- Research article
- Open Access
The extracellular EXO protein mediates cell expansion in Arabidopsis leaves
© Schröder et al; licensee BioMed Central Ltd. 2009
- Received: 09 December 2008
- Accepted: 13 February 2009
- Published: 13 February 2009
The EXO (EXORDIUM) gene was identified as a potential mediator of brassinosteroid (BR)-promoted growth. It is part of a gene family with eight members in Arabidopsis. EXO gene expression is under control of BR, and EXO overexpression promotes shoot and root growth. In this study, the consequences of loss of EXO function are described.
The exo loss of function mutant showed diminished leaf and root growth and reduced biomass production. Light and scanning electron microscopy analyses revealed that impaired leaf growth is due to reduced cell expansion. Epidermis, palisade, and spongy parenchyma cells were smaller in comparison to the wild-type. The exo mutant showed reduced brassinolide-induced cotyledon and hypocotyl growth. In contrast, exo roots were significantly more sensitive to the inhibitory effect of synthetic brassinolide. Apart from reduced growth, exo did not show severe morphological abnormalities. Gene expression analyses of leaf material identified genes that showed robust EXO-dependent expression. Growth-related genes such as WAK1, EXP5, and KCS1, and genes involved in primary and secondary metabolism showed weaker expression in exo than in wild-type plants. However, the vast majority of BR-regulated genes were normally expressed in exo. HA- and GFP-tagged EXO proteins were targeted to the apoplast.
The EXO gene is essential for cell expansion in leaves. Gene expression patterns and growth assays suggest that EXO mediates BR-induced leaf growth. However, EXO does not control BR-levels or BR-sensitivity in the shoot. EXO presumably is involved in a signalling process which coordinates BR-responses with environmental or developmental signals. The hypersensitivity of exo roots to BR suggests that EXO plays a diverse role in the control of BR responses in the root.
- Expression Profile Experiment
- Spongy Parenchyma Cell
- EXOga Construct
- Altered Transcript Level
- Det2 Mutant
Multiple pathways control growth and development. BRs received particular attention when BR-deficient and BR-insensitive mutants were identified . Loss of BR action results in extreme dwarfism. Leaves, internodes, and roots of BR-mutants show reduced size and growth of reproductive organs can be impaired . The growth-promoting effect of BR is largely based on the promotion of cell expansion, though BR may also enhance cell proliferation in leaves . The most prominent direct BR-effect is the modification of gene expression patterns. In fact, BR action requires genomic events, and numerous approaches have identified BR-regulated genes [4, 5]. The identified genes and physiological studies suggest that BR controls cell wall modifications, organisation of microtubules and cellulose microfibrils, aquaporin activity, and photosynthesis [2, 6]. BR-regulated genes also include putative signalling components, among these the EXO protein (At4g08950) [7, 8]. EXO gene expression is a strong indicator of BR-responses in vegetative tissues. BR-deficient mutants showed weak EXO expression, whereas BR application to the wild-type resulted in elevated EXO transcript levels . The BR-hypersensitive bes1-D mutant exhibited constitutive EXO expression . EXO overexpression resulted in stronger shoot and root growth in wild-type plants . However, overexpression of EXO in the BR-deficient dwf1-6 mutant did not normalize dwarfism . EXO action apparently requires the presence of further BR-dependent factors.
The transgenic line AtEM201 contains a T-DNA insertion in the EXO promoter. The EXO mRNA level was strongly reduced in these plants. However, the plants did not show an abnormal phenotype . Likewise, inhibition of EXO expression by means of RNA interference did not result in an abnormal phenotype . The lack of phenotypic changes in either approach could be due to genetic redundancy, or the exo mutant phenotype could become evident only under certain growth conditions. Alternatively, the remaining EXO mRNA in the AtEM210 and RNAi lines could be sufficient to maintain proper protein levels [7, 8].
Here we report on the characterization of an exo knock-out mutant that shows dwarfism. We show that diminished growth of exo is due to reduced cell expansion rather than impaired cell proliferation. EXO is an extracellular protein that modifies BR-induced growth responses. Expression profiling experiments identified EXO-regulated genes. The potential molecular mode of action of EXO is discussed.
The EXO/EXL protein family
Eight homologous proteins including EXO form a protein family in Arabidopsis (see Additional file 1, Figure S1). Structurally conserved proteins were identified in dicots such as tobacco , potato , wine grape and black cottonwood, and monocots such as rice and Sorghum bicolor, the conifer Picea sitchensis, and the moss Physcomitrella patens. The genome of the soil bacterium Solibacter usitatus also encodes a putative PHI1/EXO-like protein of 317 amino acids (see Additional file 2). No further homologs were identified in bacteria, archaea, fungi, animals, and protists. A phylogenetic tree is shown in Figure S2. The conserved region comprises almost the complete primary structure of about 300 amino acids (Interpro entry IPR006766; PFAM entry PF04674).
EXO and EXLexpression patterns
The EXO protein is required for shoot and root growth
Growth parameters of the exo mutant.
Soil 28 d
Soil 33 d
Soil 35 d
Fresh weight [mg]
Dry weight [mg]
0.5 × MS 10 d
0.5 × MS 15 d
0.5 × MS 25 d
Fresh weight [mg]
Root length of the exo mutant.
Root length [cm]
Root length [cm]
Root length [cm]
Another T-DNA insertion line (SALK 098601) was supposed to carry a T-DNA insertion in the EXO coding sequence. In fact, PCR using the LBb1 primer (for left border of T-DNA insertion, see http://signal.salk.edu/tdnaprimers.2.html) and an antisense primer for the 3'UTR of EXO resulted in a 0.7 kb fragment and confirmed an insertion in the EXO coding sequence. However, no homozygous mutant plants were identified though all plants were resistant to kanamycin. Therefore, the mutant was back-crossed with the Col-0 wild-type and the F2 generation was screened for homozygous plants. PCR analysis of 96 plants indicated that none carried an insertion in the EXO gene in both chromosomes. This observation may suggest that a second insertion close to the EXO gene impaired development of homozygous plants.
Loss of EXO results in reduced cell size
Leaf thickness, palisade and spongy parenchyma cell areas.
Leaf thickness [μm]
Palisade parenchyma cell area [μm2]
Spongy parenchyma cell area [μm2]
Dwarf mutants are frequently characterized by both smaller cells and a decrease in the total number of palisade cells [14, 15]. The total number of palisade cells of the 5th or 6th rosette leaf was estimated. The exo mutant showed a tendency to fewer cells in comparison to the wild-type, but differences were not consistent in independent experiments (data not shown). Thus, EXO has no major effect on leaf cell number of soil-grown plants.
EXO is an apoplastic protein
EXO modifies BR responses
The BR-response of roots was also tested. Inhibition of root growth by BL was significantly increased in exo compared to the wild-type (Figure 5C and Additional file 1). Introduction of the 35S::EXOga construct normalized exo root growth. Thus, loss of EXO results in BR-hypersensitive roots. Since this finding does not hold true for shoot organs, which are less BR-responsive in exo plants, the EXO protein may play a diverse and tissue-specific role in the control of BR responses in the root.
EXO-dependent gene expression
Genes with altered transcript levels in the exo mutant.
Weaker expression in exo
At1g21250, WALL-ASSOCIATED KINASE 1 (WAK1)
Signalling, receptor kinase
At5g40760, cytosolic glucose-6-phosphate dehydrogenase 6
At2g39800, At3g55610, delta1-pyrroline-5-carboxylate synthase
At1g54100, aldehyde dehydrogenase
Oxidation of aldehydes
At1g53310, PEP carboxylase 1
At4g02480, AAA-type ATPase
Energy-dependent unfolding of macromolecules
At5g39320, UDP-glucose 6-dehydrogenase 2
Cell wall precursor synthesis
At4g37870, PEP carboxykinase 1
At1g17050, solanesyl diphosphate synthase 2
Stronger expression in exo
At1g23410, 40S ribosomal protein S27A
Protein degradation, ubiquitin cycle
At4g15680, monothiol glutaredoxin
Real-time RT-PCR analysis of EXP5 and KCS1 gene expression.
38.4 ± 0.02
37.0 ± 0.02
39.0 ± 0.02
37.9 ± 0.01
37.2 ± 0.19
36.2 ± 0.01
37.7 ± 0.43
36.4 ± 0.01
35.1 ± 0.12
34.4 ± 0.17
32.0 ± 0.12
31.5 ± 0.06
However, the vast majority of known BR-regulated genes (including genes involved in BR-biosynthesis, BR-catabolism, and BR-signalling) did not show significantly altered transcript levels in exo. In line with this finding, we previously showed that EXO overexpression does not result in altered transcript levels of BR-regulated genes such as DWF4 and CPD . Thus, EXO is not a key control element for BR-responsive gene expression in the shoot.
Structure and subcellular localisation
EXO:GFP and EXO:HA fusion proteins were detected in the apoplast (Figure 4). Other extracellular proteins such as arabinogalactan-proteins (AGPs) are attached to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. However, analysis of the EXO primary structure did not reveal a GPI modification site, and plasmolysis experiments showed that the EXO protein was not associated with the plasma membrane (Figure 4). Cell wall proteins are embedded in a polysaccharide matrix and it can be difficult to extract them. Bayer et al.  identified the EXO, EXL1 (At1g35140), and EXL2 (At5g64260) proteins in extensively washed cell wall preparations. This finding suggests a tight association with the wall. On the other hand, Borderies et al.  recovered extracellular proteins by washing Arabidopsis cell suspension cultures with salts and chelating agents. They aimed to identify loosely bound cell wall proteins and results were critically evaluated with respect to the integrity of the plasma membrane of the cells. EXO was among the 50 identified proteins. In line with the observation of Borderies et al. , HA-tagged EXO protein could be extracted readily from 35S::EXO:HA transgenic plants using a standard method for the isolation of soluble proteins (Figure 2E). Thus, a fraction of the EXO protein is loosely bound to the cell wall, though another fraction may strongly interact with cell wall components.
EXO mediates cell expansion
The exo mutant showed reduced leaf size, root length, and biomass production. Reduced leaf size in exo is due to diminished expansion of epidermis and parenchyma cells. It was shown that EXO gene expression is BR-dependent  and under control of the BES1 transcription factor . Different experiments were performed to test the role of EXO in BR-responses. The exo mutant showed diminished cotyledon and hypocotyl elongation in response to exogenous BL (Figure 5 and Additional file 1) indicating that EXO is involved in BR-promoted cell expansion. It was shown before that EXO overexpression in the BR-deficient dwf1-6 mutant did not normalize growth . Similarly, introduction of the 35S::EXOga construct did not normalize the phenotype of the BR-deficient det2 mutant (, data not shown). Thus, EXO is necessary but not sufficient to mediate BR-promoted growth. Expression profiling experiments demonstrated that EXO controls only a subset of BR-regulated, growth-related genes such as KCS1 and EXP5. BR-deficiency and BR-insensitivity go along with changes in transcript levels of numerous genes . Since most BR-regulated genes were normally expressed in the exo mutant, it appears that EXO does not generally affect BR sensitivity and BR levels in the shoot. Our expression profiling experiments revealed further genes with altered expression levels in exo compared with the wild-type (Table 4). For example, At5g39320 and WAK1 are expressed at lower levels in the mutant. At5g39320 encodes an UDP-glucose 6-dehydrogenase that could be involved in the synthesis of cell wall precursors. WAK1 is a transmembrane protein containing a cytoplasmic Ser/Thr kinase domain and an extracellular domain which interacts with cell wall pectins . The wall-associated kinases (WAKs) are likely to be involved in signalling between the cell wall and the cytoplasm, and could play a role in development and cell expansion .
Thus, EXO is likely to act downstream of the known BR-signalling pathway in the shoot. The protein may mediate BR-induced growth via modifications of cell wall properties and metabolism. BR-hypersensitivity of exo roots suggests that EXO plays a root-specific role in the control of BR-responses. The molecular basis of this finding is unknown.
The plant extracellular proteome may comprise 2000 different proteins [21, 28]. The PHI1/EXO proteins do not show similarities to proteins with known functions, and thus may have enzymatic or signalling functions that are unknown to date. Our hypothesis is that EXO integrates cellular, metabolic, and/or environmental factors, and feeds this information into an unknown signalling pathway which controls cell wall properties and metabolic pathways. The BR-hypersensitivity of exo roots contrasts with the diminished BR-response of exo shoot organs. Further studies will address the root-specific phenotype and a potential role of EXO in the control of BR-responses in roots.
The EXO, EXL1, EXL3, and EXL5 genes showed associated expression in different plant tissues (Figure 1). The similar structure of the EXO, EXL1, EXL3, and EXL5 proteins, the associated expression in different organs, and the common control of expression by BR suggests that all four proteins may play a role in growth control. Genetic redundancy of the EXO/EXL genes could account for the relative mild phenotypic alterations of exo in comparison to BR-deficient or BR-insensitive plants. The generation and analysis of mutants in several EXO/EXL genes may address this issue.
The EXO protein represents a class of proteins that occurs widely in the plant kingdom. It is localized to the cell wall and mediates cell expansion. EXO presumably is involved in signalling processes that coordinate BR-responses with environmental or developmental signals.
Screen for mutants
The SALK_098602 line  carried a T-DNA insertion in the EXO coding sequence and was named exo. The DNA insertion site was confirmed by sequencing and is highlighted in Figure S1 (see Additional file 1). Homozygosity of T-DNA insertions was confirmed by PCR on genomic DNA using T-DNA border-specific and gene-specific primers. Impaired gene expression in the knock-out mutant was confirmed using RT-PCR (with primers spanning the respective T-DNA insertion sites) and northern-blot analyses (Figure 2C and data not shown).
Establishment of transgenic lines
The 35S::EXO overexpression construct, based on a modified pGREEN vector, was described before . A second overexpression construct was established using a Gateway-compatible vector. The EXO coding sequence was amplified using the primers EXO_GA_fw 5' CAC CCC TCT TTC ACT ATT ACA CTT TTC CT 3' and EXO_GA_rev 5' GAC CAT AGT AGA GCA AGC CGA C 3'. The PCR fragment was cloned into the pENTR/D-TOPO (Invitrogen, Karlsruhe, Germany) vector, and inserted into the pH7WG2 vector for expression under control of the 35S promoter . The resulting construct was termed 35S::EXOga and used for complementation of the exo mutant and transformation of the det2 mutant. The pENTR/D-TOPO vector carrying the EXO coding sequence was also used to establish the 35S::EXO:GFP and 35S::EXO:HA fusion constructs using the pK7FWG2  and pGWB14  vectors, respectively. All constructs were transformed into Arabidopsis plants using the floral-dip method.
Seeds for growth experiments (i.e., wild-type, exo, and 35S::EXOga in exo) were derived from plants grown in parallel in a greenhouse under identical conditions. Plants were grown in one-half concentrated Murashige and Skoog medium supplemented with 1% sucrose and solidified with 0.7% agar. After two to three days in a cold room (4°C), plants were transferred into a growth chamber with a long day light regime (16 h day, 140 μmol m-2 s-1, 22°C; 8 h night, 22°C) and grown in a randomized manner. For monitoring root growth, plants were grown on vertical plates. Alternatively, plants were established in soil (type 'GS-90 Einheitserde', Gebrüder Patzer, Germany). Seeds were allowed to germinate and to grow for two weeks in controlled growth chambers (7 days: 16 h light [140 μmol m-2 s-1], 20°C, 75% relative humidity; 8 h night, 6°C, 75% relative humidity; thereafter 7 days: 8 h light [140 μmol m-2 s-1], 20°C, 60% relative humidity; 16 h night, 16°C, 75% relative humidity). Subsequently, plants were transferred to long-day conditions in a greenhouse with artificial light (16 h light [high pressure sodium and metal halide lamps], 21°C, 50% relative humidity; 8 h night, 19°C, 50% relative humidity). All genotypes were grown in the same chamber at the same time in a randomized manner.
Gene expression analysis and protein extraction
RNA for Northern-blot analysis was isolated using the Trizol reagent (Invitrogen). Northern-blot and real-time RT-PCR analysis was performed as described . Generation of labelled cRNA and hybridisation of ATH1 oligonucleotide microarrays were performed using standard protocols in cooperation with Atlas biolabs (Berlin, Germany) as described . Profiles were normalized with RMA (Table 4), MAS5.0/GCOS (Table S2, see Additional file 1), or RMA-Express (Figure 1, Additional file 1: Figure S3, Figure S4, Table S1). Differences between wild-type and exo were tested with the LIMMA software package  using a moderated paired t-test. FDR-adjusted P-values were calculated using the approach of Benjamini & Hochberg.
Protein for Western-blot analysis was isolated using ice cold extraction buffer (50 mM Tris (pH 7.2), 100 mM NaCl, 10% glycerol, Complete Protease Inhibitor Cocktail (Roche, Grenzach, Germany)).
Leaf cross sections and microscopy
The fifth or sixth leaf of 35-day old plants was used for microscopic analysis. Leaves were embedded in 4% agarose and sectioned at 40 μm through the widest part of the blade for transverse sections using a vibratome (Leica VT 1000S, Bensheim, Germany). Leaf thickness and leaf cell area were analyzed using the cellP software (Olympus, Hamburg, Germany). At least eight leaves per genotype and ten cross sections of each leaf were measured. Cells surrounding the leaf vein were excluded. Images of sections were generated using an Olympus BX41 microscope. Subepidermal cell layers were analyzed using an Olympus AX70 microscope after bleaching of leaves with 1 M KOH for 24 hours. For scanning electron microscopy (SEM), leaf samples were fixed in 3% paraformaldehyde and 0.25% glutaraldehyde in phosphate buffer (pH 7.1) and dehydrated. A gold/palladium (80:20) coat of 2 nm was applied in a cool sputter coater SCD 050 (Bal-tec, Balzers, Liechtenstein). Images of the leaf surface were observed on a LEO 1550 (LEO, Oberkochen, Germany) microscope. GFP-fluorescence was visualized in roots of 27-day old transgenic plants grown in sterile media with a Leica TCS SP5 confocal microscope.
For immunocytochemistry leaves were fixed in 3% paraformaldehyde and 0.25% glutaraldehyde in phosphate buffer (pH 7.1). The samples were dehydrated and infiltrated in Technovit 8100 resin (Heraeus Kulzer, Wehrheim, Germany) according to the manufacturer's protocol. The leaves were sectioned at 1.2 μm using a Leica RM2255 Rotary Microtome and mounted on charged glass slides. The sections were treated with 0.1 M NH4Cl in phosphate buffered saline (PBS) for 5 min followed by a washing step in PBS for 5 min. Slides were incubated with 5% bovine serum albumin (BSA) in PBS for 30 min and incubated over night at 4°C in primary antibody (anti-HA, mouse IgG clone 12CA5, Roche) diluted in 5% BSA/PBS at a ratio of 1:60. Three washing steps with 0.1% BSA in PBS for 10 min were followed by one with 1% BSA in PBS for 10 min. Subsequently, the slides were incubated for 1 hour at RT with the secondary antibody (FITC goat anti-mouse IgG (H+L), ZYMED, San Francisco, USA) diluted in 5% BSA/PBS at a ratio of 1:100. After four washing steps with PBS for 10 min, images were generated using an Olympus BX41 microscope.
This work was supported by a grant of the Deutsche Forschungsgemeinschaft to CM (DFG, MU 1738/4-1). We thank Jürgen Hartmann and Eugenia Maximova for technical support of the microscopic analyses. We thank Martin Steup for comments on immunocytochemistry. We are grateful to Björn Usadel for assistance with statistical analysis of expression profiles. We thank Ina Talke for critical reading.
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