Reactive oxygen species (ROS) as early signals in root hair cells responding to rhizobial nodulation factors

Abstract

Reactive oxygen species (ROS) are involved in supporting polar growth in pollen tubes, fucoid cells and root hair cells. However, there is limited evidence showing ROS changes during the earliest stages of the interaction between legume roots and rhizobia. We recently reported using Phaseolus vulgaris as a model system, the occurrence of a transient increase of ROS, within seconds, at the tip of actively growing root hair cells after treatment with Nod factors (NFs).¹ This transient response is NFs-specific, and clearly distinct from the ROS changes induced by a fungal elicitor, with which sustained increases in ROS signal, is observed. Since ROS levels are transiently elevated after NFs perception, we propose that this ROS response is specific of the symbiotic interaction. Furthermore, the observed ROS changes correlate spatially and temporarily with the reported transient increases in calcium levels suggesting key roles for calcium and ROS during the early NF perception.

The symbiotic interaction between rhizobia and plant legumes entails a molecular dialogue. Legume roots exude flavonoids that induce the expression of bacterial nodulation genes, which encode proteins involved in the synthesis and secretion of Nod factors (NFs) (reviewed in ref. 2). NFs are perceived by the plant root, which in turn exhibit several responses such as ion fluxes (K⁺, Cl⁻, Ca²⁺, H⁺), cytoplasmic alkalinization, cytoplasmic calcium oscillations ([Ca²⁺] c), and gene expression,³ leading to bacterial invasion and nodule formation.

In the last decade, convincing evidences have appeared indicating that in plants as well as in animals, an elevated production and accumulation of reactive oxygen species (ROS) accompanies various processes, such as: development, hypersensitive response, hormone action, gravitropism and stress responses.⁴ In support of these findings, Ca²⁺-permeable channel modulation by ROS was demonstrated in Vicia faba guard cells and recently in Arabidopsis, where ROS regulated calcium channels are also active in sustaining plant cell growth.^5,6

It has been widely reported that in plants, the ROS production accumulate in the apical region of tip growing cells such as pollen tubes and root hair cells, as well as Fucus and fungal hyphae.^6–9 Furthermore, patch-clamp studies showed that ROS can regulate calcium channels.⁶ Arabidopsis mutants in NADPH oxidase are characterized by stunted or absence of root hairs,¹⁰ and fail to exhibit the tip-localized ROS gradient. Since NADPH oxidase contains calcium ion-binding EF hand domains, it is plausible that its regulation is calcium dependent. However, the role of calcium ions in NADPH oxidase activation in root hair cells remains unknown.

In legumes, ROS levels are elevated during the rhizobial infection, specifically in the developed nodules,¹¹ as well as during the preceding stages, such as the infection thread formation,^12,13 a process that is usually initiated after 72 h after Rhizobium inoculation. Furthermore, it has been proposed that the failure to produce and maintain proper ROS concentrations results in infection thread abortion.¹⁴ Conversely, other studies reported a decrease in intracellular ROS levels in root hair cells treated with NFs.^15,16 However, these studies were done at different time scales and the processes observed were different; while Shaw et al., (2003) and Lohar et al., (2007) made the observations on ROS levels in root hair cells several minutes after exposure to NFs during the swelling response, Santos et al., (2001) and Ramu et al., (2002) looked at the root hairs forming infection threads induced by Rhizobium inoculation. Using Phaseolus vulgaris, we recently demonstrated that living root hair cells show changes in ROS levels within few seconds after NFs addition.¹ This work was carried out by using a ROS sensitive dye (CM-H2DCFDA, Invitrogen). This fluorescent dye has been widely used as a ROS indicator dye; nevertheless, limitations are encountered when it is used as a single wavelength dye. This is due to the accessible volume in root hair cells, which usually present an increased cytoplasmic accumulation at the tip region. However, we have circumvented this problem by using the ROS sensitive CM-H2DCFDA dye in combination with a reference dye (Cell Tracker Red, Invitrogen) to establish a pseudo-ratiometric analysis. This allowed us to visualize the subcellular distribution of the ROS signal in living root hair cells, which now can be described as a tip-localized gradient.

In summary, the production and distribution of intracellular ROS levels were analyzed in P. vulgaris growing root hair cells as well as their responses to NFs. We found that ROS levels were dramatically and transiently increased within a few seconds after NFs treatment. This response is specific for NFs, and clearly distinct from those observed after the addition of chitin oligomers (pentamers).¹ It is possible that the modulation of ROS production in epidermal cells enables rhizobia 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). Root hair cells responded to the presence of NFs with a transient ROS signature signal, in a different way than observed after chitosan treatment. These results suggest that within seconds these cells are able to perceive differentially, symbiotic signals from pathogenic fungal elicitors. Therefore, this transient ROS signature specifically triggered by NFs could play a key role in modulating the rhizobia-legume interaction.

In root hairs, the NF-induced transient increase in the ROS levels, which is initiated 15 sec after NFs treatment clearly precedes the onset of the observed [Ca²⁺]c oscillations in the perinuclear region that occur 10–15 min later. Therefore, the transient ROS response can be considered as one of the earliest responses to NFs. However, these ROS changes correlate spatially and temporally with the described calcium influx at the tip region of root hair cells responding to NFs (Fig. 1), suggesting a connection for both the players during the NFs signal perception. So far we do not know if the ROS response is earlier or subsequent to the [Ca²⁺]c increases (fluxes) observed at the tip region of legume root hair cells treated with NFs. However, it is possible that a feed back response is established as suggested in Arabidopsis root hair cells.¹⁷ In this scenario, the transiently induced ROS changes observed after NFs treatment could stimulate the calcium channels located at the tip region (Fig. 1, inset). This would result in the transiently increased [Ca²⁺]c levels observed at the tip region after NFs treatment, which in turn could activate the NAD(P)H oxidase leading to the generation of more ROS. The generated ROS could then stimulate the calcium channels at the tip region triggering signalling cascade.

Figure 1.

Model on the earliest responses observed at the tip of root hair cells after NFs treatment. Note that [Ca²⁺]c and ROS at the tip region respond with a transient changes in their levels within a few seconds after NFs treatment, while [Ca²⁺]c oscillations in the perinuclear region follow after 10–15 min. Inset depicts a model where the generated ROS could activate the calcium channels located at the tip region and this in turn could generate an increase in [Ca²⁺]c. Increased [Ca²⁺]c then activates the NAD(P)H oxidase maintaining increased ROS levels.

Addendum to: Cárdenas L, Martinez A, Sanchez F, Quinto C. Fast transient and specific intracellular ROS Changes in root hair cells responding to Nod factors (NFs) Plant J. 2008 doi: 10.1111/j.1365-313X.2008.03644.x. In press.