New ideas on root hair growth appear from the flanks

Plant root hairs are experimental model systems for research on plant cell growth. The involvement of protons and reactive oxygen species (ROS) in root hair growth and development has been the subject of significant research effort (e.g., refs. 1–4). pH specifically has been implicated in the control of plant cell expansion (5), including that in root hair cells (6). ROS have recently come to the fore with the report that a specific respiratory burst oxidase homologue (RBOHC) of Arabidopsis is necessary for the establishment of the tip-based calcium gradient known to be essential for root hair polar growth (1, 7–11). In this issue of PNAS, Monshausen et al. (12) take great strides in furthering understanding of the specific roles played by both protons and ROS in root hair growth in Arabidopsis.

Monshausen et al. (12) report that root hair growth is not a constant process; instead it occurs as discrete episodes of growth followed by stasis. By measuring the external pH around the surface of the root hair apex, the researchers demonstrated that each period of growth correlated with alkalization of external pH. This external alkalization coincided with internal cellular acidification, suggesting an influx of protons into the root hair cell. Because this influx was coincidental with the growth phase, the authors advance the hypothesis that acidification of the apoplast leads to the capacity for polar growth during the elongation phase. This growth occurs through the loosening of cell wall components in specific zones of the growing root hair, i.e., just behind the root hair apex. In this way, root hair growth can be limited to only the tip, with the majority of the cell wall at the flanks of the root hair reinforced to prevent expansion. Consistent with this idea was the demonstration that an artificially generated elevation of external pH led to cessation of growth (caused by global rigidification of the cell wall), whereas reduction in pH led to bursting of hair cells (caused by nonlocalized/unregulated cell expansion). These data are consistent with previous work on the pH regulation of cell wall properties (5, 6). The spatiotemporal dissection of dynamic growth and internal/external pH changes are elegantly demonstrated (12) and represent important advances in thinking about root hair growth.

Monshausen et al. (12) also reveal a new role for ROS in root hair growth. Here, a situation analogous to that with pH was demonstrated. In this case, oscillations of apoplastic ROS concentration at the top of the flanks of the root hair (not at the apex itself) were detected. The troughs of ROS oscillations were correlated to the growth phases of the root hair. Again, artificial manipulation of ROS concentration revealed the mechanism and causality of ROS action. Increasing or reducing apoplastic ROS levels led to inhibition of root hair growth or bursting, respectively (12). These data are consistent with the reported role for ROS in regulating cell wall extensibility (13, 14). The authors naturally decided to investigate the role of RBOHC in these processes, because it had been previously implicated in ROS-dependent root hair growth (1, 4, 11). They found that, as reported previously, rbohc mutants showed reduced numbers of elongated root hairs under standard conditions (1, 4, 7, 10, 11). Some previous reports suggest that root hair development in rbohc is arrested after initiation (1, 7, 11), but others report that rbohc root hairs burst after initiation (4, 12). Monshausen et al. advance the hypothesis that the bursting of rbohc root hairs that they observed is due to reduced apoplastic ROS levels, in turn leading to uncontrolled/unfocused cell expansion. These observations also lead to a critically important conclusion: RBOHC, in addition to its signaling role in mediating tip-based calcium gradients upon which root hair growth depends (1, 7, 8, 10), is also involved in regulating cell wall extensibility. Unexpectedly, however, the rbohc mutant phenotype could be rescued by growing root hairs at alkali pH (i.e., under these conditions rbohc mutants produced root hairs equivalent in length and density to those of wild type) (12). The rescued mutant root hairs were also shown to have normal tip-based calcium gradients. This observation leads to the conclusion that RBOHC is not actually an absolute requirement for either root hair growth or calcium influx (see ref. 12 and below).

It is important to take time to consider the two distinctly different reported roles for ROS/RBOHC—signaling (1) and cell wall regulation (12)—and to consider how they may be related to each other and how one might rationalize both ideas into a single model. Fig. 1A summarizes the previously reported role for ROS/RBOHC in root hair development (1) and compares it to the role for ROS/RBOHC revealed by Monshausen et al. (12) (Fig. 1B). In the former case (model A shown in Fig. 1A), RBOHC acts to generate ROS that are recognized by calcium channels in the plasma membrane of the growing root tip (1). ROS provoke the opening of these channels, leading to the establishment of a tip-based calcium gradient, which is maintained throughout the growing phase of the root hair (with a continuous production of ROS to maintain this gradient) (1, 4, 11). Under these circumstances, both ROS and RBOHC are necessary for the calcium gradient and root hair growth. This situation contrasts with the more recent model (model B shown in Fig. 1B), whereby the target of action of the ROS is the apoplast and not the plasma membrane. In the apoplast, ROS is postulated to regulate extensibility of the cell wall as described above, and it regulates oscillating phases of growth via the oscillating activity of ROS-generating enzymes such as RBOHC.

Fig. 1.

Two models for the role of ROS and RBOHC in root hair growth. (A) RBOHC produces ROS (hydroxyl radical specifically in this case) at the tip (a and b) of the root hair cell [for this diagram, a tip-based gradient of ROS is assumed (4, 11), as is a tip-based concentration of RBOHC activity]. ROS (hydroxyl radical)-gated calcium channels are stimulated to produce a tip-based calcium gradient (c), driving elongation growth. (B) Calcium gradient is independent of ROS (a), at least at alkali pH. RBOHC generates ROS (superoxide in this case) specifically in the apoplast just behind the apex (b). This ROS is responsible for rigidifying the cell wall during nongrowing phases (c). The subsequent growth phase correlates with reduced ROS (superoxide) levels.

Can these two models be rationalized together? There is one consistent aspect between them: a necessity for both ROS and RBOHC for normal root hair growth and development under standard pH conditions. After this point, there are six important differences:

1. Growth and ROS changes were demonstrated to be oscillatory in model B. This result was not tested in previous work but would need to be taken

   Root hair growth is not a constant process: instead it occurs as discrete episodes.

   into account in later models that use the model A as a platform.

2. The site of ROS accumulation is the apex of growing hairs in model A (4, 11) and is the region just behind the apex (12) in model B. These are seemingly mutually exclusive observations. One has to bear in mind that measurements leading to model A were made by using nitro blue tetrazolium (NBT), and the measurements in model B were made by using OxyBURST Green H₂HFF-BSA. There are technical differences between the two methods. With NBT staining, it is hard to ensure that the root hair is actively growing, whereas the most recent data report tracking hair cell growth during imaging (12).

3. One model involves superoxide, and the other involves the hydroxyl radical, as the specific ROS fulfilling function. As the ROS are controlling different targets (i.e., calcium channels versus cell walls), it is quite possible that two different ROS, both produced by RBOHC, are affecting two different processes separately. Therefore, there is no major conflict between these two observations.

4. RBOHC is required for root hair elongation specifically in model A, but it regulates cell wall extensibility in model B. Nuances in the growth conditions in the two sets of experiments [e.g., in one case rbohc mutant root hairs burst (4, 12), and in the other, they arrest development (1, 11)] might explain the different observations that have led to these apparently contradictory findings. Increasing pH can rescue burst cells in rbohc, changing them into actively growing cells (12), so it may be that the standard conditions used in previous work (1, 11) allowed for rescue up to the point of root hair initiation, which is followed by stasis.

5. In one model, the calcium gradient depends on RBOHC, but in the other model, it does not; in fact, normal calcium gradients were detected in a rbohc mutant background (12). ROS itself is not an absolute requirement in this model because interference of the ROS signal by mutation can be “cured” by alkaline pH. Again, it may be that under the conditions used previously, the calcium gradient is truly dependent on RBOHC (1), but under other conditions, it may be redundant to other mechanisms (e.g., other ROS-generating enzymes). It must be pointed out that it was the rbohc mutant under alkali conditions that demonstrated the lack of necessity for RBOHC. Formally, one cannot assert that this is the case at “normal” pH.

6. Fundamentally, increased ROS/RBOHC activity is a positive factor for root hair growth by promoting cell elongation in model A, and it is a negative factor by restricting growth through reduced cell wall extensibility in model B.

A ROP GTPase (4) and RhoGDP (11) have recently been found to be important in ROS/RBOHC-mediated root hair cell growth. Now we must ask whether they play a role in regulating cell wall properties as well. It will be very important for researchers in this area to try to integrate past and present data such as these to produce a combined model, so that the most imperative targets for future research in this area can be identified.