Planta (1997) 203: 289±294
A role for arabinogalactan-proteins in root epidermal cell expansion
Lei Ding, Jian-Kang Zhu
Department of Plant Sciences, University of Arizona, Tucson, AZ 85721, USA
Received: 13 February 1997 / Accepted: 1 April 1997
Abstract. Arabinogalactan-proteins (AGPs) are abundant plant proteoglycans that react with (b-D-Glc)3 but
not (b-D-Man)3 Yariv reagent. We report here that
treatment with (b-D-Glc)3 Yariv reagent caused inhibition of root growth of Arabidopsis thaliana (L.) Heynh.
seedlings. Moreover, the treated roots exhibited numerous bulging epidermal cells. Treatment with (b-D-Man)3
Yariv reagent did not have any such e€ects. These
results indicate a role for AGPs in root growth and
control of epidermal cell expansion. Because treatment
with (b-D-Glc)3 Yariv reagent phenocopies the reb1 (root
epidermal cell bulging) mutant of Arabidopsis, AGPs
were extracted from the reb1-1 mutant and compared
with those of the wild type. The reb1-1 roots contained
an approximately 30% lower level of AGPs than the
wild type. More importantly, while the pro®le of AGPs
from wild-type roots showed two major peaks upon
crossed electrophoresis, the pro®le of AGPs from reb1-1
roots exhibited only one of the major peaks. Therefore,
the reb1 phenotype appears to be a result of defective or
missing root AGPs. Taken together, this pharmacological and genetic evidence strongly indicates a function of
AGPs in the control of root epidermal cell expansion.
Key words: Arabidopsis (reb1 mutant) ± Arabinogalactanprotein ± Cell expansion ± Mutant (reb1) ± root epidermal
cell bulging ± Root growth
Introduction
Arabinogalactan-proteins (AGPs) are a family of
proteoglycans that are widely distributed throughout
the plant kingdom (Fincher et al. 1983; Basile and Basile
1987). They are found predominantly in the plasma
Abbreviations: AGP = arabinogalactan protein; Gal = galactose;
Glc = glucose; Man = mannose
Correspondence to: J.K. Zhu; E-mail: [email protected];
Fax: 1 (520) 621 7186
membrane, cell wall and intercellular spaces (Fincher
et al. 1983; Komalavilas et al. 1991; Serpe and Nothnagel
1995). The carbohydrate moiety of AGPs consists of
mainly arabinose and galactose with minor amounts of
other sugars including uronic acids (Fincher et al. 1983;
Komalavilas et al. 1991). The protein moieties of AGPs
are typically rich in hydroxyproline, serine, alanine,
threonine and glycine (Fincher et al. 1983; Showalter
and Varner 1989). The primary structures of several
AGP core proteins have recently been elucidated via
gene cloning (Chen et al. 1994; Du et al. 1994; Cheung
et al. 1995; Mau et al. 1995; Du et al. 1996).
The expression of AGPs is highly regulated during
plant development (Knox et al. 1989; Pennell and
Roberts 1990; Stacey et al. 1990; Schindler et al. 1995).
Experiments with monoclonal antibodies to particular
AGP epitopes have demonstrated that the expression of
AGPs correlates with cell di€erentiation (Pennell and
Roberts 1990; Knox et al. 1991). For example, the AGP
epitope recognized by the monoclonal antibody
MAC207 is present in all cells of vegetative meristems,
primordia and organs, and throughout undeveloped
¯ower buds, but not in speci®c cells of developing
stamens and carpels (Pennell and Roberts 1990). Therefore, AGPs have been proposed to serve as speci®c cell
surface markers.
Several functions of AGPs have been established
recently. Speci®c AGPs have been found to be important
for somatic embryogenesis of Daucus carota L., because
addition of AGPs extracted from carrot seeds to a twoyear-old, non-embryogenic cell line resulted in the reinduction of embryogenic potential (Kreuger and van
Holst 1993). Serpe and Nothnagel (1994) observed that
treatment of rose cell suspension with (b-D-Glc)3 Yariv
reagent, a chromophoric molecule that selectively binds
AGPs, caused inhibition of cell proliferation. More
recently, secreted AGPs in the tobacco style have been
demonstrated to stimulate and guide pollen tube growth
(Cheung et al. 1995; Wu et al. 1995).
It has long been known that AGPs bind to Yariv
reagents, synthetic molecules containing phenyl-b-glycosides (Yariv et al. 1962; Clarke et al. 1978). This
290
L. Ding and J.-K. Zhu: Arabinogalactan-proteins and root cell expansion
property has been used extensively in the detection,
quanti®cation and isolation of AGPs from plant tissues
and cultured cells (Clarke et al. 1978; van Holst and
Clarke 1986; Komalavilas et al. 1991; Zhu et al. 1993).
Because the glycosidic linkage in Yariv reagents is
required to be in the b-anomeric conjugation to react
with AGPs, AGPs sometimes have also been referred to
as b-lectins (Jermyn and Yeow 1975; Clarke et al. 1978).
Yariv phenylglycosides having sugars with cis-hydroxyl
or other substitutions on C2 of the b-D-glycopyranosyl
determinant also do not bind to AGPs (Nothnagel and
Lyon 1986). For example, while (b-D-Glc)3 Yariv
reagent can bind AGPs, (b-D-Man)3 or (a-D-Gal)3
Yariv reagents do not react with AGPs. These latter
Yariv reagents can serve as valuable controls to distinguish AGP-related e€ects from non-speci®c e€ects when
using Yariv reagents in biological systems.
In the course of initiating a genetic screen for possible
Arabidopsis thaliana mutants with an altered response to
Yariv reagent, we made a fortuitous observation that
exposure to (b-D-Glc)3 Yariv reagent causes the bulging
of root epidermal cells. The morphological alterations
resemble the phenotype of the reb (root epidermal cell
bulging) mutant of Arabidopsis isolated by Baskin et al.
(1992). We then found that the root of the reb1-1 mutant
contains AGPs that are electrophoretically di€erent
from those of the wild type. These results strongly
indicate a role of AGPs in controlling root epidermal cell
expansion.
Materials and methods
Treatment with Yariv reagents. Wild-type Arabidopsis thaliana (L.)
Heynh. (ecotype Columbia) seeds were surface-sterilized and sown
onto agar media containing Murashige and Skoog (Murashige and
Skoog 1962) salts, 3% sucrose, 1.2% agar, pH 5.7 and supplemented with various amounts of Yariv reagents. Yariv reagents
were prepared according to the method of Yariv et al. (1962).
Plants were grown at 23 ‹ 2 °C under constant white light
(approx. 70 lmol photons á m)2 á s)1). Roots were observed and
photographed directly inside the agar media using an inverted
microscope.
Extraction and electrophoretic analysis of AGPs. Wild type and
reb1-1 mutant seedlings were grown for 10 d on vertical agar plates.
Roots were separated from the shoots at the base of hypocotyl with
a razor blade and collected and frozen immediately in liquid
nitrogen. To extract AGPs, 5 g of frozen root or shoot samples was
ground in 5 ml of extraction bu€er containing 50 mM Tris-HCl
(pH 8.0), 10 mM EDTA, 2 mM Na2SO5, and 1% (v/v) Triton X100. The extraction procedure is essentially as described in van
Holst and Clarke (1986) except that the desalting step was omitted.
Brie¯y, the homogenate was vortexed for 10 min, incubated at 4 °C
for 2.5 h and then centrifuged at 14 000 g for 10 min. The
supernatant was mixed with ®ve volumes of ethanol and the
mixture was incubated overnight at 4 °C. The precipitate was
collected by centrifugation at 14 000 g for 10 min and then
resuspended in 50 mM Tris-HCl (pH 8.0). The ethanol precipitation step was repeated once before the sample was analyzed by
rocket and crossed gel electrophoresis. Rocket gel electrophoresis
was run with 1% agarose containing 15 lM (b-D-Glc)3 Yariv
reagent. The gel and running bu€er consisted of 25 mM Tris and
200 mM glycine, pH 8.4. For crossed electrophoresis, the ®rst
dimension was run with 1% agarose in TBE bu€er (90 mM Tris,
90 mM boric acid and 2 mM EDTA, pH 8.3). The running bu€er
was the same as the gel bu€er. The second dimension was run with
1% agarose gel containing 15 lM (b-D-Glc)3 Yariv reagent. The gel
and running bu€er consisted of 25 mM Tris and 200 mM glycine,
pH 8.4. After completion of electrophoresis, gels were washed
overnight with 2% (w/v) NaCl and dried onto ®lter paper.
Results
b-glucosyl Yariv reagent treatment of Arabidopsis phenocopies the reb1 root epidermal cell bulging mutant.
Because (b-D-Glc)3 Yariv reagent can inhibit cell growth
in rose suspension cultures (Serpe and Nothnagel 1994),
we were interested in determining whether it may also
alter the growth of Arabidopsis thaliana seedlings, with
the aim of exploring a molecular genetic approach to the
study of AGP function. When Arabidopsis seeds were
sown onto media containing up to 50 lM b-glucosyl
Yariv reagent, their germination was not a€ected.
However, subsequent root growth was substantially
inhibited by as low as 10 lM b-glucosyl Yariv reagent.
Figure 1A illustrates this inhibition on roots grown on
an agar surface when the plates were placed vertically.
For comparison, no root inhibition was observed on
seedlings grown on media containing (b-D-Man)3 Yariv
reagent, which does not bind to AGPs (Fig. 1A). To
evaluate how fast the root inhibiton could occur, 4-d-old
seedlings grown on vertical agar plates without Yariv
reagent were transferred onto a medium containing
10 lM (b-D-Glc)3 Yariv reagent, or as controls onto
medium containing (b-D-Man)3 Yariv reagent (Fig. 1B)
or to medium without Yariv reagent (not shown). While
root growth was visible less than 1 h after the transfer on
seedlings transferred to control medium without Yariv
reagent, or with (b-D-Man)3 Yariv reagent, seedlings
transferred to (b-D-Glc)3 Yariv reagent failed to exhibit
appreciable root growth. Figure 1B shows that 2 d after
the seedling transfer, the roots on (b-D-Glc)3 Yariv
reagent had only a slight growth compared with
substantial growth on (b-D-Man)3 Yariv reagent. The
roots transplanted on (b-D-Glc)3 Yariv reagent also
showed a series of sharp twists (Fig. 1B). Shoot growth
of the seedlings on (b-D-Glc)3 Yariv reagent was not
inhibited until much later, which was probably an
indirect consequence of the reduced root growth.
A close scrutiny of roots grown inside agar medium
containing 10 lM (b-D-Glc)3 Yariv reagent revealed
surprising morphological changes. While root epidermal
cells including root hairs and non-hair cells are elongated in untreated plants, we found that many of them
became round cells when plants were grown on (b-DGlc)3 Yariv reagent-containing media (Fig. 2). Normally, roots grown inside agar media develop no or very few
root hairs (Fig. 2A). However, roots grown inside agar
media which contain (b-D-Glc)3 Yariv reagent show
numerous bulging cells (Fig. 2B,C). Thus, the epidermal
cell bulging caused by b-glucosyl Yariv reagent is
probably not restricted to hair cells. The epidermal cell
bulging occurred primarily in the elongation zone
(Fig. 2B) and in developed regions of the roots
(Fig. 2C). The root cap and meristematic region
L. Ding and J.-K. Zhu: Arabinogalactan-proteins and root cell expansion
291
Fig. 1A,B. (b-D-Glc)3 but not
(b-D-Man)3 Yariv reagent inhibits Arabidopsis root growth.
A Arabidopsis seeds were germinated and grown for 7 d on
medium containing 10 lM
(b-D-Glc)3 or (b-D-Man)3 Yariv
reagent. B Four-day-old
seedlings grown on Murashige
and Skoog (1962) medium were
transferred onto medium
containing 10 lM (b-D-Glc)3
or (b-D-Man)3 Yariv reagent
for 2 d
appeared not to be a€ected (Fig. 2B). Examination of
cross-sections of the (b-D-Glc)3 Yariv reagent-treated
roots indicated that only the epidermal layer of cells
underwent abnormal radial expansion (data not shown).
The Yariv reagent-treatment appears to phenocopy
the reb1 mutants which exhibit root epidermal cell
bulging (Fig. 2D) and reduced root growth (Baskin et al.
1992). Two alleles of reb1 are known; reb1-1 has bulging
root epidermal cells constitutively while reb1-2 only
exhibits cell bulging when roots are grown at elevated
temperatures (Baskin et al. 1992). In addition to
epidermal cell bulging, roots exposed to (b-D-Glc)3
Yariv reagent exhibited sharp twists (Fig. 1B). The
twisted growth resembles the temperature-sensitive reb1
allele (i.e. reb1-2) when it is shifted to a restrictive
temperature (Baskin et al. 1992). The sharp twists are
probably caused by asymmetric epidermal cell bulging
(Fig. 2C). More bulging cells are present on the outside
of the turn than on the inside (Fig. 2C). Roots grown
inside or on media containing up to 60 lM (b-D-Man)3
Yariv reagent did not exhibit any epidermal cell bulging
or twisted growth pattern. In fact, (b-D-Man)3 Yariv
reagent did not have any e€ect on either the morphology
or growth of Arabidopsis roots.
The reb1-1 mutant contains modi®ed arabinogalactanproteins. The fact that (b-D-Glc)3 but not (b-D-Man)3
Yariv reagent could alter the morphology and growth of
Arabidopsis roots strongly indicates that these e€ects are
a result of interference with AGPs. To determine
whether the reb1 phenotype is correlated with defective
AGPs, wild-type and reb1-1 plants were grown for 7 d in
vertical agar plates and harvested. Total cellular AGPs
were extracted from the roots and shoots, and analyzed
by rocket gel electrophoresis. Arabinogalactan-proteins
were detected in extracts from both the roots and shoots
of reb1-1 plants (Fig. 3). However, the total level of
AGPs in reb1-1 roots was only about 70% of that in
wild-type roots (Fig. 3). In contrast, reb1-1 shoots did
not contain less AGPs than the wild type (Fig. 3).
The extracted AGPs were subsequently analyzed by
crossed gel electrophoresis. The AGPs from wild-type
roots could be resolved into two major peaks, while only
one peak was detected in reb1-1 root AGPs (Fig. 4).
Therefore, reb1-1 roots contain altered AGPs. When
AGPs extracted from the shoots were subjected to the
same crossed electrophoresis, no di€erence was detected
between reb1-1 and the wild type (data not shown).
Discussion
We have shown that (b-D-Glc)3 Yariv reagent-treated
Arabidopsis seedlings exhibit reduced root growth and
inappropriate cell expansion at the root epidermis.
Treatment with (b-D-Man)3 Yariv reagent which does
not react with AGPs, did not have any of these e€ects.
This Yariv reagent speci®city points to AGPs as the
target of (b-D-Glc)3 Yariv reagent in causing the growth
and morphological alterations. Because Yariv reagents
are known to form large aggregates (Nothnagel and
Lyon 1986) and are unable to penetrate the plasma
membrane, their targets are thus limited to the cell
surface AGPs, i.e. plasma membrane and cell wall
292
L. Ding and J.-K. Zhu: Arabinogalactan-proteins and root cell expansion
Fig. 3. reb1-1 mutant Arabidopsis roots contain lower amounts of
AGPs. Gum-arabic AGPs or AGPs from root and shoot extracts of
Arabidopsis were run on an agarose-containing 15 lM (b-D-Glc)3
Yariv reagent. Lanes 1±4, 0.4, 0.3, 0.2 and 0.1 lg of gum arabic as
AGP standard, respectively; lanes 5±8, extract from 8.5 mg (fresh
weight) of wild-type root, reb1-1 root, wild-type shoot and reb1-1
shoot, respectively
Fig. 2A±D. Treatment with (b-D-Glc)3 Yariv reagent induces epidermal cell bulging in Arabidopsis roots. A wild-type root grown inside
agar medium without (b-D-Glc)3 Yariv reagent; B, C, wild type roots
grown inside agar medium containing 10 lM (b-D-Glc)3 Yariv
reagent; D reb1-1 root grown inside agar medium without (b-D-Glc)3
Yariv reagent
AGPs. These results clearly indicate a role of cell surface
AGPs in root growth and control of root epidermal cell
expansion. The conclusion is further supported by the
®nding that the root epidermal cell bulging mutant,
reb1-1, contains defective AGPs. Taken together, our
results strongly suggest a function of cell surface AGPs
in the control of cell expansion and root growth.
The inhibition of Arabidopsis root growth by (b-DGlc)3 Yariv reagent was not unexpected because this
compound has already been shown to inhibit the growth
of cultured rose cells (Serpe and Nothnagel 1994).
However, our observation of altered root epidermal cell
morphology was surprising. During the preparation of
this manuscript, Willats et al. (1996) reported similar
growth and morphological e€ects of (b-D-Glc)3 Yariv
reagent on Arabidopsis plant roots. However, these
authors did not examine AGPs in the reb1-1 mutant.
The root-inhibition phenotype could be a convenient
assay to select Arabidopsis mutants which do not
respond to (b-D-Glc)3 Yariv reagent treatment. This
type of mutant would be valuable for elucidating the
interaction of cell surface AGPs with cell wall macromolecules. Arabinogalactan-proteins have been proposed to interact with other cell surface molecules
(Roberts 1989). Although the exact mechanism of the
binding between Yariv reagents and AGPs is not
understood, it is generally believed that this interaction
indicates the ability of AGPs to bind to certain b-linked
glucans in the cell wall. Several b-linked polysaccharides
are very abundant in plant cell walls. The most abundant
one is cellulose, a b-1,4-glucan (Carpita and Gibeaut
1993). Structures similar to phenyl-b-glycosides (Yariv
reagent) might also be present in the cell wall polysaccharides and phenolics, and AGPs may be able to bind
to these wall components.
Multiple modes of action could be proposed to
account for the role of AGPs in cell expansion. For
example, cell surface AGPs could be involved in wall
biosynthesis or assembly. Loss of these AGPs or
interference with their function by Yariv reagents may
Fig 4. Arabinogalactan-protein pro®les of wild-type and reb1-1
Arabidopsis roots as revealed by crossed gel electrophoresis. The
AGPs were extracted from 280 mg (fresh weight) each of wild-type
and reb1-1 roots. The agarose gel in the second dimension contained
15 lM (b-D-Glc)3 Yariv reagent
L. Ding and J.-K. Zhu: Arabinogalactan-proteins and root cell expansion
disrupt or weaken cell wall structure which is necessary
for controlled cell expansion. Plasma-membrane AGPs
have also been proposed to interact with the actin
cytoskeleton (Roberts 1989). Defects in AGPs could
thus lead to a disorganized actin network and subsequent loss of directional cell expansion. Alternatively,
Yariv reagent binding could activate a signal-transduction cascade through cell surface AGPs and their
interacting cellular components, and indirectly lead to
growth inhibition and other responses.
The results in Fig. 4 show that there is apparent loss
of a subgroup of AGPs in reb1-1 roots. That subgroup is
represented by the smaller peak upon crossed electrophoresis (Fig. 4). Cell surface AGPs are known to be
extremely heterogeneous in size and charge (Komalavilas
et al. 1991; Serpe and Nothnagel 1995; Serpe and
Nothnagel 1996; Stohr et al. 1996). Crossed electrophoresis can only reveal major changes in AGPs. Subtle
changes in AGPs which might be of immense functional
signi®cance are dicult to detect. The apparent loss of a
major AGP peak could correspond to the failure of root
epidermal cells to synthesize AGPs in the reb1-1 mutant.
The REB1 gene may encode an AGP core protein, or an
enzyme directly involved in its glycosylation. Alternatively, the REB1 gene product could indirectly regulate
AGP biosynthesis or modi®cation such as glycosylation.
It is also conceivable that the apparent di€erence in
AGP pro®le between wild-type and reb1-1 roots involves
some molecule to which AGPs bind, thereby altering the
extractability of AGPs.
The molecular and cell biological basis for the reb1
mutant phenotypes is not known. It has been suggested
that a breakdown in the organization of cortical
microtubules or a cell wall component may be responsible (Baskin et al. 1992). Alternatively, the mutants
could be defective in establishing or maintaining the
polarity of root epidermal cell growth. Gene cloning of
the REB1 locus might provide insights into the molecular nature of the gene product and its mode of action.
We thank Dr. Tobias Baskin (Univ. of Missouri, Columbia, MO)
for kindly providing us with the reb1 mutant seeds, and Dr. Eugene
A. Nothnagel (Univ. of California, Riverside, CA) for providing us
with the Yariv reagents used in the early phase of this work. This
work is supported by a grant from the National Science Foundation (IBN-9507191).
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