Sarah J. Swanson

Changes in root cap pH are required for the gravity response of the Arabidopsis root

Although the columella cells of the root cap have been identified as the site of gravity perception, the cellular events that mediate gravity signaling remain poorly understood. To determine if cytoplasmic and/or wall pH mediates the initial stages of root gravitropism, we combined a novel cell wall pH sensor (a cellulose binding domain peptide–Oregon green conjugate) and a cytoplasmic pH sensor (plants expressing pH-sensitive green fluorescent protein) to monitor pH dynamics throughout the graviresponding Arabidopsis root. The root cap apoplast acidified from pH 5.5 to 4.5 within 2 min of gravistimulation. Concomitantly, cytoplasmic pH increased in columella cells from 7.2 to 7.6 but was unchanged elsewhere in the root. These changes in cap pH preceded detectable tropic growth or growth-related pH changes in the elongation zone cell wall by 10 min. Altering the gravity-related columella cytoplasmic pH shift with caged protons delayed the gravitropic response. Together, these results suggest that alterations in root cap pH likely are involved in the initial events that mediate root gravity perception or signal transduction.

In vivo imaging of Ca2+, pH, and reactive oxygen species using fluorescent probes in plants.

Changes in the levels of Ca(2+), pH, and reactive oxygen species (ROS) are recognized as key cellular regulators involved in diverse physiological and developmental processes in plants. Critical to understanding how they exert such widespread control is an appreciation of their spatial and temporal dynamics at levels from organ to organelle and from seconds to many hours. With appropriate controls, fluorescent sensors can provide a robust approach with which to quantify such changes in Ca(2+), pH, and ROS in real time, in vivo. The fluorescent cellular probes available for visualization split into two broad classes: (a) dyes and (b) an increasingly diverse set of genetically encoded sensors based around green fluorescent proteins (GFPs). The GFP probes in particular can be targeted to well-defined subcellular locales, offering the possibility of high-resolution mapping of these signals within the cell.

ROS in plant development

Reactive oxygen species (ROS) are now recognized as important regulators of plant developmental programs and recent work on tip-growing systems has revealed a central role for the NADPH oxidases in generating such developmentally important ROS. Tip-growing cells have also shown that the functions of cytosolic ROS, acting as regulators of activities such as ion channel gating, are closely linked to those of ROS produced to the apoplast, where they act to modulate cell wall properties. Thus, coordination of ROS production and their activities between compartments is emerging as an important theme in our understanding of how growth and developmental programs are integrated.

Extracellular nucleotides elicit cytosolic free calcium oscillations in Arabidopsis

Extracellular ATP induces a rise in the level of cytosolic free calcium ([Ca2+]cyt) in plant cells. To expand our knowledge about the function of extracellular nucleotides in plants, the effects of several nucleotide analogs and pharmacological agents on [Ca2+]cyt changes were studied using transgenic Arabidopsis (Arabidopsis thaliana) expressing aequorin or the fluorescence resonance energy transfer-based Ca2+ sensor Yellow Cameleon 3.6. Exogenously applied CTP caused elevations in [Ca2+]cyt that displayed distinct time- and dose-dependent kinetics compared with the purine nucleotides ATP and GTP. The inhibitory effects of antagonists of mammalian P2 receptors and calcium influx inhibitors on nucleotide-induced [Ca2+]cyt elevations were distinct between CTP and purine nucleotides. These results suggest that distinct recognition systems may exist for the respective types of nucleotides. Interestingly, a mutant lacking the heterotrimeric G protein Gβ-subunit exhibited a remarkably higher [Ca2+]cyt elevation in response to all tested nucleotides in comparison with the wild type. These data suggest a role for Gβ in negatively regulating extracellular nucleotide signaling and point to an important role for heterotrimeric G proteins in modulating the cellular effects of extracellular nucleotides. The addition of extracellular nucleotides induced multiple temporal [Ca2+]cyt oscillations, which could be localized to specific root cells. The oscillations were attenuated by a vesicle-trafficking inhibitor, indicating that the oscillations likely require ATP release via exocytotic secretion. The results reveal new molecular details concerning extracellular nucleotide signaling in plants and the importance of fine control of extracellular nucleotide levels to mediate specific plant cell responses.

Touch induces ATP release in Arabidopsis roots that is modulated by the heterotrimeric G-protein complex

Amongst the many stimuli orienting the growth of plant roots, of critical importance are the touch signals generated as roots explore the mechanically complex soil environment. However, the molecular mechanisms behind these sensory events remain poorly defined. We report an impaired obstacle‐avoiding response of roots in Arabidopsis lacking a heterotrimeric G‐protein. Obstacle avoidance may utilize a touch‐induced release of ATP to the extracellular space. While sequential touch stimulation revealed a strong refractory period for ATP release in response to mechano‐stimulation in wild‐type plants, the refractory period in mutants was attenuated, resulting in extracellular ATP accumulation. We propose that ATP acts as an extracellular signal released by mechano‐stimulation and that the G‐protein complex is needed for fine‐tuning this response.

Physiology of the aleurone layer and starchy endosperm during grain development and early seedling growth: new insights from cell and molecular biology

Cereal grain germination and early seedling growth involve the co-ordinated action of endosperm and embryo tissues to mobilize the storage reserves of the starchy endosperm. This mobilization is accomplished by hydrolases secreted from the aleurone and scutellar tissues. The breakdown products are then transported to the growing seedling by the scutellum. This resource-harvesting system is regulated at multiple levels. One well-defined aspect of control is brought about by the hormone gibberellin (GA). Gibberellin is released from the embryo upon imbibition and activates the aleurone cells. The secretory apparatus of the aleurone then proliferates, supporting increased hydrolase synthesis and secretion to degrade the starchy endosperm. The molecules that regulate this response to GA are now being increasingly characterized. Elements such as cGMP, calcium, calmodulin and protein kinases are well known as regulators in other eukaryotic cell types and are emerging as key control factors in the aleurone hormone response. However, superimposed upon this molecular regulatory system is another level of control, the structural pattern of tissues and stored macro- molecules that was laid down during grain development. It is the interaction of these structural motifs combined with the molecular regulatory mechanisms that ensure the appropriate timing and positioning of hydrolase production and endosperm reserve mobilization. This integrated control system ensures an extended release of nutrients to fuel early seedling growth.

Rapid, Long-Distance Electrical and Calcium Signaling in Plants

Plants integrate activities throughout their bodies using long-range signaling systems in which stimuli sensed by just a few cells are translated into mobile signals that can influence the activities in distant tissues. Such signaling can travel at speeds well in excess of millimeters per second and can trigger responses as diverse as changes in transcription and translation levels, posttranslational regulation, alterations in metabolite levels, and even wholesale reprogramming of development. In addition to the use of mobile small molecules and hormones, electrical signals have long been known to propagate throughout the plant. This electrical signaling network has now been linked to waves of Ca(2+) and reactive oxygen species that traverse the plant and trigger systemic responses. Analysis of cell type specificity in signal propagation has revealed the movement of systemic signals through specific cell types, suggesting that a rapid signaling network may be hardwired into the architecture of the plant.

Imaging Changes in Cytoplasmic Calcium Using the Yellow Cameleon 3.6 Biosensor and Confocal Microscopy

Changes in the concentration of cytoplasmic calcium, [Ca(2+)]cyt are central regulators in many cellular signal transduction pathways including many lipid-mediated regulatory networks. Given this central role that [Ca(2+)] has during plant growth, monitoring spatial and temporal [Ca(2+)] dynamics can reveal a critical component of cellular physiology. Here, we describe the measurement of [Ca(2+)]cyt in Arabidopsis root cells using plants expressing Yellow Cameleon 3.6 (YC 3.6). YC3.6 is a Ca(2+)-sensitive biosensor where the intensity of its fluorescence resonance energy transfer (FRET) signal changes as the Ca(2+) level within the cell rises and falls. The FRET from this calcium reporter can be visualized using confocal microscopy and the resultant images converted to a quantitative map of the levels of Ca(2+) using an approach called ratio analysis.

Environmental and Genetic Factors Regulating Localization of the Plant Plasma Membrane H+-ATPase

Cellular dynamics and localization of plasma membrane H+-ATPase is determined by cell type, light condition, and a receptor kinase, FERONIA, which regulates cell elongation growth. A P-type H+-ATPase is the primary transporter that converts ATP to electrochemical energy at the plasma membrane of higher plants. Its product, the proton-motive force, is composed of an electrical potential and a pH gradient. Many studies have demonstrated that this proton-motive force not only drives the secondary transporters required for nutrient uptake, but also plays a direct role in regulating cell expansion. Here, we have generated a transgenic Arabidopsis (Arabidopsis thaliana) plant expressing H+-ATPase isoform 2 (AHA2) that is translationally fused with a fluorescent protein and examined its cellular localization by live-cell microscopy. Using a 3D imaging approach with seedlings grown for various times under a variety of light intensities, we demonstrate that AHA2 localization at the plasma membrane of root cells requires light. In dim light conditions, AHA2 is found in intracellular compartments, in addition to the plasma membrane. This localization profile was age-dependent and specific to cell types found in the transition zone located between the meristem and elongation zones. The accumulation of AHA2 in intracellular compartments is consistent with reduced H+ secretion near the transition zone and the suppression of root growth. By examining AHA2 localization in a knockout mutant of a receptor protein kinase, FERONIA, we found that the intracellular accumulation of AHA2 in the transition zone is dependent on a functional FERONIA-dependent inhibitory response in root elongation. Overall, this study provides a molecular underpinning for understanding the genetic, environmental, and developmental factors influencing root growth via localization of the plasma membrane H+-ATPase.

The Rice E3-Ubiquitin Ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE1 Modulates the Expression of ROOT MEANDER CURLING, a Gene Involved in Root Mechanosensing, through the Interaction with Two ETHYLENE-RESPONSE FACTOR Transcription Factors

Rice root curling, a response to a mechanical barrier that involves the plant hormone jasmonic acid, is modulated by an E3-ubiquitin ligase. Plant roots can sense and respond to a wide diversity of mechanical stimuli, including touch and gravity. However, little is known about the signal transduction pathways involved in mechanical stimuli responses in rice (Oryza sativa). This work shows that rice root responses to mechanical stimuli involve the E3-ubiquitin ligase rice HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE1 (OsHOS1), which mediates protein degradation through the proteasome complex. The morphological analysis of the roots in transgenic RNA interference::OsHOS1 and wild-type plants, exposed to a mechanical barrier, revealed that the OsHOS1 silencing plants keep a straight root in contrast to wild-type plants that exhibit root curling. Moreover, it was observed that the absence of root curling in response to touch can be reverted by jasmonic acid. The straight root phenotype of the RNA interference::OsHOS1 plants was correlated with a higher expression rice ROOT MEANDER CURLING (OsRMC), which encodes a receptor-like kinase characterized as a negative regulator of rice root curling mediated by jasmonic acid. Using the yeast two-hybrid system and bimolecular fluorescence complementation assays, we showed that OsHOS1 interacts with two ETHYLENE-RESPONSE FACTOR transcription factors, rice ETHYLENE-RESPONSIVE ELEMENT BINDING PROTEIN1 (OsEREBP1) and rice OsEREBP2, known to regulate OsRMC gene expression. In addition, we showed that OsHOS1 affects the stability of both transcription factors in a proteasome-dependent way, suggesting that this E3-ubiquitin ligase targets OsEREBP1 and OsEREBP2 for degradation. Our results highlight the function of the proteasome in rice response to mechanical stimuli and in the integration of these signals, through hormonal regulation, into plant growth and developmental programs.