This is a repository copy of Pectic galactan affects cell wall architecture during secondary
cell wall deposition.
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http://eprints.whiterose.ac.uk/160385/
Version: Accepted Version
Article:
Moneo-Sánchez, M, Vaquero-Rodríguez, A, Hernández-Nistal, J et al. (5 more authors)
(2020) Pectic galactan affects cell wall architecture during secondary cell wall deposition.
Planta, 251 (5). 100. ISSN 0032-0935
https://doi.org/10.1007/s00425-020-03394-2
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1
María Moneo-Sánchez
1
; Andrea Vaquero-Rodríguez
1
; Josefina Hernández-Nistal
2
; Lucía
Albornos
1
; Paul Knox
3
; Berta Dopico
1
; Emilia Labrador
1
; Ignacio Martín
1*
.
Pectic Galactan Affects Cell Wall Architecture During Secondary Cell Wall Deposition.
1
Departamento de Botánica y Fisiología Vegetal, Centro Hispano Luso de Investigaciones
Agrarias (CIALE), Universidad de Salamanca. Salamanca 37007. Spain.
2
Departamento de Biología Funcional. Universidad de Santiago de Compostela. Lugo 27002.
Spain.
3
Centre for Plant Sciences, Faculty of Biological Sciences, University of Leeds, LS2 9JT (United
Kingdom).
*
Corresponding Author: Ignacio Martín. Email: a56562@usal.es; Fax: +34 923 294515.
Main conclusion
β-(1,4)-galactan determines the interactions between different matrix polysaccharides and
cellulose during the cessation of cell elongation
Abstract
Despite recent advances regarding the role of pectic β-(1,4)-galactan neutral side chains
in primary cell wall remodelling during growth and cell elongation, little is known about the
specific function of this polymer in other developmental
processes. We have used transgenic
Arabidopsis plants overproducing chickpea βI-Gal β-galactosidase under the 35S CaMV
promoter (35S::βI-Gal) with reduced galactan levels in the basal non-elongating floral stem
internodes to gain insight into the role of β-(1,4)-galactan in cell wall architecture during the
cessation of elongation and the beginning of secondary growth. The loss of galactan mediated
by βI-Gal in 35S::βI-Gal plants is accompanied by a reduction in the levels of KOH-extracted
xyloglucan and an increase in the levels of xyloglucan released by a cellulose specific
endoglucanase. These variations in cellulose-xyloglucan interactions cause an altered xylan
and mannan deposition in the cell wall that in turn
results in a deficient lignin deposition.
Considering these results, we can state that β-(1,4)-galactan plays a key structural role in the
correct organization of the different domains of the cell wall during the cessation of growth and
the early events of secondary cell wall development. These findings reinforce the notion that
there is a mutual dependence between the different polysaccharides and lignin polymers to
form an organized and functional cell wall.
Keywords: β-(1,4)-galactan, β-galactosidase, Arabidopsis, Cell Wall, Hemicellulose
2
Introduction
Plant cell walls are highly organized and dynamic structures primarily composed of a mixture
of polysaccharides, proteins and phenolic compounds. The main polysaccharides of the cell wall,
namely cellulose, hemicelluloses and pectins, interact with each other and with other cell wall
components through covalent and non-covalent bonds. These complex interactions, along with
the relative proportions of the different polymers, define the physicochemical properties of the
wall and the distinct features of individual cells throughout development (Carpita and Gibeaut
1993; Park and Cosgrove 2015).
The specific nature of these interactions and the precise cell wall polymers involved have
not been completely elucidated in all developmental processes. However, several models have
been proposed to explain the structural organization of the cell wall, mainly focusing on primary
cell wall structure and its remodelling during cell elongation. Although classical cell wall models
assume that the cellulose-xyloglucan network is the main load-bearing structure, more recent
studies suggest pectins play a major role in the maintenance of cell wall architecture (Carpita and
Gibeaut 1993; Cosgrove 2005; Peaucelle et al. 2012). Several interactions between pectic
polysaccharides and cellulose, hemicelluloses and cell wall proteins have been proposed
(Peaucelle et al. 2012; Broxterman and Schols 2018; Cornuault et al. 2018).
More specifically, among pectic polysaccharides, rhamnogalacturonan-I (RG-I) neutral
galactan and arabinan side chains seem to play crucial roles in cell wall architecture. In addition,
they have been proposed to be determining factors in interactions with other components, such
as cellulose and hemicelluloses, during various processes (Popper and Fry 2008; Zykwinska et
al. 2008; Wang et al. 2019). Most of these studies are aimed at determining the role of these
pectic side chains in primary cell wall architecture and cell elongation. In a recent study Moneo-
Sánchez et al. (2019), by means of altering β-(1,4)-galactan levels in Arabidopsis thaliana
elongating organs, provided evidence that this polymer is directly involved in etiolated hypocotyl
and apical floral stem internode elongation. Moreover, it was shown that the amount of this pectic
side chain may be controlling the degree of interaction between cellulose and XG.
The prior results suggest that pectic neutral side chains, and more specifically β-(1,4)-
galactans, have a role in primary cell wall remodelling during growth and cell elongation. However,
little is known about the specific function of these polymers in the cell wall and during the cessation
of elongation and the onset of secondary growth and extensive secondary wall deposition.
Depending on the organ, and even on the specific cell type, the cessation of elongation occurs in
a series of phases, starting from the first moments of the transition from primary to secondary
growth to the synthesis and deposition of cell wall components. This therefore implies the marked
reorganization of the cell wall, including a shift in the pectin/cellulose balance, increased
deposition of xylan and mannan polysaccharides, and in some cases, lignification of the cell wall
(Hao et al. 2014; Hao and Mohnen 2014; Hernández-Gómez et al. 2015).
Given the aforementioned involvement of β-(1,4)-galactan in XG interactions with cellulose
(Moneo-Sánchez et al. 2019), and taking into account previous reports about the possible
3
interactions of pectic polysaccharides with other cell wall components, the main objective of this
work was to study the function of pectic galactan in cell wall remodelling during the transition
between primary and secondary cell wall formation. For this purpose, we have used previously
generated transgenic Arabidopsis plants overproducing chickpea βI-Gal β-galactosidase, under
the control of the 35S CaMV promoter (35S::βI-Gal), with reduced galactan levels in apical
stem internodes (Moneo-Sánchez et al. 2019). The characterization of the basal stem internodes
precisely at the moment of cell elongation cessation, during the first stages of secondary growth
and secondary cell wall deposition, has shed light on the interactions between the cell wall
polymers at this transition stage.
Materials and Methods
Plant material and growth conditions
Arabidopsis thaliana Columbia-0 (Col-0) ecotype and transgenic Arabidopsis plants
overproducing chickpea βI-Gal β-galactosidase (coded by CarBGal1) under the 35S CaMV
promoter (35S::βI-Gal plants) were used. The 35S::βI-Gal plants were generated in previous
work. Detailed information on vector construction, plant transformation and line selection is
available in Moneo-Sánchez et al. (2019). Seeds from WT and 35S::βI-Gal plants were surface
sterilized and grown in solid MS medium (Murashige and Skoog 1962) as described in Izquierdo
et al. (2018) in a growth chamber (Aralab, Portugal) at 22ºC with 16-h photoperiod (80-100 μmol
m
-2
s
-1
irradiance). Ten-d-old green seedlings were transferred to plastic pots containing a 3:1
mixture of potting soil and Vermiculite and grown under the same conditions until basal floral stem
internodes were collected from 32-d-old plants, when elongation of the basal internode has
already ceased. Pieces of 1 cm were cut using a razorblade from the most basal zone of the floral
stem, and were immediately frozen in liquid nitrogen for cell wall analyses or immersed in a
fixative solution for carrying out the staining/immunolocalization studies.
For agroinfiltration experiments, Nicotiana benthamiana seeds were sown in potting soil and
allowed to grow for 6 weeks in a growth chamber (Aralab, Portugal) at 25°C and 16-hour (h)
photoperiod.
Preparation of cell wall extracts
Basal internodes from WT and 35S::βI-Gal plants were used for cell wall extracts
preparation, according to the method described by Cornuault et al. (2014). Freeze-dried material
was ground for 2 min in a Retsch mixer mill MM400 (Sarstedt, Germany) at 30 oscillations/s and
washed with 70% and 90% ethanol/H
2
O (v/v), chloroform methanol (1:1) and acetone to obtain
the alcohol insoluble residue (AIR) material. Cell wall components were extracted sequentially
from 1 mg of AIR with 500 µl H
2
O during 20 min in a mixer at 30 oscillations per second. After
centrifugation at 14000 g for 15 min, the supernatant was collected and the remaining material
was further extracted with 500 µl of 50 mM CDTA, pH 7.5 and 4 M KOH containing 1% w/v NaBH
4
with the same conditions. The pH of KOH extract was neutralized with 80% v/v acetic acid. All
4
extracts were stored at 20ºC until use. The remaining residue was considered the cellulosic
fraction.
Analysis of the cellulosic fraction
The cellulosic fraction was washed sequentially two times with 70% ethanol and H
2
O for 20
min and centrifuged at 14000 g for 12 min after each wash. The cellulosic residue was incubated
with 18 U of a cellulose-specific endoglucanase from Aspergillus niger (Megazyme, Ireland) 0.1
M Na-acetate buffer pH 4.5 (containing 0.02% sodium azide) at 37ºC for 48 h. For all samples, a
control with no enzyme was performed under the same conditions. The sugars released to the
incubation media were collected (14000 g for 20 min), neutralised with 1 M Na
2
CO
3
and analysed
by ELISA as described below.
Anion-exchange epitope detection chromatography (AE-EDC)
For anion exchange epitope detection chromatography (AE-EDC) of the polysaccharides
present in H
2
O, CDTA and KOH extracts, the method described in Cornuault et al. (2014) was
followed. For anion-exchange chromatography, 50 µl aliquots of the H
2
O, CDTA or KOH extracts
were diluted in 2.5 ml H
2
O and eluted through a 1 ml Hi-Trap ANX FF column (GE Healthcare,
UK) using a BioLogic LP system (Bio-Rad, USA). Samples were eluted with 20 mM sodium
acetate buffer, pH 4.5, from 0-2 min, followed by a linear gradient from 0% to 50% 0.6 M NaCl in
50 mM sodium acetate buffer, pH 4.5 (25 min), followed by 50% to 100% 0.6 M NaCl (31 min)
and 0.6 M NaCl to 48 min. All steps were conducted at 1 ml/min flow-rate. The fractions were
neutralized with 50 µl of 1 M Na
2
CO
3
. One hundred µl aliquots were incubated in NUNC Maxisorp
microtiter plate wells (Thermo Fisher Scientific, USA) overnight at 4°C and used in ELISA
analysis. ELISA assays were conducted as described in Moneo-Sánchez et al. (2019) using a
1:25 dilution of primary antibodies and 1:1000 dilution of secondary antibody (anti-rat or anti-
mouse horseradish peroxidase-conjugated IgG; Sigma, USA) in 5% w/v milk powder/PBS (137
mM NaCl, 2.7 mM KCl, 10 mM Na
2
HPO
4
, 2 mM KH
2
PO
4
). Plates were developed using 100 µl of
substrate per well (0.1 M sodium acetate buffer, pH 6, 1% tetramethyl benzidine, 0.006% H
2
O
2
).
The reaction was stopped with 50 µl of 2.5 M H
2
SO
4
and the absorbance read at 450 nm.
Antibodies used
Monoclonal antibodies against pectic polysaccharides used in this study include LM5, which
recognizes a minimum of three sugar residues of β-(1,4)-galactan (Jones et al. 1996; Andersen
et al. 2016) and JIM7, which recognizes methyl-esterified epitopes of homogalacturonan (HG)
but does not bind to un-esterified homogalacturonan (Verhertbruggen et al. 2009). Regarding
xyloglucan (XG), three antibodies were used: anti-fucosylated XG CCRC-M1, that recognizes
terminal fucosyl residues linked α-(1,2) to a galactosyl residue (Puhlmann et al. 1994); LM25, that
binds the XG backbone motif XXXG and galactosylated XXLG/XLLG epitopes (Pedersen et al.
2012); and CCRC-M100, which recognizes the unsubstituted motif XXXG in XG backbone
(Pattathil et al 2010; Zabotina et al. 2012). The nomenclature proposed by Fry et al. (1993) for
XG substitutions is used in this work. For xylan analyses we used two antibodies specific to the