal level by 10 min

after addition; this coincided with the arrest of root hair

growth, and with the typical swelling response observed at

the apical dome (data not shown). ROS signals could not be

detected in young (emerging) root hair cells from root zone I

ROS changes in root hairs responding to NFs 803

ª 2008 Universidad Nacional Autonoma de MexicoJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 56, 802–813

Figure 1. Intracellular reactive oxygen species

(ROS) levels observed in growing Phaseolus

vulgaris root hairs.

Inset: an overlay of a DIC and the corresponding

fluorescent image from a root hair cell; the tip-

localized intracellular ROS distribution was

revealed using an ROS-sensitive dye (CM-

H2DCFDA). The drawing indicates the region of

interest (ROI) where the measurement was

performed. Red and blue colors indicate high

and low intensities, respectively. Scale bar:

15 lm. The graph illustrates the relative ROS

values pooled from 15 different individual root

hair cells, and shows that the fluorescent signal

remained invariable over time.

(b)

(a) Figure 2. Intracellular reactive oxygen species

(ROS) changes in living Phaseolus vulgaris root

hairs after treatment with Nod factors (NFs).

(a) The left-hand image shows a growing root

hair cell loaded with CM-H2DCFDA dye and

presenting a tip-localized ROS signal (t = 0).

The middle image depicts the same root hair cell

treated with Rhizobium etli NFs (t = 1 min): the

intracellular ROS levels rapidly increased within

the first 10–15 s, and peaked after 1 min; there-

after, basal ROS levels were achieved after 3 min,

as observed in the right-hand image. Red and

blue colors indicate high and low ROS concen-

tration levels, respectively. Scale bar: 15 lm.

(b) Time course analysis of ROS production in

root hair cells treated with growth medium

containing NFs (10)9M) or chitin oligomers

(10)9M); a control with only growth medium

was also included. Images were analyzed for

relative intensity values at the tip of each root

hair cell. Pooled data from 15 different cells are

shown.

804 Luis Cardenas et al.


(data not shown), because this root region has particularly

elevated ROS levels (Shin et al., 2005) that mask single-cell

measurements.

The observed transient ROS responses were specifically

induced by NFs, as the addition of growth medium alone or

growth medium containing chitin oligomers (pentamers)

did not induce any response (Figure 2b). Accordingly, chitin

oligomers were used as a negative control for the rest of

the study.

H2O2 and UV radiation induced similarly increased ROS

levels in root hair cells

In order to test if intracellular ROS levels could be artificially

increased in root hairs, exogenous hydrogen peroxide

(50 lM) was added to the growth medium. Under these

conditions, a sustained increase in intracellular ROS levels

was observed after 3 min (Figure 3). Intracellular ROS levels

were also artificially increased in root hair cells after a 5-s

pulse of UV light (340 nm), as previously reported (Dixit and

Cyr, 2003; Mackerness et al., 2001) (Figure 3). This con-

tinuously increasing ROS signal was similar to that observed

after the addition of H2O2. Apparently, root hair cells could

only overcome UV and H2O2 treatment within the first 3 min;

after this time there was a dramatic increase in intracellular

ROS levels (Figure 3).

In contrast, the transient ROS response induced in root

hairs after treatment with NFs (Figure 2) was different from

that observed with exogenously applied H2O2, or after UV

irradiation (Figure 3). A sustained and increasing ROS signal

that eventually saturated the probe was observed instead

(Figure 3), suggesting that these cells were dying. At the

cellular level, root hair cells treated in this way became

vacuolated, and a swelling response was usually observed

(data not shown). Collectively, these data confirm that the

conditions and the fluorescent dye concentration used

adequately probed the levels of intracellular H2O2 and free

radicals in living root hair cells under different conditions.

Chitosan induced sustained increases in intracellular

ROS levels

In order to assess whether the ROS levels observed in root

hairs after treatment with NFs were specific for the symbiotic

interaction, a fungal elicitor was added instead. Chitosan,

which is widely used to induce a hypersensitive response in

plants, was added to root hair cells that were previously

treated with the ROS-sensitive dye (Figure 4, left-hand

image), and ROS levels were analyzed under the aforemen-

tioned conditions. ROS levels were dramatically increased

(Figure 4, middle image) by 5 min after treatment with chit-

osan, and reached their highest level by 9 min (Figure 4,

right-hand image). This sustained and increased response

was similar to that induced by UV light radiation and H2O2

treatments (Figure 3). It is worth mentioning that besides

causing a dramatic increase in intracellular ROS levels, chit-

osan also markedly reduced cytoplasmic streaming by

10 min after its addition (data not shown). Intracellular ROS

levels in root hair cells were entirely different when com-

paring transient increases after NF treatment (Figure 4b) with

sustained increases after elicitor treatment (Figure 4a,b).

Ratio-imaging of intracellular ROS levels reveals a

tip-focused signal that responds to treatment with NFs,

and is inhibited by diphenylene iodonium (DPI)

The ROS-sensitive dye (CM-H2DCFDA) used in the experi-

ments previously described is a single-wavelength dye that

Figure 3. Reactive oxygen species (ROS)

changes in response to hydrogen peroxide

(H2O2) and UV radiation.

Root hair cells loaded with CM-H2DCFDA dye

and treated with 50 lM exogenously applied

H2O2 (black squares) or for 5 s with UV radiation

at 340 nm (grey circles) were immediately im-

aged after treatment. These cells responded with

sustained increased ROS levels, and both treat-

ments induced similar responses.



does not allow ratio-imaging. In order to account for the

variably accessible cytoplasmic volume of root hair cells, we

introduced a reference dye (see Experimental procedures for

the pseudo-ratio-imaging analysis. This approach was used

to depict, in a semi-quantitative way, the subcellular ROS

distribution in growing root hair cells. We found that in-

tracellular ROS levels were indeed focused to the apical

dome (Figure 5, inset, left-hand image), and were drastically

increased in response to NFs with a transient pattern that

peaked after 1 min (Figure 5, inset middle image; Figure S2).

This response resembled that obtained previously with the

non-ratio-imaging approach (Figure 2, middle image), and

was very consistent in 10 of 12 cells; moreover, this ratio

analysis allowed the visualization of the subcellular dis-

tribution before and after treatment with NFs. It is interesting

that at the end of the transient response, the intracellular

ROS levels remained higher than the initial values observed

before treatment with NFs (Figure 5).

DPI is an inhibitor of NADPH oxidases and flavin-contain-

ing enzymes, and has been widely used to reduce intracel-

lular levels of ROS in plant cells (Foreman et al., 2003; Lohar

et al., 2007; Shaw and Long, 2003b). Growing root hair cells

have constant intracellular levels of ROS that did not change

when chitin pentamers were used as a control (Figure 6).

However, with DPI treatment, the intracellular levels of ROS

were drastically reduced after 4 min, although they did not

completely disappear, and remained at the basal level after

10 min of treatment with DPI (Figure 6). When root hairs

(a)

(b)

Figure 4. Differential intracellular reactive oxy-

gen sepcies (ROS) responses obtained with

treatment with chitosan and Nod factors (NFs).

(a) The left-hand image depicts a growing root

hair cell loaded with CM-H2DCFDA and showing

the typical tip ROS distribution (t = 0). The

middle image represents the same root hair cell

after treatment with chitosan: the cell presented

increased ROS levels. The right-hand image

illustrates elevated ROS levels observed after

9 min of chitosan treatment. The red color

indicates high levels of ROS. Scale bar: 15 lm.

(b) Graph showing that treatment with chitosan

induced a continuous increase in intracellular

ROS. The ROS level did not return to the initial

values (red squares). Conversely, the addition of

NFs induced transient ROS induction (black

diamonds). Chitosan or NFs were added at the

beginning of the series (t = 0).



showed basal levels of ROS (after 10 min of treatment with

DPI) they were challenged with NFs, but specific transient

changes in ROS were not observed (Figure 5). The absence

of transient ROS induction after treatment with NFs in root

hairs previously treated with DPI suggests that the activation

of NADPH oxidases is closely linked to the perception and

signaling of NFs.

Root hair cells treated with DPI showed arrested growth

and, eventually, root hair swelling (data not shown). As

organelles such as chloroplasts and mitochondria with

highly oxidizing metabolic activity or high electron flow

are also important sources of ROS production in plant cells,

we explored the subcellular distribution of mitochondria

in growing root hair cells using a mitochondria-specific

fluorescent probe (Mitotracker Green). Mitochondria were

distributed throughout the cell, but were more abundantly

localized at the apical region of the root hair cells (Figure 6,

see inset), which coincides with the region in which the ROS

signal was observed. Thus, we propose that these mito-

chondria could partially contribute to ROS production in this

region, including basal production observed after DPI

treatment.

Extracellular ATP modulates intracellular levels of ROS

Extracellular ATP has been described as inducing changes in

intracellular levels of ROS (Song et al., 2006); however, the

subcellular distribution of these ROS have not been

described. Root hair cells previously loaded with ROS-sen-

sitive and reference dyes were challenged with 10 mM ATP.

Under these conditions a continuous increase of apical ROS

levels was observed (Figure 7), which was eventually

Figure 5. Root hairs pre-treated with dipheny-

lene iodonium (DPI) did not display the transient

intracellular reactive oxygen species (ROS)

response.

Root hair cells were treated with a reference dye

(Cell Tracker Red) and ROS-sensitive dye (CM-

H2DCFDA) for ratio-imaging of the spatial dis-

tribution of ROS. Root hair cells that were

preliminarily treated with DPI and then chal-

lenged with Nod factors (NFs) did not show the

transient ROS response (gray trace). Root hair

cells under the same conditions without DPI

showed the typical transient response after

treatment with NFs. This transient response

was observed in 10 of 12 cells. The inset shows

the typical subcellular distribution before treat-

ment with NFs (left image), at the peak of the

response (middle image) and after the transient

response (right-hand image). Note that the

intracellular ROS levels remained slightly ele-

vated at the end of the experiment. Scale

bar: 15 lm.

Figure 6. Diphenylene iodonium (DPI) gradually

reduced the levels of intracellular reactive oxy-

gen species (ROS).

Root hair cells previously treated with DPI were

closely monitored for production of intracellular

ROS after 4 min (we did not observe a change

prior to that time); as depicted, DPI treatment

resulted in gradually decreased levels of cyto-

plasmic ROS after 4 min, but ROS production

was not abolished the (black circles). Root hairs

treated with pentamers as a control did not show

any response (gray diamonds). Inset, a DIC

image of a living root hair cell (left-hand image)

treated with Mitotracker Green to follow the

mitochondrial distribution (right-hand image).

Note that the apical region is enriched with

mitochondria. Scale bar: 15 lm.



accompanied by a higher cytoplasmic accumulation at the

tip region that sometimes resulted in cell bursting (data not

shown). Furthermore, repeated local delivery of 5 mM ATP in

the vicinity of the root hair cell using a blunt needle resulted

in transient increases in the intracellular levels of ROS

(Figure 7).

Discussion

In the present study, the production and distribution of

intracellular ROS, as well as the specific response to NFs,

were analyzed in growing P. vulgaris root hair cells. Inter-

estingly, ROS levels were dramatically and transiently

increased within a few seconds after treatment with NFs.

The response was specific for NFs, and was clearly different

to that observed after the addition of H2O2, ATP or chitosan,

or after exposure to UV radiation.

As ROS can be used as intracellular signals, all organisms

have developed several ways to counteract their deleterious

effects (Mittler, 2002; Neill et al., 2002; Vieira Dos Santos and

Rey, 2006). The ability of cells to suppress ROS toxicity, and

to decode the strength and amplitude of the signal, requires

many genes involved in regulating the ROS homeostatic

response (Mittler et al., 2004). The functions of ROS signal-

ing in eukaryotic cells include the regulation of cell

migration, growth modulation, opening and closing of ion

channels, gene expression, development and programmed

cell death (Foreman et al., 2003; Mittler et al., 2004; Ushio-

Fukai, 2006). As ROS easily diffuse across the plasma

membrane, and have a short half-life, rapid localization at

the subcellular level is crucial for understanding the nature

of the signaling events after a given stimulus. As the ROS-

sensitive dye (CM-H2DCFDA) is a non-ratiometric and single-

wavelength dye, it was necessary to use an additional dye as

a reference for ratio-imaging. Using this approach, we were

able to register transient ROS changes induced by NFs in a

semi-quantitative way. In this work, we showed that normal

root hair cells from P. vulgaris present a tip-localized ROS

signal when analyzed with a pseudo-ratio-imaging app-

roach. These ROS changes transiently occurred within a few

seconds, and lasted up to 3 min, on average, after root hairs

were challenged with NFs. In addition, we found that this

response was localized to the tip of the growing cell. This

apical response suggests an important role for ROS during

the perception of NFs, probably in regulating polar growth,

as reported in Arabidopsis, pollen tubes and focus zygotes

(Cardenas et al., 2006; Coelho et al., 2008; Foreman et al.,

2003; Potocky et al., 2007; Takeda et al., 2008). Transient

ROS changes might participate in decoding the rhizobial

signal as a part of symbiosis. Although this idea has been

previously proposed (Ramu et al., 2002), responses at the

subcellular level have not been observed. By using a

pseudo-ratiometric approach, it was possible to subcellu-

larly visualize transiently increased ROS levels in root hair

cells responding to NFs. Interestingly, intracellular ROS

levels remained higher than the initial values. This suggests

that increased ROS levels could be necessary for inducing

expression of some early nodulins, as previously suggested

(Ramu et al., 2002). Early nodulin expression could be

involved in morphological changes that anticipate infection

thread formation, or in the regulation of infection thread

number, thereby allowing bacteria to enter root hair cells

when they are able to circumvent the high basal levels

of ROS.

Multiple sources for ROS production inside cells (Ushio-

Fukai, 2006) have been described. In particular, it is known

that mitochondria and chloroplasts are major sources

for generation of ROS as a result of intense membrane

Figure 7. ATP induced an increase in the levels

of intracellular reactive oxygen species (ROS).

Root hair cells loaded with the ROS-sensitive dye

and the reference dye were exposed to 10 mM

ATP; intracellular ROS increased constantly until

reaching a constant value (black circles). Root

hair cells under the same conditions, but

exposed to rapid 5 mM ATP perfusion (3 s)

showed transiently increased ROS levels (gray

diamonds). This response was reproduced when

cells were perfused again with 5 mM ATP.

Arrows indicate when ATP was added.



electron flow (Mittler et al., 2004). Plant NADPH oxidases

have been shown to be the main source of ROS during

pathogen attack and UV irradiation (Rao et al., 1996; Sagi

and Fluhr, 2001, 2006; Torres et al., 2005). In this work, we

showed that root hairs from P. vulgaris showed decreased

intracellular ROS levels when previously treated with DPI

(Figure 6, black trace). However, a basal level is main-

tained, indicating that other sources such as plant cell wall

diamine oxidase and mitochondria could engage in

cytoplasmic production of ROS (Wisniewski et al., 2000).

In fact, we observed that mitochondria were located more

abundantly at the tip region of the root hair (Figure 6,

inset), and might potentially contribute to generation of

ROS in the apical zone. Further analysis revealed that root

hair cells previously treated with DPI and subsequently

challenged with NFs failed to respond with transient

increases in intracellular levels of ROS (this work). This

result suggests that NADPH oxidases are most likely to be

the main source of intracellular ROS. The evidence above,

in addition to previous reports (D’Haeze et al., 2003; Ramu

et al., 2002), suggest that ROS production is intimately

related with the NFs perception pathway. Although it has

been shown recently that phosphatidyl inositol 3-Kinase

(PI3K) and phosphatidyl inositol-specific phospholipase C

(PI-PLC) inhibitors suppress ROS production, a clear

analysis of subcellular ROS distribution has not yet been

provided (Peleg-Grossman et al., 2007). Furthermore, it is

not known how early ROS homeostasis is executed at the

subcellular level, or what the differences are between

symbiotic and pathogenic responses (Levine et al., 1994).

As the ROS response was transiently observed after

treatment with NFs, root hair cells must not only have a

mechanism to increase the production of ROS, but must

also have a mechanism to efficiently remove or neutralize

them. ROS homeostasis can be achieved by scavenging

intracellular ROS or inhibiting their production. The

present finding of increased ROS production only seconds

after exposure to NFs seems to contradict the findings of

Lohar et al. (2007) recently reported in M. truncatula, at 1 h

after treatment with NFs. That study found decreased

expression of NADPH oxidase genes, which could account

for the reduced levels of ROS observed after 1 h (Lohar

et al., 2007; Shaw and Long, 2003b). However, it should be

noted that the previous experiments were started several

minutes after exposure to NFs, and did not explore the

immediate response. This consideration leads us to

suggest that NFs may both positively and negatively

modulate the intracellular levels of ROS. According to our

results, NFs might rapidly stimulate NADPH oxidase either

directly or indirectly in the early stages. This could be

followed later by inhibition, as demonstrated by Lohar

et al. (2007). It has been reported that DPI reduces

intracellular levels of ROS, and mimics the swelling

response induced by NFs (Lohar et al., 2007). It is possible

that decreased ROS levels are required later on during the

swelling response, but that higher concentrations might be

required during the very early stages of the symbiotic

process.

It has been reported that an Arabidopsis NADPH oxidase

(RBOH) has EF-hand motifs that are involved in calcium

binding (Keller et al., 1998; Sagi and Fluhr, 2001), and that

ROS can activate calcium channels (Foreman et al., 2003;

Mori and Schroeder, 2004). Furthermore, it has been

reported that the oscillating growth pattern of root hair cells

is coupled to extracellular pH and changes in ROS (Mon-

shausen et al., 2007): this suggests that ROS could be

involved in cell wall property changes that allow polarized

growth. The observed ROS gradient (this work) in the apical

region spatially correlates with the recently described

localization of NADPH oxidase at the apical plasma mem-

brane of root hair cells (Takeda et al., 2008). These data

support the idea that ROS and calcium are two key

interacting signaling elements required to modulate polar-

ized tip growth. It is plausible that the previously reported

elevated calcium influx observed after treatment with NFs

(Cardenas et al., 1999; Shaw and Long, 2003a) could be

involved in the transient increases in ROS observed in the

present study. In general, the EF-hand and calmodulin

binding domains in some NADPH oxidases are thought to

be well suited for this kind of regulation (Banfi et al., 2004;

Tirone and Cox, 2007). In fact, some calmodulins have been

shown to be differentially expressed in response to NFs

(Camas et al., 2002). However, there may be other mechan-

isms responsible for downregulating their function, as

NADPH oxidases, which are upregulated during a pathogen

attack, are modulated by different kinases, acting in a

sequential or parallel manner (Benschop et al., 2007). It will

be interesting to determine how this fine regulation is

accomplished, and whether ROS changes activate calcium

responses or vice versa. In this scenario, calcium and ROS

play synergistic key roles in modulating the early stages of

the symbiotic interaction.

That the signal of the ROS-sensitive dye (CM-H2DCFDA)

was decreased, even though it is irreversibly photooxidized,

might be the result of at least two factors. Firstly, the ROS-

generating mechanism is tip localized, which means that it

occurs in a very limited region of the root hair cells, and

could be downregulated after the transient response to NFs,

as suggested above, with the consequence that there is a

reduction in the generation of ROS. Secondly, the limited

region with increased ROS levels could be rapidly dissipated

by the strong cytoplasmic streaming and diffusion.

In animal cells, ATP induces ROS production by activating

NADPH oxidase (Dichmann et al., 2000; Pines et al., 2005). A

regulatory role for extracellular ATP in modulating cellular

ROS levels via activation of NADPH oxidases has been

recently suggested to occur in plant cells (Roux and

Steinebrunner, 2007), including root hair cells (Kim et al.,



2006; Song et al., 2006). Our results add weight to the idea

that ATP induces changes in intracellular ROS levels in root

hair cells, as previously suggested (Song et al., 2006), and

thus also favors the idea that ATP could be involved in polar

growth, as it can increase intracellular levels of ROS. Analysis

of the spatiotemporal correlation between extracellular ATP,

calcium and ROS changes in the early stages of symbiosis,

and also during pathogen attack, will help in the under-

standing of the signal pathways involved in each response.

Based on the findings of the present study, we propose that

the early production and distribution of ROS are closely

related to ATP and calcium signaling in legume root hair cells.

Indeed, increased levels of ROS (this work) and calcium

spatially and temporally coincide after treatment of root hair

cells with NFs (Cardenas et al., 1999; Shaw and Long, 2003a).

Pathogenesis and symbiosis have many common fea-

tures (Baron and Zambryski, 1995). The precise regulation of

ROS production seems to play a key role in successful

Rhizobium–legume interaction (Perotto et al., 1994; Santos

et al., 2001; Vasse et al., 1993). It is possible that by

regulating ROS production to occur at the right time and

place, rhizobia are allowed to enter the host plant without

triggering a hypersensitive response. A failure to control

ROS elevation might provoke an infection thread abortion

(Vasse et al., 1993). The response of root hair cells to NFs

with a transient ROS signature signal different from that

observed after treatment with chitosan, UV and H2O2

suggests that these cells can differentiate symbiotic from

pathogenic signals within seconds. The inability of intracel-

lular ROS to reach initial basal levels after the transient

response suggests that increased ROS levels could play a

role in the cellular responses. This idea is supported by

experiments involving treatment with DPI that showed an

inhibition of root hair curling and infection thread formation

(Peleg-Grossman et al., 2007).

Using a semiquantitative ratiometric approach in living

root hair cells, we established that specific transient

elevations in ROS occur at the tip within seconds of

treatment with NFs. This transient ROS signature could be

the earliest downstream signal after a symbiotic or patho-

genic molecular stimulus is perceived by the plant. It is

important to unravel the mechanisms underlying ROS

signaling during the early symbiotic interaction, in addition

to the intimate connections between calcium signaling,

extracellular ATP, cell wall peroxidase activities and poten-

tial cytoskeleton rearrangements triggered by transient

Ca2+ elevation, and ROS production.

Experimental procedures

Seed germination

Phaseolus vulgaris cv. Negro Jamapa seeds were surface-sterilizedwith sodium hypochlorite for 5 min, followed by five rinses with

sterile water, subsequent treatment with pure ethanol for 1 min andfive more rinses with water, as described in Cardenas et al. (1995).Sterile bean seeds were transferred to special plates with wet papertowels, covered with foil and then transferred to a growth chamberat 27�C. After 48 h, seeds were germinated and ready to use in ourexperiments.

Mounting the living root hairs

The 2-day-old seedlings were placed in liquid Fahreus medium atpH 6.0. After 8 h root hairs were usually well adapted to the med-ium. Intact seedlings containing the growing root hairs weremounted in chambers constructed from large Petri dishes. Thesewere perforated in the center, and the hole was covered with a largeglass cover slip glued with silicon. Seedlings were visualized undera Diaphot 300 (Nikon, http://www.nikon.com) inverted microscopewith an 40·/1 NA water immersion lens (Nikon). The chambercontained a layer of solid Fahreus medium (phytagel 0.8%), and theseedlings were set over this layer. At this point, growth medium wascontinuously perfused with a peristaltic pump at 0.2 ml min)1 (Bio-Rad, http://www.bio-rad.com). The total volume in the chamber wasmaintained in 2.5–3 ml, on average, and the root region wascovered with cellophane paper. The solid Fahreus medium andthe cellophane paper helped to prevent root movement.

Treatment of root hair cells with an ROS-sensitive

(CM-H2DCFDA) and reference dye (CMTPX)

In brief, the ROS-sensitive probe CM-H2DCFDA [5-(and-6)-chlor-omethyl-2¢,7¢-dichlorodihydrofluorescein diacetate, acetyl ester](Molecular Probes, http://probes.invitrogen.com) was dissolved inDMSO (Sigma-Aldrich, http://www.sigmaaldrich.com) and cen-trifuged for 2 min at 8000 g to remove non-dissolved particles.Then, the dye solution was diluted with Fahreus medium at a finalconcentration of 30–50 lM and then added to the plate, replacing theoriginal medium. After 15 min, the medium was replaced with freedye and measurements were performed. This procedure was car-ried out carefully to avoid any mechanical stress to cells. Whenperforming ratio-imaging, the reference dye Cell tracker� redCMTPX (Invitrogen, http://www.invitrogen.com) was dissolved inDMSO as a stock solution of 30 lM. Special plates containing theseedlings with the growing root hair cells were treated with theCMTPX dye for 2–3 h. After this time, the dyes were clearly locatedin the cytoplasm, and were excluded from the vacuoles (Figure S3).Then, the ROS-sensitive dye was added and observed 60 min laterunder the fluorescence microscope.

Treatment of root hair cells with DPI, H2O2, chitosan,

UV and ATP

Root hairs were treated with DPI (Sigma-Aldrich), which is an in-hibitor of NADPH oxidase and other flavin-containing enzymes. DPIwas dissolved in DMSO and used at 40 lM. H2O2 (Sigma-Aldrich)was freshly prepared each time and used at a concentration of50 lM. Chitosan (Sigma-Aldrich) was prepared according to themethod of Hadwiger and Beckman (1980), dissolved in water andthen cleaved by exposure to glacial acetic acid for more than 12 h.Then, the fragments were neutralized by adjusting the pH to 6.0(Hadwiger and Beckman, 1980). The resulting mixture was used at afinal concentration of 100 mg L)1 by diluting the stock solution withFahreus medium. Treatment with UV was automatically performedas follows: the UV light source was set to open for 5 s, and then the



shutter was closed so that we could open the path for excitation at485 nm, take an image and visualize the intracellular ROS level ateach point. ATP was prepared as a 0.1 M stock solution in Tris bufferat 180 nM as follows: 45 ll of 1 M Tris base plus 205 ll of dH2O weremixed carefully and then transferred to an Eppendorf tube con-taining 14 mg of ATP with shaking for 30 s until all of the ATP wasdissolved; aliquots were stored at )80�C.

Incubation of root hairs with NFs

Rhizobium etli NFs were purified by HPLC and applied to plant rootsas previously described (Cardenas et al., 1995). Before treatment,NFs were mixed with 0.5 ml of the same medium that the seedlingshad been growing in, and were then added to the growing root hairswith a peristaltic pump to replace the NF-free medium. As a nega-tive control, 10)8

M penta-N-acetylchitopentaose (Seikagaku, http://www.seikagaku.co.jp) dissolved in CHAPS {non-denaturing, zwit-terionic detergent [3-(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate; Sigma-Aldrich} was used.

Image acquisition and processing

All images were acquired with a CCD camera (Sensys; RoperScientific, http://www.roperscientific.com) attached to a NikonTE300 inverted microscope with a 40·/1 NA water immersionobjective lens. Loaded cells were excited using a xenon illumina-tion source (DG-4; Sutter Instruments, http://www.sutter.com),which contained a 175 W ozone-free xenon lamp (330–700 nm) anda galvanometer for a wavelength switch. The ROS-sensitive dyeexcitation was 485 nm and the emission was collected at 535 nm(bandpass: 25 nm). The CMTPX dye was excited at 555 nm and theemission was collected at 605 nm (bandpass: 30 nm). The besttemporal and spatial resolutions were achieved when cells wereexposed for 15–20 ms at a 490-nm excitation wavelength, with a2 · 2 binning of the CCD camera. Images were acquired eachsecond, which was fast enough to detect immediate changes inROS levels. These short exposure times were crucial; otherwisephoto-oxidation would have contributed a continuous signal in-crease (Figure S4). All of the filters used were from ChromaTechnology (http://www.chroma.com), and emission filters werelocated in a filter wheel (Sutter Instruments, http://www.sutter.com). The set-up was automatically controlled by METAMORPH/METAFLUOR software (Universal Imaging, http://www.moleculardevices.com). Finally, images were prepared for publication usingAdobe PHOTOSHOP software (http://www.adobe.com).

Acknowledgements

This work was supported by grants from Direccion General deAsuntos del Personal Academico (DGAPA), Universidad NacionalAutonoma de Mexico, Nos IN228903 (LC) and IN204305 (CQ);CONACyT 58323 (LC), 42560-Q (CQ) and 42562-Q (FS). We thankLiliana Martinez, Maria Luisa Barroso, Olivia Santana and NoreideNava for technical support. We also thank Dr Otto Geiger, ChrisWood and Peter K. Hepler for their critical reading of the manuscript.

Supporting Information

Additional Supporting Information may be found in the onlineversion of this article:Figure S1. A time series of a living root hair cell from Phaseolusvulgaris responding to Nod factors (NFs) from Rhizobium etli.

Figure S2. A time series of a living root hair cell from Phaseolusvulgaris responding to Nod factors (NFs) using a pseudo-ratio-imaging approach.Figure S3. Root hair cells showing the cytoplasmic distribution ofthe reactive oxygen species (ROS)-sensitive (left-hand image) andreference dyes (middle image). The DIC image that depicts thecytoplasm distribution is also included (right-hand image).Figure S4. Effect of exposure time in root hair cells loaded with areactive oxygen species (ROS)-sensitive dye and a reference dye forratiometric imaging of intracellular ROS levels.Please note: Wiley-Blackwell are not responsible for the content orfunctionality of any supporting materials supplied by the authors.Any queries (other than missing material) should be directed to thecorresponding author for the article.

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