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Running Head: PRX01, PRX44 and PRX73 are peroxidases active in root hair growth
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Authors for Correspondence:
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José M. Estevez
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Fundación Instituto Leloir, Av. Patricias Argentinas 435, Buenos Aires C1405BWE, Argentina. TE: 54-
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115238-7500 EXT. 3206
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Centro de Biotecnología Vegetal, Facultad de Ciencias de la Vida, Universidad Andrés Bello and
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Millennium Institute for Integrative Biology (iBio), Santiago CP 8370146, Chile.
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Email: jestevez@leloir.org.ar / jose.estevez@unab.cl
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Research area most appropriate for paper: Plant Biology
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Text Word count: 6,668
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Figures 1-4
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Table 1
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Experimental procedures
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References: 74
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.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted April 29, 2020. ; https://doi.org/10.1101/2020.02.04.932376doi: bioRxiv preprint
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RAPID REPORT
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Class III peroxidases PRX01, PRX44, and PRX73 potentially target extensins during root hair
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growth in Arabidopsis thaliana
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Eliana Marzol
1,#
, Cecilia Borassi
1,#
, Philippe Ranocha
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, Ariel. A. Aptekman
3,4
, Mauro Bringas
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, Janice
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Pennington
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, Julio Paez-Valencia
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, Javier Martínez Pacheco
1
, Diana Rosa Rodríguez Garcia
1
,
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Yossmayer del Carmen Rondón Guerrero
1
, Mariana Carignani
1
, Silvina Mangano
1
, Margaret
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Fleming
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, John W. Mishler-Elmore
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, Francisca Blanco-Herrera
9,10
, Patricia Bedinger
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, Christophe
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Dunand
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, Luciana Capece
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, Alejandro D. Nadra
3,4
, Michael Held
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, Marisa S. Otegui
6,11
&
José M.
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Estevez
1,9,†
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1
Fundación Instituto Leloir and IIBBA-CONICET. Av. Patricias Argentinas 435, Buenos Aires C1405BWE,
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Argentina.
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2
Université de Toulouse, UPS, UMR 5546, Laboratoire de Recherche en Sciences Végétales, F-31326
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CNRS, UMR 5546 Castanet-Tolosan, France.
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Departamento de Fisiología, Biología Molecular y Celular, Instituto de Biociencias, Biotecnología y
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Biología Traslacional (iB3). Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires,
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Ciudad Universitaria, Buenos Aires C1428EGA, Argentina.
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Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de
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Buenos Aires (IQUIBICEN-CONICET), Ciudad Universitaria, Buenos Aires C1428EGA, Argentina.
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Departamento de Química Inorgánica, Analítica y Química Física, Facultad de Ciencias Exactas y
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Naturales, Universidad de Buenos Aires (INQUIMAE-CONICET), Buenos Aires, CP. C1428EGA,
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Argentina.
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Laboratory of Cell and Molecular Biology, University of Wisconsin, Madison, WI, USA.
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Department of Biology, Colorado State University, Fort Collins, Colorado 80523-1878, USA.
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Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45701, USA.
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Centro de Biotecnología Vegetal, Facultad de Ciencias de la Vida, Universidad Andrés Bello and
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Millennium Institute for Integrative Biology (iBio), Santiago, Chile.
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Center of Applied Ecology and Sustainability (CAPES), Chile.
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Departments of Botany and Genetics, University of Wisconsin, Madison, WI, USA.
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#
co-first authors
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†
Correspondence should be addressed. Email: jestevez@leloir.org.ar / jose.estevez@unab.cl (J.M.E).
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Key words: Arabidopsis, cell walls, extensins, root hairs, ROS, class-III peroxidases.
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Word count: 4,295
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Abstract
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Root hair cells are important sensors of soil conditions. Expanding several hundred times their
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original size, root hairs grow towards and absorb water-soluble nutrients. This rapid growth is
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oscillatory and is mediated by continuous remodelling of the cell wall. Root hair cell walls contain
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polysaccharides and hydroxyproline-rich glycoproteins including extensins (EXTs).
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Class-III peroxidases (PRXs) are secreted into the apoplastic space and are thought to trigger either
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cell wall loosening, mediated by oxygen radical species, or polymerization of cell wall components,
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including the Tyr-mediated assembly of EXT networks (EXT-PRXs). The precise role of these EXT-
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PRXs is unknown.
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Using genetic, biochemical, and modeling approaches, we identified and characterized three root
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hair-specific putative EXT-PRXs, PRX01, PRX44, and PRX73. The triple mutant prx01,44,73 and the
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PRX44 and PRX73 overexpressors had opposite phenotypes with respect to root hair growth,
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peroxidase activity and ROS production with a clear impact on cell wall thickness.
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Modeling and docking calculations suggested that these three putative EXT-PRXs may interact with
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non-O-glycosylated sections of EXT peptides that reduce the Tyr-to-Tyr intra-chain distances in EXT
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aggregates and thereby may enhance Tyr crosslinking. These results suggest that these three
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putative EXT-PRXs control cell wall properties during the polar expansion of root hair cells.
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Word count: 200
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was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted April 29, 2020. ; https://doi.org/10.1101/2020.02.04.932376doi: bioRxiv preprint
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Introduction
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Primary cell walls, composed by a diverse network containing mainly polysaccharides and a small
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amount of structural glycoproteins, regulate cell elongation, which is crucial for several plant growth
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and developmental processes. Extensins (EXTs) belong to hydroxyproline (Hyp)-rich glycoprotein
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(HRGP) superfamily and broadly include related glycoproteins such as proline-rich proteins (PRPs) and
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leucine-rich repeat extensins (LRXs) with multiple Ser-(Pro)
3–5
repeats that may be O-glycosylated and
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contain Tyr (Y)-based motifs (Lamport et al. 2011; Marzol et al. 2018). EXTs require several
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modifications before they become functional (Lamport et al., 2011; Marzol et al. 2018). After being
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hydroxylated and O-glycosylated in the secretory pathway, the secreted O-glycosylated EXTs are
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crosslinked and insolubilized in the plant cell wall by the oxidative activity of secreted class-III
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peroxidases (PRXs) on the Tyr-based motifs (Baumberger 2001, 2003; Ringli 2010; Held et al. 2004;
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Lamport et al., 2011; Chen et al. 2015; Marzol et al. 2018). PRXs are thought to facilitate both intra
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and inter-molecular covalent Tyr–Tyr crosslinks in EXT networks, possibly through the assembly of
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triple helices (Velasquez et al. 2015a; Marzol et al. 2018) by generating isodityrosine units (IDT) and
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pulcherosine, or di-isodityrosine (Di-IDT), respectively (Brady et al., 1996; 1998; Held et al. 2004). In
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addition, O-glycosylation levels in EXTs also affect their insolubilization process in the cell wall (Chen
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et al. 2015; Velasquez et al. 2015a) since it might influence the EXT interactions with other cell wall
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components (Nuñez et al., 2009; Valentin et al., 2010). However, the underlying molecular
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mechanisms of EXT crosslinking and assembly have not been fully determined. It is proposed that O-
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glycosylation levels as well as the presence of Tyr-mediated crosslinking in EXT and related
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glycoproteins allow them to form a dendritic glycoprotein network in the cell wall. This EXT network
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affects de novo cell wall formation during embryo development (Hall and Cannon 2002; Cannon et al.,
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2008), they are also implicated in roots, petioles and rosette leaves growth (Saito et al 2014; Møller
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et al. 2017) and in polar cell expansion processes in root hairs (Baumberger 2001, 2003; Ringli 2010;
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Velasquez et al. 2011; 2012; 2015a,b) as well as in pollen tubes (Fabrice et al. 2018; Sede et al. 2018;
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Wang et al. 2018).
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Apoplastic class-III PRXs are heme-iron-dependent proteins, members of a large multigenic family in
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land plants, with 73 members in Arabidopsis thaliana (Passardi et al. 2004; Weng and Chapple, 2010).
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These PRXs catalyze several different classes of reactions. PRX activities coupled to
apo
ROS molecules
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(
apo
H
2
O
2
) directly affect the degree of cell wall crosslinking (Dunand et al. 2007) by oxidizing cell wall
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compounds and leading to stiffening of the cell wall through a peroxidative cycle (PC) (Passardi et al.
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2004, Cosio & Dunand 2009; Lamport et al. 2011). By constrast,
apo
ROS coupled to PRX activity
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enhances non-enzymatic cell wall-loosening by producing oxygen radical species (e.g.,
●
OH) and
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promoting growth in the hydroxylic cycle (HC). In this HC cycle, PRXs catalyze the reaction in which
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hydroxyl radicals (
●
OH) are produced from H
2
O
2
after O
2
●-
dismutation. In this manner, some PRXs
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(e.g. PRX36) may function in weaken plant cell walls by the generated
●
OH that cleave cell wall
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polysaccharides in seed mucilage extrusion in epidermal cells in the Arabidopsis seed coat (Kunieda
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was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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et al., 2013). It is unclear how these opposite effects on cell wall polymers are coordinated during
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plant growth (Passardi et al. 2004, Cosio & Dunand 2009; Lee et al. 2013; Ropollo et al. 2011; Lee et
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al 2018; Francoz et al. 2019). Finally, PRXs also contribute to the superoxide radical (O
2
●-
) pool by
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oxidizing singlet oxygen in the oxidative cycle (OC), thereby affecting
apo
H
2
O
2
levels. Thus, several PRXs
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are involved in the oxidative polymerization of monolignols in the apoplast of the lignifying cells in
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xylem (e.g. PRX017, Cosio et al 2017; PRX72, Herrero et al. 2013), in the root endodermis (e.g. PRX64;
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Lee et al. 2013; Ropollo et al. 2011), and in petal detachment (Lee et al 2018). In addition, PRXs are
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able to polymerize other components of the plant cell wall such as suberin (Bernards et al., 1999),
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pectins (Francoz et al. 2019), and EXTs (Schnabelrauch et al., 1996; Jackson et al., 2001). Although
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several candidates of PRXs have been associated specifically with EXT-crosslinking (EXT-PRXs) by in
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vitro studies (Schnabelrauch et al., 1996; Wojtaszek et al., 1997; Jackson et al., 2001; Price et al., 2003;
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Pereira et al. 2011; Dong et al., 2015) or based on an immunolabelling extensin study linked to a
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genetic profile (Jacobowitz et al. 2019), the in vivo characterization and mode of action of these EXT-
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PRXs remain largely unknown. In this work, we used a combination of reverse genetics, molecular and
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cell biology, computational molecular modeling, and biochemistry to identify three apoplastic PRXs,
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PRX01, PRX44 and PRX73, as key enzymes possibly potentially involved in Tyr-crosslinking of cell wall
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EXTs in growing root hair cells. In addition, we propose a hypothetical model in which O-glycosylation
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levels on the triple helixes of EXTs might regulate the degree of Tyr-crosslinking affecting the
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expansion properties of cell walls as suggested before based on the extended helical polyproline-II
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conformation state of EXTs (Stafstrom & Staehelin 1986; Owen et al., 2010; Ishiwata et al., 2014)
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together with an experimental Atomic Force Microscopic (AFM) analysis of crosslinked EXT3
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monomers (Cannon et al. 2008) linked to modelling approaches (Velasquez et al. 2015a; Marzol et al
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2018). Our results open the way for the discovery of similar interactions in EXT assemblies during root
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hair development and in response to the environmental changes, such fluctuating nutrient availability
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in the soil.
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Results and Discussion
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In this work, we have chosen to analyze root hair cells because they are an excellent model for tracking
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cell elongation and identifying PRXs involved in EXT assembly. In previous work, the phenotypes of
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mutants for PRX01, PRX44 and PRX73 suggested that these PRXs are involved in root hair growth and
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ROS homeostasis, although their mechanisms of action remained to be characterized (Mangano et al.
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2017). All three PRXs are under the transcriptional regulation of the root hair specific transcription
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factor RSL4 (Yi et al. 2010; Mangano et al. 2017). As expected, these three PRXs are also highly co-
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expressed with other root hair-specific genes encoding cell wall EXTs (e.g., EXT6-7, EXT12-14, and
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EXT18) and EXT-related glycoproteins (e.g. LRX1 and LRX2), which functions in cell expansion (Ringli
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2010; Velasquez et al. 2011; Velasquez et al. 2015b) (Figure S1). Based on this evidence, we
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hypothesized that these three PRXs might be EXT-PRXs and catalyze Tyr-crosslinks to assemble EXTs
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in root hair cell walls.
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