Gabriele B. Monshausen

Oscillations in extracellular pH and reactive oxygen species modulate tip growth of Arabidopsis root hairs

Root hairs show highly localized cell expansion focused to their growing tips. This growth pattern is accomplished through restriction of secretion to the elongating apex and modulation of cell wall properties, with the wall just behind the tip becoming rigidified to resist the lateral expansive forces of turgor. In this report we show that root hairs exhibit oscillating growth that is associated with oscillating increases in extracellular pH and reactive oxygen species (ROS), which lag growth by ≈7 s. Consistent with a role for these changes in growth control, artificially increasing extracellular pH arrested root hair elongation, whereas decreasing pH elicited bursting at the tip. Similarly, application of exogenous ROS arrested elongation, whereas scavenging of ROS led to root hair bursting. Roots hairs of the root hair-defective rhd2-1 mutant, which lack a functional version of the NADPH oxidase ATRBOH C, burst at the transition to tip growth. This phenotype could be rescued by elevating the pH of the growth medium to ≥6.0. Such rescued root hairs showed reduced cytoplasmic ROS levels and a lack of the oscillatory production of ROS at the tip. However, they exhibited apparently normal tip growth, including generation of the tip-focused Ca2+ gradient thought to drive apical growth, indicating that ATRBOH C is not absolutely required to sustain tip growth. These observations indicate that root hair elongation is coupled to spatially distinct regulation of extracellular pH and ROS production that likely affect wall properties associated with the polarized expansion of the cell.

Ca2+ Regulates Reactive Oxygen Species Production and pH during Mechanosensing in Arabidopsis Roots

Mechanical stimulation of plants triggers a cytoplasmic Ca2+ increase that is thought to link the touch stimulus to appropriate growth responses. We found that in roots of Arabidopsis thaliana, external and endogenously generated mechanical forces consistently trigger rapid and transient increases in cytosolic Ca2+ and that the signatures of these Ca2+ transients are stimulus specific. Mechanical stimulation likewise elicited an apoplastic alkalinization and cytoplasmic acidification as well as apoplastic reactive oxygen species (ROS) production. These responses showed the same kinetics as mechanically induced Ca2+ transients and could be elicited in the absence of a mechanical stimulus by artificially increasing Ca2+ concentrations. Both pH changes and ROS production were inhibited by pretreatment with a Ca2+ channel blocker, which also inhibited mechanically induced elevations in cytosolic Ca2+. In trichoblasts of the Arabidopsis root hair defective2 mutant, which lacks a functional NADPH oxidase RBOH C, touch stimulation still triggered pH changes but not the local increase in ROS production seen in wild-type plants. Thus, mechanical stimulation likely elicits Ca2+-dependent activation of RBOH C, resulting in ROS production to the cell wall. This ROS production appears to be coordinated with intra- and extracellular pH changes through the same mechanically induced cytosolic Ca2+ transient.

Imaging of the Yellow Cameleon 3.6 Indicator Reveals That Elevations in Cytosolic Ca2+ Follow Oscillating Increases in Growth in Root Hairs of Arabidopsis

In tip-growing cells, the tip-high Ca2+ gradient is thought to regulate the activity of components of the growth machinery, including the cytoskeleton, Ca2+-dependent regulatory proteins, and the secretory apparatus. In pollen tubes, both the Ca2+ gradient and cell elongation show oscillatory behavior, reinforcing the link between the two. We report that in growing root hairs of Arabidopsis (Arabidopsis thaliana), an oscillating tip-focused Ca2+ gradient can be resolved through imaging of a cytosolically expressed Yellow Cameleon 3.6 fluorescence resonance energy transfer-based Ca2+ sensor. Both elongation of the root hairs and the associated tip-focused Ca2+ gradient show a similar dynamic character, oscillating with a frequency of 2 to 4 min−1. Cross-correlation analysis indicates that the Ca2+ oscillations lag the growth oscillations by 5.3 ± 0.3 s. However, growth never completely stops, even during the slow cycle of an oscillation, and the concomitant tip Ca2+ level is always slightly elevated compared with the resting Ca2+ concentration along the distal shaft, behind the growing tip. Artificially increasing Ca2+ using the Ca2+ ionophore A23187 leads to immediate cessation of elongation and thickening of the apical cell wall. In contrast, dissipating the Ca2+ gradient using either the Ca2+ channel blocker La3+ or the Ca2+ chelator EGTA is accompanied by an increase in the rate of cell expansion and eventual bursting of the root hair tip. These observations are consistent with a model in which the maximal oscillatory increase in cytosolic Ca2+ is triggered by cell expansion associated with tip growth and plays a role in the subsequent restriction of growth.

Feeling green: mechanosensing in plants

Owing to the sessile nature of their lifestyle, plants have to respond to a wide range of signals, such as the force of the wind or the impedance of the soil, to entrain their development to prevailing environmental conditions. Indeed, mechanically responsive growth has been documented in plants for many years but new work on lateral root formation strongly supports the idea that biophysical forces can elicit complete de novo developmental programs. In addition, only recently have molecular candidates for plant mechanosensors emerged. Such advances in understanding plant mechanoresponsive development have relied heavily on comparison with mechanosensors characterized in organisms such as Saccharomyces cerevisiae and Escherichia coli, but key questions remain about the cellular basis of the plant mechanosensory system.

Mechanical Stimuli Modulate Lateral Root Organogenesis

Unlike mammals, whose development is limited to a short temporal window, plants produce organs de novo throughout their lifetime in order to adapt their architecture to the prevailing environmental conditions. The production of lateral roots represents one example of such postembryonic organogenesis. An endogenous developmental program likely imposes an ordered arrangement on the position of new lateral roots. However, environmental stimuli such as nutrient levels also affect the patterning of lateral root production. In addition, we have found that mechanical forces can act as one of the triggers that entrain lateral root production to the environment of the Arabidopsis (Arabidopsis thaliana) plant. We observed that physical bending of the root recruited new lateral root formation to the convex side of the resultant bend. Transient bending of 20 s was sufficient to elicit this developmental program. Such bending triggered a Ca2+ transient within the pericycle, and blocking this change in Ca2+ also blocked recruitment of new lateral root production to the curved region of the root. The initial establishment of the mechanically induced lateral root primordium was independent of an auxin supply from the shoot and was not disrupted by mutants in a suite of auxin transporters and receptor/response elements. These results suggest that Ca2+ may be acting to translate the mechanical forces inherent in growth to a developmental response in roots.

A force of nature: molecular mechanisms of mechanoperception in plants

The ability to sense and respond to a wide variety of mechanical stimuli-gravity, touch, osmotic pressure, or the resistance of the cell wall-is a critical feature of every plant cell, whether or not it is specialized for mechanotransduction. Mechanoperceptive events are an essential part of plant life, required for normal growth and development at the cell, tissue, and whole-plant level and for the proper response to an array of biotic and abiotic stresses. One current challenge for plant mechanobiologists is to link these physiological responses to specific mechanoreceptors and signal transduction pathways. Here, we describe recent progress in the identification and characterization of two classes of putative mechanoreceptors, ion channels and receptor-like kinases. We also discuss how the secondary messenger Ca(2+) operates at the centre of many of these mechanical signal transduction pathways.

The Receptor-like Kinase FERONIA Is Required for Mechanical Signal Transduction in Arabidopsis Seedlings

Among the myriad cues that constantly inform plant growth and development, mechanical forces are unique in that they are an intrinsic result of cellular turgor pressure and also imposed by the environment. Although the key role of mechanical forces in shaping plant architecture from the cellular level to the level of organ formation is well established, the components of the early mechanical signal transduction machinery remain to be defined at the molecular level. Here, we show that an Arabidopsis mutant lacking the receptor-like kinase FERONIA (FER) shows severely altered Ca(2+) signaling and growth responses to different forms of mechanical perturbation. Ca(2+) signals are either abolished or exhibit qualitatively different signatures in feronia (fer) mutants exposed to local touch or bending stimulation. Furthermore, mechanically induced upregulation of known touch-responsive genes is significantly decreased in fer mutants. In addition to these defects in mechanical signaling, fer mutants also exhibit growth phenotypes consistent with impaired mechanical development, including biased root skewing, an inability to penetrate hard agar layers, and abnormal growth responses to impenetrable obstacles. Finally, high-resolution kinematic analysis of root growth revealed that fer mutants show pronounced spatiotemporal fluctuations in root cell expansion profiles with a timescale of minutes. Based on these results, we propose that FER is a key regulator of mechanical Ca(2+) signaling and that FER-dependent mechanical signaling functions to regulate growth in response to external or intrinsic mechanical forces.

Regulation of root‐wave response by extra large and conventional G proteins in Arabidopsis thaliana

Heterotrimeric G proteins composed of alpha, beta and gamma subunits regulate a number of fundamental processes concerned with growth and development in plants. In addition to the canonical heterotrimeric G proteins, plants also contain a small family of extra large G proteins (XLGs) that show significant similarity to the G-protein alpha subunit in their C-terminal regions. In this paper we show that one of the three XLG genes, XLG3, and the Gbeta subunit (AGB1) of the Arabidopsis G-protein heterotrimer are specifically involved in the regulation of a subset of root morphological and growth responses. Based on analysis of T-DNA insertional mutant phenotypes, XLG3 and AGB1 each positively regulate root waving and root skewing. Since these responses are regulated by physical as well as physiological cues, we assessed the roles of AGB1 and XLG3 in gravitropism, thigmotropism and hormonal responses. Our data show that mutants lacking either XLG3 or AGB1 genes are hypersensitive to ethylene and show growth responses consistent with alterations in auxin transport, while maintaining an essentially wild-type response to the physical cues of gravity and touch. These results suggest that XLG3 and AGB1 proteins regulate the hormonal determinants of root-waving and root-skewing responses in plants and possibly interact in a tissue-specific or signal-specific manner. Because plants harboring knockout mutations in the Galpha subunit gene, GPA1, exhibit wild-type root waving and skewing, our results may indicate that the AGB1 subunit functions in these processes without formation of a classic Galphabetagamma heterotrimer.

The exploring root — root growth responses to local environmental conditions

Because of their sessile lifestyle, the areas which plants can access to forage for resources are confined to those which can be explored by growth. High sensitivity to environmental conditions coupled to the appropriate readjustment of growth and developmental responses are thus critical to plant survival. In this review, we focus on how roots perceive physical cues such as soil water status and mechanical properties and translate them into physiological signals to redirect organ growth and modulate root system architecture. Because the precise molecular identity of most of the sensors used by the root to sample the soil environment remain to be determined, the mechanisms underlying similar processes in microbes are providing important models for how these receptor systems may be functioning in plants.

A cytoplasmic Ca2+ functional assay for identifying and purifying endogenous cell signaling peptides in Arabidopsis seedlings: identification of AtRALF1 peptide.

Transient increases in the cytoplasmic Ca(2+) concentration are key events that initiate many cellular signaling pathways in response to developmental and environmental cues in plants; however, only a few extracellular mediators regulating cytoplasmic Ca(2+) singling are known to date. To identify endogenous cell signaling peptides regulating cytoplasmic Ca(2+) signaling, Arabidopsis seedlings expressing aequorin were used for an in vivo luminescence assay for Ca(2+) changes. These seedlings were challenged with fractions derived from plant extracts. Multiple heat-stable, protease-sensitive peaks of calcium elevating activity were observed after fractionation of these extracts by high-performance liquid chromatography. Tandem mass spectrometry identified the predominant active molecule isolated by a series of such chromatographic separations as a 49-amino acid polypeptide, AtRALF1 (the rapid alkalinization factor protein family). Within 40 s of treatment with nanomolar concentrations of the natural or synthetic version of the peptides, the cytoplasmic Ca(2+) level increased and reached its maximum. Prior treatment with a Ca(2+) chelator or inhibitor of IP 3-dependent signaling partially suppressed the AtRALF1-induced Ca(2+) concentration increase, indicating the likely involvement of Ca(2+) influx across the plasma membrane as well as release of Ca(2+) from intracellular reserves. Ca(2+) imaging using seedlings expressing the FRET-based Ca(2+) sensor yellow cameleon (YC) 3.6 showed that AtRALF1 could induce an elevation in Ca(2+) concentration in the surface cells of the root consistent with the very rapid effects of addition of AtRALF1 on Ca(2+) levels as reported by aequorin. Our data support a model in which the RALF peptide mediates Ca(2+)-dependent signaling events through a cell surface receptor, where it may play a role in eliciting events linked to stress responses or the modulation of growth.