Accepted Manuscript

Complex regulation of Prolyl-4-hydroxylases impacts root hair expansion

Silvia M. Velasquez, Martiniano M. Ricardi, Christian Peter Poulsen, Ai Oikawa,
Adiphol Dilokpimoladil, Adnan Halim, Silvina Mangano, Silvina Paola Denita Juarez,
Eliana Marzol, Juan D. Salgado Salter, Javier Gloazzo Dorosz, Cecilia Borassi,
Svenning Rune Mller, Rafael Buono, Yukiko Ohsawa, Ken Matsuoka, Marisa S.
Otegui, Henrik V. Scheller, Naomi Geshi, Bent Larsen Petersen, Norberto D. Iusem,
Jos M. Estevez
PII:

S1674-2052(14)00038-0

DOI:

10.1016/j.molp.2014.11.017

Reference:

MOLP 37

To appear in:

MOLECULAR PLANT

Received Date: 10 July 2014

Revised Date:

17 November 2014

Accepted Date: 30 November 2014

Please cite this article as: Velasquez, S.M., Ricardi, M.M., Poulsen, C.P., Oikawa, A., Dilokpimoladil, A.,
Halim, A., Mangano, S., Denita Juarez, S.P., Marzol, E., Salgado Salter, J.D., Dorosz, J.G., Borassi, C.,
Mller, S.R., Buono, R., Ohsawa, Y., Matsuoka, K., Otegui, M.S., Scheller, H.V., Geshi, N., Petersen,
B.L., Iusem, N.D., and Estevez, J.M. (2014). Complex regulation of Prolyl-4-hydroxylases impacts root
hair expansion. Mol. Plant. doi: 10.1016/j.molp.2014.11.017.
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P4H5 role on peptidyl-proline hydroxylation

Complex regulation of Prolyl-4-hydroxylases impacts root hair expansion

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Instituto de Fisiologa, Biologa Molecular y Neurociencias (IFIBYNE-CONICET), Facultad de

Ciencias Exactas y Naturales, Universidad de Buenos Aires C1428EGA, Argentina.
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VKR Research Centre, Department of Plant and Environmental Sciences, University of
Copenhagen, DK-1871 Frederiksberg C, Denmark.
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Joint BioEnergy Institute, Feedstocks Division, Lawrence Berkeley National Laboratory, 5885
Hollis St., Emeryville, CA94608, USA.
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Copenhagen Center for Glycomics, Department of Cellular and Molecular, Faculty of Health
and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark.
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Department of Botany, University of Wisconsin, Madison, WI53706, USA.
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RIKEN Plant Science Center, Tsurumi-ku, Yokohama,230-0045, Japan.
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Laboratory of Plant Nutrition, Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1,
Higashi-ku, Fukuoka, 812-8581, Japan.
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Departamento de Fisiologa, Biologa Molecular y Celular, Facultad de Ciencias Exactas y
Naturales, Universidad de Buenos Aires, Buenos Aires C1428EGA, Argentina.
*Current address: Fungal Physiology, CBS-KNAW Fungal Biodiversity Center, Uppsalalaan 8,
Utrecht 3584 CT, The Netherlands.

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Silvia M. Velasquez1, Martiniano M. Ricardi1*, Christian Peter Poulsen2*, Ai Oikawa3*,

Adiphol Dilokpimoladil2*, Adnan Halim4, Silvina Mangano1, Silvina Paola Denita Juarez1, Eliana
Marzol1, Juan D. Salgado Salter1, Javier Gloazzo Dorosz1, Cecilia Borassi1, Svenning Rune
Mller2, Rafael Buono5, Yukiko Ohsawa6, Ken Matsuoka7, Marisa S. Otegui5, Henrik V.
Scheller3, Naomi Geshi2, Bent Larsen Petersen2, Norberto D. Iusem1,8 & Jos M. Estevez1,

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* These authors contributed equally to this paper.

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Correspondence should be addressed. Email: jestevez@fbmc.fcen.uba.ar

Short Summary

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.

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P4H5 role on peptidyl-proline hydroxylation

Abstract

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 proved that P4H5, and to a lower extent

P4H2 and P4H13, are pivotal for root hair tip growth. Second, we demonstrate that P4H5 has

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in vitro preferred specificity for EXT substrates rather than for other HRGPs. Third, by P4H

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promoter and protein swapping approaches, we show that P4H2 and P4H13 have

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interchangeable functions but cannot replace P4H5. These three P4Hs are shown to be

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targeted to the secretory pathway, where P4H5 forms dimers with P4H2 and P4H13. Finally,

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we explore the impact of deficient proline hydroxylation on the cell wall architecture. Taken

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together, our results support a model in which correct peptidyl-proline hydroxylation on EXTs,

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and possibly in other HRGPs, is required for proper cell wall self-assembly and hence root hair

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elongation in Arabidopsis thaliana.

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Keywords:

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hydroxylation; root hairs

cell

expansion;

cell

walls;

protein

targeting;

proline

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enzymology;

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P4H5 role on peptidyl-proline hydroxylation

Introduction

Plant root hairs have been used as a single-cell model to study cell wall biosynthesis during tip

growth (Park et al., 2011; Velasquez et al., 2011). They can reach high growth rates and to

expand their cell walls have to accommodate extensive structural changes (Somerville et al.,

2004; Cosgrove, 2005; Ishida et al., 2008). Cell walls at the root hair tip are comprised of low-

crystalline cellulose, xyloglucans and pectins, together with hydroxyproline (Hyp)-rich O-

glycoproteins (HRGPs) that comprises several groups of O-glycoproteins including extensins

(EXTs), proline rich proteins (PRPs), and arabinogalactan proteins (AGPs) as well as small

peptides (e.g. CLAVATA3). In the case of EXTs, they are modular and structural O-

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glycoproteins with partial structural similarity to collagen, which contains tyrosine (Tyr)-

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crosslinking motifs. Unlike collagen, EXTs also include plant-specific post-translational O-

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glycosylation on the Ser-(Hyp)4 repeats. The Arabidopsis genome encodes up to 63 putative

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EXTs, some of which are classical EXTs while others are EXT-like chimeras and hybrid-EXTs

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that contain additional protein domains (Showalter et al., 2010; Lamport et al., 2011). Despite

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the high number of proteins with predicted EXT domains in the plant cell wall (Lamport et al.,

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2011), only one related mutation, ext3, has been reported to be lethal (Cannon et al., 2008).

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Several other EXT-related mutants are not lethal but rather exhibit shorter root hairs, as

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reported for ext6-7,10 and 12 (Velasquez et al., 2011; Velasquez et al., 2012), prp1, prp3and

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prp-like1 (prpl1; (Fowler et al., 1999; Bernhardt and Tierney, 2000; Boron et al., 2014) and the

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lrx1 and lrx2 mutants lacking leucine-rich repeat extensin proteins 2 (Baumberger et al., 2001;

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Baumberger et al., 2003; Ringli, 2010). This suggests that location and function of each EXT

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and EXT-related proteins within the hypothetical network at the root hair cell wall is relevant

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to the whole EXT assembly and performance. However, the molecular basis of the

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coordinated assemblage is completely unknown.

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EXT domain-containing structural O-glycoproteins are ultimately secreted into plant cell walls

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but are first shaped by several and complex PTMs such as signal peptide processing in the ER,

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conversion by hydroxylation of peptidyl-proline residues into hydroxyproline (Hyp) and O-

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glycosylation on Hyp and Ser. Detailed studies show that EXTs are usually O-glycosylated with

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P4H5 role on peptidyl-proline hydroxylation

chains of up to 4-5 linear arabinosyl units on each Hyp (Velasquez et al., 2011; Velasquez et

al., 2012; Ogawa-Ohnishi et al., 2013) and mono-galactosylated on Ser residues (Lamport et

al., 1973; Saito et al., 2014). Specifically, three arabinosyltransferases (AraT) HPAT1-HPAT3,

RRA1-RRA3, and XEG113 have recently been implicated in the sequential addition of the

innermost three L-arabinose units (Egelund et al., 2007; Gille et al., 2009; Velasquez et al.,

2011;

galactosyltransferase, named SERGT1, has been recently reported to add a single -galactose

residue to serine in EXTs (Saito et al., 2014). Finally, glycosylated EXTs are possibly crosslinked

by putative type-III peroxidases (PERs) at the Tyr residues (Cannon et al., 2008; Lamport et al.,

et

al.,

2013).

In

addition,

2011), although an in vivo evidence remains elusive.

novel

peptidyl-serine

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one

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Ogawa-Ohnishi

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Prior to O-glycosylation, prolyl 4-hydroxylases (P4Hs) hydroxylate peptidyl-proline in HRGPs,

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including EXTs. Thus, the conversion of Pro to Hyp (Lamport, 1963) in the secretory pathway

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provides reactive hydroxyl groups for further modification such as O-glycosylation. Plant P4Hs

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are monomeric membrane-bound enzymes that belong to a family of 2-oxoglutarate-

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dependent dioxygenases (Hieta and Myllyharju, 2002; Koski et al., 2007; Koski et al., 2009).

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Partial in vitro and in vivo characterization of plant P4Hs has been carried out in several plant

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model systems (Hieta and Myllyharju, 2002; Tiainen et al., 2005; Yuasa et al., 2005; Keskiaho

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et al., 2007; Vlad et al., 2007; Asif et al., 2009; Vlad et al., 2010; Velasquez et al., 2011;

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Velasquez et al., 2012). However, only the activity of two (Hieta and Myllyharju, 2002; Tiainen

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et al., 2005) out of the 13 putative AtP4H homologs and one P4H from tobacco (Yuasa et al.,

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2005) have been characterized in vitro, and only one P4H (CrP4H1 from Chlamydomonas) has

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been crystallized (Koski et al., 2007; Koski et al., 2009). Here, we demonstrate that prolyl-4-

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hydroxylase 5 (P4H5), and to a lower extent P4H2 and P4H13, are pivotal for EXT-mediated

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root hair tip-growth. By P4H promoter and protein swap approaches, we show that P4H2 and

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P4H13 have interchangeable functions but they cannot replace P4H5. These three P4Hs are

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targeted to the secretory pathway, most specifically to the ER and Golgi compartments,

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where P4H5 forms dimers with P4H2 and P4H13, suggesting the existence of P4Hs protein

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complexes. These results imply that peptidyl-proline hydroxylation in root hairs is a complex

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P4H5 role on peptidyl-proline hydroxylation
1

process that requires these three P4Hs to define the O-glycosylation sites on EXTs, and

possibly on other HRGPs to accommodate cell expansion at their apical ends.

Results

Role of P4H5 on peptidyl-proline hydroxylation of EXTs associated with root hair tip growth.

Despite P4H2,5,13 were identified as the most relevant P4Hs for root hair elongation

(Velasquez et al., 2011) only the p4h5 mutant showed a drastic root hair growth defect. To

explore the molecular basis of P4H5 predominant role on root hair cell elongation, we first

identified four residues in the oxoglutarate (OG) binding site of P4H5 by homology alignment

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with the crystallized Chlamydomonas reinhardtii CrP4H1 that could be crucial for its activity. It

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is well known that to hydroxylate peptidyl-proline residues, P4Hs needs to bind the 1- and 5-

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carboxylate groups of 2-oxoglutarate (Koski et al., 2007; Koski et al., 2009). A P4H5 variant

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(P4H5OG) with point mutations at four residues that proved to completely inactivate AtP4H1

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(Lys277Arg, Ser279Ala, Thr281Ala, Trp283Phe) (Hieta and Myllyharju, 2002) was

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used to test complementation of the insertional p4h5 mutant. As expected, unlike its WT

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counterpart, P4H5OG failed (Fig. 1B, Fig. S1B-C) to rescue p4h5, thus confirming that the

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catalytic activity of P4H5 through its intact active site, is required for root hair tip expansion.

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To test substrate specificity, we purified recombinant P4H5 without its transmembrane

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domain and found that was able to hydroxylate both AGP and EXT peptide substrates in vitro,

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albeit the latter more efficiently than the former in a given time span (Fig. 1C). In addition, 21

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peptides containing proline residues in different contexts were not hydroxylated, indicating,

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again, a high-specificity for the EXT sequence and to a lower extent for an AGP substrate

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(Table S1). Moreover, in a P4H5 substrate competition assay, increasing the AGP

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concentration up to 4:1 AGP:EXT molar ratio did not affect proline hydroxylation of EXT,

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showing the same values as EXT alone (ratio 0:1), whereas an increase in EXT concentration

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significantly diminished the hydroxylation of AGP as seen at ratios 1:1 and 1:4 AGP:EXT

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compared to AGP alone (ratio 1:0) (Fig. 1D). These results strongly suggest a preference of the

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P4H5 enzyme for EXT-substrates.

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P4H5 role on peptidyl-proline hydroxylation

We further explored whether P4H5 Pro-hydroxylation follows a particular positional order

within the Ser-(Pro)4 EXT-repeat. Based on partially hydroxylated species, we were able to

find that the reaction can start on prolines at positions 1 or 2 but not at positions 3 or 4.

Subsequently, the P4H5 hydroxylated prolines at positions 1, 2 or 3 but never at position 4,

resulting in a final product with Hyp at positions 1, 2 and 3 (Fig. 1E and Fig. S2A-D). Clearly,

P4H5 displayed a defined order of in vitro Pro hydroxylation of the EXT protein sequence in

vitro, although it is unknown if P4H5 follows the same sequential order on EXTs substrates in

vivo.

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Previously, it was determined that GFP-tagged P4H2 and P4H5 were present only in

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trichoblast cells, whereas P4H13 was expressed in both trichoblast and atrichoblast cells

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(Velasquez et al., 2011). Although all three P4Hs were localized to the apical zone of actively

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growing root hairs, P4H5 showed a higher expression than the others two P4Hs. To assess the

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degree of functional redundancy of P4H5 in comparison to P4H2 and P4H13 in root hair

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growth, we analysed multiple p4h mutants. The p4h2,5,13 triple mutant did not display a

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more drastic phenotype than the severely impaired single p4h5 mutant (Fig. 2A). No changes

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were observed for root hair density (not shown). To test whether there is a contribution of

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P4Hs other than P4H2,5,13 in the p4h mutant root hairs, we treated roots with the P4H

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inhibitors with EDHB (ethyl-3,4-dihydroxybenzoate) and DP (,-dipyridyl). EDHB interacts

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with the active oxoglutarate binding site of some P4Hs (Majamaa et al., 1986; Vlad et al.,

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2010) whereas DP chelates the cofactor Fe2+ (Barnett, 1970) (Fig. 1A). Previously to this work,

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the inhibitory concentration 50 (IC50) was determined for both inhibitors, EDHB (219 nM) and

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DP (48 nM), at which the root hair growth inhibition in Wt Col-0 roots was reduced 50% in

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comparison to non-treated root hairs (Velasquez et al., 2011). Whereas the single p4h2 and

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p4h13 mutants responded to these inhibitors, none of the other p4h mutant multiple

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combinations showed additional cell expansion inhibition indicating that besides P4H2,5,13,

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no other hair-expressed P4H contributes significantly to root hair growth (Fig. 2B). These

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results highlight that the three herein explored P4Hs are required for root hair elongation but

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not required for its initial development as the triple p4h mutant still has root hairs.

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P4H5 role on peptidyl-proline hydroxylation
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Functional interchangeability of P4H and its impact on root hair growth.

To test whether the overexpression of one P4H could rescue root hair defects in a mutant

deficient for another P4H, each P4H was GFP-tagged and expressed under the control of the

constitutive 35S promoter (35Spro) into the two other single p4h mutant backgrounds (Fig. 2C-

D). At least two to three independent transgenic lines with the highest expression levels of a

single P4H were analysed in each case. Neither 35Spro::P4H2 nor 35Spro::P4H13 were able to

rescue the p4h5 mutant. The failure in restoring the root hair growth in p4h5 mutant was not

due to an eventual low level of protein expression of P4H2 or P4H13, since similar or even

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higher levels of the P4H-GFP fluorescence were detected in conditions they fully rescued the

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corresponding mutants phenotypes (Fig. S3). Conversely, 35Spro::P4H5 could fully restore both

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p4h2 and p4h13 defective phenotypes whereas the overexpression of P4H2 in p4h13 and

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P4H13 in p4h2 also rescued root hair growth defects. Taken together, these results first

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indicate that P4H2 and P4H13 enzymatic functions are similar and thus interchangeable

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whereas P4H5 is essential and irreplaceable by the other two P4Hs.

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To better understand the observed phenotypic results in terms of expression patterns vs.

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protein sequences of P4H2, P4H5, and P4H13, root hair growth was measured in several

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independent P4H mutant lines transformed with P4H genes with swapped endogenous

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promoters. First, P4H gene expression was modified by swapped promoters fused with P4H2,

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P4H5, and P4H13 coding regions (Fig. 2E). The expression of P4H13pro::P4H2 in the p4h2 and

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P4H2pro::P4H13 in p4h13 mutant backgrounds as well as the expression of P4H5pro::P4H13 in

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p4h13 mutants rescued root hair growth defects. In addition, P4H13pro::P4H5 partially

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rescued p4h5 mutant. These results indicate partial or complete overlap in the temporal and

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spatial expression patterns of P4H2/P4H13 and P4H5/P4H13 proteins during root hair

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expansion. Next, functional interchangeability of root hair P4Hs was tested by a full P4H

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protein swap experiment to test functional specificity measured as rescue of root hair growth

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(Fig. 2F). The expression of full length P4H2 or P4H5 driven by the P4H13 promoter rescued

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the p4h13 mutant, and the expression of P4H5 protein under the control of the P4H2

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P4H5 role on peptidyl-proline hydroxylation

promoter rescued the p4h2 mutant root hair growth deficiency. In contrast, neither P4H2 nor

P4H13 proteins driven by the P4H5 promoter were able to restore the root hair growth of the

p4h5 mutant. This clearly highlights the unique and essential role of P4H5 in root hair tip

growth.

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To test whether a single P4H could complement more than one P4H at the same time, we

overexpressed individual P4Hs in double p4h mutant backgrounds (Fig. S4). P4H13 and to a

lesser extent P4H2 were able to rescue the p4h2/p4h13 double mutant but any of both P4Hs

failed to rescue the double mutant containing a p4h5 mutant allele. Conversely, high levels of

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P4H5 expression partially rescued (by 70%) the root hair growth defects in the p4h2-p4h5

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double mutant (Fig. S4). Taken together, these results reinforce the notion that P4H2 and

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P4H13 activities are likely similar and interchangeable whereas P4H5 can partially to fully

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substitute for P4H2 and P4H13 but not vice versa. Also, certain amounts of both proteins,

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P4H2 and P4H13, seem to be required for a correct cell expansion process in the root hairs.

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To corroborate the high degree of functional specificity of Arabidopsis P4H5, we

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overexpressed Nicotiana tabaccum (Nt) NtP4H1.1 and NtP4H2.1, the closest homologs of

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Arabidopsis P4H2 and P4H13, respectively, in the p4h5 mutant background (Fig. S5B). We

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then tested if any of these NtP4Hs could rescue the root hair tip-growth in the absence of

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P4H5 (Fig. S5C). Consistent with our experiments using AtP4H2 and AtP4H13, neither

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NtP4H1.1 nor NtP4H2.1 was able to complement the Atp4h5 mutant.

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Subcellular localization and protein-protein interactions of P4Hs in the secretory pathway.

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P4H2, P4H5, and P4H13 were previously shown to localize to the secretory endomembrane

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system (Velasquez et al., 2011). To investigate their spatial patterns in more detail, we

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expressed fluorescently tagged P4H fusion proteins in Nicotiana benthamiana and tested co-

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localization with several ER and cis-, medial-, and trans-Golgi markers (Fig. 3A). P4H2 localized

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to the ER but not Golgi whereas P4H5 and P4H13 were not localized to the ER but rather to

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the cis- to trans-Golgi apparatus (Fig. 3A). We next assayed for co-localization between

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fluorescently-tagged P4H2, P4H5, and P4H13 in the three possible pair-wise combinations.

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Surprisingly, the pairs P4H2-P4H5 and P4H5-P4H13 were found to localize together to

punctuated compartments, most likely the Golgi stacks (Fig. 3B). Therefore, in the presence of

P4H5, localization of P4H2 changed dramatically from the ER compartment (where P4H2 is

present when overexpressed by itself), to Golgi. However, co-expression of P4H13 with P4H2

did not have the same effect on P4H2 as the two proteins did not co-localize (Fig. 3C).

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protein interaction between P4H2 and P4H5. To determine whether the P4Hs are able to

form quaternary structures as homodimers and heterodimers, we employed bimolecular

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fluorescence complementation (BiFC) as well as Frster resonance energy transfer (FRET)

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approaches (Fig. 4; Table S2). BiFC constructs were co-expressed along with the ER or the

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Golgi marker to define the compartments where BiFC occurs. Homodimer formation between

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P4H5 and P4H13 in the Golgi rather than ER was visualized by both BiFC (Fig. 4A) and FRET

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(Fig. 4B). On the other hand, BiFC showed weak self-assembly of P4H2 homo-dimers in the ER

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but not in Golgi, although the existence of P4H2 as homodimers could not be confirmed by

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FRET (Fig. 4B). In addition, P4H5 showed a strong heterodimeric interaction with P4H2 and a

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weaker interaction with P4H13 in the Golgi, whereas no interaction was observed between

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P4H2 and P4H13 (Fig. 4C) Thus, localization of P4H2 changed depending on the co-expressed

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partner; namely when co-expressed with P4H5, P4H2 was found in the Golgi-like

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compartments, while P4H2 was found in the ER when expressed alone (Fig. 3C) or co-

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expressed with marker proteins or P4H13. In addition, P4H2 interacted with P4H5 but not

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with P4H13. All together this implies a central role of P4H5 in the formation of P4H

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complex(es) (Fig. 4C).

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ERGP4H5 domain regulates P4H5 subcellular targeting and function.

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All 13 Arabidopsis P4Hs share a well-conserved central 2-oxoglutarate dioxygenase domain

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(Hieta and Myllyharju, 2002; Tiainen et al., 2005) (Fig. S5A). In addition, P4H5 and P4H13 have

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a [RK]X[RK] motif (ERG) on the N-terminus (Fig. S5A), proposed to be responsible for ER-Golgi

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transport (Yuasa et al., 2005). On the other hand, P4H2 contains an exclusive C-terminal

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P4H5 role on peptidyl-proline hydroxylation

cysteine (Cys)-rich domain (CD) with six non-contiguous Cys residues (Fig. S5A) with no

assigned function, which is absent in P4H5 and P4H13. We then explored the functional

contribution of these domains by expressing chimeric variants of P4H2 and P4H5 (Fig. 5A,E) in

p4h2-1 and p4h5 mutant backgrounds (Fig. 5B-C,F-G) and assessing root hair growth. Neither

ERG-less P4H5 (P4H5-ERG) nor P4H5-ERG fused to the P4H2 CD domain (P4H5-ERG+CD(P4H2)), were

able to rescue the p4h5 mutant (Fig. 5C-D). Moreover, expression of P4H2+ERG(P4H5) did not

rescue root hair growth in p4h5, suggesting a key role of the ERG domain but only within its

own sequence context. On the other hand, P4H2-CD as well as P4H2-CD+ERG(P4H5) were able to

rescue both the p4h2 root hair defects (Fig. 5F-G, S6B-C). Based on these results, we

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hypothesized that the ERG domain directs P4H5 to its proper subcellular localization but is

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dispensable for P4H2. We next examined and compared the degree of co-localization of full-

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length P4H5 and P4H5-ERG with ER and Golgi markers. Truncated P4H5-ERG localized only to the

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ER whereas the full length P4H5 protein was found only in Golgi (Fig. 5H), confirming that the

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ERG-domain of P4H5 is necessary for its Golgi targeting, which in turn seems to be important

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for its function. Consistently, P4H2 that lacks the ERG domain is localized mostly in the ER

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compartment and P4H13 that contains an ERG domain is targeted to the Golgi apparatus, as

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observed for P4H5 (Fig. 3A).

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Cell wall architecture in p4h5 mutant.

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Finally, to determine whether deficient peptidyl-proline hydroxylation and subsequent O-

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glycosylation in EXTs and in other HRGPs triggers structural differences in the cell wall

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architecture, root hair tip cell walls were imaged by transmission electron microscopy (TEM)

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(Fig. 6A). Approximately, 68% of the cell wall fibrils in p4h5 and 25% of the fibrils in Wt root

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hair tips appear at angles between +20 and -20 degrees, close to being parallel to the plasma

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membrane (Fig. 6B). Fibrils are visualized as linear electrodense structures present in the cell

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wall. This analysis highlights that cell walls of Wt root hair tips are composed of differentially

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oriented fibrils (closed to +60 or -60 degrees) rather than parallel layers of fibrils like those

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observed in the p4h5 mutant. However, no significant changes at the level of root hair wall

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thickness (t-test, P<0.88) or surface wall morphology were observed (not shown). These

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findings link, for the first time, defective PTMs in EXTs and possibly in other HRGPs to changes

in the cell wall architecture at the root hair tip.

Discussion

Localized secretion and modification of extracellular matrices including plant cell walls is a

highly regulated process that is crucial for the correct development and function of

multicellular organisms. In plants cells, secretion, assembly, crosslinking and loosening of cell

wall components drive morphogenesis and maintenance of the correct cell shape in individual

cells. Profound cell wall changes take place in the endodermis lignification in the Casparian

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strips (Lee et al., 2013), in xylem vessels development (Oda and Fukuda, 2012), in pollen tube

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release of sperm cells during fertilization (Escobar-Restrepo et al., 2007; Boisson-Dernier et

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al., 2009) and in the opening and closure of stomata cells (Bergmann and Sack, 2007). Overall,

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it is still largely unknown how chemical changes on specific cell wall polymers relate to their

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functions in single-cell development, and more importantly, which molecular mechanisms

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underlie these localized changes.

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Here we show, by using the model plant Arabidopsis thaliana, that peptidyl-proline

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hydroxylation of HRGPs is required for correct root hair growth, most likely through the

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establishment of a platform for subsequent O-glycosylation. Previously, we provided evidence

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that P4H5 is a key enzyme that, together with P4H2/P4H13, converts peptidyl-Pro into Hyp,

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preferentially in EXT backbone contexts (Velasquez et al., 2011) although other HRGPs

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substrates cannot be excluded. The preference of P4H5 for EXT-type substrates was here

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confirmed by an in vitro enzymatic activity assay. In addition, by promoter and catalytic P4H

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swap approaches, we showed that P4H2 and P4H13 are interchangeable but cannot replace

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P4H5, which is absolutely required for normal root hair growth. Full protein swaps showed

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that the P4H5 catalytic domain has a distinctive function that cannot be replaced by P4H2 and

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P4H13. It is then tempting to propose that P4H5 is required for the initiation of peptidyl-

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proline hydroxylation in a specific order, and subsequently, P4H2 and/or P4H13 would

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continue this enzymatic task to achieve the fully modified Ser-Hyp-Hyp-Hyp-Hyp repeats

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commonly found in EXTs. In a similar fashion, AtP4H2 was shown to specifically hydroxylate

the last residue at the C-terminal PPP in an EXT-like peptide (Tiainen et al., 2005) suggesting a

preference in the order of peptidyl-proline hydroxylation as well. In addition, it is expected

that P4H5 would also hydroxylate proline in several contexts other than polyproline contigs of

EXTs and PRPs. What remains unclear however is the final fate of the EXT molecules that are

not properly hydroxylated and O-glycosylated during their transit throughout the ER-Golgi

compartments and final secretion into the cell wall (e.g. in the p4h5 mutants as well as in the

glycosyltransferase mutants rra3 and xeg113). Nevertheless, in some of these mutants, EXTs

do not seem to be completely degraded as it usually occurs with N-glycosylated proteins due

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to the ER-associated protein degradation (ERAD) pathway. This assertion is based on the fact

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that abnormal O-glycosylation states were clearly detected in a previous study (Velasquez et

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al., 2011) .

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The three P4Hs (P4H2, P4H5 and P4H13) studied in this work are targeted to the ER-Golgi

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compartments where P4H5 forms homo-/hetero-dimers with P4H2 and P4H13 in the Golgi,

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required for proper peptidyl-proline hydroxylation. The existence of even more complex P4Hs

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oligomers cannot be ruled out by the techniques here employed. It is then plausible that

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more than one type of P4H hetero-oligomer would be assembled in the ER-Golgi

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compartment, where the presence of P4H5 may be required for either the correct

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recruitment or scaffolding of the other P4Hs. In this context, previous studies in mammalian

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and plant cells have shown similar localization changes through direct protein-protein

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interaction, for example between: 1) GAUT1 and GAUT7 for pectin synthesis, 2) UDP-

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galactose transporter and ceramide galactosyltransferase for lipid galactosylation, and 3)

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exostosin1 and exostosin2 for heparan sulphate synthesis, suggesting a common mechanism

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of protein localization and interaction across species (Sprong et al., 2003; Oikawa et al., 2013).

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It is thus attractive to speculate that the presence of P4H protein complexes is necessary for

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the in vivo proline hydroxylation of EXT substrates. Still, it remains unclear which is the

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precise domain required for the P4H-P4H interaction. Regarding amino acid motifs for

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intracellular localization, the cytosolic, N-terminal ERG domain of P4H5 and P4H13, containing

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the [RK]X[RK] dibasic motif, is required for these enzymes to be transported and retained in

the Golgi apparatus. This dibasic motif is also found in many members of glycosyltransferases

(Giraudo and Maccioni, 2003). In addition, the role of the short cytoplasmic tail of

glycosylation enzymes as a key feature for Golgi-retention has been shown in detail for yeast

(Schmitz et al., 2008; Tu et al., 2008). It is also possible that other domains and protein

features would be important for P4H targeting such as the length and specific sequence of the

transmembrane domain (TM) as it was shown for the N-glycan enzymes N-

acetylglucosaminyltransferase I (GnT1), beta-1,4-galactosyltransferase (GALT), and -2,6-

sialyltransferase (ST) (Nilsson et al., 1991; Munro, 1995; Schoberer et al., 2014). Overall, these

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results together suggest that proline hydroxylation in root hairs is a complex process, in which

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P4H5 is indispensable for its function at both a biochemical and physiological level. More

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experimental evidence is necessary to elucidate the molecular mechanism that fine-tunes the

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peptidyl-proline hydroxylation of EXTs by P4Hs during their transit through the ER-Golgi

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compartments.

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As far as the spatial organization of the cell wall is concerned, there are a few pieces of

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evidence -by means of Transmission Electro Microcopy (TEM) and Atomic Force Microscopy

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(AFM)- demonstrating an in vitro EXT-assembly into a 3D- network (Cannon et al. 2008;

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Valentin et al. 2010). At least under these experimental conditions, EXT monomers are self-

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assembled into a dendritic-like network. The dimensions of the smallest EXT unit within this

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putative network suggest that several EXT chains are bound together by lateral alignment of

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up to six molecules (average width of around 10 nm and average length of 127 nm) (Cannon

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et al. 2008). These results allow us to hypothesize that the absence of peptidyl-proline

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hydroxylation and following O-glycosylation in EXTs -and possibly other HRGPs- would impose

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changes in the way EXT polypeptides interact with themselves and the surrounding cell wall

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environment. In agreement with that idea, we have also observed noticeable alterations in

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the overall cell wall architecture in the p4h5 mutant that would reflect EXT particular self-

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assembly patterns. If these EXT-structures are amalgamated just after secretion into the cell

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wall or, alternatively, they are first assembled during vesicular transport is something for

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which there is no evidence that we are aware of. In this context, a direct interaction of EXTs

with pectins was shown in vitro by AFM (Valentin et al., 2010) and specific chemical linkages

between them have even been suggested (Qi et al., 1995). Nevertheless, there are no

convincing models yet for in vivo mechanisms accounting for this putative macromolecular

association during cell wall assembly.

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1

EXPERIMENTAL PROCEDURES
Plant Materials. Arabidopsis thaliana Columbia-0 (Col-0) was used as the wild type (Wt)
genotype. Seedlings were germinated on half-strength MS agar plates in a Percival incubator
at 22C in a growth room with 16h light/8h dark cycles for 7-10 days. Plants were transferred
to soil for growth under the same conditions as previously described.

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Sequence homology searches. Amino acid sequences of Arabidopsis P4Hs were searched
with the BLASTP program (http://www.arabidopsis.org/Blast) and aligned using ClustalW
software. Alignment was manually corrected based on the secondary structure. A
phylogenetic tree of AtP4Hs was obtained using with MEGA 5.0.

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BiFC protein-protein interaction. Agrobacterium tumefaciensstrain C58C1-pGV3101 carrying

the BiFC-compatible gene constructs(Gehl et al., 2009)containing P4H2, P4H5 and P4H13
genes were incubated on solid LB Medium containing tetracycline (20 g/l), kanamycin (50
g/l), and rifampicin (50 g/l) at 28C for 3 days. The different BiFC partner strains were
grown in LB media with appropriate antibiotics overnight at 28C with agitation at 200rpm.
After centrifugation, cell pellets were resuspended in 1ml infiltration buffer containing 10mM
MES, pH 5.6, 100mM acetosyringone and 10mM MgCl2 at a final optical density at 600nm of
0.05. The viral suppressor of gene silencing, p19 strain(Voinnet et al., 2003) was co-infiltrated
at an final optical density at 600 nm of 0.1 in combinations containing P4H2, -5, and -13 as
well as one ER marker (HDEL-mCherry) or one Golgi marker, a cis-marker (ManI-mCherry).

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T-DNA mutant analysis. For identification of T-DNA knockout lines, genomic DNA was
extracted from rosette leaves(Weigel and Glazebrook, 2002). Confirmation by PCR of a single
and multiple T-DNA insertions in the target gene were performed using an insertion-specific
LBb1 (for SALK lines) or Lb3 (for SAIL lines) primer in addition to one gene-specific primer. To
ensure gene disruptions, PCR was also run using two gene-specific primers, expecting bands
corresponding to fragments larger than in WT. In this way, we isolated homozygous (for all
the genes mentioned above)p4h2.1, p4h2.2, p4h5, andp4h13 T-DNA mutants (Velasquez et
al., 2011) were isolated previously.

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P4H localization analysis. P4H2, -5, and -13 C-terminally tagged with monomeric CFP or eYFP
were transiently co-expressed in Nicotiana benthamiana leaf with one of the organelle
markers: HDEL fused to mCherry (ER marker); N-acetyl-glucosaminyltransferase I (GNTI) fused
to mRFP (Schoberer et al., 2009); -mannosidase-II (GMII) fused to mRFP (medial-Golgi
marker,Strasseret al., 2006); sialyl transferase (ST) fused to GFP (trans-Golgi marker, SaintJore-Dupaset al., 2004). P4H2 is mainly localized to the ER, while P4H5 and P4H13 are
localized to Golgi apparatus. Additionally, combination of P4H5-mCer + P4H2-EYFP, P4H5mCer + P4H13-EYFP and P4H13-mCer + P4H2-EYFP fusion constructs were transiently coexpressed in N. benthamiana. Leaves were analyzed using a Leica TCS SP5 inverted confocal
laser scanning microscope (CLSM) LeicaTM 63x water immersion objective, NA=1.2.A. The
following argon laser lines and emission filter settings were used: mCer3: excitation (ex) 458
nm, emission (em) 475-505nm;GFP:ex 488nm,em 501-528nm; EYFP:ex 514nm,em 525600nm; mRFP:ex 543nm,em 595-650nm.

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Transient expression of the fusion proteins were carried out in 4-week-old N. benthamiana
plants that have been grown in greenhouses at 24C/17C day/night temperatures, 16h
photoperiod. Each strain combination was infiltrated into a separate leaf, in two independent
plants. Infiltrations were done by injection on the bottom face of the leaf using a syringe
without a needle, with a fingertip providing counter pressure. After infiltration, the plants
were placed in the greenhouse for 23 days until observations by a Zeiss LSM 710 confocal
microscope. The Zeiss Zen software was used for image processing.

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FRETanalysis. The following combinations were analyzed for FRET; P4H2-mCer3 + P4H2-EYFP,
P4H2-mCer3 + P4H5-EYFP, P4H5-mCer3 + P4H2-EYFP,P4H5-mCer3 + P4H5-EYFP, P4H13mCer3 + P4H13-EYFP, and P4H13-mCer3 + P4H5-EYFP. Aquadric leaf piece of 1cm2 from each
infiltrated plant was incubated in 0.1 M PBS pH 7.4, 30 g/ml Cytochalasin D solution for 2h at
20 C or in 0.1M PBS pH 7.4, 4% PFA for 10 min and subsequently washed 2 x 10min in water.
PFA fixation was solely used for combination P4H13-mCer3 + P4H5-EYFP. Each leaf piece was
analyzed using a Leica TCS SP5 inverted confocal laser scanning microscope (CLSM) LeicaTM
63x water immersion objective, NA=1.2. A region of interest (ROI) was drawn around every
visible circular fluorescent signal or around areas with ER pattern for P4H2-mCer3 + P4H2EYFP combination, in each image. A parameter proportional to Frster resonance energy
transfer efficiency was calculated as FRET ratio = (Dpost-Dpre)/Dpost. ROIs were rejected from
further analysis if (a), the summarized intensity of a ROI was reduced more than 25% or
increased more than 5% during the acquisition. (b), ROIs that showed out-of-range reduction
in YFP channel post bleaching, hence not between 60% and 100%. FRET measurements:
P4H5-mCer3 + P4H5-EYFP (Fr =5.6 1.3, z test 0 = 1.5E-05, n=39, 5 cells).P4H13-mCer3 +
P4H13-EYFP (Fr =11.9 2.3, z test 0 = 6.9E-08, n=24, 4 cells).P4H2-mCer3 + P4H5-EYFP (Fr
=12.1 2.6, z test 0 = 2.2E-06, n=34, 3 cells).P4H13-mCer3 + P4H5-EYFP (Fr =8.6 3.4, z test
0 = 0,0062, n=10, 3 cells). P4H2-mCer3 + P4H2-YFP (Fr =-5.0 1.4, z test 0 = 0.9999, n=36,
3 cells).

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Root hair phenotypic analysis. For quantitative analysis of root hair phenotypes in p4hs
mutants, the corresponding genetically complemented lines, and in the overexpressing lines,
200 fully elongated root hairs were measured (n roots= 20-30) from seedlings grown on
vertical plates for 7 days under continuous light. Values are reported as the mean SD using
the Image J software. For root-hair density measurement, 7-day-old roots were imaged using
differential interference contrast microscopy in the root maturation zone. For measurements
of root hair inhibition, P4H inhibitors ethyl-3,4-dihydrohydroxybenzoate (EDHB) (Barnett,

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FRET protein-protein interaction. Monomeric Cerulean3 (3) was PCR amplified and used to
replace EYFP fluorophore in pEarleyGate 102 vector to generate pEarleyGate mCer3
expression vector (Earley et al., 2006). Full length P4H2, P4H5 and P4H13 in pDONOR-D-TOPO
was cloned into pEarleyGate 101 and pEarleyGate mCer3 respectively using LR reaction
(Invitrogen). Briefly, N. benthamiana leaves of 3-week-old plants were infiltrated with a mix
of Agrobacterium lines (final OD600 = 0.2 each) harboring constructs. An Agrobacterium line
harboring P19 (final OD600 = 0,1) was included in all mixtures. The infiltrated tobacco plants
were placed in the greenhouse for 50 h before analysis.

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1970) and ,-dipyridyl (DP)(Majamaa et al., 1986) were added to half-strength MS medium
and the Inhibitory Concentration 50 (IC50) was used for DP compound at 48 nM and IC50 for
EDHB compound at 219 nM as previously determined (Velasquez et al., 2011). IC50 for both
compounds was only measured in Wt Col-0 root hairs growth. Measurements were made
after 7 days. Fully elongated root hairs (n= 150-200; n roots= 20-30) were analyzed at each
P4H inhibitors concentration. Twenty seedlings of each genotype were measured. Genetic
rescue of p4h mutants were carried out by overexpression of P4Hs under the control of 35S
promoter (35Spro::P4H2-GFP, 35Spro::P4H5-GFP, or 35Spro::P4H13-GFP). Three independient
transgenic lines were scored in T2 seedlings from lines exhibiting robust and consistent GFP
expression. An average value for three independent lines in each case is shown in the analysis.
The GFP expression analysis was carried out by using the ImageJ software (Abramoff et al.,
2004). The mean gray area was determined for a same fixed area in 10 independent root hairs
for each transgenic line. Images were captured with the following excitation (ex) and emission
(em) wavelengths (ex/em): GFP 488nm/501528nm and mCherry 561nm/600620nm.
Images were processed in Image J and Photoshop (Adobe), and figures were assembled in
Illustrator (Adobe). Images were captured with a Leica DM5000 microscope equipped with a
Retiga Exi digital camera.

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DNA Manipulations. Vectors based on the Gateway cloning technology (Invitrogen) were
used for most manipulations. Description of the plant binary vectors can be found in(Curtis
and Grossniklaus, 2003; Nelson et al., 2007). Constitutive expression of P4Hs was achieved in
plant destination vector pMDC83. Translational fusions of P4Hs driven by the native promoter
(Prom) plus cDNA sequence of P4Hs were done in pGWB4 destination vectors. P4H5pro:P4H5,
P4H5-ERG, P4H2-CD, P4H2+ERG, P4H2+ERG-CD were synthesized by Genscript (NJ, USA;
http://www.genscript.com) on pENTRY/D-TOPO and then recombined in pGWB4. For the
overexpressing lines with 35S promoter, 35S::P4H-GFP constructs, cDNA P4Hs sequences
were PCR-amplified with AttB recombination sites. PCR products were then recombined first
in pDONOR201, linearized with PvuII and transferred into pGWB83 using the Gateway LR
Clonase II enzyme mix (Invitrogen). Primers used for assembling each construct are listed in
Table S3.
Recombinant P4H5 assay. P4H5 CDS was PCR amplified removing the putative TMD
(transmembrane domain) from 1-67 aa (P4H5TMD). After sequencing P4H5TMD was cloned by
gateway LR recombination into Pichia pastoris destination vector pPICZ_aplpha_1B containing
in its C-terminus a His-tag giving P4H5TMD-His6. Pichia pastoris was transformed according to
protocol from Invitrogen and 25 colonies were tested for recombinant expression. The
highest expression clone was used for P4H5TMD production. Briefly 30 C ON BMGY starter
cultures were launched and inoculated into 100 ml of BMGY at an OD600 of 0.5. After 24hs
incubation at 30C the cells were washed and resuspended into 100 ml of BMMY. Cells were
incubated 24 hs at 22C 150 R.P.M. shaking for induction. After induction the culture media
was recovered and P4H5TMD was purified by Ni2+ affinity cromatography (GE Hitrap columns).
Purified P4H5TMD was run in an SDS PAGE to verify enzyme MW and possible contaminants in
the eluate. 0.5 l of enzyme were used in 10 l assays containing 25 mM Tris-HCl ph 8, 50 M
FeSO4, 300 M 2-oxoglutaric acid, 200 M Ascorbic acid, 100 M DTT, and typically 50 M of

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substrates shown in Table S1. Reactions were performed at 25C and stopped by adding
EDTA to a 2mM final concentration. Samples were mixed with a 2,5-dihydroxybenzoic acid
matrix and loaded into a Bruker autoflex speed LRF MALDI TOFF for hydroxylation analysis.
EXT substrate (SPPPPYVYSSPPPPYYHis6) and AGP peptide (ATVEAPAPAPTSHis6) were used to
measure P4H5 activity. Competitive assays were performed by triplicate. For calculation, we
used the percentage of hydroxylation obtained taking into account all the peaks
corresponding to the peptide (including unmodified peptide, all the different hydroxylated
products and all the complexes with Na+ and K+ salts). Proline-hydroxylation activity was
determined as the % of peptides with either one or two, due the higher activity,
hydroxylations on AGP and EXT peptides, respective. For hydroxylation site mapping on EXT
substrates, we analyzed partially hydroxylated species at two different times, 20 min and 100
min. All present hydroxylated peptides were purified by Stage Tips (Thermo Scientific) and
analyzed by nano-flow liquid chromatography-tandem mass spectrometry (nLC-MS/MS). The
setup was composed of an EASY-nLC II (Thermo Scientific) interfaced via a nanoSpray Flex ion
source to an LTQ-Orbitrap XL hybrid mass spectrometer. Peptides were separated using a
single analytical column (PicoFrit Emitters, New Objectives, 75 m inner diameter) packed inhouse with Reprosil-Pure-AQ C18 phase (Dr. Maisch, 3 m particle size). MS1 precursor ion
scan (m/z 300-1,700) was performed in the orbitrap using a nominal resolution setting of
30,000 followed by HCD-MS2 fragmentation of the five most abundant multiply charged
precursor ions. A minimal signal threshold of 50,000 ions was used for triggering HCD-MS2
events. MS data was processed by Proteome Discoverer 1.4 (Thermo Scientific) using
hydroxyproline as variable modification. All spectra were inspected to verify the accuracy of
the hydroxyproline assignments.

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Transmission Electron Microscopy (TEM) of root hair cell walls. Seeds were germinated on
0.2X MS, 1% sucrose, 0.8% agar. Seven days after germination, 1mm root segments with root
hairs embedded in 0.8% agar were transferred to freezing planchettes containing 0.1M of
sucrose and high-pressure frozen in a Baltec HPM 010 unit (Technotrade, Manchester, NH,
USA). Substitution was performed in 2% OsO4 in anhydrous acetone at 80C for 72h, and
followed by slow warming to room temperature over a period of 2 days. After several
acetone rinses, samples were removed from the holders and infiltrated in Epon resin (Ted
Pella Inc., Redding, CA) according to the following schedule: 5% resin in acetone (4h), 10%
resin (12h), 25% resin (12h) 50%, 75%, and 100% (24h for each concentration). Polymerization
was performed at 60C. Sections were stained with 2% uranyl acetate in 70% methanol for
10min followed by Reynolds lead citrate (2.6% lead nitrate and 3.5% sodium citrate (pH
12.0)) and observed in a FEI CM120 electron microscope. Quantitative analysis of cell wall
thickness and fibril angles were performed using FIJI (Schindelin et al.). For fibril angles
analysis, the angle of the discernible fibrils in the flanking regions of 3 mutant and 3 WT root
hairs were performed. Fibrils were identified as linear electron dense structures in the cell
wall. Boxes of 0.25 square microns randomly located along the cell walls on the root hair tip
were defined and the angles of the fibrils inside the box were measured with respect to the
cell surface. Angles varied from 0 (parallel to the surface) to -90 or +90 (perpendicular
to the cell surface). The results are presented as a percent histogram and the sample size is
184 fibril angles measured for Wt Col-0 and 117 fibril angles for the mutant p4h5.

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ACKNOWLEDGEMENTS

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We would like to thank S. Maldonado and S. Petrucelli for providing access to a stereoscope,
A. Nebenfhr and R. Strasser for providing vectors of the ER- and Golgi-markers, and J.P.
Muschietti for critical reading of the manuscript. We thank ABRC (Ohio State University) for
providing T-DNA lines seed lines. A.D.N., N.D.I., and J.M.E. are investigators of the National
Research Council (CONICET) from Argentina. This work was supported by grants from ANPCyT
(PICT2011-054, PICT2013-003 to J.M.E and PICT 2011-967 to N.D.I.), CONICET (PIP0071 to
J.M.E. and PIP0342 to N.D.I.), Mizutani Foundation for Glycoscience (Grant 130004 to J.M.E.),
Fulbright-CONICET and Fulbright-Bunge & Born Fellowships (to J.M.E and S.M.V, respectively),
EMBO short term fellowship (to M.M.R.), the Danish Agency for Science and Technology (27409-0082, 2101-07-0071) to N.G., The Danish Council for Independent Research/Natural
Sciences (12-125709) & The Danish Council for Strategic Research (12-131859) to B.L.P and
Danish National Research Foundation (DNRF107) to A.H., and NSF 1157824 (to M.S.O.).

AUTHOR CONTRIBUTION

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S.M.V. performed most of the experiments and analyzed the data. M.M.R., J.S.S, and J.G.D.
analyzed and performed some of the experiments and analyzed the data. R.B. and M.O.
performed the imaging of root hair tip by TEM. M.M.R., S.R.M, A.H, A.D., N.G. and B.L.P.
performed and analysed the in vitro peptide hydroxylation assays. S.M.V. performed P4H
swapps and P4H overexpression experiments and analyzed the data. A.O. and H.V.S.
performed the BiFC and some of the P4H co-localization experiments. C.P.P. and N.G.
performed FRET analysis and some of the P4H co-localization experiments. N.D.I. analyzed the
data. J.M.E. designed research, performed some of the experiments, analyzed the data,
supervised the project, and wrote the paper. All authors commented on the results and the
manuscript. This manuscript has not been published and is not under consideration for
publication elsewhere. All the authors have read the manuscript and have approved this
submission.

Competing financial interest

The authors declare no competing financial interests. Correspondence and requests for
materials should be addressed to J.M.E. (Email: jestevez@fbmc.fcen.uba.ar).

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LEGENDS TO FIGURES

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Figure 1. Characterization of P4H5. (A) Chemical reaction catalyzed by P4Hs yielding a trans4-hydroxyprolyl residue (Hyp). The compound ethyl-3,4-dihydroxybenzoate (EDHB)
competitively binds to 2-oxoglutarate-binding site of P4Hs whereas ,-dipyridyl (DP)
chelates Fe2+ ions, thus both inhibit the formation of Hyp. (B) P4H5 with point mutations
introduced in the OG binding site (P4H5OG) cannot complement p4h5 mutant. L1-L3=
independent transgenic lines expressing P4H5pro::P4H5OG-GFP construct in p4h5. NS= not
significant. (C-E) In vitro activity of recombinant P4H5. (C) Enzyme was incubated for 0 min
(top) or 90 min (bottom) before reaction termination. Reactions were performed with AGPlike (left) or EXT-like (right) peptides. The molecular weight and corresponding species,
determined by MALDI-TOFF are indicated for each peak. (D) Competition assay of AGP/EXT
substrates. Competition assay with different AGP:EXT ratios were performed during 90
minutes. Controls with only one of the substrates were performed (ratio 0:1 and 1:0). For AGP
peptides we measured % of single-site hydroxylated peptides (black, left axis) and for EXT we
measured % of two hydroxylated sites peptides (red, right axis). Tested by ANOVA (*P<0.01
and **P<0.001 Bon Ferroni post test compared against 1:0). (E) Determination of possible
sequential steps in the hydroxylation of EXT-peptide by MS/MS. See also Supplementary
Figures S1-S2.

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Figure 2. P4H5 is a key player on root hair cell growth. (A) Multiple p4h mutant analysis. The
double p4h mutants as well as the triple p4h2,5,13 mutant are compared to the
corresponding single p4h2.1, p4h2.2, p4h5 and p4h13 mutants (mean s.e.m., n= 200). Pvalue of one-way anova, (*) P<0.01, (**) P<0.001. NS= not significant. (B) Assessment of
residual P4H activity in p4h multiple mutants. EDHB or DP at inhibitory concentration 50 (IC50)
were used to inhibit P4H activity. P-value of one-way anova, (*) P<0.01, (**) P<0.001. NS= not
significant. (C) Genetic complementation by over expression with CaMV 35S promoter of
single GFP-tagged P4Hs (35Spro::P4H2, 35Spro::P4H5, or 35Spro::P4H13) in p4h mutant
background. Only P4H2 and P4H13 showed equivalent functions based on their root hair
phenotype. Two-three independent transgenic lines are included in each measurement
(mean s.e.m., n= 200). NS= not significant. (D) Root hair phenotypes in the
complementation lines. Scale bar = 600 m. In the bottom panels are shown the GFP
fluorescence of the GFP tagged P4Hs. (E) P4H promoter swapping experiments and effect on
root hair growth. Complementation of each p4h mutant line was tested using the
corresponding P4H driven by the other two promoters. Scale bar, 600 m. In the bottom
panels are shown the GFP fluorescence of the GFP tagged P4Hs. (F) P4H full protein swapping
experiments and effect on root hair growth. Complementation of each p4h mutant line was
tested by the expression of the other two P4H proteins driven by the promoter of the
corresponding mutant line. Scale bar, 600 m. P values of one-way analysis of variance
(ANOVA) test, (*) P < 0.01, (**) P < 0.001, (***) P < 0.0001. In the bottom panels are shown
the GFP fluorescence of the GFP tagged P4Hs. White bars indicate measurements on the
corresponding mutant background (C, E and F panels). See also Supplementary Figures S3-S5.

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Figure 3. P4H-P4H co-expression analysis in theER and Golgi compartments. (A) Transient
expression in N. benthamiana leaves of 35S-P4H2,5,13-Cer3/GFP/EYFP (in green) and cis- to
trans-Golgi markers. HDEL-mCherry was used as ER-marker, GNTI-mRFP (N-acetylglucosaminyl-transferase I) was used as cis-marker, GMII (Golgi--mannosidase-II) as medialmarker, and ST-GFP (Sialyl-transferase) as a trans-marker (markers in magenta). Scale bar, 10
m. On the right, putative ER and intra-Golgi subcellular localization of P4Hs are inferred from
co-localization results. (B) Co-expression of P4H pairs. cis-trans-Golgi P4H5-P4H13 overlap and
cis-Golgi P4H2-P4H5 co-localization are indicated (in yellow). No or low overlap is detected for
P4H2-P4H13. (C) P4H2 showed a different localization when co-expressed with P4H5. When
P4H2 or P4H5 were expressed alone, localization to ER-like and Golgi-like structures are
observed, respectively (cell in the middle and on the right). While both P4H2 and P4H5 were
expressed, two proteins were co-expressed in the Golgi-like structure (cell on the left).

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Figure 4. P4H5 protein-protein interactions. P4H-P4H protein interaction assessed by

bimolecular fluorescent complementation (BiFC) and Frsterresonance energy transfer (FRET)
in Nicotiana benthamiana. (A) BiFC P4H protein-protein interactions. Interactionsbetween
P4H proteins fused to C-terminal YFP (cY) and N-terminal YFP (nY) areshown as green signals.
Magenta signals represent ER marker: HDEL-mCherry and cis-Golgi marker: ManI-mCherry.
Co-localized signals between the interaction (green) and the organelle marker (magenta)
aredisplayed as white dots. Scale bar, 10m. (B) FRET ratio of P4H-P4H (%) calculated as
((Dpost - Dpre)/Dpost ). The combination of P4H2/P4H13 was not investigated since the two
proteins were not co-localized. (C) Summary scheme of P4H interactions and localizations.
P4H2 localization can shift by associating with P4H5. P4H5-P4H5, P4H5-P4H13, P4H5-P4H2
interactions suggest a large Golgi P4H complex consisting of P4H2, P4H5, and P4H13.
Thickness of each black line between P4H proteins represents the interaction strength based
on the BiFC-FRET results. See also Supplementary Table S2.

Figure 5. ERG-domain targets P4H5 to the Golgi apparatus. (A) Diagram of GFP-tagged P4H5
variants used in rescue experiments to analyze domain function. ERG= N-terminal domain
from P4H5; CD= cysteine-rich C-terminal domain from P4H2. (B) Detection of GFP-tagged
P4H5 variants in roots. (C) Quantitative analysis of root hair length (mean s.e.m., n= 200) in
p4h5 mutants expressing truncated versions of P4H5 under regulation by of its own promoter
(P4H5pro). Both P4H5pro::P4H5-ERG and P4H5pro::P4H5-ERG+CD(P4H2), failed to complement p4h5
root hair growth. In addition, P4H2pro::P4H2+ERG(P4H5) (under the control of P4H2 promoter
with ERG domain from P4H5) did not rescue p4h5 mutant. NS= not significant. Two
independent transgenic lines are included in each measurement. (D) Root hair phenotypes in
p4h5 roots expressing GFP-tagged, truncated P4H5 variant and also the chimeric
P4H2+ERG(P4H5). Scale bar, 600 m. (E) Diagram of GFP-tagged P4H2 variants used in rescue
experiments to analyse domain function. ERG= N-terminal domain from P4H5; CD= cysteinerich C-terminal domain from P4H2. (F) Quantitative analysis of root hair length (mean
s.e.m., n= 200) in p4h2-1 mutants expressing truncated versions of P4H2. All versions

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complemented p4h2-1 root hair growth. NS= not significant. Two independent transgenic
lines are included in each measurement. (G) Detection of GFP-tagged P4H2 variants in roots.
(H) The ERG domain affects P4H5 Golgi localization. Coexpression of full length P4H5 and
truncated P4H5-ERG both GFP tagged with an ER-Cherry marker and two Golgi markers (ManImCherry and XT-Cherry; only co-expressed to P4H5-ERG) in a transient expression system. Scale
bar, 10 m. (Zoomed image, Scale bar, 2 m). See also Supplementary Figure S6.

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Figure 6. Impact of deficient peptidyl-proline hydroxylation in the cell wall structure on root
hair tips. (A) Cell wall architecture in root hairs tips of Wt and p4h5 mutant by transmission
electron microscopy (TEM). Scale bar, 250 nm. One whole root hair tip section (left) and
detailed cell walls areas are shown (right). Electrodense fibrillar layers are indicated in yellow
lines in Wt Col-0 (left) and in the p4h5 mutant cell walls (right). PM= plasma membrane. Scale
bar, 100 nm. (B) Analysis of fibril angle relative to the cell surface is shown as percent
histogram. n= 184 fibril angles for Wt Col-0 and n= 117 fibril angles for the p4h5 were
analyzed.

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ACKNOWLEDGEMENTS

We would like to thank S. Maldonado and S. Petrucelli for providing access to a stereoscope,

A. Nebenfhr and R. Strasser for providing vectors of the ER- and Golgi-markers. We thank

ABRC (Ohio State University) for providing T-DNA lines seed lines. J.M.E. is an investigator of

the National Research Council (CONICET) from Argentina. This work was supported by grants

from ANPCyT (PICT2010-658, PICT2011-054, and PICT2013-003 to J.M.E.), CONICET (PIP0071

to J.M.E.), Mizutani Foundation for Glycoscience (130004to J.M.E.) and Fulbright-CONICET

and Fulbright-Bunge & Born Fellowships (to J.M.E and S.M.V, respectively), EMBO short term

fellowship (to M.M.R.), the Danish Agency for Science and Technology (274-09-0082, 2101-

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07-0071 to N.G), The Danish Council for Independent Research/Natural Sciences (12-

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125709to N.G) and Danish National Research Foundation (DNRF107 to N.G), U.S. Department

12

of Energy, Office of Science (DE-AC02-05CH11231 to H.V.S.).

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