Transcription factor NAC1 activates expression of peptidase-encoding AtCEPs in roots to limit root hair growth.

Plant genomes encode a unique group of papain-type Cysteine EndoPeptidases (CysEPs) containing a KDEL endoplasmic reticulum (ER) retention signal (KDEL-CysEPs or CEPs). CEPs process the cell-wall scaffolding EXTENSIN (EXT) proteins that regulate de novo cell-wall formation and cell expansion. Since CEPs cleave EXTs and EXT-related proteins, acting as cell-wall-weakening agents, they may play a role in cell elongation. The Arabidopsis (Arabidopsis thaliana) genome encodes 3 CEPs (AtCPE1-AtCEP3). Here, we report that the genes encoding these 3 Arabidopsis CEPs are highly expressed in root-hair (RH) cell files. Single mutants have no evident abnormal RH phenotype, but atcep1-3 atcep3-2 and atcep1-3 atcep2-2 double mutants have longer RHs than wild-type (Wt) plants, suggesting that expression of AtCEPs in root trichoblasts restrains polar elongation of the RH. We provide evidence that the transcription factor NAC1 (petunia NAM and Arabidopsis ATAF1, ATAF2, and CUC2) activates AtCEPs expression in roots to limit RH growth. Chromatin immunoprecipitation indicates that NAC1 binds to the promoter of AtCEP1, AtCEP2, and, to a lower extent, AtCEP3 and may directly regulate their expression. Inducible NAC1 overexpression increases AtCEP1 and AtCEP2 transcript levels in roots and leads to reduced RH growth while the loss of function nac1-2 mutation reduces AtCEP1-AtCEP3 gene expression and enhances RH growth. Likewise, expression of a dominant chimeric NAC1-SRDX repressor construct leads to increased RH length. Finally, we show that RH cell walls in the atcep1-3 atcep3-2 double mutant have reduced levels of EXT deposition, suggesting that the defects in RH elongation are linked to alterations in EXT processing and accumulation. Our results support the involvement of AtCEPs in controlling RH polar growth through EXT processing and insolubilization at the cell wall.

Ethylene signaling increases reactive oxygen species accumulation to drive root hair initiation in Arabidopsis.

Root hair initiation is a highly regulated aspect of root development. The plant hormone ethylene and its precursor, 1-amino-cyclopropane-1-carboxylic acid, induce formation and elongation of root hairs. Using confocal microscopy paired with redox biosensors and dyes, we demonstrated that treatments that elevate ethylene levels lead to increased hydrogen peroxide accumulation in hair cells prior to root hair formation. In the ethylene-insensitive receptor mutant, etr1-3, and the signaling double mutant, ein3eil1, the increase in root hair number or reactive oxygen species (ROS) accumulation after ACC and ethylene treatment was lost. Conversely, etr1-7, a constitutive ethylene signaling receptor mutant, has increased root hair formation and ROS accumulation, similar to ethylene-treated Col-0 seedlings. The caprice and werewolf transcription factor mutants have decreased and elevated ROS levels, respectively, which are correlated with levels of root hair initiation. The rhd2-6 mutant, with a defect in the gene encoding the ROS-synthesizing RESPIRATORY BURST OXIDASE HOMOLOG C (RBOHC), and the prx44-2 mutant, which is defective in a class III peroxidase, showed impaired ethylene-dependent ROS synthesis and root hair formation via EIN3EIL1-dependent transcriptional regulation. Together, these results indicate that ethylene increases ROS accumulation through RBOHC and PRX44 to drive root hair formation.

Class III Peroxidases PRX01, PRX44, and PRX73 Control Root Hair Growth in Arabidopsis thaliana.

Root hair cells are important sensors of soil conditions. They grow towards and absorb water-soluble nutrients. This fast and oscillatory growth is mediated by continuous remodeling of the cell wall. Root hair cell walls contain polysaccharides and hydroxyproline-rich glycoproteins, including extensins (EXTs). Class-III peroxidases (PRXs) are secreted into the apoplastic space and are thought to trigger either cell wall loosening or polymerization of cell wall components, such as Tyr-mediated assembly of EXT networks (EXT-PRXs). The precise role of these EXT-PRXs is unknown. Using genetic, biochemical, and modeling approaches, we identified and characterized three root-hair-specific putative EXT-PRXs, PRX01, PRX44, and PRX73. prx01,44,73 triple mutation and PRX44 and PRX73 overexpression had opposite effects on root hair growth, peroxidase activity, and ROS production, with a clear impact on cell wall thickness. We use an EXT fluorescent reporter with contrasting levels of cell wall insolubilization in prx01,44,73 and PRX44-overexpressing background plants. In this study, we propose that PRX01, PRX44, and PRX73 control EXT-mediated cell wall properties during polar expansion of root hair cells.

Apoplastic class III peroxidases PRX62 and PRX69 promote Arabidopsis root hair growth at low temperature.

Root Hairs (RHs) growth is influenced by endogenous and by external environmental signals that coordinately regulate its final cell size. We have recently determined that RH growth was unexpectedly boosted when Arabidopsis thaliana seedlings are cultivated at low temperatures. It was proposed that RH growth plasticity in response to low temperature was linked to a reduced nutrient availability in the media. Here, we explore the molecular basis of this RH growth response by using a Genome Wide Association Study (GWAS) approach using Arabidopsis thaliana natural accessions. We identify the poorly characterized PEROXIDASE 62 (PRX62) and a related protein PRX69 as key proteins under moderate low temperature stress. Strikingly, a cell wall protein extensin (EXT) reporter reveals the effect of peroxidase activity on EXT cell wall association at 10 °C in the RH apical zone. Collectively, our results indicate that PRX62, and to a lesser extent PRX69, are key apoplastic PRXs that modulate ROS-homeostasis and cell wall EXT-insolubilization linked to RH elongation at low temperature.

Proline-rich extensin-like receptor kinases PERK5 and PERK12 are involved in pollen tube growth.

Proline-rich extensin-like receptor kinases (PERKs) belong to the hydroxyproline-rich glycoprotein (HRGP) superfamily known to be involved in many plant developmental processes. Here, we characterized two pollen-expressed PERKs from Arabidopsis thaliana, PERK5 and PERK12. Pollen tube growth was impaired in single and double perk5-1 perk12-1 loss of function mutants, with an impact on seed production. When the segregation was analysed, a male gametophytic defect was found, indicating that perk5-1 and perk12-1 mutants carry deficient pollen transmission. Furthermore, perk5-1 perk12-1 displayed an excessive accumulation of pectins and cellulose at the cell wall of the pollen tubes. Our results indicate that PERK5 and PERK12 are necessary for proper pollen tube growth, highlighting their role in cell wall assembly and reactive oxygen species homeostasis.

A cell surface arabinogalactan-peptide influences root hair cell fate.

Root hairs (RHs) develop from specialized epidermal trichoblast cells, whereas epidermal cells that lack RHs are known as atrichoblasts. The mechanism controlling RH cell fate is only partially understood. RH cell fate is regulated by a transcription factor complex that promotes the expression of the homeodomain protein GLABRA 2 (GL2), which blocks RH development by inhibiting ROOT HAIR DEFECTIVE 6 (RHD6). Suppression of GL2 expression activates RHD6, a series of downstream TFs including ROOT HAIR DEFECTIVE 6 LIKE-4 (RSL4) and their target genes, and causes epidermal cells to develop into RHs. Brassinosteroids (BRs) influence RH cell fate. In the absence of BRs, phosphorylated BIN2 (a Type-II GSK3-like kinase) inhibits a protein complex that regulates GL2 expression. Perturbation of the arabinogalactan peptide (AGP21) in Arabidopsis thaliana triggers aberrant RH development, similar to that observed in plants with defective BR signaling. We reveal that an O-glycosylated AGP21 peptide, which is positively regulated by BZR1, a transcription factor activated by BR signaling, affects RH cell fate by altering GL2 expression in a BIN2-dependent manner. Changes in cell surface AGP disrupts BR responses and inhibits the downstream effect of BIN2 on the RH repressor GL2 in root epidermis.

High Auxin and High Phosphate Impact on RSL2 Expression and ROS-Homeostasis Linked to Root Hair Growth in Arabidopsis thaliana.

Root hair size determines the surface area/volume ratio of the whole roots exposed to the nutrient and water pools, thereby likely impacting nutrient and water uptake rates. The speed at which they grow is determined both by cell-intrinsic factors like hormones (e.g., auxin) and external environmental signals like nutrient availability in the soil (e.g., phosphate). Overall root hair growth is controlled by the transcription factors RSL4 and RSL2. While high levels of auxin promote root hair growth, high levels of inorganic phosphate (Pi) in the media are able to strongly repress RSL4 and RSL2 expression linked to a decreased polar growth. In this work, we inquired the mechanism used by root hairs to integrate conflicting growth signals like the repressive signal of high Pi levels and a concomitant high auxin exposure that promotes growth and questioned whether these complex signals might activate known molecular players in root hair polar growth. Under these conditions, RSL2 expression (but not RSL4) is activated linked to ROS production and root hair growth. On the other hand, by blocking ROS production derived from the NADPH Oxidase C (or RBOHC for RESPIRATORY BURST OXIDASE HOMOLOG C) and ROS production from Secreted type-III Peroxidases (PERs), it was possible to repress the auxin growth-promoting effect. This study identifies a new layer of complexity between auxin, Pi nutrient availability and RSL2/RSL4 transcription factors all acting on ROS homeostasis and growth at the root hair level.

Filling the Gaps to Solve the Extensin Puzzle.

Extensins (EXTs) are highly repetitive plant O-glycoproteins that require several post-translational modifications (PTMs) to become functional in plant cell walls. First, they are hydroxylated on contiguous proline residues; then they are O-glycosylated on hydroxyproline and serine. After secretion into the apoplast, O-glycosylated EXTs form a tridimensional network organized by inter- and intra-Tyr linkages. Recent studies have made significant progress in the identification of the enzymatic machinery required to process EXTs, which includes prolyl 4-hydroxylases, glycosyltransferases, papain-type cysteine endopeptidases, and peroxidases. EXTs are abundant in plant tissues and are particularly important in rapidly expanding root hairs and pollen tubes, which grow in a polar manner. Small changes in EXT PTMs affect fast-growing cells, although the molecular mechanisms underlying this regulation are unknown. In this review, we highlight recent advances in our understanding of EXT modifications throughout the secretory pathway, EXT assembly in cell walls, and possible sensing mechanisms involving the Catharanthus roseus cell surface sensor receptor-like kinases located at the interface between the apoplast and the cytoplasmic side of the plasma membrane.

Calcium dynamics in tomato pollen tubes using the Yellow Cameleon 3.6 sensor.

KEY MESSAGE: 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 ([Ca2+]cyt) gradient and the actin cytoskeleton. This [Ca2+]cyt 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 [Ca2+]cyt 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 [Ca2+]cyt gradient with the same period as growth. Then, we used a pharmacological approach to disturb the intracellular Ca2+ homeostasis, evaluating how the [Ca2+]cyt gradient, pollen germination and in vitro pollen tube growth were affected. We found that cyclopiazonic acid (CPA), a drug that inhibits plant PIIA-type Ca2+-ATPases, increased [Ca2+]cyt in the subapical zone, leading to the disappearance of the Ca2+ oscillations and inhibition of pollen tube growth. In contrast, 2-aminoethoxydiphenyl borate (2-APB), an inhibitor of Ca2+ released from the endoplasmic reticulum to the cytoplasm in animals cells, completely reduced [Ca2+]cyt at the tip of the tube, blocked the gradient and arrested pollen tube growth. Although both drugs have antagonistic effects on [Ca2+]cyt, both inhibited pollen tube growth triggering the disappearance of the [Ca2+]cyt 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 Ca2+ 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 [Ca2+]cyt gradient. All these results indicate that tomato pollen tube is an excellent model to study calcium dynamics.

Molecular link between auxin and ROS-mediated polar growth.

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.

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.

An update on cell surface proteins containing extensin-motifs.

In recent years it has become clear that there are several molecular links that interconnect the plant cell surface continuum, which is highly important in many biological processes such as plant growth, development, and interaction with the environment. The plant cell surface continuum can be defined as the space that contains and interlinks the cell wall, plasma membrane and cytoskeleton compartments. In this review, we provide an updated view of cell surface proteins that include modular domains with an extensin (EXT)-motif followed by a cytoplasmic kinase-like domain, known as PERKs (for proline-rich extensin-like receptor kinases); with an EXT-motif and an actin binding domain, known as formins; and with extracellular hybrid-EXTs. We focus our attention on the EXT-motifs with the short sequence Ser-Pro(3-5), which is found in several different protein contexts within the same extracellular space, highlighting a putative conserved structural and functional role. A closer understanding of the dynamic regulation of plant cell surface continuum and its relationship with the downstream signalling cascade is a crucial forthcoming challenge.

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 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.