A comprehensive, improved protocol for generating common bean (Phaseolus vulgaris L.) transgenic hairy roots and their use in reverse-genetics studies.

Generating transgenic hairy roots has been the preferred strategy for molecular studies in common bean (Phaseolus vulgaris L.), since generating stable knockout lines in this species is challenging. However, the number of plants producing hairy roots following the original protocol published in 2007 is usually low, which has impeded progress. Since its initial publication, the original protocol has been extensively modified, but these modifications have not been adequately or systematically reported, making it difficult to assess the reproducibility of the method. The protocol presented here is an update and expansion of the original method. Importantly, it includes new, critical steps for generating transgenic hairy roots and using them in molecular analyses based on reverse-genetics approaches. Using this protocol, the expression of two different genes, used as an example, was significantly increased or decreased in approximately 30% of the transformed plants. In addition, the promoter activity of a given gene was observed, and the infection process of rhizobia in transgenic hairy roots was monitored successfully. Thus, this improved protocol can be used to upregulate, downregulate, and perform promoter activity analysis of various genes in common bean transgenic hairy roots as well as to track rhizobia infection.

The Phaseolus vulgaris Receptor-Like Kinase PvFER1 and the Small Peptides PvRALF1 and PvRALF6 Regulate Nodule Number as a Function of Nitrate Availability.

Legumes associate with Gram-negative soil bacteria called rhizobia, resulting in the formation of a nitrogen-fixing organ, the nodule. Nodules are an important sink for photosynthates for legumes, so these plants have developed a systemic regulation mechanism that controls their optimal number of nodules, the so-called autoregulation of nodulation (AON) pathway, to balance energy costs with the benefits of nitrogen fixation. In addition, soil nitrate inhibits nodulation in a dose-dependent manner, through systemic and local mechanisms. The CLE family of peptides and their receptors are key to tightly controlling these inhibitory responses. In the present study, a functional analysis revealed that PvFER1, PvRALF1, and PvRALF6 act as positive regulators of the nodule number in growth medium containing 0 mM of nitrate but as negative regulators in medium with 2 and 5 mM of nitrate. Furthermore, the effect on nodule number was found to be consistent with changes in the expression levels of genes associated with the AON pathway and with the nitrate-mediated regulation of nodulation (NRN). Collectively, these data suggest that PvFER1, PvRALF1, and PvRALF6 regulate the optimal number of nodules as a function of nitrate availability.

Phospholipase Ds in plants: Their role in pathogenic and symbiotic interactions.

Phospholipase Ds (PLDs) are a heterogeneous group of enzymes that are widely distributed in organisms. These enzymes hydrolyze the structural phospholipids of the plasma membrane, releasing phosphatidic acid (PA), an important secondary messenger. Plant PLDs play essential roles in several biological processes, including growth and development, abiotic stress responses, and plant-microbe interactions. Although the roles of PLDs in plant-pathogen interactions have been extensively studied, their roles in symbiotic relationships are not well understood. The establishment of the best-studied symbiotic interactions, those between legumes and rhizobia and between most plants and mycorrhizae, requires the regulation of several physiological, cellular, and molecular processes. The roles of PLDs in hormonal signaling, lipid metabolism, and cytoskeletal dynamics during rhizobial symbiosis were recently explored. However, to date, the roles of PLDs in mycorrhizal symbiosis have not been reported. Here, we present a critical review of the participation of PLDs in the interactions of plants with pathogens, nitrogen-fixing bacteria, and arbuscular mycorrhizal fungi. We describe how PLDs regulate rhizobial and mycorrhizal symbiosis by modulating reactive oxygen species levels, hormonal signaling, cytoskeletal rearrangements, and G-protein activity.

Metallothionein1A Regulates Rhizobial Infection and Nodulation in Phaseolus vulgaris.

Metallothioneins (MTs) constitute a heterogeneous family of ubiquitous metal ion-binding proteins. In plants, MTs participate in the regulation of cell growth and proliferation, protection against heavy metal stress, oxidative stress responses, and responses to pathogen attack. Despite their wide variety of functions, the role of MTs in symbiotic associations, specifically nodule-fabacean symbiosis, is poorly understood. Here, we analyzed the role of the PvMT1A gene in Phaseolus vulgaris-Rhizobium tropici symbiosis using bioinformatics and reverse genetics approaches. Using in silico analysis, we identified six genes encoding MTs in P. vulgaris, which were clustered into three of the four classes described in plants. PvMT1A transcript levels were significantly higher in roots inoculated with R. tropici at 7 and 30 days post inoculation (dpi) than in non-inoculated roots. Functional analysis showed that downregulating PvMT1A by RNA interference (RNAi) reduced the number of infection events at 7 and 10 dpi and the number of nodules at 14 and 21 dpi. In addition, nodule development was negatively affected in PvMT1A:RNAi transgenic roots, and these nodules displayed a reduced nitrogen fixation rate at 21 dpi. These results strongly suggest that PvMT1A plays an important role in the infection process and nodule development in P. vulgaris during rhizobial symbiosis.

The CrRLK1L subfamily: One of the keys to versatility in plants.

Catharanthus roseous kinase 1L receptors (CrRLK1Ls) are a subfamily of membrane receptors unique to plant cells that perceive internal and external signals, integrate metabolic, physiological, and molecular processes, and regulate plant development. Recent genomic studies have suggested that this receptor subfamily arose during the emergence of terrestrial plants and has since diversified, preserving its essential functions. Participation of some of these CrRLK1Ls in different processes is presented and discussed herein, as well as the increasing number of interactors necessary for their function. At least five different responses have been detected after activating these receptors, such as physiological changes, formation or disassembly of protein complexes, metabolic responses, modification of gene expression, and modulation of phytohormone activity. To date, a common response mechanism for all processes involving CrRLK1Ls has not been described. In this review, the information available on the different functions of CrRLK1Ls was compiled. Additionally, the physiological and/or molecular mechanisms involved in the signaling processes triggered by these receptors are also discussed. In this review, we propose a possible common signaling mechanism for all processes regulated by CrRLK1Ls and pose questions to be answered in the future.

An NADPH oxidase regulates carbon metabolism and the cell cycle during root nodule symbiosis in common bean (Phaseolus vulgaris).

BACKGROUND: Rhizobium-legume symbiosis is a specific, coordinated interaction that results in the formation of a root nodule, where biological nitrogen fixation occurs. NADPH oxidases, or Respiratory Burst Oxidase Homologs (RBOHs) in plants, are enzymes that generate superoxide (O2 •-). Superoxide produces other reactive oxygen species (ROS); these ROS regulate different stages of mutualistic interactions. For example, changes in ROS levels are thought to induce ROS scavenging, cell wall remodeling, and changes in phytohormone homeostasis during symbiotic interactions. In common bean (Phaseolus vulgaris), PvRbohB plays a key role in the early stages of nodulation. RESULTS: In this study, to explore the role of PvRbohB in root nodule symbiosis, we analyzed transcriptomic data from the roots of common bean under control conditions (transgenic roots without construction) and roots with downregulated expression of PvRbohB (by RNA interference) non-inoculated and inoculated with R. tropici. Our results suggest that ROS produced by PvRBOHB play a central role in infection thread formation and nodule organogenesis through crosstalk with flavonoids, carbon metabolism, cell cycle regulation, and the plant hormones auxin and cytokinin during the early stages of this process. CONCLUSIONS: Our findings provide important insight into the multiple roles of ROS in regulating rhizobia-legume symbiosis.

Genome-Wide Identification of the CrRLK1L Subfamily and Comparative Analysis of Its Role in the Legume-Rhizobia Symbiosis.

The plant receptor-like-kinase subfamily CrRLK1L has been widely studied, and CrRLK1Ls have been described as crucial regulators in many processes in Arabidopsis thaliana (L.), Heynh. Little is known, however, about the functions of these proteins in other plant species, including potential roles in symbiotic nodulation. We performed a phylogenetic analysis of CrRLK1L subfamily receptors of 57 different plant species and identified 1050 CrRLK1L proteins, clustered into 11 clades. This analysis revealed that the CrRLK1L subfamily probably arose in plants during the transition from chlorophytes to embryophytes and has undergone several duplication events during its evolution. Among the CrRLK1Ls of legumes and A. thaliana, protein structure, gene structure, and expression patterns were highly conserved. Some legume CrRLK1L genes were active in nodules. A detailed analysis of eight nodule-expressed genes in Phaseolus vulgaris L. showed that these genes were differentially expressed in roots at different stages of the symbiotic process. These data suggest that CrRLK1Ls are both conserved and underwent diversification in a wide group of plants, and shed light on the roles of these genes in legume-rhizobia symbiosis.

Actin Depolymerizing Factor Modulates Rhizobial Infection and Nodule Organogenesis in Common Bean.

Actin plays a critical role in the rhizobium-legume symbiosis. Cytoskeletal rearrangements and changes in actin occur in response to Nod factors secreted by rhizobia during symbiotic interactions with legumes. These cytoskeletal rearrangements are mediated by diverse actin-binding proteins, such as actin depolymerization factors (ADFs). We examined the function of an ADF in the Phaseolus vulgaris-rhizobia symbiotic interaction (PvADFE). PvADFE was preferentially expressed in rhizobia-inoculated roots and nodules. PvADFE promoter activity was associated with root hairs harbouring growing infection threads, cortical cell divisions beneath root hairs, and vascular bundles in mature nodules. Silencing of PvADFE using RNA interference increased the number of infection threads in the transgenic roots, resulting in increased nodule number, nitrogen fixation activity, and average nodule diameter. Conversely, overexpression of PvADFE reduced the nodule number, nitrogen fixation activity, average nodule diameter, as well as NODULE INCEPTION (NIN) and EARLY NODULIN2 (ENOD2) transcript accumulation. Hence, changes in ADFE transcript levels affect rhizobial infection and nodulation, suggesting that ADFE is fine-tuning these processes.

Transcriptome analysis of the differential effect of the NADPH oxidase gene RbohB in Phaseolus vulgaris roots following Rhizobium tropici and Rhizophagus irregularis inoculation.

BACKGROUND: Reactive oxygen species (ROS) are generated by NADPH oxidases known as respiratory burst oxidase homologs (RBOHs) in plants. ROS regulate various cellular processes, including the mutualistic interactions between legumes and nitrogen-fixing bacteria or arbuscular mycorrhizal (AM) fungi. Rboh is a multigene family comprising nine members (RbohA-I) in common bean (Phaseolus vulgaris). The RNA interference-mediated silencing of RbohB (PvRbohB-RNAi) in this species diminished its ROS production and greatly impaired nodulation. By contrast, the PvRbohB-RNAi transgenic roots showed early hyphal root colonization with enlarged fungal hypopodia; therefore, we proposed that PvRbohB positively regulates rhizobial infection (Rhizobium tropici) and inhibits AM colonization by Rhizophagus irregularis in P. vulgaris. RESULTS: To corroborate this hypothesis, an RNA-Seq transcriptomic analysis was performed to identify the differentially expressed genes in the PvRbohB-RNAi roots inoculated with Rhizobium tropici or Rhizophagus irregularis. We found that, in the early stages, root nodule symbioses generated larger changes of the transcriptome than did AM symbioses in P. vulgaris. Genes related to ROS homeostasis and cell wall flexibility were markedly upregulated in the early stages of rhizobial colonization, but not during AM colonization. Compared with AM colonization, the rhizobia induced the expression of a greater number of genes encoding enzymes involved in the metabolism of auxins, cytokinins, and ethylene, which were typically repressed in the PvRbohB-RNAi roots. CONCLUSIONS: Our research provides substantial insights into the genetic interaction networks in the early stages of rhizobia and AM symbioses with P. vulgaris, as well as the differential roles that RbohB plays in processes related to ROS scavenging, cell wall remodeling, and phytohormone homeostasis during nodulation and mycorrhization in this legume.

Differential tetraspanin genes expression and subcellular localization during mutualistic interactions in Phaseolus vulgaris.

Arbuscular mycorrhizal fungi and rhizobia association with plants are two of the most successful plant-microbe associations that allow the assimilation of P and N by plants, respectively. These mutualistic interactions require a molecular dialogue, i.e., legume roots exude flavonoids or strigolactones which induce the Nod factors or Myc factors synthesis and secretion from the rhizobia or fungi, respectively. These Nod or Myc factors trigger several responses in the plant root, including calcium oscillations, and reactive oxygen species (ROS). Furthermore, superoxide and H2O2 have emerged as key components that regulate the transitions from proliferation to differentiation in the plant meristems. Similar to the root meristem, the nodule meristem accumulates superoxide and H2O2. Tetraspanins are transmembrane proteins that organize into tetraspanin web regions, where they recruit specific proteins into platforms required for signal transduction, membrane fusion, cell trafficking and ROS generation. Plant tetraspanins are scaffolding proteins associated with root radial patterning, biotic and abiotic stress responses, cell fate determination, and hormonal regulation and recently have been reported as a specific marker of exosomes in animal and plant cells and key players at the site of plant fungal infection. In this study, we conducted transcriptional profiling of the tetraspanin family in common bean (Phaseolus vulgaris L. var. Negro Jamapa) to determine the specific expression patterns and subcellular localization of tetraspanins during nodulation or under mycorrhizal association. Our results demonstrate that the tetraspanins are transcriptionally modulated during the mycorrhizal association, but are also expressed in the infection thread and nodule meristem development. Subcellular localization indicates that tetraspanins have a key role in vesicular trafficking, cell division, and root hair polar growth.

RbohA coordinates lateral root emergence in common bean.

Respiratory burst oxidase homologs (RBOHs) constitute a multigene family in plants. These reactive oxygen species (ROS)-generating enzymes participate in diverse biological processes. We previously demonstrated that PvRbohB plays an important role in lateral root (LR) development in Phaseolus vulgaris. However, little is known about the roles of other Rboh members in root development. Here, we report that PvRbohA is involved in LR emergence in P. vulgaris. PvRbohA was expressed in wild-type P. vulgaris root tissues, e.g., the radicle, inter LR zone, and LR zone, and its expression increased during LR formation. Analysis of the spatio-temporal expression patterns of a reporter construct under the control of the PvRbohA promoter (PvRbohA::GUS-GFP) in transgenic roots showed that PvRbohA was active at all three stages of LR development, but its spatial expression pattern varied at each stage. The relative expression levels of PvRbohA during LR formation correlated with the activity of PvRbohA promoter. Furthermore, upon PvRbohA transcript silencing, LR growth was significantly altered in transgenic hairy roots. These findings suggest that RbohA participates in LR initiation, emergence, and development in the legume P. vulgaris by delimiting the region for LR emergence.

Respiratory Burst Oxidase Homolog Gene A Is Crucial for Rhizobium Infection and Nodule Maturation and Function in Common Bean.

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.

The Class II Trehalose 6-phosphate Synthase Gene PvTPS9 Modulates Trehalose Metabolism in Phaseolus vulgaris Nodules.

Legumes form symbioses with rhizobia, producing nitrogen-fixing nodules on the roots of the plant host. The network of plant signaling pathways affecting carbon metabolism may determine the final number of nodules. The trehalose biosynthetic pathway regulates carbon metabolism and plays a fundamental role in plant growth and development, as well as in plant-microbe interactions. The expression of genes for trehalose synthesis during nodule development suggests that this metabolite may play a role in legume-rhizobia symbiosis. In this work, PvTPS9, which encodes a Class II trehalose-6-phosphate synthase (TPS) of common bean (Phaseolus vulgaris), was silenced by RNA interference in transgenic nodules. The silencing of PvTPS9 in root nodules resulted in a reduction of 85% (± 1%) of its transcript, which correlated with a 30% decrease in trehalose contents of transgenic nodules and in untransformed leaves. Composite transgenic plants with PvTPS9 silenced in the roots showed no changes in nodule number and nitrogen fixation, but a severe reduction in plant biomass and altered transcript profiles of all Class II TPS genes. Our data suggest that PvTPS9 plays a key role in modulating trehalose metabolism in the symbiotic nodule and, therefore, in the whole plant.

A Legume TOR Protein Kinase Regulates Rhizobium Symbiosis and Is Essential for Infection and Nodule Development.

The target of rapamycin (TOR) protein kinase regulates metabolism, growth, and life span in yeast, animals, and plants in coordination with nutrient status and environmental conditions. The nutrient-dependent nature of TOR functionality makes this kinase a putative regulator of symbiotic associations involving nutrient acquisition. However, TOR's role in these processes remains to be understood. Here, we uncovered the role of TOR during the bean (Phaseolus vulgaris)-Rhizobium tropici (Rhizobium) symbiotic interaction. TOR was expressed in all tested bean tissues, with higher transcript levels in the root meristems and senesced nodules. We showed TOR promoter expression along the progressing infection thread and in the infected cells of mature nodules. Posttranscriptional gene silencing of TOR using RNA interference (RNAi) showed that this gene is involved in lateral root elongation and root cell organization and also alters the density, size, and number of root hairs. The suppression of TOR transcripts also affected infection thread progression and associated cortical cell divisions, resulting in a drastic reduction of nodule numbers. TOR-RNAi resulted in reduced reactive oxygen species accumulation and altered CyclinD1 and CyclinD3 expression, which are crucial factors for infection thread progression and nodule organogenesis. Enhanced expression of TOR-regulated ATG genes in TOR-RNAi roots suggested that TOR plays a role in the recognition of Rhizobium as a symbiont. Together, these data suggest that TOR plays a vital role in the establishment of root nodule symbiosis in the common bean.

An Autophagy-Related Kinase Is Essential for the Symbiotic Relationship between Phaseolus vulgaris and Both Rhizobia and Arbuscular Mycorrhizal Fungi.

Eukaryotes contain three types of lipid kinases that belong to the phosphatidylinositol 3-kinase (PI3K) family. In plants and Saccharomyces cerevisiae, only PI3K class III family members have been identified. These enzymes regulate the innate immune response, intracellular trafficking, autophagy, and senescence. Here, we report that RNAi-mediated downregulation of common bean (Phaseolus vulgaris) PI3K severely impaired symbiosis in composite P. vulgaris plants with endosymbionts such as Rhizobium tropici and Rhizophagus irregularis Downregulation of Pv-PI3K was associated with a marked decrease in root hair growth and curling. Additionally, infection thread growth, root-nodule number, and symbiosome formation in root nodule cells were severely affected. Interestingly, root colonization by AM fungi and the formation of arbuscules were also abolished in PI3K loss-of-function plants. Furthermore, the transcript accumulation of genes encoding proteins known to interact with PI3K to form protein complexes involved in autophagy was drastically reduced in these transgenic roots. RNAi-mediated downregulation of one of these genes, Beclin1/Atg6, resulted in a similar phenotype as observed for transgenic roots in which Pv-PI3K had been downregulated. Our findings show that an autophagy-related process is crucial for the mutualistic interactions of P. vulgaris with beneficial microorganisms.

Regulation of Small RNAs and Corresponding Targets in Nod Factor-Induced Phaseolus vulgaris Root Hair Cells.

A genome-wide analysis identified the set of small RNAs (sRNAs) from the agronomical important legume Phaseolus vulgaris (common bean), including novel P. vulgaris-specific microRNAs (miRNAs) potentially important for the regulation of the rhizobia-symbiotic process. Generally, novel miRNAs are difficult to identify and study because they are very lowly expressed in a tissue- or cell-specific manner. In this work, we aimed to analyze sRNAs from common bean root hairs (RH), a single-cell model, induced with pure Rhizobium etli nodulation factors (NF), a unique type of signal molecule. The sequence analysis of samples from NF-induced and control libraries led to the identity of 132 mature miRNAs, including 63 novel miRNAs and 1984 phasiRNAs. From these, six miRNAs were significantly differentially expressed during NF induction, including one novel miRNA: miR-RH82. A parallel degradome analysis of the same samples revealed 29 targets potentially cleaved by novel miRNAs specifically in NF-induced RH samples; however, these novel miRNAs were not differentially accumulated in this tissue. This study reveals Phaseolus vulgaris-specific novel miRNA candidates and their corresponding targets that meet all criteria to be involved in the regulation of the early nodulation events, thus setting the basis for exploring miRNA-mediated improvement of the common bean-rhizobia symbiosis.

Legume NADPH Oxidases Have Crucial Roles at Different Stages of Nodulation.

Plant NADPH oxidases, formerly known as respiratory burst oxidase homologues (RBOHs), are plasma membrane enzymes dedicated to reactive oxygen species (ROS) production. These oxidases are implicated in a wide variety of processes, ranging from tissue and organ growth and development to signaling pathways in response to abiotic and biotic stimuli. Research on the roles of RBOHs in the plant's response to biotic stresses has mainly focused on plant-pathogen interactions; nonetheless, recent findings have shown that these oxidases are also involved in the legume-rhizobia symbiosis. The legume-rhizobia symbiosis leads to the formation of the root nodule, where rhizobia reduce atmospheric nitrogen to ammonia. A complex signaling and developmental pathway in the legume root hair and root facilitate rhizobial entrance and nodule organogenesis, respectively. Interestingly, several reports demonstrate that RBOH-mediated ROS production displays versatile roles at different stages of nodulation. The evidence collected to date indicates that ROS act as signaling molecules that regulate rhizobial invasion and also function in nodule senescence. This review summarizes discoveries that support the key and versatile roles of various RBOH members in the legume-rhizobia symbiosis.

Effect of Rhizobium and arbuscular mycorrhizal fungi inoculation on electrolyte leakage in Phaseolus vulgaris roots overexpressing RbohB.

Respiratory oxidative burst homolog (RBOH)-mediated reactive oxygen species (ROS) regulate a wide range of biological functions in plants. They play a critical role in the symbiosis between legumes and nitrogen-fixing bacteria or arbuscular mycorrhizal (AM) fungi. For instance, overexpression of PvRbohB enhances nodule numbers, but reduces mycorrhizal colonization in Phaseolus vulgaris hairy roots and downregulation has the opposite effect. In the present study, we assessed the effect of both rhizobia and AM fungi on electrolyte leakage in transgenic P. vulgaris roots overexpressing (OE) PvRbohB. We demonstrate that elevated levels of electrolyte leakage in uninoculated PvRbohB-OE transgenic roots were alleviated by either Rhizobium or AM fungi symbiosis, with the latter interaction having the greater effect. These results suggest that symbiont colonization reduces ROS elevated electrolyte leakage in P. vulgaris root cells.

Hyper, a hydrogen peroxide sensor, indicates the sensitivity of the Arabidopsis root elongation zone to aluminum treatment.

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.