603
Expansins and cell growth$
Yi Li, Louise Jones and Simon McQueen-Mason
Expansins are now generally accepted to be key regulators of
wall extension during growth. Several alternative roles for
expansins have emerged in which the emphasis of their action is
on wall breakdown or softening in processes such as fruit
ripening, pollination, germination and abscission. Expansins are
commonly encoded by substantial gene families and have
classically been divided into two subfamilies, referred to as
a- and b-expansins. Two further subfamilies have now been
identified: g-expansins, which were first described in
Arabidopsis, and d-expansins, which were identified in rice and
are absent from Arabidopsis. Both are truncated versions of
a- and b-expansins, with g-expansins representing the amino-
terminal half of a mature expansin and d-expansins the carboxy-
terminal half of a b-expansin. Functional roles for g- and
d-expansins have yet to be defined, although recent data
indicate a signalling role for g-expansins.
Addresses
CNAP, Biology Department, University of York, PO Box 373,
York YO10 4YW, UK
 between cellulose microfibrils and cross-linking glycans in
e-mail: sjmm1@york.ac.uk
Current Opinion in Plant Biology 2003, 6:603–610
This review comes from a themed issue on
Cell Biology
Edited by Takashi Hashimoto and Dirk Inzé
1369-5266/$ – see front matter
ß 2003 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.pbi.2003.09.003
Abbreviations
EST expressed sequence tag
ORF open reading frame
ROS reactive oxygen species
XTH xyloglucan endotransglucosylase/hydrolase
Introduction
Cell growth and cell walls are intimately connected in
plants. Cell walls have to be extremely strong to bear the
stresses imposed by the internal turgor pressure of plant
cells, but must also maintain the ability to extend during
cell growth. The cellulose–hemicellulose network plays a
leading role in determining the extensibility of cell walls,
and enzymes that act on this network control the process
of cell growth. Throughout the 1970s and 80s, it was
generally held that glucanases loosen the structure of
the wall to allow growth to occur. In the early 1990s,
two new groups of proteins, xyloglucan endotransgluco-
$
Supplementary material associated with this article can be found at
doi: 10.1016/j.pbi.2003.09.003
www.current-opinion.com
sylases (now known as xyloglucan endotransglucosylase/
hydrolases [XTHs]) [1,2] and expansins [3], almost
simultaneously entered the pantheon of potential wall-
loosening enzymes. XTHs are attractive wall-loosening
candidates because their action involves breaking lin-
kages in the xyloglucan–cellulose network and then re-
forming the same linkage in a new position. This action
allows the breakage of stress-bearing bonds in the wall
(thus increasing extensibility) without impairing overall
wall integrity. This review focuses on the role of expan-
sins in cell growth and other wall-associated events, and
also provides an update on expansin sequence diversity.
Expansins induce wall extension during
growth
Expansins were identified because of their ability to
restore long-term extension to cell walls that were isolated
from growing plant tissues [3]. The mechanism of expansin
action appears to involve the disruption of hydrogen bonds
the wall [4,5]. Expansins directly induce wall extension
whereas XTHs and glycanases generally do not [6,7],
as such a model involving primary and secondary wall-
loosening mechanisms has been proposed [8]. According
to this model, primary wall-loosening factors such as
expansins directly induce turgor-driven wall extension,
whereas secondary wall-loosening factors (such as XTHs
and glucanases) modify the structure of the wall, rendering
it more responsive to primary wall-loosening events.
The expansin gene family
The elucidation of the first expansin sequences [9] led to
the realisation that these proteins are encoded by a sub-
stantial multigene family. Indeed the Arabidopsis genome
contains 38 open reading frames (ORFs) that encode
expansin-like proteins, two of which appear to be pseu-
dogenes [10]. Phylogenetic analysis showed that the 38
ORFs could be separated into three clear subgroups on
the basis of protein sequence similarity. The largest sub-
group in Arabidopsis, with 26 members, is the a-expansins.
These are highly similar in sequence to the first expansins
cloned from cucumber that can induce wall extension in
several different plant tissues [3]. The second largest
subfamily is the b-expansins, containing eight ORFs that
fall into three clear subgroups (b1–b3). b-expansins share
several conserved features with the a-group, and at least
one b-expansin has been shown to induce the extension of
cell walls in maize coleoptiles and silks [11].
The g-expansins form the smallest subgroup in Arabidop-
sis with only two representative ORFs. These genes
encode proteins that are significantly shorter than
Current Opinion in Plant Biology 2003, 6:603–610
604 Cell biology

Figure 1

osaEXPα1.1
0.1
osaEXPα1.2
osaEXPα1.3
osaEXPα1.4
osaEXPα1.5
96 osaEXPα1.6
osaEXPα1.7
osaEXPα1.8
osaEXPα1.9
osaEXPα1.10
osaEXPα1.11
osaEXPα1.12
osaEXPα1.13
74 osaEXPα1.14
82 csaAAB37749
athEXPα1.14
osaEXPα1.15
osaEXPα1.16
62 osaEXPα1.17
99 osaEXPα1.18
osaEXPα1.19
athEXPα1.13
athEXPα1.24
athEXPα1.2
athEXPα1.1
athEXPα1.3
athEXPα1.4
osaEXPα1.20
osaEXPα1.21
athEXPα1.5
athEXPα1.11
csaAAB37746
ptaAAD47901
α
athEXPα1.12
osaEXPα1.22
osaEXPα1.23
osaEXPα1.24
osaEXPα1.25
osaEXPα1.26
athEXPα1.8
athEXPα1.6
95 athEXPα1.7
athEXPα1.9
athEXPα1.10
92 mquAAF17571
mquAAF17570
ppaEXP1
90 osaEXPα1.27
100 athEXPα1.23
100 osaEXPα1.28
athEXPα1.22
athEXPα1.18
athEXPα1.17
athEXPα1.16
athEXPα1.15
99 100 athEXPα1.20
athEXPα1.19
athEXPα1.21p
87 100 osaEXPα1.29
osaEXPα1.30
75 osaEXPα1.31
90 osaEXPα1.32
98 athEXPα1.26
athEXPα1.25
osaEXPδ1.1
osaEXPδ1.2p
osaEXPδ1.3
osaEXPδ1.4
osaEXPδ1.5
osaEXPδ1.6p
osaEXPδ1.7
osaEXPδ1.8p

δ
0.1 osaEXPδ1.9p
osaEXPδ1.10
osaEXPδ1.11
osaEXPδ1.12
osaEXPδ1.13
osaEXPδ1.14
99 osaEXPδ1.15
osaEXPδ1.16
osaEXPδ1.17
100 osaEXPδ1.18
osaEXPβ1.1
osaEXPβ1.2
0.1 100 osaEXPβ1.3
osaEXPβ1.4p
100 osaEXPβ1.5
osaEXPβ1.6
100 osaEXPβ1.7
osaEXPβ1.8
osaEXPβ1.9
97 osaEXPβ1.10
osaEXPβ1.11
osaEXPβ1.12
100 osaEXPβ1.13 β1
100 osaEXPβ1.14
85 athEXPβ1.5
63 athEXPβ1.6
athEXPβ1.2p
athEXPβ1.1
athEXPβ1.3
β
athEXPβ1.4
osaEXPβ1.15
osaEXPβ1.16
osaEXPβ1.17
73 91 osaEXPβ1.18
osaEXPβ1.19
osaEXPβ1.20
osaEXPβ2.1
100 osaEXPβ2.2
64
athEXPβ2.1
osaEXPβ2.3 β2
89 99 athEXPβ2.3
100 athEXPβ2.2
praT08115
84 osaEXPβ3.1
athEXPβ3.1 β3
osaEXPγ1.1
93 osaEXPγ1.2
osaEXPγ1.3
osaEXPγ1.4p
γ
72
osaEXPγ1.5
100 athEXPγ1.2
osaEXPγ1.6
athEXPγ1.1

0.1
Current Opinion in Plant Biology

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 Expansins and cell growth Li, Jones and McQueen-Mason 605

a- and b-expansins, lacking most of the carboxy-terminal
half of the mature protein. A g-expansin-like protein from
Citrus jambhiri lacks expansin activity [12]. A recent
publication indicates that these proteins might function
as natriuretic peptides. Natriuretic proteins function as
peptide hormones that regulate water balance in verte-
brates, and plant analogues that correspond to g-expan-
sins induced osmotic changes in plant protoplasts and
stomatal opening, suggesting that they may regulate
water balance in plant cells [13,14].
Eighty expansin-like ORFs in the rice
genome
Lee and Kende [15,16] recently examined the rice expan-
sin gene family and found a total of 40 expansin-encoding
genes in rice. Now that the complete genome sequence of
rice is publicly available, we have been able to ascertain
the full extent of the rice expansin gene family and to
compare it with that of Arabidopsis. BLAST searches of the
rice genome revealed a total of 80 ORFs that encode
expansin-like proteins, compared to the 38 in Arabidopsis.
Sequence alignment and phylogenetic analysis revealed
that the majority of the predicted rice proteins fit into the
subfamilies and subgroups previously defined for the
Arabidopsis expansin family (Figure 1). The biochemistry
of only a few expansins has been characterised, and so we
use a purely sequence-based systematic nomenclature for
expansins [10]. This nomenclature uses a three-letter
species identification (e.g. Osa for Oryza sativa) followed
by the group annotation (a, b, g or d) and a number
representing the subgroup. A table listing accession num-
bers for the rice ORFs presented in Figure 1 is available
online as Supplementary Material. The a-expansin sub-
family is of similar size in rice and Arabidopsis (32 and 27
members, respectively). Within the a-subfamily, there are
some clearly defined orthologous subsets; for example, the
subset formed by OsaExpa1.27 and OsaExpa1.28 is ortho-
logous to that formed by AthExpa1.22 and AthExpa1.23.
The b-expansins in rice fall within the three recognised
subgroups (b1–b3) [10]. Rice and Arabidopsis both have
just a single orthologous member of the b3 group and
both have three paralogous members of the b2 group. Rice
possesses 20 b1-expansins, however, compared to just six
in Arabidopsis.
A new subfamily of expansin sequences
Rice possesses a hitherto unrecognised group of expansin-
like sequences that are not found in Arabidopsis. On
the basis of sequence similarity (Figure 2) and phy-
logenetic analysis, this subfamily (named d-expansins)
appears to have arisen as a result of recent duplication
events associated with a distinct clade of b1-expansins
(Figure 1). We have designated this group as a distinct
subfamily because they encode truncated expansins that
are similar in size to those of the g-subfamily. By contrast,
the d-expansins encode truncated proteins that have high
sequence similarity to the carboxy-terminal half of ex-
pansins but that lack the amino-terminal half of these
proteins. We suggest that d-expansins might represent a
monocot-specific subgroup that has recently diverged
from the b1-subfamily, a hypothesis that is supported
by Bootstrap analysis. Of the 18 d-expansins in the rice
genome, ten occur as a cluster on chromosome 6 (Figure 3)
and six appear to be the result of a recent chromosomal
duplication event on chromosome 4. d-expansins possess
a short amino-terminal signal peptide of about 24 amino
acids, as predicted by SignalP V2.0, suggesting that they
are secreted proteins that have potential wall-related
functions. It has been proposed that the carboxy-terminal
half of canonical expansins share similarity with cellulose-
binding domains [9], and thus it seems likely that d-
expansins interact with wall components. At present, the
function of d-expansins is unknown, but it is worth noting
that all but four are represented in expressed sequence
tags (ESTs) from a rice panicle cDNA library. The most
abundant d-expansins in this library were OsaExpd1.14
and OsaExpd15, which are represented by 21 ESTs
(these two genes are difficult to distinguish as they differ
by just one nucleotide).
The existence of the g- and d-expansins is intriguing
because the g-expansins are truncated proteins that
lack the carboxy-terminal half of the canonical a- and
b-expansin groups, whereas the d-expansins lack the
amino-terminal half. Is it possible that canonical expan-
sins incorporate two functional domains that maintain
biological activity of some sort when expressed indepen-
dently? Alternatively, these truncated expansin-like pep-
tides, g-expansins in particular, might have evolved
signalling functions [9]. The ancestral form of expansins
appears to have existed as the full-length protein exhib-
ited by the a- and b-subfamilies, which are similar in size
to the small expansin-like gene family identified in
Dictyostelium discoideum [10]. From the phylogenetic
analysis, it is clear that the g-expansins are an ancient
subgroup that have representatives in all land plants,

(Figure 1 Legend) Phylogenetic analysis of the expansin gene family in rice and Arabidopsis. Datasets were created from a master alignment by
removing the regions containing gaps (i.e. ambiguous alignment regions). Distance methods used Protdist with a PAM250 substitution matrix,
and trees were constructed from the matrices using Fitch (Fitch-Margoliash method) in the Phylip3.5c software package. Bootstrap analyses were
carried out with 1000 replicates using the neighbour-joining algorithm. Three sub-trees were constructed on the basis of maximum amino-acid
positions shared in each group, and were appended to the backbone tree as indicated by broken lines. Only bootstrap values of more than 60% are
indicated. All branches are drawn to scale, as indicated by the scale bar showing 0.1 substitutions per site. Major plant taxonomic divisions are
indicated by colour: Arabidopsis thaliana and two additional cucumber sequences, black; Oryza sativa (japonica), green; pines, blue; ferns,
orange; and bryophytes, red. Species-specific identifiers are as follows: Ath, Arabidopsis thaliana; Csa, Cucumis sativa; Mqu, Marsilea quadrifolia;
Osa, rice (Oryza sativa); Ppa, Physcomitrella patens; Pra, Pinus radiata and Pta, Pinus taeda.

www.current-opinion.com Current Opinion in Plant Biology 2003, 6:603–610

606 Cell biology

Figure 2

(a)
Signal peptide
___________________________________________________

 AthExpα1.3 ---MGLLG--------IAL------FCFAAMVCSVHG------YDA---G-WVNAHATFYGGSD--ASGTMGGACGYG-NLYSQG-YGTNTAALSTALFNNGLSCGACFEIKCQSDG—-
α  OsaExpα1.18 ---MLSGMEKP----AMLLVLV----TLCAF-----A------CKR---SVAQSAFATFYGGKD--GSGTMGGACGYG-NLYNAG-YGLYNAALSSALFNDGAMCGACYTITCDTSQT-
 AthExpβ1.5 ---MQLFP------VILPTLCVFLHLLISGSGSTPPLTHS---NQQVAATRWLPATATWYGSAE--GDGSSGGACGYGSLVDVKP-FKARVGAVSPILFKGGEGCGACYKVRCLDKT--
 OsaExpβ1.9 ---MAFSISK------KAAVAALFSFLVVTCVAGARPGNFSASDFTADPN-WEVARATWYGAPTGAGPDDDGGACGFK-NTNQYP-FSSMTSCGNEPIFKDGKGCGSCYQIRCVNHP--
 AthExpβ2.3 --------------MRSFLYLIVVIFLFSSSVNACDRC-------------LHRSKASYFSSAS---AL-SSGACAYG-PMATSF-FAGHIAAAIPSIYKDGAGCGACFQVRCKNPK--
 OsaExpβ2.1 MAVSVRCCFGSSLSHHARLLLVIVALLAPRLASGCDRC-------------VRRSRAAYYTSS----LTLTAGSCGYG-TAAATFNGGGFLAAAGPALYRGGVGCGACYQVRCKDKK--
 AthExpβ3.1 -------MKH----SHVLLLLFVQVIVLLPLLCLSDD--------------FVNSRATYYGSPD-CKAN-PRGHCGYG-EFGRDI-NNGEVSGVSWRLWNNGTGCGACYQVRCKIPP—-
 OsaExpβ3.1 MAQLLR-----------RHLPVILSLILFLSKATADAN-------------FTVSRAAYYPNSD--IKGTENGACEYG-AFGATL-NNGDV-SASASLYRDGVGCGACYQVRCTNPY—-
β  OsaExpβ1.2 MASSS---------LLLACVVVAAMVSAVSCGPPKVPPGPNITTSYGDK--WLEAKATWYGAPKGAGPKDNGGACGYK-DVDKAP-FLGMNSCGNDPIFKDGKGCGSCFEIKCSKPE--
δ  OsaExpδ1.14 --------------MASLSSFRLAVAVAALLVVGSCA----------------------------------------------------------------------------------
 AthExpγ1.1 ----------------MAVKFVVVMIVFAQILAPIAE--------------AAQGKAVYYDPPY------TRSAC-YGTQR------ETLVVGVKNNLWQNGRACGRRYRVRCIGATYN
γ  OsaExpγ1.5 ----------------MARVGILLAAALLLGLVSASQ--------------AIEGTATFYTVYT-------PSAC-YGFQDQ-----GTMIAAASDGLWDGGRACGRMYTVRCVRGTNA-
W C C C C

 AthExpα1.3 --AWCPG--AIIVTATNFCPPNNALPNNAGGWCNPPLHHFDLSQPVFQRIAQY---------KAGVPVSYRRVPCMRRG-GIRFTINGHS---YFNLVLVTNVGGAGDVHSVAVKGSRT
α  OsaExpα1.18 --KWCPGGNSITITATNLCPPNWALPSNSGGWCNPPLQHFDMSQPAWENIAVY---------QAGIPVNYKRVPCQRSG-GIRFAISGHD---YFELVTVTNVGGSGVVAQMSIKGSNT
 AthExpβ1.5 ---ICKR--AVTIIATDQSPSGPSAK--------AKHTHFDLSGAAFGHMAIP--GHNGVIRNRGLNILYRRTACKYRGKNIAFHVNAGS-TDYWLSLLIEYEDGEGDIGSMHIRQAGS
 OsaExpβ1.9 ---ACGN--PETVIITDMNYYP------------VSKYHFDLSGTAFGAMAKP--GQNDQLRHAGIDIQFKRVPCNFPGLKVTFHVEEGS-NPVYFAVLVEYEDGDGDVVQVDLMEANS
 AthExpβ2.3 ---LCSK--GTIVMVTDLNT--------------SNQTDLVLSSRAFRAMAKPVVGVDKYLLKQGIDVEYQRVPCNYGKRNLNVRVEEASKKPNYLAIKLLYQGGQTEVVGIDIAPVGS
 OsaExpβ2.1 ---LCNA--GARVVVTDRAR--------------TNRTGLVLSSPAFAAMARP--GMAASLTELAADVEYKRVPCEYRHRSLSVRVDERSRGPNELTISFLYQGGQTDIVAVDVAQVGS
 AthExpβ3.1 ---HCEE--GVYVVATDSGE--------------GDGTDFILSPKAYGRMARP--GTENQLYSFGVNVEYQRIPCRYAGYNLVYKIHEKSYNPHYLAILVLYVGGVNDILAVEVWQEDC
 OsaExpβ3.1 ---YCPN--GVTIVITDSGA--------------SDGTDFILSQHAFTRMAQST-DAGTALLTLGVGIEYRRVSCTYPNKNIVFKITESSNFPNYLEFEIWYQQGNQDIIAVQLCETVN
β  OsaExpβ1.2 ---ACSDK-PALIHVTDMNDEP------------IAAYHFDLSGLAFGAMAKD—GKDEELRKAGIIDTQFRRVKCKYPADTKITFHIEKASNPNYLALLVKYVAGDGDVVEVEIKEKGS
δ  OsaExpδ1.14 -------------------------------------------------------------------------------TELTFKVAEGS--SASSLELVTNVA----ISEVEIKEKGG
 AthExpγ1.1 FDRACGR--TVDVKVVDFCRE-------------PCNGDLNLSRDAFRVIANT---------DAGNRVVYTPI----------------------------------------------
γ  OsaExpγ1.5 VPNPCGG--TVTVKIVDRCPSP------------GCTSTLDLSREAFAAIGNL---------DAGRVIDYNQV----------------------------------------------
C C C HFDLS C

 AthExpα1.3 R-WQQMSRNWGQNWQSNNLLNG-QALSFKVTAS-DGR---TVVSNNIAPASWSFGQTF--TGRQFR----------------
α  OsaExpα1.18 G-WMAMSRNWGANWQSNAYLAG-QSLSFIVQLD-DGR---KVTAWNVAPSNWFFGATYSTSWVQF-----------------
 AthExpβ1.5 KEWISMKHIWGANWCIVE-GPLKGPFSVKLTTLSNNK---TLSATDVIPSNWVPKATY-TSRLNFSPVL-------------
 OsaExpβ1.9 QSWTPMRESWGSIWRLDSNHRLTAPFSLRITNE-SGK---QLVASQVIPANWAPMAVY-RSFVQYSS---------------
 AthExpβ2.3 SQWSYMSRSHGAVWATDKVPTGALQFKFTVTGGYDGK---TVWSKRVLPANWNSGRIY-DAGVQITDIAQEGCDTCG-HIWN
 OsaExpβ2.1 SSWKFMTREHGPSWSMANAPPGPLQMRLVVTGGYDGKWV-WADR-EVLPRRWRAGEVY-DTGVQITDIAQEGCFPCDTHEWK
 AthExpβ3.1 KEWRRMRRVFGAVHDLQNPPRGTLTLRFLVYGSAGIN---WIQSPNAIPADWTAGATY-DSNILLT----------------
 OsaExpβ3.1 LTCQLLSRTHGAVWAAVSPPSGPLSIRMLFSSGAPRGGDTWLVPTNIVPQNWTAGATY-DSGVQVQLQ--------------
β  OsaExpβ1.2 EEWKALKESWGAIWRIDTPKPLKGPFSVRVTTE-GGE---KIIAEDAIPDGWKADSVY-KSNVQAK----------------
δ  OsaExpδ1.14 KDWVALKESSSNTWTIKSEAPLKGPFSVRFLVK-NGG---YRVVDDVIPESFTAGSEY-KSGIQL-----------------
 AthExpγ1.1 ----------------------------------------------------------------------------------
γ  OsaExpγ1.5 ----------------------------------------------------------------------------------
W W W W
(b)
Canonical expansin (α or β)

γ-expansin

δ-expansins

Signal peptide

Current Opinion in Plant Biology

(a) Protein sequence alignment of selected members of the various subgroups of expansins. (b) Diagrammatic representation of the relative
sizes of the g- and d- groups compared to those of the canonical a- and b-expansins.

whereas the d-expansins have a recent origin having
arisen from a subgroup of the b1-expansins.
Expansin biochemistry
Studies of expansin activity have generally involved the
use of fairly crude protein extracts, which reveal little
about the molecular details of expansin action. Several
factors have limited the research carried out in this area.
Expansins are difficult to purify, they are not typically
abundant and most tissues express more than one closely
related isoform. This is compounded by expansin assays
that require a dedicated apparatus, are time-consuming
and require considerable amounts of protein. The pro-
duction of active recombinant expansins has proven lar-
gely unsuccessful. Most information on expansin activity
originates from the cucumber a-expansin CsExp1, which
appears to induce wall extension by disrupting hydrogen
bonds between cellulose fibrils and cross-linking glycans
[4,5]. CsExp1 has been shown to induce the extension of
composites of cellulose microfibrils and xyloglucans but
not of composites containing other cross-linking glucans,
suggesting that it has a high degree of substrate specificity
[17]. Whether all a-expansins operate in the same way
and are specific for the cellulose/xyloglucan network is not

Current Opinion in Plant Biology 2003, 6:603–610 www.current-opinion.com


Figure 3
(a)
δ1.3 δ1.5
45 kb
(b)
δ1.11 δ1.13
15 kb 9 kb
95% 97%
15 kb 8 kb
δ1.9p δ1.12
Diagrammatic representations of the chromosomal locations of d-expansin genes in the rice genome. All are present on either chromosome 4 or 6.
(a) Ten d-expansin genes are located in a cluster on chromosome 6. (b) There are two nearly identical clusters of 25 kb on chromosome 4, each
having three d-expansin genes, indicating a local chromosomal duplication event. The percentage predicted amino acid similarities are indicated.
yet clear, but we have successfully detected expansin
activity from a range of dicot [18,19,20] and grass species
[21] using pure cellulose/xyloglucan composites.
Even less is known about the b-expansins, which were
initially investigated as group I pollen allergens from
grasses because they are abundantly released from
hydrating pollen grains and partly responsible for pollen
allergies. Cosgrove et al. [11] showed that extracts
enriched for group I pollen allergens possess expansin
activity, inducing the extension of maize silks and wheat
coleoptiles but not cucumber hypocotyl walls. This
strongly suggests that b-expansins have a similar activity
but different substrate specificity to a-expansins. In this
context, it is notable that there are generally greater
numbers of b-expansins in grass species than in dicots,
and it has been suggested that this may reflect the
differences in wall composition between these groups
of species [15].
Expansins and growth
Expansins were identified because of their ability to
induce wall extension, and the role of expansins in growth
has been generally accepted. Initial studies [3] showed
that expansin activity in cucumber hypocotyls was only
extractable from actively growing tissues. Subsequent
studies have generally supported this observation, high-
lighting a positive correlation between the presence of
expansin activity, epitopes or transcripts and growth
rates [22–27].
Some of the most compelling evidence for the role of
expansins in growth has been provided by a series of
experiments that examined the effects of expansins on
the production of leaf primordia in vegetative meristems.
Fleming et al. [28] showed that topical applications of
expansins to discrete regions on the flanks of tomato
vegetative meristems could lead to the initiation of aber-
www.current-opinion.com
Expansins and cell growth Li, Jones and McQueen-Mason 607
δ1.7 δ1.6p
δ1.1 δ1.8p δ1.4 δ1.2p δ1.16 δ1.17
3 kb 2 kb 7 kb 10 kb 4 kb
δ1.15
AL606599
99%
AL662972
δ1.14
Current Opinion in Plant Biology
rant primordia, although these primordia did not grow on
to form normal leaves. It was subsequently shown that the
localised induction of expansin transgene expression on
the flanks of tobacco vegetative meristems not only
induced the appearance of primordia but also reiterated
the whole process of leaf development, giving rise to
phenotypically normal leaves [20]. In addition, expansin
transcripts appear in the early stages of primordium
development in tomato plants [28], suggesting that they
play a similar role in initiating this process in vivo.
Recent reports have also indicated a similar role for
expansins in the initiation and development of root hairs.
The presence of expansin in the bulges initiating root hair
outgrowth from root epidermal cells in maize was shown
using immuno-localisation [29], and a similar pattern was
observed using reporter fusions to promoters from two
Arabidopsis expansin genes [30].
If expansins play a key role in plant growth, then it might
be predicted that increasing or decreasing expansin
expression will affect this process. Several reports have
shown increased growth of transgenic plants that over-
express expansins [31,32]. This is not always apparent,
however. For example, Rochange et al. [33] reported
decreased growth in tomato plants that expressed high
levels of recombinant cucumber expansins, and this was
apparently associated with compensatory decreases in
wall extensibility.
Expansin mutants
We have obtained mutants lines that have T-DNA inser-
tions in several different Arabidopsis expansin genes and
have yet to see a pronounced phenotype in any of these
plants. Similar outcomes have been reported elsewhere
[34]. Moreover, it has been reported that knockout
mutations in several expansin genes in the moss Physco-
mitrella patens caused no clear phenotype [35]. One
Current Opinion in Plant Biology 2003, 6:603–610
608 Cell biology
clear reason that may account for this apparent lack of
phenotype is the possibility of functional redundancy
within the expansin family, as has been reported in other
multi-gene families. Hence, it may be necessary to stack
mutations in several related expansin genes to generate
phenotypic changes.
Growth without expansins?
A recent report has indicated that expansins may not
be required for plant growth in the grass Festuca pratensis,
in which transcript accumulation for neither a- nor
b-expansins correlated with regions of leaf cell expansion
[21]. Similarly, in F. pratensis, assays of a-expansin
activity and Western analysis indicated low expansin
concentrations in the most rapidly growing tissues, with
levels actually increasing as growth rates decreased.
In-situ RNA hybridisation studies of a- and b-expansin
genes in the grass leaves indicated that the expression of
these genes was largely confined to developing vascular
tissues. Interestingly, reactive oxygen species (ROS) are
capable of inducing substantial wall extension in maize
(i.e. another grass) coleoptiles in vitro, and inhibitors of
ROS production can block auxin-induced growth and
wall extension in these tissues [36]. Similarly, ROS have
been shown to be necessary for the growth of maize
leaves [37]. The mechanism of ROS-induced wall
extension probably involves the scission of wall poly-
saccharides [38]. Taken together, these data suggest
that growth (and wall extension) may occur without
expansins in some organs of some plants.
Wall softening and breakdown
Several roles of expansins that do not involve wall expan-
sion have emerged. Cosgrove et al. [11] showed that a
b-expansin from maize pollen has expansin activity on
maize silks. This protein is released from hydrating pollen
grains onto the surface of the stigma and is proposed to
soften stigmatic tissues, thus facilitating penetration by
the pollen tube. Interestingly, it was recently shown that a
similar (but in this case pistil-specific) b-expansin is
expressed at high concentrations at the stigmatic surface
of tobacco during pollination [39], suggesting that this
role for some b-expansins is conserved between dicots
and grasses.
Furthermore, several reports have shown that expansins
are expressed abundantly in softening fruit, differentiat-
ing vascular tissues and abscission zones. Rose et al. [40]
characterised an expansin that is expressed exclusively
during fruit softening in tomato. Reduced expression of
this expansin in transgenic plants gave rise to fruit that
softened more slowly, whereas overexpressing the gene
led to more rapid softening [41]. It was suggested that the
role of the expansin in fruit softening might be to facil-
itate the breakdown of glucan wall components; how-
ever, analysis of the transgenic tomatoes offered no clear
evidence of enhanced or reduced wall breakdown as a
Current Opinion in Plant Biology 2003, 6:603–610
result of changes in expansin expression. Although the
mechanisms of expansin-induced fruit softening remain
obscure, ripening-associated expansins have now been
identified in a range of different fruits [19,42–44].
At least six different expansin genes are expressed in
differentiating tracheary elements in Zinnia cell cultures
[45,46]. The expansins described by Im et al. [45] appear
to be associated with the intrusive growth of protoxylem
elements in Zinnia stems. In contrast, those reported
by Milioni et al. [46] appear to be expressed during
the differentiation of the cultured mesophyll cells
into tracheary elements, suggesting that they may be
involved either in secondary wall formation or primary
wall disassembly.
Abscission involves wall breakdown, and Cho and
Cosgrove [30] showed that the AthExpa1.1 (AtExp10)
promoter drove the expression of the b-glucuronidase
(GUS) reporter gene at the base of the petioles (as
well as in other organs) of transgenic Arabidopsis. These
authors therefore suggested that this expansin might be
involved in petiole abscission, although there appears to
be no petiole abscission zone in Arabidopsis. In support of
this, we have identified an expansin transcript that is
induced in response to ethylene specifically in the petiole
abscission zones of Sambucus nigra. The expression of this
transcript is accompanied by an increase in expansin
activity in the same tissues (S McQueen-Mason, J
Roberts, E Belfield, unpublished data). Similarly, Chen
and Bradford [47] showed that an expansin is closely
associated with endosperm weakening during the germi-
nation of tomato seeds. Once again, this process involves
wall breakdown and weakening rather than growth. As
previously noted [48], there appears to be a common
posse of wall-modifying enzymes, be they associated with
wall expansion or breakdown, that are expressed in tis-
sues undergoing wall modifications that involve the cel-
lulose hemicellulose network. These enzymes include
expansins, XTHs and glucanases.
Conclusions and future directions
It is over a decade since expansins were first identified.
These past years have seen a consolidation of the view that
expansins play a major role in cell-wall extension during
growth, although there are tantalising indications that they
might not always be completely necessary for this process.
It has also become increasingly apparent that expansins
are involved in a range of other wall-related processes
beyond cell expansion. Several serious challenges remain
to be tackled in this area. First, we need to know more
about expansin biochemistry: do all expansins work by
disrupting bonds between wall polymers? Do they all work
on cellulose-associated cross-linking glycans? What are the
substrate specificities of different expansins? Second, we
need to determine the reasons for the existence of such a
large gene family encoding these proteins.
www.current-opinion.com

Finally, some intriguing questions relating to the evolu-
tionary origins of expansins have been raised. Li et al.
[10] identified a small family of genes that encode
convincingly expansin-like proteins in the mycetozoan
(slime mould) Dictyostelium discoideum, which produces a
cellulose-based cell wall during some of the multicellular
stages of its life cycle. This suggests a common origin for
expansins very early in Eukaryotic evolution. It also begs
the question of whether all organisms that have cellulosic
cell walls might not also have expansins to help to
manipulate these walls during development. Although
many bacteria make cellulose, a true cell wall is found
only in the Eukarya. It is therefore not surprising to find
that, at the time of writing, no convincingly expansin-like
sequences can be detected by homology searches of the
wide range of partial and complete bacterial genomic
sequences. Comprehensive genomic, or even EST, data
are available for a few organisms that have cellulosic walls
outside of land plants and Dictyostelium. As more sequence
information becomes available, it will be fascinating to
see if expansin-like genes are present in other lower
Eukaryotic species.
References and recommended reading
Papers of particular interest, published within the annual period of
review, have been highlighted as:
 of special interest
 of outstanding interest
1. Fry SC, Smith RC, Renwick KF, Martin DJ, Hodge SK, Matthews KJ:
Xyloglucan endotransglycosylase, a new wall-loosening
enzyme-activity from plants. Biochem J 1992, 282:821-828.
2. Nishitani K, Tominaga R: Endoxyloglucan transferase, a novel
class of glycosyltransferase that catalyzes transfer of a
segment of xyloglucan molecule to another xyloglucan
molecule. J Biol Chem 1992, 267:21058-21064.
3. McQueen-Mason SJ, Durachko DM, Cosgrove DJ: Two
endogenous proteins that induce cell-wall extension in plants.
Plant Cell 1992, 4:1425-1433.
4. McQueen-Mason SJ, Cosgrove DJ: Disruption of hydrogen-
bonding between plant-cell wall polymers by proteins that
induce wall extension. Proc Natl Acad Sci USA 2000,
91:6574-6578.
5. McQueen-Mason SJ, Cosgrove DJ: Expansin mode of action on
cell walls: analysis of wall hydrolysis, stress-relaxation, and
binding. Plant Physiol 1995, 107:87-100.
6. McQueen-Mason SJ, Fry SC, Durachko DM, Cosgrove DJ:
The relationship between xyloglucan endotrans-glycosylase
and in vitro cell wall extension in cucumber hypocotyls.
Planta 1993, 190:327-331.
7. Cosgrove DJ, Durachko DM: Autolysis and extension of isolated
walls from growing cucumber hypocotyls. J Exp Bot 1994,
45:1711-1719.
8. Cosgrove DJ: Expansive growth of plant cells. Plant Physiol
Biochem 2000, 38:109-124.
9. Shcherban TY, Shi J, Durachko DM, Guiltinan MJ,
McQueen-Mason SJ, Shieh M, Cosgrove DJ: Molecular-cloning
and sequence-analysis of expansins — a highly conserved,
multigene family of proteins that mediate cell-wall extension in
plants. Proc Natl Acad Sci USA 1995, 92:9245-9249.
10. Li Y, Darley CP, Ongaro V, Fleming A, Schipper O, Baldauf SL,
 McQueen-Mason SJ: Plant expansins are a complex multigene
family with an ancient evolutionary origin. Plant Physiol 2002,
128:854-864.
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Expansins and cell growth Li, Jones and McQueen-Mason 609
The authors elucidate the complete expansin gene family in Arabidopsis
by comprehensive phylogenetic analysis and demonstrate the presence
of three distinct expansin subgroups.
11. Cosgrove DJ, Bedinger P, Durachko DM: Group I allergens of
grass pollen as cell wall-loosening agents. Proc Natl Acad Sci
USA 1997, 94:6559-6564.
12. Ceccardi TL, Barthe GA, Derrick KS: A novel protein associated
with citrus blight has sequence similarities to expansin.
Plant Mol Biol 1998, 38:775-783.
13. Maryani MM, Bradley G, Cahill DM, Gehring CA: Natriuretic
 peptides and immunoreactants modify osmoticum-dependent
volume changes in Solanum tuberosum L. mesophyll cell
protoplasts. Plant Sci 2001, 61:443-452.
Natriuretic plant peptides, corresponding to g-expansins, are shown to
induce the swelling of mesophyll cell protoplasts, indicating a potential
role for this subfamily of expansins in regulating cell water relations.
14. Ludidi NN, Heazlewood JL, Seoighe C, Irving HR, Gehring CA:
Expansin-like molecules: novel functions derived from
common domains. J Mol Evol 2002, 54:587-594.
15. Lee Y, Choi D, Kende H: Expansins: ever-expanding numbers
and functions. Curr Opin Plant Biol 2001, 4:527-532.
16. Lee Y, Kende H: Expression of alpha-expansin and
 expansin-like genes in deepwater rice. Plant Physiol 2002,
130:1396-1405.
The authors describe the expression patterns of expansins in the inter-
nodes, leaves, coleoptiles, and roots of deepwater rice. Ten a-expansins
and three expansin-like genes were detected. Correlations between
expansin expression and growth, gibberellin treatment and wounding
are discussed.
17. Whitney SEC, Gidley MJ, McQueen-Mason SJ: Probing expansin
action using cellulose/hemicellulose composites. Plant J 2000,
22:327-334.
18. Rochange SF, McQueen-Mason SJ: Expression of a
heterologous expansin in transgenic tomato plants.
Planta 2000, 211:583-586.
19. Harrison EP, McQueen-Mason SJ, Manning K: Expression of six
expansin genes in relation to extension activity in
developing strawberry fruit. J Exp Bot 2001, 52:1437-1446.
20. Pien S, Wyrzykowska J, McQueen-Mason S, Smart C, Fleming A:
 Local expression of expansin induces the entire process of
leaf development and modifies leaf shape. Proc Natl Acad Sci
USA 2001, 98:11812-11817.
The induction of expansin transgene expression on the flanks of the
vegetative meristem induces the formation of leaf primordia that develop
into phenotypically normal leaves.
21. Reidy B, McQueen-Mason S, Nosberger J, Fleming A: Differential
 expression of alpha- and beta-expansin genes in the
elongating leaf of Festuca pratensis. Plant Mol Biol 2001,
46:491-504.
Expansin activity and expression is low in rapidly growing leaves of the
grass species Festuca pratensis but increases during the cessation of
growth.
22. Lee Y, Kende H: Expression of beta-expansins is correlated with
internodal elongation in deepwater rice. Plant Physiol 2001,
127:645-654.
23. Vriezen WH, De Graaf B, Mariani C, Voesenek LACJ:
Submergence induces expansin gene expression in flooding-
tolerant Rumex palustris and not in flooding-intolerant R.
acetosa. Planta 2000, 210:956-963.
24. Wu YJ, Cosgrove DJ: Adaptation of roots to low water potentials
by changes in cell wall extensibility and cell wall proteins.
J Exp Bot 2000, 51:1543-1553.
25. Wu YJ, Thorne ET, Sharp RE, Cosgrove DJ: Modification of
expansin transcript levels in the maize primary root at low
water potentials. Plant Physiol 2001, 126:1471-1479.
26. Ruan YL, Llewellyn DJ, Furbank RT: The control of single-
celled cotton fiber elongation by developmentally reversible
gating of plasmodesmata and coordinated expression of
sucrose and KR transporters and expansin. Plant Cell 2001,
13:47-60.
Current Opinion in Plant Biology 2003, 6:603–610
610 Cell biology
27. Harmer SE, Orford SJ, Timmis JN: Characterisation of six alpha-
expansin genes in Gossypium hirsutum (upland cotton).
Mol Gen Genomics 2002, 268:1-9.
28. Fleming AJ, McQueen-Mason S, Mandel T, Kuhlemeier C:
Induction of leaf primordia by the cell wall protein expansin.
Science 1997, 276:1415-1418.
29. Baluska F, Salaj J, Mathur J, Braun M, Jasper F, Samaj J, Chua NH,
Barlow PW, Volkmann D: Root hair formation: F-actin-
dependent tip growth is initiated by local assembly of profiling-
supported F-actin meshworks accumulated within expansin-
enriched bulges. Dev Biol 2000, 227:618-632.
30. Cho HT, Cosgrove DJ: Regulation of root hair initiation and
 expansin gene expression in Arabidopsis. Plant Cell 2002,
14:3237-3253.
The authors demonstrate that expansins are expressed at the sites of
root hair initiation in Arabidopsis using promoter analyses of two expansin
genes involving both b-glucuronidase (GUS) and green fluorescent pro-
tein (GFP) reporter constructs.
31. Cho HT, Cosgrove DJ: Altered expression of expansin
modulates leaf growth and pedicel abscission in Arabidopsis
thaliana. Proc Natl Acad Sci USA 2000, 97:9783-9788.
32. Lee DK, Ahn JH, Song SK, Choi YD, Lee JS: Expression of an
 expansin gene is correlated with root elongation in soybean.
Plant Physiol 2003, 131:985-997.
The overexpression of an expansin from soybean increases growth in
transgenic tobacco.
33. Rochange SF, Wenzel CL, McQueen-Mason SJ: Impaired growth
in transgenic plants over-expressing an expansin isoform.
Plant Mol Biol 2001, 46:581-589.
34. Cosgrove DJ, Li LC, Cho HT, Hoffmann-Benning S, Moore RC,
 Blecker D: The growing world of expansins. Plant Cell Physiol
2002, 43:1436-1444.
A recent review on expansins that contains novel data on the effects of
ectopic expansin application on root hairs and hypocotyls.
35. Schipper O, Schaefer D, Reski R, Fleming A: Expansins in the
 bryophyte Physcomitrella patens. Plant Mol Biol 2002, 50:789-802.
Knockout mutations of expansins in a moss species do not have an
obvious phenotype.
36. Schopfer P, Liszkay A, Bechtold M, Frahry G, Wagner A: Evidence
 that hydroxyl radicals mediate auxin-induced extension
growth. Planta 2002, 214:821-828.
ROS are shown to be involved in auxin-induced wall extension and
growth in the coleoptiles of maize seedlings. Growth is inhibited by both
scavengers of superoxide and hydroxyl radicals and by inhibitors of
ROS production.
37. Rodriguez AA, Grunberg KA, Taleisnik EL: Reactive oxygen
 species in the elongation zone of maize leaves are necessary
for leaf extension. Plant Physiol 2002, 129:1627-1632.
Current Opinion in Plant Biology 2003, 6:603–610
High levels of ROS were detected in the expansion zone of maize leaf
blades, suggesting a potential role for ROS in growth. The authors also
demonstrate that the elongation of segments from this expansion zone
was blocked in the presence of inhibitors of ROS production.
38. Fry SC, Dumville JC, Miller JG: Fingerprinting of
polysaccharides attacked by hydroxyl radicals in vitro and
in the cell walls of ripening pear fruit. Biochem J 2001,
357:729-737.
39. Pezzotti M, Feron R, Mariani C: Pollination modulates expression
 of the PPAL gene, a pistil-specific beta-expansin.
Plant Mol Biol 2002, 49:187-197.
A pistil-specific b-expansin is expressed at high concentrations at the
stigmatic surface in tobacco plants, suggesting that it has a role in
facilitating pollen tube penetration.
40. Rose JKC, Lee HH, Bennett AB: Expression of a divergent
expansin gene is fruit-specific and ripening-regulated.
Proc Natl Acad Sci USA 1997, 94:5955-5960.
41. Brummell DA, Harpster MH, Civello PM, Palys JM, Bennett AB,
Dunsmuir P: Modification of expansin protein abundance in
tomato fruit alters softening and cell wall polymer metabolism
during ripening. Plant Cell 1999, 11:2203-2216.
42. Rose JKC, Cosgrove DJ, Albersheim P, Darvill AG, Bennett AB:
Detection of expansin proteins and activity during tomato fruit
ontogeny. Plant Physiol 2000, 123:1583-1592.
43. Hiwasa K, Rose JKC, Nakano R, Inaba A, Kubo Y: Differential
expression of seven a-expansin genes during growth and
ripening of pear fruit. Physiol Plant 2003, 117:564-572.
44. Mbeguie-A-Mbeguie D, Gouble B, Gomez RM, Audergon JM,
Albagnac G, Fils-Lycaon B: Two expansin cDNAs from
Prunus armeniaca expressed during fruit ripening are
differently regulated by ethylene. Plant Physiol Biol 2002,
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45. Im KH, Cosgrove DT, Jones AM: Subcellular localization of
expansin mRNA in xylem cells. Plant Physiol 2000,
123:463-470.
46. Milioni D, Sado PE, Stacey NJ, Domingo C, Roberts K,
McCann MC: Differential expression of cell-wall-related
genes during the formation of tracheary elements in
the Zinnia mesophyll cell system. Plant Mol Biol 2001,
47:221-238.
47. Chen F, Bradford KJ: Expression of an expansin is associated
with endosperm weakening during tomato seed germination.
Plant Physiol 2000, 124:1265-1274.
48. Rose JKC, Bennett AB: Cooperative disassembly of the
cellulose-xyloglucan network of plant cell walls: parallels
between cell expansion and fruit ripening. Trends Plant Sci 1999,
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