An α-expansin, VfEXPA1, is involved in regulation of stomatal movement in Vicia faba L.

The wall loosening of guard cells differs from other types of plant cells. However, the regulation of wall loosening during stomatal movement is poorly understood. VfEXPA1 is an α-expansin gene cloned from Vicia faba epidermal strips. Expression of VfEXPA1 is regulated by darkness and submergence, and is not affected by light and abscisic acid (ABA). In situ hybridization showed that VfEXPA1 is expressed primarily in the guard cells. Overexpression of VfEXPA1 in transgenic tobacco accelerated light-induced stomatal opening, and increased both transpiration and photosynthetic rates under favorable growth conditions. Our results indicate the guard cell-expressed expansin VfEXPA1 plays an important role in regulation of stomatal opening.

Plant stomatal pores are surrounded by guard cells in the epidermis of plant stems and leaves. Stomata are essential for oxygen-carbon dioxide exchange and release of water vapor [1]. The sensitivity of stomata is vital in the regulation of transpiration and photosynthesis. The uptake of water via osmosis is the result of K + and Cl  uptake, H + extrusion and the production of organic solutes. This process leads to guard cell swelling and subsequent stomatal opening [2].
Compared to other epidermal cells or mesophyll cells, guard cell is a highly specialized cell type. In Vicia faba L., turgor pressure within guard cells increased by as much as 5.0 MPa [3], suggesting that the guard cell wall is strong enough to support 10 times the pressure of most other plant cell types. The shape and volume of guard cells during opening and closing can be dramatically altered in a matter of minutes. Guard cells are reported to undergo a change in surface area of up to 40% [3,4]. Two opposing observations have been made regarding plant cell extension. It is widely accepted that internal turgor pressure drives cell extension; however, the cell wall restricts extension. Therefore, it is intriguing that such a rigid cell wall can undergo rapid and reversible adjustment in guard cells. The role of cell walls in the regulation of stomatal movement is poorly understood, but it is likely that a unique regulatory mechanism to complete a loosen-regeneration during a stomatal movement cycle in less than a few hours should exist. Several factors might accelerate primary wall loosening, but only the wall weaken by expansins does not gradually and progressively [5].
Expansins were identified as cell wall-loosening proteins that mediate pH-dependent plant cell wall extension and cell growth [6,7]. However, no enzymatic activity has been documented for expansin genes. A superfamily of expansin genes has been divided into four families, which consist of -expansin (EXPA), -expansin (EXPB), expansin-like A (EXLA) and expansin-like B (EXLB) subfamilies [8]. Among these, only the -expansin and -expansin families reportedly have cell wall-loosening properties [6,9]. Evidence suggests the known expansins disrupt noncovalent bonds between cellulose microfibrils and matrix glucans that adhere to microfibrils [10,11]. Therefore, expansin might be involved in adjusting guard cell wall loosening to meet the requirement of reversible and rapid movement of stomata in an energy-saving and nondestructive manner. A large number of expansins are involved in the growth of a specific cell type [12][13][14], while others are implicated in plant developmental processes such as fruit softening [15,16], abscission [17], and pollination [18]. Research on Arabidopsis indicates the expansin gene AtEXPA1 might be guard cell-specific [19]. Recently, our results indicated AtEXPA1 might be involved in the regulation of stomatal sensitivity [20]. It is intriguing whether the function of guard cell-expressed expansin is specific to Arabidopsis or is common to other plant species. In this study, Vicia faba, a model system for stomatal research, was chosen. An -expansin, VfEXPA1, was cloned from leaf epidermal strips. Our results on the expression pattern and functional analysis of VfEXPA1 indicate VfEXPA1 may play an important role in regulation of stomatal opening.

Plant materials and growth conditions
Vicia faba seeds were sterilized in 0.1% HgCl 2 for 10 min, soaked in water for 24 h, and incubated at 25°C for 2-3 d. Following germination, when the length of the root reached 0.5 cm, the seedlings were planted in a greenhouse under the following growth conditions: a 12 h/12 h (light/dark) cycle, illumination intensity of 200 μmol m 2 s 1 , day/night temperatures of 22°C/17°C, and 70% relative humidity. Epidermal strips were removed from mature leaves for the extraction of RNA and protein.
Tobacco (Nicotiana tabacum L.) plants were grown in 3 L pots with ready-mixed soil. During transformation, tobacco plants were grown in 250 mL flasks that contained 100 mL agar medium. Growth conditions were 25°C/22°C (night/day) under a 12 h photoperiod with light intensity of 200 mol m 2 s 1 .

Gene cloning
Following an analysis of known EXPA sequences, highly conserved sequence regions were selected to design primers: 5′-GTGATGCCTCTGGCACAA-3′ and 5′-GGAGGATTA-CACCAACCC-3′. Abaxial epidermal strips of mature Vicia leaves were harvested for reverse transcription. The firststrand cDNA was reverse transcribed from total RNA using the anchor primer T18GC. The resulting RT-PCR band was cut and reclaimed, then cloned into the pGEM-T vector (Promega, Madison, WI, USA) and sequenced.
The full-length cDNA was constructed in a two-step procedure using the SMART TM RACE cDNA Amplification Kit (Clontech, Palo Alto, CA, USA). Following the specified protocol, 5′-RACE clones were generated. 5′-RACE-Ready cDNA was reverse transcribed from total RNA using a 5′-CDS primer and SMART II Oligonucleotide. PCR was then executed employing the Universal Primer A Mix (UPM; Clontech) and 5GSP1 (5′-ATTGAATGAACATCA-CCAGCACC-3′, designed according to the EST of VfEXPA1). The first PCR product was used as a template for nested PCR amplification, which was carried out using the Nested Universal Primer A (NUP; Clontech) and 5GSP2 (5′-GCA-CCACCTTGGATCATCGTTGCATC-3′, designed according to the EST of VfEXPA1). The amplified fragment was cloned into the pGEM-T vector and sequenced. In the second step, 3′-RACE-Ready cDNA was constructed using a 3′-CDS primer and PCR amplification was carried out using UPM and 3GSP1 (5′-CTTCTGGCACAATGGGTGGGGC-ATG-3′, designed according to the EST of VfEXPA1). Nested PCR amplification was performed with NUP and 3GSP2 (5′-TCCATCAAAGGCTCAAGAACTTCATGGC-AAC-3′). All gene-specific primers were designed according to the EST of VfEXPA1. The amplified product was cloned into pGEM-T and sequenced. Finally, the full-length cDNA was constructed.

Plasmid construction and plant transformation
The entire sequence of VfEXPA1 was cloned by RT-PCR with primers 5′-CCTTCTAGAATGACTCAAAAGCTTA-GAATG-3′ and 5′-AATGAGCTCCTAAAATTGAGCTCC-TTGAAAG-3′. The PCR fragment was digested with XbaI and SacI and inserted into the binary vector pBI121 [21] under the control of the Cauliflower mosaic virus 35S promoter. The construct was confirmed by DNA sequencing and introduced into Agrobacterium tumefaciens strain LB4404 for tobacco plant transformation. Kanamycin-resistant seedlings were transferred to soil, and examined by genomic DNA PCR and Western blot analysis. For selection of T 2 transgenic plants, seeds were germinated on agar medium containing 50 g/mL kanamycin.

Measurement of stomatal aperture and density
Epidermal strips were removed carefully from the abaxial leaf surface of tobacco or Vicia and cut into 5 mm × 10 mm pieces. The strips were brushed to remove the mesophyll cells and destroy the epidermal cells. The efficacy of mesophyll cell removal and epidermal cell destruction was tested using Neutral Red staining. The staining showed only guard cells remained viable in the epidermal strip. Then, the strips were floated briefly in 10 mmol/L MES (pH adjusted to 5.8 with Tris base) with 50 mmol/L KCl and 100 mol/L CaCl 2 , and placed in the dark until the stomata were fully closed. The pieces were floated in the same buffer and treated with light (200 mol m 2 s 1 ) from a halogen cold source to induce stomatal opening. Five fields of view were randomly selected and 10 stomatal apertures were measured in each field. Each experiment was repeated three times and the average value ± SE was calculated. Stomatal density was determined following the standard protocol [22] by microscopic examination of nail-polished imprints from leaf surfaces.

Measurement of transpiration and photosynthesis
Both transpiration and photosynthetic rates (CO 2 uptake) were measured with a CI-510 hand-held photosynthesis system (CID, Camas, WA, USA). Measurements were recorded in the greenhouse between 9:00 and 10:00 (~180 mol m 2 s 1 ). All experiments were repeated three times.

In situ hybridization and immunohistochemical localization
Fixation, paraffin embedding, sectioning and hybridization were performed as described by Cho and Kende [23], except we used 12 m sections. Probe hybridization was detected with antidigoxigenin antibodies conjugated to alkaline phosphatase, and visualized by color development according to the manufacturer's instructions (Roche Diagnostics, Mannheim, Germany). Gene-specific 3′-untranslated regions were amplified with the primers 5′-TCCATCAAA-GGCTCAAGAACTTCATGGCAACC-3′ and 5′-CAATCA-TACTAATATCAATTGAAAAGA-3′ from Vicia faba cDNA and cloned into pGEM-T. Sense and antisense VfEXPA1specific probes were generated by in vitro transcription using a digoxigenin labeling kit (Roche Diagnostics). Probes were hydrolyzed to 100 nt by alkaline hydrolysis prior to hybridization. Sense or antisense probe hybridization was performed three times.
For immunochemical localization, tissues were prepared as described above for in situ hybridization and blocked for 2 h at room temperature in TBST (10 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.05% (v/v) Tween-20, pH 7.5) with 1% bovine serum albumin (BSA). Tissues were washed five times for 5 min each, and incubated for 2-3 h at 37°C with rabbit polyclonal antiserum against AtEXPA1 (1 : 200 dilution) [20]. After three washes in TBST, sections were incubated with anti-rabbit immunoglobulin G alkaline phosphatase conjugate (Sigma-Aldrich, St. Louis, MO, USA) for 2 h and washed three times in TBST. The color reaction was carried out in the dark with NBT/BCIP.

Western blot analysis
Western blot analysis was achieved by protein transfer from polyacrylamide gels to a polyvinylidene difluoride filter (Millipore, Bedford, MA, USA) using a gel blotter (Bio-Rad Laboratories, Richmond, CA, USA). The transfer was in 0.01 mol/L 3-cyclohexylamino-propanesulfonic acid (CAPS) and 10% (w/v) methanol at 100 V for 1 h at 4°C. After transfer, the PVDF membrane was blocked with 3% BSA and incubated with the primary polyclonal antibody against AtEXPA1 (1 : 200) for 2 h at room temperature. Cross-reaction with the antibody was revealed using an alkaline phosphatase conjugated with a secondary anti-rabbit antibody.

Northern blot analysis
For Northern blot, 20-d-old seedlings were treated with either abscisic acid (100 mol/L), darkness, light (300 mol m 2 s 1 , illumination intensity of 100 mol m 2 s 1 ), or submergence for 2 h. Total roots, stems, and mature leaves were collected from 4-week-old seedlings, and RNA isolation and gel blot analyses were performed as described by Cho and Kende [24].

An abundance of expansin proteins were located in guard cell walls
Mature leaves of plants undergo few cell wall extension events. Thus, the function of expansins in mature leaves is puzzling. To elucidate the role of expansins in plants, immunohistochemistry staining was performed on mature leaves of Vicia faba using an antibody raised against the Arabidopsis expansin AtEXPA1 [20]. Most signals were detected in guard cells. The cells surrounding the vascular bundles also generated weak signals, but the signals were markedly weaker than those of guard cells (Figure 1(a) and  (b)). Immunoassay of single stoma showed the proteins recognized by the antibody were mainly located on cell walls. However, the distribution of the immunostain signal was not uniform. Stronger signals were detected in the dorsal guard cell walls than in the ventral walls (Figure 1(c) and (d)).

Cloning and phylogenetic analysis of VfEXPA1 from epidermal cells of Vicia faba mature leaves
To clone guard cell-expressed expansins, a highly conserved EXPA region, which encodes approximately 90 amino acids in the N-terminal region of mature proteins, was used to design RT-PCR primers. RT-PCR amplification was performed using total RNA extracted from epidermal strips of mature leaves. A 536-bp fragment was obtained and sequenced. The EST sequence of Vicia faba was submitted to GenBank (accession number AF464953). The full-length cDNA was then cloned using 5′-and 3′-RACE and subsequently named VfEXPA1 (GenBank accession number EF190969). The VfEXPA1 open reading frame was 765 bp and encoded an expansin with a molecular weight of approximately 27.5 kD. The cDNA contained a 45-bp 5′-untranslated region and a 318 bp 3′-untranslated region. The presence of a signal peptide was predicted by the SignalP program (http://www.cbs.dtu.dk/services/), and the most likely cleavage site was between amino acids 27 and 28. The mature protein was approximately 25 kD after excision of the signal peptide. A phylogenetic tree was constructed from the deduced amino acid sequence of VfEXPA1 and all -expansins of Arabidopsis, which showed VfEXPA1 belongs to the -subfamily and AtEXPA8 has the highest sequence similarity (75%) to VfEXPA1. Alignment of the deduced amino acid sequence of VfEXPA1 with typical known α-expansins also indicated VfEXPA1 was an α-subfamily expansin ( Figure S1). VfEXPA1 showed a characteristic α-insertion in front of a conserved HFD (His-Phe-Asp) motif with four highly conserved 'GWCN' residues at its 3′ end [25] (Figure S1). In addition, two other conserved domains of expansins, the cellulose-binding-like domain (from amino acid 172 to 251) and the family-45 endoglucanaselike domain (from amino acid 50 to 162), were revealed in its structure.

VfEXPA1 expression patterns
To investigate the tissue expression pattern of VfEXPA1, 20-d-old green seedlings of Vicia were dissected into roots, stems, and mature leaves for Northern blot analysis. VfEXPA1 was expressed primarily in stems and leaves, especially at a higher level in leaves, and expression was not detected in the roots (Figure 2(a)). The expression of VfEXPA1 was examined under treatment with light, dark, ABA, or sub- mergence in water for 2 h. Northern blot analysis revealed VfEXPA1 was slightly upregulated by darkness and submergence, but was not affected by light and ABA ( Figure  2(b)).
In situ hybridization in mature leaves with a gene-specific probe was used to analyze the cellular location of VfEXPA1. Transcripts of VfEXPA1 accumulated in the guard cells. Weaker signals were observed in vascular bundles and some mesophyll cells (Figure 2(c), (d)).

Overexpression of VfEXPA1 accelerated stomatal opening
To examine the function of VfEXPA1, the full length sequence was fused under the control of the CaMV 35S promoter and transformed into tobacco. Six T 2 lines were selected from 60 T 2 kanamycin-resistant lines for further analysis. Proteins were isolated from epidermal strips of mature leaves of tobacco plants to perform Western blot analysis. A band with a molecular weight of 25 kD was detected in all lines, including the control plants, but was more abundant in the six transgenic lines (Figure 3). This result indicated VfEXPA1 was overexpressed in the transgenic plants. Transgenic lines 2 and 34 were selected and designated OE1 and OE2 for further phenotype and physiological studies.
To verify the involvement of VfEXPA1 in the regulation of stomatal movement, the rate of stomatal opening was investigated in transgenic and wild-type plants. Abaxial epidermal strips of transgenic and wild plants were pretreated in the dark until stomata closure, and then subjected to cold light. VfEXPA1 overexpression lines showed a much higher rate of light-induced stomatal opening (Figure 4(a)), especially in the early stage. However, the final stomatal aperture size was not altered by VfEXPA1 overexpression.

Overexpression of VfEXPA1 enhanced transpiration and photosynthetic rates
Since VfEXPA1 accelerates stomatal movement in epidermal strips in vitro, we wondered if this phenomenon has any physiological effects and could be replicated in vivo. Therefore, we measured both transpiration and photosynthetic rates of transgenic and wild-type plants after 2 h exposure to light following pretreatment in the dark. When growing under favorable conditions, the transgenic plants showed significantly higher transpiration and photosynthetic rates than the control plants (Figure 4(b) and (c)). Because the stomatal number and sensitivity both determine the transpiration and photosynthetic rates, stomata density and stomatal aperture size of mature leaves were measured. Stomata density in transgenic lines was identical to that of the wild type (Table  1). However, the stomata of transgenic plants had larger apertures (Figure 4(d)), which indicated the increased stomatal sensitivity in VfEXPA1 overexpression lines might induce higher transpiration and photosynthetic rates.

Discussion
Expansins usually function in growing regions of plants [26]. Developing leaves possess at least one expansin responsible for acid-growth properties [27,28]. Previous research found that most leaf expansins were expressed at leaf formation or in an elongation zone [14,28] and these specific expansins could be responsible for enhanced cell wall extensibility. However, several expansins are expressed in Figure 4 Effects of VfEXPA1 overexpression on photosynthesis, transpiration rate and stomatal movement. (a) Epidermal strips from transgenic and wild-type plants were pretreated with darkness until all stomata closed, and then treated with cool light (200 mol m 2 s 1 ) to induce stomatal opening. Stomatal apertures were measured every 60 min. Error bars represent the standard error of the mean of at least 150 stomata per plant. Transpiration rate (b) and photosynthesis rate (c) of wild-type (white columns) and transgenic VfEXPA1 (gray and black columns) lines in the greenhouse were measured at 11:00. Illumination intensity was 180 mol m 2 s 1 . Concurrently, stomatal aperture (d) was measured on leaves at 9:00. White columns represent the wild type, gray and black columns represent transgenic plants. Error bars represent the standard error of the mean of at least five plants per line. mature leaves [29,30]. Our data also demonstrated the presence of expansin in mature leaves of Vicia faba. Moreover, immunoblot analysis showed expansins are present also in the mature leaves of ailanthus, peach, dayflower, and wheat ( Figure S2), which suggested expression in mature leaves is a common feature of certain types of expansin. Thus, it is interesting to elucidate the specific cell types in which expansins are expressed. In the present study, an immunoassay was performed to investigate the cellular location of expansin in mature leaves. Although the specificity of the anti-AtEXPA1 antibody has not been well investigated yet, the single band in the Western blot analysis of mature-leaf proteins of 5 plant species indicated at least it could specifically recognize a group of EXPA proteins with a 25 kD molecular weight. The immunoassay showed most expansins present in mature leaves were located in guard cells. In addition, we cloned an expansin gene, VfEXPA1, from epidermal strips from mature leaves of V. faba. Because guard cells are a major component of leaf epidermal strips, these results indicate VfEXPA1 is a guard cell-expressed protein. Furthermore, in situ hybridization analysis proved VfEXPA1 accumulated in guard cells. Together with previous findings of expansin specifically expressed in guard cells in Arabidopsis [19,20], these results imply in mature leaves expansin accumulates in guard cells.
The matrix of cellulose crosslinked by glycans, such as xyloglucan and glucuronoarabinoxylan, is believed to be crucial for cell wall extensibility [31]. Congruent with a previous report [32], we found that cellulose hydrolytic enzymes, either cellulase or -glucanase, have no detectable effect on stomatal movement (data not shown). However, modification of pectins might have an effect on stomatal movement. Overexpression of a polygalacturonase, which cleaves pectins, disable stomatal closure [33]. A pectindegrading enzyme, arabinanase, can block guard cell movement while endopolygalacturonase (endoPGase), in concert with pectin methylesterase (PME), increased stomatal aperture [32,34]. However, it is important to note that the hydrolysis of pectin is slow and energy-dependent and, as a consequence of pectinase activity, the guard cells and neighboring epidermal cells are often separated by structural damage to the cell wall [33]. Therefore, enzyme-related pectin hydrolysis is unlikely to be the mechanism of the rapid and reversible cell wall loosening in guard cells.
Expansin is a potential candidate for a regulator of sto-matal opening sensitivity, because it is known to catalyze the disruption of hydrogen bonds between cellulose and the load-bearing crosslinking glycans (such as xyloglucan in dicots) [10,35]. Expansins also induce cell expansion both in vivo and in vitro [6,36]. Thus expansins show characteristics for regulation of the rapid and non-destructive cell wall expansion required in stomatal movement. Our data indicate expansins in mature leaves are expressed predominantly in guard cells. Since cell growth has almost been completed in mature leaves, the accumulation of expansins in guard cells of mature leaves indicates expansin might have potential functions in stomatal movement. Furthermore, stomatal sensitivity is increased by overexpression of the guard cell-expressed expansin gene VfEXPA1. Although VfEXPA1 in leaf tissue other than guard cells in overexpression lines might affect stomatal movement, mesophyll cells and epidermal cells were destroyed by careful brushing of epidermal strips used to test light-induced stomatal opening in vitro. In this case, the difference in stomatal sensitivity between VfEXPA1 overexpression lines and the wild type is dependent on VfEXPA1 expression in guard cells alone. Because stomatal sensitivity increased in both in vitro and in vivo tests on VfEXPA1 overexpression lines, we believe VfEXPA1 could regulate stomatal movement. Immunohistochemical localization showed the expansin level increased in the dorsal wall compared with that of ventral walls in guard cells. It is interesting to note that in sugar beet (Beta vulgaris L.) leaves, the xyloglucan epitope recognized by the CCRC-M1 antibody was more abundant in the ventral wall [37]. Xyloglucan-crosslinked cellulose can decrease the rigidity and increase the extensibility of cell walls [38]. Expansin-facilitated relaxation of the cellulose-xyloglucan complex is typical in turgor-mediated cell expansion [39]. During stomatal opening, the dorsal wall extends more than the ventral wall, which borders the stomatal pore. This could be the reason for higher accumulation of expansins and a lower quantity of xyloglucan in the dorsal wall. Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. Figure S1 Amino acid sequence comparison of VfEXPA1 with other expansins. Figure S2 Expansins are present in the mature leaves of diverse plant species.

Supporting Information
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