Silvina Mangano

The Ionic Environment Controls the Contribution of the Barley HvHAK1 Transporter to Potassium Acquisition

The control of potassium (K+) acquisition is a critical requirement for plant growth. Although HAK1 (high affinity K+ 1) transporters provide a pathway for K+ acquisition, the effect exerted by the ionic environment on their contribution to K+ capture remains essentially unknown. Here, the influence of the ionic environment on the accumulation of transcripts coding for the barley (Hordeum vulgare) HvHAK1 transporter as well as on HvHAK1-mediated K+ capture has been examined. In situ mRNA hybridization studies show that HvHAK1 expression occurs in most root cells, being augmented at the outermost cell layers. Accumulation of HvHAK1 transcripts is enhanced by K+ deprivation and transiently by exposure to high salt concentrations. In addition, studies on the accumulation of transcripts coding for HvHAK1 and its close homolog HvHAK1b revealed the presence of two K+-responsive pathways, one repressed and the other insensitive to ammonium. Experiments with Arabidopsis (Arabidopsis thaliana) HvHAK1-expressing transgenic plants showed that K+ deprivation enhances the capture of K+ mediated by HvHAK1. A detailed study with HvHAK1-expressing Saccharomyces cerevisiae cells also revealed an increase of K+ uptake after K+ starvation. This increase did not occur in cells grown at high Na+ concentrations but took place for cells grown in the presence of NH4+. 3,3′-Dihexyloxacarbocyanine iodide accumulation measurements indicate that the increased capture of K+ in HvHAK1-expressing yeast cells cannot be explained only by changes in the membrane potential. It is shown that the yeast protein phosphatase PPZ1 as well as the halotolerance HAL4/HAL5 kinases negatively regulate the HvHAK1-mediated K+ transport.

ROS Regulation of Polar Growth in Plant Cells

ROS act as a key growth signal in root hairs and pollen tubes. 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 (Ca2+) 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 Ca2+ 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.

Point mutations in the barley HvHAK1 potassium transporter lead to improved K+-nutrition and enhanced resistance to salt stress.

Members of group I KT‐HAK‐KUP transporters play an important role in K+ acquisition by plant roots, a process that is strongly affected by salt stress. A PCR‐based random mutagenesis approach on HvHAK1 allowed identification of V366I and R591C substitutions, which confer enhanced K+‐capture, and improved NaCl, LiCl and NH4Cl tolerance, to yeast cells. Improved K+‐capture was linked to an enhanced V max. Results reveal an intrinsic protective effect of K+, and assign an important role to the 8th transmembrane domain, as well as the C‐terminus, in determining the maximum capacity for the transport of K+ in KT‐HAK‐KUP transporters.

Complex Regulation of Prolyl-4-Hydroxylases Impacts Root Hair Expansion

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 inxa0vitro preferred specificity for EXT substrates rather than for other HRGPs. Third, by P4H promoter and protein swapping approaches, wexa0show 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.

Molecular link between auxin and ROS-mediated polar growth

Significance Tip-growing root hairs are an excellent model system for deciphering the molecular mechanism underlying reactive oxygen species (ROS)-mediated cell elongation. Root hairs are able to expand in response to external signals, increasing several hundred-fold their original size, which is important for survival of the plant. Although their final cell size is of fundamental importance, the molecular mechanisms that control it remain largely unknown. In this study, we propose a molecular mechanism that links the auxin–auxin response factors module to activation of RSL4, which directly targets genes encoding ROS-producing enzymes such as NADPH oxidases (or RESPIRATORY BURST OXIDASE HOMOLOG proteins) and secreted type III peroxidases. Activation of these genes impacts on apoplastic ROS homeostasis, thereby stimulating root hair cell elongation. Root hair polar growth is endogenously controlled by auxin and sustained by oscillating levels of reactive oxygen species (ROS). These cells extend several hundred-fold their original size toward signals important for plant survival. Although their final cell size is of fundamental importance, the molecular mechanisms that control it remain largely unknown. Here we show that ROS production is controlled by the transcription factor RSL4, which in turn is transcriptionally regulated by auxin through several auxin response factors (ARFs). In this manner, auxin controls ROS-mediated polar growth by activating RSL4, which then up-regulates the expression of genes encoding NADPH oxidases (also known as RESPIRATORY BURST OXIDASE HOMOLOG proteins) and class III peroxidases, which catalyze ROS production. Chemical or genetic interference with ROS balance or peroxidase activity affects root hair final cell size. Overall, our findings establish a molecular link between auxin and ROS-mediated polar root hair growth.

Low Sugar Is Not Always Good: Impact of Specific O-Glycan Defects on Tip Growth in Arabidopsis

Mutants of the O-glycosylation pathway of extensins as well as molecular dynamics simulations uncover the effects of the O-glycosylation machinery on root hair tip growth.

RSL4 Takes Control: Multiple Signals, One Transcription Factor

Root hair growth dramatically expands the root surface area, thus facilitating water and nutrient uptake. Until recently, the molecular mechanism underlying root hair growth was unknown. Recent studies have revealed that the transcription factor ROOT HAIR DEFECTIVE 6 LIKE 4 (RSL4) coordinates hormonal, environmental, and developmental factors to trigger polar growth.

How Does pH Fit in with Oscillating Polar Growth

Polar growth in root hairs and pollen tubes is an excellent model for investigating plant cell size regulation. While linear plant growth is historically explained by the acid growth theory, which considers that auxin triggers apoplastic acidification by activating plasma membrane P-type H+-ATPases (AHAs) along with cell wall relaxation over long periods, the apoplastic pH (apopH) regulatory mechanisms are unknown for polar growth. Polar growth is a fast process mediated by rapid oscillations that repeat every ∼20-40s. In this review, we explore a reactive oxygen species (ROS)-dependent mechanism that could generate oscillating apopH gradients in a coordinated manner with growth and Ca2+ oscillations. We propose possible mechanisms by which apopH oscillations are coordinated with polar growth together with ROS and Ca2+ waves.

Improved ROS Measurement in Root Hair Cells

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).

CONFLICTING AUXIN-PHOSPHATE SIGNALS IMPACT ON RSL2 EXPRESSION AND ROS-HOMEOSTASIS LINKED TO ROOT HAIR GROWTH IN ARABIDOPSIS

Here, we examined by which mechanism root hairs integrate conflicting growth-signals like the repressive high Pi-level clue and a concomitant high auxin exposure that should promote growth and questioned if these complex signals might activate known molecular players in polar growth.