Tatiana N. Bibikova

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.

Root hair growth in Arabidopsis thaliana is directed by calcium and an endogenous polarity.

Abstract. Tip growth of plant cells has been suggested to be regulated by a tip-focused gradient in cytosolic calcium concentration ([Ca2+]c). However, whether this gradient orients apical growth or follows the driving force for this process remains unknown. Using localized photoactivation of the caged calcium ionophore Br-A23187 we have been able to artificially generate an asymmetrical calcium influx across the root hair tip. This led to a change in the direction of tip growth towards the high point of the new [Ca2+]c gradient. Such reorientation of growth was transient and there was a return to the original direction within 15 min. Root hairs forced to change the direction of their growth by placing a mechanical obstacle in their path stopped, reoriented growth to the side, and grew past the mechanical blockage. However, as soon as the growing tip had cleared the obstacle, growth returned to the original direction. Confocal ratio imaging revealed that a tip-focused [Ca2+]c gradient was always centered at the site of active growth. When the root hair changed direction the gradient also reoriented, and when growth returned to the original direction, so did the [Ca2+]c gradient. This normal direction of apical growth of Arabidopsis thaliana (L.) Heynh. root hairs was found to be at a fixed angle from the root of 85 ± 6.7 degrees. In contrast, Tradescantia virginiana (L.) pollen tubes that were induced to reorient by touch or localized activation of the caged ionophore, did not return to the original growth direction, but continued to elongate in their new orientation. These results suggest that the tip-focused [Ca2+]c gradient is an important factor in localizing growth of the elongating root hair and pollen tube to the apex. However, it is not the primary determinant of the direction of elongation in root hairs, suggesting that other information from the root is acting to continuously reset the growth direction away from the root surface.

A Sec14p-nodulin domain phosphatidylinositol transfer protein polarizes membrane growth of Arabidopsis thaliana root hairs

Phosphatidylinositol (PtdIns) transfer proteins (PITPs) regulate signaling interfaces between lipid metabolism and membrane trafficking. Herein, we demonstrate that AtSfh1p, a member of a large and uncharacterized Arabidopsis thaliana Sec14p-nodulin domain family, is a PITP that regulates a specific stage in root hair development. AtSfh1p localizes along the root hair plasma membrane and is enriched in discrete plasma membrane domains and in the root hair tip cytoplasm. This localization pattern recapitulates that visualized for PtdIns(4,5)P2 in developing root hairs. Gene ablation experiments show AtSfh1p nullizygosity compromises polarized root hair expansion in a manner that coincides with loss of tip-directed PtdIns(4,5)P2, dispersal of secretory vesicles from the tip cytoplasm, loss of the tip f-actin network, and manifest disorganization of the root hair microtubule cytoskeleton. Derangement of tip-directed Ca2+ gradients is also apparent and results from isotropic influx of Ca2+ from the extracellular milieu. We propose AtSfh1p regulates intracellular and plasma membrane phosphoinositide polarity landmarks that focus membrane trafficking, Ca2+ signaling, and cytoskeleton functions to the growing root hair apex. We further suggest that Sec14p-nodulin domain proteins represent a family of regulators of polarized membrane growth in plants.

Root Hair Development

Root hairs are projections from the epidermal cells of the root that are thought to increase its effective surface area for nutrient and water uptake, enlarge the volume of exploited soil, and aid in anchoring the plant to the soil. Their formation occurs as a series of developmental processes starting with cell fate specification in the meristem. The root-hair-forming epidermal cell, or trichoblast, then participates in the diffuse growth phase associated with the elongation of the main root axis. After the fully elongated trichoblast exits the elongation zone, growth is reorganized and localized to the side in the process of root hair initiation. Initiation is then followed by a sustained phase of tip growth until the hair reaches its mature length. Thus, root hairs provide insight into a range of developmental processes from cell fate determination to growth control. The theme emerging from the molecular analysis of the control of root hair formation is that many regulators act at several stages of development. Root hair formation is also responsive to a multitude of nutrient and other environmental stimuli. Therefore, one explanation for the presence of the complex networks that regulate root hair morphogenesis may lie in the need to coordinate their highly plastic developmental program and entrain it to the current soil microenvironment being explored by the root.

Calcium in Root Hair Growth

The growth of cells as diverse as fungal hyphae, pollen tubes, algal rhizoids, and root hairs is characterized by a highly localized control of cell expansion confined to the growing tip. The cellular regulators that have been shown to maintain this spatial localization of growth range from monomeric G-proteins and the actin cytoskeleton to protein kinases and phospholipid-modulating enzymes. A central theme in the proposed mode of action of most of these factors is either regulation of, or response to, the concentration of cytoplasmic Ca2+. For example, a tip-focused Ca2+gradient is associated with the growing point of the root hair and is thought to mediate spatial control of membrane trafficking and the cytoskeleton. Key advances in our understanding of how Ca2+acts in this system have been the recent identification of some of the Ca2+channels and transporters likely responsible for modulating this Ca2+gradient and the likely central role for reactive oxygen species (ROS) in regulating these events. Similarly, molecular identification of Ca2+-responsive elements, such as Ca2+-dependent protein kinases, likely to interact with the tip-focused gradient has begun. This Ca2+-signaling system appears to interact with many of the other components of the tip growth system, including monomeric G-proteins, phospholipases, and the cytoskeleton, helping integrate these activities to facilitate the localization of growth.