The response of the actin cytoskeleton to nodulation (Nod) factors secreted by Rhizobium etli has been studied in living root hairs of bean (Phaseolus vulgaris) that were microinjected with fluorescein isothiocyanate-phalloidin. In untreated control cells or cells treated with the inactive chitin oligomer, the actin cytoskeleton was organized into long bundles that were oriented parallel to the long axis of the root hair and extended into the apical zone. Upon exposure to R. etli Nod factors, the filamentous actin became fragmented, as indicated by the appearance of prominent masses of diffuse fluorescence in the apical region of the root hair. These changes in the actin cytoskeleton were rapid, observed as soon as 5 to 10 min after application of the Nod factors. It was interesting that the filamentous actin partially recovered in the continued presence of the Nod factor: by 1 h, long bundles had reformed. However, these cells still contained a significant amount of diffuse fluorescence in the apical zone and in the nuclear area, presumably indicating the presence of short actin filaments. These results indicate that Nod factors alter the organization of actin microfilaments in root hair cells, and this could be a prelude for the formation of infection threads.

Summary: Application of Nod factors to growing, responsive root hairs of the bean Phaseolus vulgaris induces marked changes in both the intracellular cytosolic free calcium (Ca²⁺) and in the influx of extracellular [Ca²⁺]. The intracellular [Ca²⁺], which has been measured by ratiometric imaging in cells microinjected with fura‐2‐dextran (70 kDa), elevates within 5 min from approximately 400 n m to 1500 n m in localised zones in the root hair apex. Of particular note is the observation that the elevated regions of [Ca²⁺] appear to shift position during short time intervals. Increases in and fluctuations of the intracellular [Ca²⁺] are also observed in the perinuclear region after 10–15 min treatment with Nod factors. The extracellular Ca²⁺ flux, detected with the non‐invasive, calcium specific vibrating electrode, is inwardly directed and also increases quickly in response to Nod factors from 13 pmol cm^–2 s^–1 to 28 pmol cm^–2 s^–1. Chitin‐oligomers, which are structurally similar but biologically inactive when compared to the active Nod factors, fail to elicit changes in either intracellular or extracellular Ca²⁺. The similar timing and location of the intracellular elevations and the increased extracellular influx provide support for the idea that Ca²⁺ participates in secretion and cell wall remodelling, which occur in anticipation of root hair deformation and curling.

Plant NADPH oxidases [respiratory burst oxidase homologs (RBOHs)] have emerged as key players in the regulation of plant–pathogen interactions. Nonetheless, their role in mutualistic associations, such as the rhizobia–legume symbiosis, is poorly understood. In this work, nine members of the Phaseolus vulgaris Rboh gene family were identified. The transcript of one of these, PvRbohB, accumulated abundantly in shoots, roots and nodules. PvRbohB promoter activity was detected in meristematic regions of P. vulgaris roots, as well as during infection thread (IT) progression and nodule development. RNA interference (RNAi)-mediated PvRbohB down-regulation in transgenic roots reduced reactive oxygen species (ROS) production and lateral root density, and greatly impaired nodulation. Microscopy analysis revealed that progression of the ITs was impeded at the base of root hairs in PvRbohB-RNAi roots. Furthermore, the few nodules that formed in PvRbohB-down-regulated roots displayed abnormally wide ITs and reduced nitrogen fixation. These findings indicate that this common bean NADPH oxidase is crucial for successful rhizobial colonization and probably maintains proper IT growth and shape.

Reactive oxygen species (ROS) produced by respiratory burst oxidase homologs (RBOHs) regulate numerous plant cell processes, including the symbiosis between legumes and nitrogen-fixing bacteria. Rapid and transient ROS production was reported after Phaseolus vulgaris root hairs were treated with Nod factors, indicating the presence of a ROS-associated molecular signature in the symbiosis signaling pathway. Rboh is a multigene family containing nine members (RbohA–I) in P. vulgaris. RNA interference of RbohB suppresses ROS production and attenuates rhizobial infection thread (IT) progression in P. vulgaris root hairs. However, the roles of other Rboh members in symbiotic interactions are largely unknown. In this study, we characterized the role of the NADPH oxidase-encoding gene RbohA (Phvulv091020621) in the P. vulgaris–Rhizobium tropici symbiosis. The spatiotemporal activity of the RbohA promoter colocalized with growing ITs and was associated with vascular bundles in developing nodules. Subcellular localization studies indicated that RBOHA was localized in the plasma membrane of P. vulgaris root hairs. After rhizobial inoculation, PvRBOHA was mainly distributed in the infection pocket and, to a lesser extent, throughout the IT. In PvRbohA RNAi lines, the rhizobial infection events were significantly reduced and, in successful infections, IT progression was arrested within the root hair, but did not impede cortical cell division. PvRbohA-RNAi nodules failed to fix nitrogen, since the infected cells in the few nodules formed were empty. RbohA-dependent ROS production and upregulation of several antioxidant enzymes was attenuated in rhizobia-inoculated PvRbohA-RNAi roots. These combined results indicate that PvRbohA is crucial for effective Rhizobium infection and its release into the nodule cells. This oxidase is partially or indirectly required to promote nodule organogenesis, altering the expression of auxin- and cyclin-related genes and genes involved in cell growth and division.

Emerging evidence indicates that some reactive oxygen species (ROS), such as the superoxide anion radical and hydrogen peroxide (H₂O₂), are central regulators of plant responses to biotic and abiotic stresses. Thus, the cellular levels of ROS are thought to be tightly regulated by an efficient and elaborate pro- and antioxidant system that modulates the production and scavenging of ROS. Until recently, studies of ROS in plant cells have been limited to biochemical assays and the use of fluorescent probes; however, the irreversible oxidation of these fluorescent probes makes it impossible to visualize dynamic changes in ROS levels. In this work, we describe the use of Hyper, a recently developed live cell probe for H₂O₂ measurements in living cells, to monitor oxidative stress in Arabidopsis roots subjected to aluminum treatment. Hyper consists of a circularly permuted YFP (cpYFP) inserted into the regulatory domain of the Escherichia coli hydrogen peroxide-binding protein (OxyR), and is a H₂O₂-specific ratiometric, and therefore quantitative, probe that can be expressed in plant and animal cells. Now we demonstrate that H₂O₂ levels drop sharply in the elongation zone of roots treated with aluminum. This response could contribute to root growth arrest and provides evidence that H₂O₂ is involved in early Al sensing.