Root hairs are single cells that develop by tip growth, a process shared with pollen tubes, axons, and fungal hyphae. However, structural plant cell walls impose constraints to accomplish tip growth. In addition to polysaccharides, plant cell walls are composed of hydroxyproline-rich glycoproteins (HRGPs), which include several groups of O-glycoproteins, including extensins (EXTs). Proline hydroxylation, an early post-translational modification (PTM) of HRGPs catalyzed by prolyl 4-hydroxylases (P4Hs), defines their subsequent O-glycosylation sites. In this work, our genetic analyses prove that P4H5, and to a lesser extent P4H2 and P4H13, are pivotal for root hair tip growth. Second, we demonstrate that P4H5 has in vitro preferred specificity for EXT substrates rather than for other HRGPs. Third, by P4H promoter and protein swapping approaches, we show that P4H2 and P4H13 have interchangeable functions but cannot replace P4H5. These three P4Hs are shown to be targeted to the secretory pathway, where P4H5 forms dimers with P4H2 and P4H13. Finally, we explore the impact of deficient proline hydroxylation on the cell wall architecture. Taken together, our results support a model in which correct peptidyl-proline hydroxylation on EXTs, and possibly in other HRGPs, is required for proper cell wall self-assembly and hence root hair elongation in Arabidopsis thaliana.

ORCID ID: 0000-0001-6332-7738 (J.M.E.).Root hair cells and pollen tubes, like fungal hyphae, possess a typical tip or polar cell expansion with growth limited to the apical dome. Cell expansion needs to be carefully regulated to produce a correct shape and size. Polar cell growth is sustained by oscillatory feedback loops comprising three main components that together play an important role regulating this process. One of the main components are reactive oxygen species (ROS) that, together with calcium ions (Ca 2+ ) and pH, sustain polar growth over time. Apoplastic ROS homeostasis controlled by NADPH oxidases as well as by secreted type III peroxidases has a great impact on cell wall properties during cell expansion. Polar growth needs to balance a focused secretion of new materials in an extending but still rigid cell wall in order to contain turgor pressure. In this review, we discuss the gaps in our understanding of how ROS impact on the oscillatory Ca 2+ and pH signatures that, coordinately, allow root hair cells and pollen tubes to expand in a controlled manner to several hundred times their original size toward specific signals.

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Reactive oxygen species (ROS) are recognized as important signaling components in various processes in plants. ROS are produced for NADPH oxidase in different subcellular compartments and they are involved for a wide range of stimuli, such as cell cycle, growth, plant defenses, abiotic stress responses, and abscisic acid signaling in guard cells. In Arabidopsis, root hairs ROS also play a key role in root hair growth and they control the activity of calcium channels required for polar growth (Takeda et al. Science 319:1241-1244, 2008). The production of reactive oxygen species is under a specific molecular control in order to avoid detrimental side effects. Here we describe a protocol to detect ROS by oxidation of a derivative of fluorescein: 2',7-dihidro dicloro fluorescein (H2DCFDA).

In vitro tomato pollen tubes show a cytoplasmic calcium gradient that oscillates with the same period as growth. Pollen tube growth requires coordination between the tip-focused cytoplasmic calcium concentration ([Ca]) gradient and the actin cytoskeleton. This [Ca] gradient is necessary for exocytosis of small vesicles, which contributes to the delivery of new membrane and cell wall at the pollen tube tip. The mechanisms that generate and maintain this [Ca] gradient are not completely understood. Here, we studied calcium dynamics in tomato (Solanum lycopersicum) pollen tubes using transgenic tomato plants expressing the Yellow Cameleon 3.6 gene under the pollen-specific promoter LAT52. We use tomato as an experimental model because tomato is a Solanaceous plant that is easy to transform, and has an excellent genomic database and genetic stock center, and unlike Arabidopsis, tomato pollen is a good system to do biochemistry. We found that tomato pollen tubes showed an oscillating tip-focused [Ca] gradient with the same period as growth. Then, we used a pharmacological approach to disturb the intracellular Ca homeostasis, evaluating how the [Ca] gradient, pollen germination and in vitro pollen tube growth were affected. We found that cyclopiazonic acid (CPA), a drug that inhibits plant P-type Ca-ATPases, increased [Ca] in the subapical zone, leading to the disappearance of the Ca oscillations and inhibition of pollen tube growth. In contrast, 2-aminoethoxydiphenyl borate (2-APB), an inhibitor of Ca released from the endoplasmic reticulum to the cytoplasm in animals cells, completely reduced [Ca] at the tip of the tube, blocked the gradient and arrested pollen tube growth. Although both drugs have antagonistic effects on [Ca], both inhibited pollen tube growth triggering the disappearance of the [Ca] gradient. When CPA and 2-APB were combined, their individual inhibitory effects on pollen tube growth were partially compensated. Finally, we found that GsMTx-4, a peptide from spider venom that blocks stretch-activated Ca channels, inhibited tomato pollen germination and had a heterogeneous effect on pollen tube growth, suggesting that these channels are also involved in the maintenance of the [Ca] gradient. All these results indicate that tomato pollen tube is an excellent model to study calcium dynamics.