Targeted Modification of Homogalacturonan by ... - Plant Physiology

Targeted Modification of Homogalacturonan by Transgenic Expression of a Fungal Polygalacturonase Alters Plant Growth1 Cristina Capodicasa2, Donatella Vairo2, Olga Zabotina2, Lesley McCartney, Claudio Caprari, Benedetta Mattei, Cinzia Manfredini, Benedetto Aracri, Jacques Benen, J. Paul Knox, Giulia De Lorenzo, and Felice Cervone* Dipartimento di Biologia Vegetale e Laboratorio di Genomica Funzionale e Proteomica, Universita` di Roma La Sapienza, 00185 Rome, Italy (C.C., D.V., O.Z., C.C., B.M., C.M., B.A., G.D., F.C.); Centre for Plant Sciences, University of Leeds, Leeds, United Kingdom (L.M., J.P.K.); and Fungal Genomics Group, Laboratory for Microbiology, Agricultural University, NL–6703 HA Wageningen, The Netherlands (J.B.)

Pectins are a highly complex family of cell wall polysaccharides comprised of homogalacturonan (HGA), rhamnogalacturonan I and rhamnogalacturonan II. We have specifically modified HGA in both tobacco (Nicotiana tabacum) and Arabidopsis by expressing the endopolygalacturonase II of Aspergillus niger (AnPGII). Cell walls of transgenic tobacco plants showed a 25% reduction in GalUA content as compared with the wild type and a reduced content of deesterified HGA as detected by antibody labeling. Neutral sugars remained unchanged apart from a slight increase of Rha, Ara, and Gal. Both transgenic tobacco and Arabidopsis were dwarfed, indicating that unesterified HGA is a critical factor for plant cell growth. The dwarf phenotypes were associated with AnPGII activity as demonstrated by the observation that the mutant phenotype of tobacco was completely reverted by crossing the dwarfed plants with plants expressing PGIP2, a strong inhibitor of AnPGII. The mutant phenotype in Arabidopsis did not appear when transformation was performed with a gene encoding AnPGII inactivated by site directed mutagenesis.

Although biochemical events causing structural changes of cell walls are expected to influence plant growth and development (Pilling et al., 2000; Bouton et al., 2002), the degradation and remodeling of cell wall constituents during development appear to be of considerable complexity and far from being fully understood. For example, the remodeling of pectin, which is thought to impact upon processes such as adhesion between cells and plant growth, requires the action of several different enzyme classes (Ridley et al., 2001). The complex role of pectin in the dynamics of cell walls is further indicated by the presence of large families of these pectic enzymes in plant genomes. For example, more than 50 genes encoding putative polygalacturonases (PGs) have been identified in Arabidopsis (Arabidopsis Genome Initiative, 2000). Indications that the pectic polymers homogalacturonan (HGA), rhamnogalacturonan-I (RG-I), and rhamnogalacturonan-II (RG-II) play an important role in plant development have been obtained by analyzing several cell wall mutants, most of which have been 1

This work was supported by the European Community (grant no. QLK3–CT99–089), by a MURST-FIRB grant, and by the Armenise-Harvard Foundation and Fondazione Cenci Bolognetti. 2 These authors contributed equally to the paper. * Corresponding author; e-mail [email protected]; fax 390649912446. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.042788. 1294

obtained by mutagenesis of Arabidopsis. Among these, Arabidopsis emb30 mutants, having a mutation in a gene putatively involved in the secretory pathway, show an abnormal localization and accumulation of pectin in intercellular/interstitial spaces rather than in the corners. Cells of emb30 mutants are larger than those of the wild type and do not adhere well to each other; the seeds are impaired in the control of cell division, expansion, and polarity and do not develop as wild type (Shevell et al., 2000). The Arabidopsis quartet mutants have microspores that fail to separate during pollen development as a result of the persistence of pectin in the pollen mother cell wall (Rhee and Somerville, 1998). The Arabidopsis mur1 mutation, leading to a deficiency in fucosyl residues, affects RG-II, reducing its capacity to dimerize through the formation of boron diester cross-links, and this disrupts plant growth (O’Neill et al., 2001). The Arabidopsis quasimodo mutants, with a T-DNA insertion in a gene that encodes a putative membrane-bound glycosyltransferase, are dwarfed and show a reduced cell adhesion associated with a 25% reduction in GalUA content (Bouton et al., 2002). In the Arabidopsis parvus mutants, characterized by reduced levels of RG-I branching, growth and fertility are influenced by humidity (Lao et al., 2003). The tomato (Lycopersicon esculentum) Cnr mutant has an altered deposition of both HGA and arabinan in the fruit pericarp, affecting cell wall properties and intercellular adhesion (Thompson et al., 1999; Orfila et al., 2001).

Plant Physiology, July 2004, Vol. 135, pp. 1294–1304, www.plantphysiol.org Ó 2004 American Society of Plant Biologists

Homogalacturonan and Plant Growth

Lorenzo et al., 2001). Here, we report the use of a fungal endopolygalacturonase to explore the impact of the specific degradation of HGA on plant growth and development. The transformation of tobacco and Arabidopsis with PG resulted in altered cell wall composition and reduced the growth of both species. Our results demonstrate an important function for HGA in plant growth.

RESULTS AnPGII Is Expressed at a Low Level in Tobacco

Figure 1. Immunoblot analyses of AnPGII transgenic tobacco plants. Western-blot analysis of leaf total protein extracts (A) and intercellular fluids (B) of tobacco primary transformants numbers 5, 7, and 16 using an AnPGII-specific polyclonal antibody; AnPGII, 2 ng of purified AnPGII from A. niger. C, Western-blot analysis of leaf total protein extracts (10 mg) of 10-week-old transgenic R2 progeny plants of lines 5, 7, and 16 using the same antibody as in A.

For detailed knowledge of the role of pectic polymers in cell development, their specific manipulation by plant transformation with pectic enzymes represents a useful alternative to the characterization of mutants. Transgenic expression of pectic enzymes with a well-characterized mode of action provides a direct approach to modify specific pectin domains in planta. Galactanase, rhamnogalacturonan lyase, and arabinanase from Aspergillus aculeatus, which attack RG-I, have been used to transform potatoes (Solanum tuberosum). Plants expressing galactanase displayed no altered phenotype (Oxenboll et al., 2000), while those expressing rhamnogalacturonan lyase had more wrinkled tubers than the wild type (Oomen et al., 2002) and those expressing arabinanase suffered serious developmental disruption and produced no side shoots, flowers, stolons, or tubers (Skjot et al., 2001). Plant-derived PGs often have specialized functions and act in a tissue-specific manner (Peretto et al., 1992; Allen and Lonsdale 1993; Kalaitzis et al., 1997; Wang et al., 2000). Either up- and down-regulation in tomato of an endogenous gene encoding an endopolygalacturonase involved in fruit softening had no apparent effect on growth and development (Giovannoni et al., 1989; Smith et al., 1990). The expression of a tomato PG in tobacco (Nicotiana tabacum) resulted in no phenotype (Osteryoung et al., 1990), whereas the overexpression of an apple (Malus domestica) fruit PG in apple resulted in novel phenotypes involving changes in cell adhesion (Atkinson et al., 2002). PGs derived from microbes are often components of their offensive arsenal necessary to invade and colonize plant tissues. They are usually more active than plant-derived PGs and more efficiently catalyze the fragmentation and solubilization of pectin (De Plant Physiol. Vol. 135, 2004

Transgenic tobacco plants expressing the pgII gene encoding the endopolygalacturonase II of Aspergillus niger (AnPGII) were prepared by Agrobacteriummediated transformation. To target AnPGII into the apoplast, the sequence encoding the secretion signal peptide of pgII was replaced with that of pgip1 of Phaseolus vulgaris, and the chimeric gene was placed under the control of the cauliflower mosaic virus (CaMV) 35S promoter for constitutive expression. Sixteen independent primary transformants (T0) were obtained compared with about 100 transformants obtained in a parallel transformation experiment using the empty vector. The low number of plants obtained upon transformation with the pgII gene was due to a low number of plantlets generated from tobacco calli, although the amount of calli was comparable to that obtained in the experiment with the empty vector. This result as well as the low level of PG activity detected in the transformed plants (see later) indicate a strong selective pressure against plants expressing AnPGII. Screening of primary transformants was performed by western-blot analysis of leaf total protein extracts using a polyclonal antibody against AnPGII. The expression of AnPGII was very low, and the highest expression levels, in plants 5, 7, and 16, resulted in

Figure 2. Purification by affinity chromatography and in-gel activity of AnPGII from transgenic tobacco plants. SDS-PAGE (A) and western-blot analysis using the AnPGII-specific antibody (B) of proteins retained by a bean PGIP2-Sepharose affinity column and eluted with PBS. Affinity chromatography was performed as reported in ‘‘Materials and Methods.’’ Lane 1, AnPGII eluted from the affinity column; lane 2, 30 ng (A) and 1 ng (B) of purified AnPGII from A. niger. C, IEF gel of purified AnPGII stained by silver nitrate. D, IEF gel of AnPGII stained for enzymatic activity. 1295

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only a few nanograms of enzyme per milligram of total protein. Two bands were detected by the anti-AnPGII antibody: the larger band had the expected molecular mass of 38 kD, close to that of the enzyme produced by A. niger; the smaller band had a molecular mass of 33 kD (Fig. 1A). Both bands were present in the extracellular fluid, indicating that the enzyme had been correctly delivered into the apoplast (Fig. 1B). Southern-blot analysis revealed a single integrated copy of the PG transgene, and northern-blot analysis showed a detectable amount of PG transcript in each of the

three lines (data not shown). Homozygous R1 and R2 progeny plants, which produced amounts of AnPGII comparable to that produced by the primary transformants, were obtained by self-crossing (Fig. 1C). AnPGII was purified from leaves of the homozygous R2 progeny plants of line 16 by anion-exchange chromatography and affinity chromatography on a P. vulgaris PGIP2-Sepharose column. Both the 38-kD and the 33-kD bands interacted with PGIP2 and were eluted from the affinity column with phosphatebuffered saline (PBS) as detected by SDS-PAGE

Figure 3. Morphological, anatomical, and immunoblot analyses of transgenic tobacco plants. A, Growth characteristics of 10-weekold untransformed(SR1), transgenic homozygous tobacco plants of lines 5, 7, and 16 and a plant obtained by crossing line 16 with transgenic plants expressing P. vulgaris PGIP2 (line 16 3 PGIP2). B, Transverse sections of stems of SR1, line 7, and line 16. Arrows in A indicate the approximate region from which the transverse stem sections were taken. c, cortical parenchyma; p, pith parenchyma. Scale bars 5 1 mm. C, Western-blot analysis of leaf protein extracts from the same tobacco plants as for A, using the AnPGII-specific antibody, or D, a PGIP-specific antibody. AnPGII, 4 ng of purified AnPGII from A. niger. PGIP2, 2 ng of purified PGIP2 from P. vulgaris.

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Table I. PG and PGIP activity in protein extracts (80 mg) of leaves from 10-week-old transgenic tobacco plants PG activity was measured by agarose diffusion assay and is expressed in APU. PGIP was assayed against purified AnPGII and is expressed in inhibitory units as reported in ‘‘Materials and Methods.’’ Plants

PG Activity

PGIP Activity

SR1 Line 5 Line 7 Line 16 Line 16 3 PGIP2 PGIP2

0 0.45 0.75 1.2 0 0

0 0 0 0 400 1,380

(Fig. 2, A and B) and isoelectric focusing (IEF) gel (Fig. 2C). The in-gel PG assay showed enzymatic activity associated with one of the IEF bands but not with the other (Fig. 2D). The matrix-assisted laser-desorption ionization-mass spectrometry (MALDI-MS) spectrum of the peptides obtained from the in-gel digest of the 38-kD band showed nine peaks corresponding to AnPGII sequences. Among them, a very intense peak with m/z of 790.4 was subjected to postsource decay sequencing and checked to correspond to the amino acids 83 to 88 (DGARWW) of AnPGII. The MALDI-MS spectrum of the peptides obtained from the in-gel di-

gest of the 33-kD band showed five peaks corresponding to AnPGII sequences among which the C-terminal peptide was present. This indicates that the 33-kD band is an N-truncated form of AnPGII. Activity of AnPGII in Tobacco Causes Reduced Growth and Altered Stem Anatomy of Transgenic Plants

In comparison with the untransformed plants (SR1), both primary transformants and R2 homozygous plants expressing AnPGII showed reduced size (Fig. 3A). Size reduction was particularly severe in line 16, which expresses the highest amount of AnPGII, indicating a correlation between PG activity and intensity of phenotype. At 10 weeks the plants had the same number of nodes/internodes, indicating that stem extension was specifically affected. Analysis of transverse sections of 10-week-old stems from both SR1 and R2 homozygous plants (lines 7 and 16) indicated altered anatomy was an aspect of this phenotype. In comparison to SR1, line 16 displayed an increase in the relative amount of cortical parenchyma and a decrease of the pith parenchyma. Line 7, with an intermediate expression of the transgene, displayed an intermediate alteration in its stem anatomy (Fig. 3B). Figure 4. Anatomical analysis of transgenic tobacco plants. Stem transverse sections at the level of fourth internode from 8-week-old SR1, line 7, and line 16 plants. A, Cortical region. Line 16 shows smaller and rounder epidermal and subepidermal cells, compared to SR1 plants. B, Central region. Pith parenchyma cells of line 16 are smaller than SR1 and line 7 cells. The number of phloem-associated parenchyma cells in line 16 is greater than in SR1 and line 7 plants. C, Vascular tissue. Along the radial axis in the region of the cambium, line 16 has more cell layers. Cells of line 16 are rounder and smaller than SR1 and line 7 cells. Bars 5 100 mm.


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sensitive to dehydration than wild-type plants (Fig. 5). Unlike quasimodo, mutants cells of our transformed plants did not easily detach from each other, and no apparent effect on cell adhesion could be detected. Our plants were not more fragile than SR1 plants. Activity of AnPGII Modifies Cell Wall Composition of Transgenic Tobacco Plants

Figure 5. Dehydration of leaves. A, Leaves of 8-week-old SR1 and line 16 plants were collected, transferred in the hood, and their fresh weight was determined at different time intervals. Three independent samples, each formed by three leaves from three different plants, were used. Bars indicate the SE for each data point. B, Scanning electron micrographs of abaxial midrib of SR1, line 7, and line 16 leaves of 8-week-old tobacco plants. Cells of line 16 quickly dehydrated when transferred into the vacuum chamber of the electron microscope. A slight dehydration is also evident in line 7 plants. Bars 5 100 mm.

In order to demonstrate that AnPGII enzymatic activity is responsible for the reduced growth and altered morphology, activity was measured in transgenic plants (Table I). Moreover, transgenic tobacco plants expressing PGIP2 of P. vulgaris, an efficient inhibitor of AnPGII (Leckie et al., 1999), were prepared and crossed with plants of line 16. Progeny plants, expressing both AnPGII and PGIP2 (Fig. 3, C and D), exhibited no detectable PG activity (Table I) and showed normal growth rates and regular morphology (Fig. 3A), indicating that a reversion of the phenotype is caused by the inhibition of the enzyme activity. Interestingly, western-blot analysis of progeny plants showed the presence of the 38-kD band corresponding to the intact AnPGII but not of the 33 kD AnPGII degradation product (Fig. 3C), indicating that the interaction of AnPGII with PGIP2 in planta protects the enzyme from degradation. In addition to the alterations discussed above, the cells from transgenic plants were smaller than those of the wild type, and their shape appeared rounder in comparison with the slightly polygonal SR1 cells. Moreover, more cells were associated with the conducting tissues in line 16 than in SR1 and line 7 plants (Fig. 4). Like the quasimodo mutants described by Bouton et al. (2002), the transformed plants were more 1298

The modifications that had occurred in the pectin composition and structure of transformed tobacco plants as a consequence of the expression of AnPGII were investigated by chemical and immunocytological analysis. Cell wall polysaccharides were isolated, fractionated, and analyzed for monosaccharide composition. Total wall material (alcohol insoluble solids [AIS]) and a fraction enriched in polyuronides (chelating agent soluble solids [ChASS]), prepared from stems of transgenic tobacco lines 5, 7, and 16, revealed a content of uronic acids lower than that of stems from SR1 plants with the difference being most significant for line 16. In parallel with the reduced level of uronic acids, a slightly higher amount of Rha, Ara, and Gal was observed in the ChASS fraction of transgenic plants. The level of Glc, Xyl, and Man was not significantly different in untransformed and transgenic plants (Fig. 6). Monoclonal antibodies can be used to assay precise structural features of pectic polysaccharides in both

Figure 6. Chemical analysis of cell walls of transgenic tobacco plants expressing AnPGII. Monosaccharide composition of AIS (A) and ChASS (B) fractions prepared from cell wall material of stems of 30-d-old SR1 and transgenic lines 5, 7, and 16. Results are expressed in percentage of total moles. Bars indicate SE (n 5 3). Plant Physiol. Vol. 135, 2004


Figure 7. Immunodot analysis of cell wall fractions. ChASS fractions were extracted from cell wall material of untransformed and stems of transgenic tobacco lines 7 and 16 and applied to nitrocellulose in the dilution series indicated. Immunodot analysis with PAM1 indicated that large unesterified blocks of HGA are present in all ChASS fractions, but were 5-fold less abundant in line 16.

isolated preparations of cell walls and in situ. A range of anti-HGA monoclonal antibodies have been used here. PAM1 is specific to large deesterified blocks of HGA (Willats et al., 1999) and thus recognizes the same regions of HGA that can act as targets for AnPGII action. LM7 recognizes a partially methyl-esterified epitope of HGA that is also degraded by AnPGII (Willats et al., 2001; Clausen et al., 2003). JIM5 binds to unesterified HGA in addition to partially methylesterified epitopes (Willats et al., 2000; Clausen et al., 2003), and JIM7 binds to a range of partially methylesterified epitopes of HGA that are not directly degraded by AnPGII action (Willats et al., 2000; Clausen et al., 2003). ChASS fractions of SR1, lines 7 and 16, and that of a tobacco line transformed with the 35S:GUS alone as an additional control were analyzed with the antiHGA antibodies using immunodot assays (IDAs) as shown in Figure 7. Equivalent amounts of carbohydrates from each fraction were applied to nitrocellu-

lose in a 5-fold dilution series and probed with PAM1, LM7, JIM7, and JIM5. IDA analyses with PAM1 indicated that large deesterified HGA blocks were present in all ChASS fractions and were at least 5-fold less abundant in line 16. The LM7 epitope is extremely labile (Willats et al., 2001), and no LM7 binding was found to any of the fractions. No significant differences in the levels of the abundant JIM7 HGA epitope in the control and AnPGII expressing lines were detected, although a slight reduction in the strength of the signal for JIM5 epitope in ChASS fraction isolated from line 16 plants was observed (Fig. 7). Immunolocalization of the PAM1 epitope in transverse sections of stems and leaf midribs of 10-weekold tobacco plants indicated that in both organs the abundance of the epitope was reduced in the presence of AnPGII. The PAM1 epitope is largely restricted to cell wall lining intercellular spaces (Fig. 8, A–C). Similar labeling patterns were observed in tobacco stem sections (data not shown). In tobacco stems the

Figure 8. Immunofluorescent labeling of pectic HGA components in transgenic tobacco plants expressing AnPGII. Hand-cut transverse sections of midrib of 10-week-old plants (A–C) and Steedman’s wax transverse sections of stems of 9-week-old plants (D–F) were labeled with PAM1 (A–C) and LM7 (D–F). Arrows indicate a corner of an intercellular space (is) in each case. Scale bar 5 10 mm.


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LM7 epitope was evident in very discrete locations of the cell walls of SR1 and line 7 and was not detected in line 16. In SR1 and line 7, the epitope was restricted to the corners of the expanded triangular intercellular spaces (Fig. 8, D–F). In nonexpanded intercellular spaces of SR1 and line 7, the LM7 epitope was present in the developing space and was not found in other regions of the cell wall (data not shown). Similar results were obtained by using tobacco midrib material. No differences in immunolabeling patterns or levels were found between the wild type and lines 7 and 16 using anti-HGA probes JIM5 and JIM7 (data not shown). Transgenic Arabidopsis Plants Expressing AnPGII Also Exhibit a Dwarf Phenotype

The coding sequence of the mature AnPGII, fused with the sequence encoding the signal peptide of PGIP1 of P. vulgaris and placed under the control of the CaMV 35S promoter, was introduced in Arabidopsis by Agrobacterium-mediated transformation. Five independent plants (lines 1, 2, 5, 6, and 13) exhibited different levels of AnPGII expression. Selfed seeds (R2) were collected from all these plants and used for subsequent analysis. The level of PG expression was higher for lines 1 and 2 and lower for lines 5, 6, and 13. Arabidopsis plants were also transformed with the gene encoding the mutated form of AnPGII (AnPGII

Figure 9. Morphological and immunoblotting analyses of transgenic Arabidopsis plants expressing AnPGII. A, Growth characteristics of transgenic Arabidopsis R2 plants of lines 1 and 5 compared to SR1 (control). B, Western-blot analysis of leaf total protein extracts of line 1, line 5, and control using the AnPGII-specific polyclonal antibody. 1300

D201N) in which the substitution of Asp-201 by Asn causes a complete loss of enzyme activity (Armand et al., 2000; Federici et al., 2001). Western-blot analysis of protein extracts with antibodies against AnPGII showed the presence of a 38-kD band in the plants transformed with either the wild type or the mutated AnPGII gene. Unlike in tobacco, no AnPGII degradation products were observed in the transgenic Arabidopsis plants (Fig. 9). In comparison to the untransformed plants, those expressing the active AnPGII showed reduced growth with multifoliaceous rosette, small, and slightly curled leaves. The phenotype was particularly severe in the lines 1 and 2 expressing the highest amount of PG and less pronounced in lines 5, 6, and 13 (Fig. 9). The morphological alterations were clearly due to the PG activity since plants expressing the inactive AnPGII had a normal phenotype and did not show any difference compared to the wild type.

DISCUSSION

In this study, we have produced transgenic tobacco plants expressing the gene encoding PGII of A. niger. The number of transformants obtained was very low, suggesting that the activity of a microbial enzyme probably has harmful effects on the plant cell wall and therefore on the fitness of the transformed plants. Not only was the number of transformants low but also the level of enzyme expression in the transformed plants was low, indicating a strong selective pressure against plants expressing AnPGII. Similarly, the transformation efficiency of potatoes with the genes encoding rhamnogalacturonan lyase and arabinanase of A. aculeatus has been reported to be very low (Skjot et al., 2001; Oomen et al., 2002). Interestingly in a situation where we did not limit the effects of the enzyme activity by using tissue-specific promoters and/or specific targeting signals, the plant was capable of performing its own control of AnPGII by proteolytically processing and inactivating the enzyme. In those plants where AnPGII is expressed together with PGIP2 and, therefore, the enzyme is inhibited, the plant does not need to cleave proteolytically the enzyme, which consequently remains intact in the tissue. Among the transformed tobacco plants obtained, the three lines analyzed in this study had the highest level of expressed enzyme and showed a dwarfed phenotype. One of the transformants (line 16) was severely affected in the growth rate and had a reduced height and length between leaf internodes. In comparison to the wild type, line 16 also showed anatomical differences in the reduced stems with a larger cortical parenchyma and a reduced pith parenchyma. However, our analyses with antibodies to cell wall components have indicated no differences between these two tissues, and, so far, we have no evidence that the observed altered cell wall composition is specific to a particular Plant Physiol. Vol. 135, 2004


cell type. The ability of AnPGII to confer a dwarfed phenotype was also maintained in Arabidopsis. An active PG was sufficient and necessary to confer the dwarfed phenotype to both tobacco and Arabidopsis. The inhibitor PGIP was successfully used to inhibit the outcome of the dwarfed phenotype in tobacco. Similarly, the transformation of Arabidopsis with a gene encoding an enzyme inactivated by site-directed mutagenesis produced plants with a normal phenotype. Long regions of HGA without methyl esterification are the optimal substrate for PGII (Limberg et al., 2000). It is likely that the capacity of AnPGII to confer a dwarfed phenotype is a direct consequence of its ability to modify the HGA region of pectin by cleavage of unesterified regions in muro. Transgenic line 16 exhibited a significant reduction in GalUA content and an absence of deesterified blocks of HGA (recognized by PAM1) and also an absence of the LM7 nonblockwise deesterified HGA epitope. Concomitant with the decrease of uronic acids and AnPGII-sensitive HGA epitopes, an apparent increase of other sugars characteristic of RG-I occurred, which may reflect a compensatory response. Plants are known to have the capacity to compensate for the decrease of a cell wall component by increasing another (Bouton et al., 2002). Compared with the quasimodo mutants, which exhibit a similar decrease in uronic acid content, our plants showed a similar propensity to dehydrate. However, they did not appear to be fragile like the quasimodo plants and neither did they tend to fall apart as a consequence of an altered cell cohesion. The partial similarity among quasimodo and our plants indicates a similar but not identical alteration of the cell wall structure. It is likely that two different pectin domains with distinct functions are modified, and a detailed comparison among quasimodo and our transformed plants may elucidate this point. How a low content of deesterified HGA affects plant growth is not clear. In this context our transgenic plants represent a useful tool for future detailed studies. HGA forms Ca21-dependent bonds that promote crosslinked gel formation at defined locations in the cell wall and influence the porosity of the cell wall (BaronEpel et al., 1988). This may therefore affect the mobility of proteins and enzymes that control the extensibility of the wall and/or the incorporation of new polymers into the expanding cell wall (Cosgrove, 1999). Alternatively, the low content of HGA may influence the interactions of the different pectin components and interfere with the correct assembly of the hemicellulosecellulose network (Jarvis, 1992). Strengthening the pectin-hemicellulose-cellulose network may lead to a reduced extensibility of the wall and to a slower rate of growth. The possibility that expression of PG in plants produces an excess of oligogalacturonides, which in turn induce a sort of defense reaction and increase rigidity of cell walls, is not supported by the finding that crosses between plants expressing AnPGII and those expressing the inhibitor PGIP have no phenotype. It is well known that PGIPs, by interacting Plant Physiol. Vol. 135, 2004

with fungal PGs, favor the release of elicitor-active oligogalacturonides (De Lorenzo et al., 2001). In conclusion, we show that expression of a fungal PG in transgenic plants modifies cell wall HGA and plant growth. HGA is synthesized and incorporated into the cell wall in a highly methyl-esterified form, and during cell development methyl groups are removed by pectin methylesterases, thereby creating a substrate for endogenous PGs (Willats et al., 2001). The relative amounts of methyl-esterified and unesterified HGA are likely to be critical for determining many processes occurring during plant development (Kim and Carpita, 1992; Steele et al., 1997; Femenia et al., 1998; Wen et al., 1999). The observations reported here indicate that the deesterified form of HGA is a critical factor in plant growth.

MATERIALS AND METHODS Plant Transformation The gene encoding AnPGII was fused with the sequence encoding the signal peptide of PGIP1 from Phaseolus vulgaris (Toubart et al., 1992) by using the technique of splicing by overlap extension in which two sets of PCR reactions and four primers are required. The oligonucleotides used were as follows: primers A (5#-GTTAGGGATCCAATGACTCAATTCAATATCCCAG-3#), B (5#-TCTTTGAGAACTGCACTCTCAGACAGCTGCACGTTCACC-3#), C (5#-GGTGAACGTGCAGCTGTCTGAGAGTGCAGTTCTCAAAGA-3#), and D (5#-TGCTAGAGCTCAAGCTTCTAACAAGAGGCCACCGAA-3#). The primers A and D contained flanking regions with BamHI and SacI sites at the 5# termini, respectively. Two separate PCR reactions were performed to amplify the sequence encoding the signal peptide of P. vulgaris pgip1 using the primers A and C and the sequence encoding the mature AnPGII using the primers B and D. The PCRs were run for 10 cycles at 94°C for 1 min, and 58°C and 72°C for 45 s. The products of the two polymerase reactions were purified by gel electrophoresis and used as a template in a second PCR using the primers A and D. The second PCR was run for 12 cycles at 94°C for 1 min, and 58°C and 72°C for 45 s. The full-length DNA product was digested with BamHI and SacI and ligated into pBI121 digested with BamHI and SacI. The gene fusion cloned in pBI was sequenced to verify the construct integrity. The pBI121 vector containing the pgII gene was mobilized into Agrobacterium tumefaciens strain LBA4404 by electroporation. Leaf discs from tobacco plants (Nicotiana tabacum) cv Petit Havana were transformed (Hansen et al., 1994), and kanamycin-resistant plants were regenerated on Murashige and Skoog (MS) medium (SigmaAldrich, St. Louis) by standard methods. Primary transformants were allowed to self-fertilize, and R0 seeds were collected and germinated on MS medium with 300 mg mL21 kanamycin. Selected seedlings were transferred into soil and grown for a few weeks. The same construct described before and a pgII gene carrying the mutation D201N were used for Arabidopsis transformation. The pBI121 vectors containing the genes pgII and pg D201N were introduced into A. tumefaciens strain GV3101 carrying the pADI289 plasmid conferring overexpression of virG (Hansen et al., 1994) by electroporation. Vectors were introduced into 7-week-old Arabidopsis ecotype Wassilewskija by the vacuum infiltration method as described (Clough and Bent, 1998). To identify transgenic plants overexpressing PGII, seeds harvested from infiltrated plants (T0) were grown on MS germinating medium agar plate with Gamborg’s B5 vitamins supplemented with 50 mg L21 kanamycin. The plates were transferred under 16 h light, 8 h dark. After 14 d, seedlings resistant to kanamycin (T1 progeny) were transplanted into potting mix and grown in a controlled environmental chamber maintained at 22°C, 70% humidity, under 12-h light-dark cycle. T2 progeny was subjected to the same selection, and lines showing a segregation ratio 3:1 for resistance to kanamycin were selected for subsequent analysis. The gene encoding PvPGIP2 of P. vulgaris was used for the construction of the binary vector pBIVPGIP2, derived from pBI121 vector, which contained the pgip2 gene under the control of the CaMV promoter 35S and the translation enhancer V from tobacco mosaic virus. Transgenic tobacco plants were obtained and selected as described above for the plant transformation with the gene pgII.

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PG and PGIP Assay PG activity was measured either by reducing end-group analysis as described (Leckie et al., 1999) or by using a modified agarose diffusion assay (Taylor and Secor, 1988). A solution containing purified AnPGII or crude extracts from tobacco or Arabidopsis transgenic plants were added to 0.5-cm wells in plates containing 0.5% polygalacturonic acid, 100 mM sodium acetate, pH 4.6, and 0.8% agarose. Plates were incubated for 12 h at 30°C, and haloes formed by the enzyme activity were visualized after 5 min of treatment with 6 M HCl. PG activity was expressed as agarose diffusion units (APU); 1 APU is the amount of enzyme producing a halo of 0.5-cm radius (external to the inoculation well) after 12 h at 30°C. PGIP was measured in the agarose diffusion assay against purified AnPGII and was expressed as inhibitory units. One inhibitory unit is the amount of PGIP that reduces by 50% the halo of 1 APU of enzyme.

Preparation of Intercellular Washing Fluids Intercellular washing fluids were collected as described previously (Bellincampi et al., 1995). Stem sections (3-cm long) of transgenic tobacco plants were excised, washed with water to remove cytosolic contaminants, and stacked upright on a 105-mm nylon mesh at the bottom of a 50-mL plastic syringe. The packed sections were vacuum-infiltrated for 10 min with 50 mM sodium acetate, pH 5.0. Intercellular washing fluid was recovered by centrifuging the vacuum-infiltrated stems at 800g for 20 min a 4°C.

SDS-PAGE, Western Blot, and Isoelectric Focusing SDS-PAGE and immunoblotting were performed as described previously (Desiderio et al., 1997). Polyclonal antibodies against PGII from Aspergillus niger were used for immunoblotting experiments. IEF was performed on a mini cell 111 Bio-Rad (Hercules, CA) apparatus according to the manufacturer’s instructions. A pH gradient was generated in a 6% polyacrylamide gel containing carrier ampholytes (Amersham, Buckinghamshire, UK; pH 3–8) by pre-electrophoresis for 15 min at 125 V. Samples were subjected to electrophoresis for 15 min at 100 V, 15 min at 200 V, and 60 min at 450 V. Proteins were stained with silver, and PG activity was visualized by a polygalacturonateagarose overlay gel. Overlays were incubated for 30 min in 0.5% polygalacturonic acid and stained in 0.06% ruthenium red (Ried and Collmer, 1986).

Purification of AnPGII from Transgenic Tobacco Plants Leaves of homozygous tobacco plants were homogenized in 1 M NaCl (2 mL g21), incubated under gentle shaking for 1 h, and centrifuged for 20 min at 10,000g. The supernatant was filtered through Miracloth (Calbiochem, San Diego) and dialized against 20 mM sodium acetate, pH 4.7, overnight at 4°C. The dialyzed extract was loaded on a DEAE cellulose (DE52; Whatman, Kent, UK) column pre-equilibrated with 20 mM sodium acetate, pH 4.7. The nonabsorbed proteins were loaded on a Sepharose (Pharmacia, Piscataway, NJ)-P. vulgaris PGIP2 column pre-equilibrated with 20 mM sodium acetate, pH 4.7, and bound proteins were eluted with PBS.

column containing Poros R2 column material (Applied Biosystems, Foster City, CA) as described by Gobom et al. (1999). For analyses by MALDI-MS, the peptides were eluted with 0.8 mL of matrix solution (10 mg mL21 a-cyanohydroxycinnamic acid in 70% [v/v] CH3CN and 0.1% [w/v] trifluoroacetate) and deposited directly onto the MALDI target. MALDI mass spectra were acquired on a Voyager DE-STR MALDI-TOF mass spectrometer (Applied Biosystems), operated in positive ion reflector mode. The instrument was equipped with delayed ion extraction. The delay time used was 175 ns. Mass spectra were recorded as single-shot spectra by using a UV laser operated at 337 nm and an acceleration voltage of 20 kV. Single-shot spectra (200–600) were averaged to give the final spectrum. Internal mass calibration of peptide masses was performed using porcine trypsin autolysis products.

Antibodies The polyclonal antibody against AnPGII has been described (Cervone et al., 1987). The antibody against PGIP of P. vulgaris has been described (Bergmann et al., 1994). The monoclonal antibodies used in this study have all been described previously. Three rat monoclonal antibodies have been used: JIM5 and JIM7, which recognize a range of partially methyl-esterified epitopes of HGA (Knox et al., 1990; Willats et al., 2000; Clausen et al., 2003); and LM7, which recognizes HG domains with nonblockwise patterns of methyl esterification (Willats et al., 2001). PAM1 is a phage display monoclonal antibody specific to large blocks of deesterified HGA (Willats et al., 1999; Willats et al., 2000). Soluble PAM1 single chain synthetic antibody fragments (scFvs) were used in this study (also known as PAM1scFv) and have the same specificity as PAM1 scFvs when attached to phage particles (W.G.T. Willats, personal communication).

Preparation of Cell Wall Fractions and Monosaccharide Composition Analysis Cell walls were purified and fractionated as described (Stolle-Smits et al., 1997). AIS were prepared by adding 80% ethanol to 2 g of plant material, homogenized in liquid nitrogen. After heating at 80°C for 30 min, the mixture was blended and centrifuged at 7,000g for 15 min. The pellet was washed four times in 80% ethanol and twice in 95% ethanol. AIS was subsequently washed with acetone and air dried in a fume cupboard. AIS was then washed three times with 50 mM sodium acetate buffer, pH 5.2, at 70°C for 1 h, resuspended in 50 mM sodium acetate buffer, pH 5.2, containing 10 mg of a-amylase (1,000 units mL21), and incubated overnight at 30°C. After inactivation of the enzyme in boiling water for 10 min, the suspension was cooled, centrifuged at 7,000g for 20 min, and the pellet washed twice with SDS (1.5%) in 50 mM sodium metabisulfate. A fraction enriched in polyuronides and ChASS was extracted from the deproteinated pellets using 50 mM sodium acetate buffer containing 50 mM of cyclohexane diamine tetraacetic acid and ammonium oxalate. Extraction was carried out by stirring at room temperature for 6 h and was repeated three times. All supernatants were dialyzed against 100 mM ammonium acetate buffer, pH 5.2, concentrated by rotary evaporation, dialyzed against distilled water, and lyophilized. AIS and ChASS were treated for 1 h at 30°C with 72% sulfuric acid and then with 1 M sulfuric acid for 3 h at 100°C. Total sugar content was estimated using orcinol assay and uronic acids were determined by using the m-hydroxybiphenyl assay (Schols et al., 1995). To determine the neutral monosaccharide composition, both fractions were hydrolyzed by using 2 M aqueous trifluoroacetic acid for 2 h at 121°C and the monosaccharides analyzed by highperformance anion-exchange chromatography on a Carbo-Pac PA1 column (Dionex, Sunnyvale, CA) using 16 mM NaOH as an eluant. Sugars were monitored by pulsed-amperometric detection.

Dehydration Experiments For dehydration experiments, 8-week-old plants were harvested and placed in a horizontal laminar air-flow unit. Dehydration was followed by weighing four independent samples of three leaves from each plant at different times.

Peptide Mass Mapping by MALDI-TOF-MS Bands were cut out from Coomassie-stained gels and subjected to in-gel digestion with endoproteinase Asp-N. After soaking the endoproteinase into the gel, the gel pieces were incubated at 37°C overnight. The supernatant was then used for micropurification of peptides by application to a micro RP

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Immunodot Assays ChASS fractions (1 mg mL21) were applied as 1-mL aliquots to nitrocellulose in a 5-fold dilution series and allowed to air dry. The procedure for the development of tissue prints and IDAs was identical. After blocking for 1 h with 5% (w/v) milk protein in PBS (PBS/MP), the nitrocellulose sheets were incubated with a 10-fold dilution of the appropriate rat monoclonal antibody hybridoma supernatant or a 20-fold dilution of PAM1scFv for at least 1 h. The nitrocellulose sheets were then washed extensively in water prior to incubation in a 1,000-fold dilution of secondary antibody in PBS/MP. For JIM5, JIM7, and LM7, anti-rat horseradish peroxidase conjugate (Sigma, Poole, UK)



was used and for PAM1scFv, anti-polyHistidine horseradish peroxidase conjugate (Sigma). After washing, freshly prepared substrate solution was used to detect antibody binding (25 mL of deionized water, 5 mL of MeOH containing 10 mg mL21 4-chloro-1-naphthol, and 30 mL of 6% H2O2). When the reaction was complete, the nitrocellulose sheets were washed extensively in water.

Preparation of Plant Material for Microscopy and Immunolabeling Tobacco material was either sectioned by hand to a thickness of approximately 100 to 300 mm or cut to a thickness of 12 mm in Steedman’s wax (see below). Hand-sectioned material was immediately fixed for 1 h in PEM buffer (50 mM PIPES, 5 mM EGTA, 5 mM MgSO4,, pH 6.9) containing 4% paraformaldehyde at room temperature. Following fixation, the seedlings were washed extensively in 1 3 PBS (prepared from a 10 3 stock solution containing 80 g NaCl, 2 g KCl, 28.6 g Na2HPO4 3 12H2O and 2 g KH2PO4 in 1 L of deionized water, pH 7.2) and immunolabeled as described below. For sectioning in Steedman’s wax tobacco, material was fixed for 1 h in PEM buffer containing 4% paraformaldehyde at room temperature, with an initial vacuum infiltration. Following fixation, the seedlings were washed extensively in 1 3 PBS. Samples were then dehydrated in an ethanol series consisting of 30%, 50%, 70%, 90%, and 97% ethanol (30 min each, at 4°C), moved to 37°C, allowed to warm up, and transferred to Steedman’s wax and ethanol (1:1) and left O/N at 37°C. The samples were transferred to 100% wax (2 3 1 h, at 37°C) and then placed in sample molds filled with molten wax and left at room temperature to solidify (for at least 12 h). Sections were cut to a thickness of 12 mm and collected on polysine-coated microscope slides (BDH Laboratory Supplies, Dorset, UK), dewaxed and rehydrated in a series consisting of 97% ethanol (3 3 10 min), 90% ethanol (10 min), 50% ethanol (10 min), PBS (10 min), and PBS (90 min), and immunolabeled as described below. For labeling with rat monoclonal antibody LM7, tobacco sections were incubated in PBS containing 3% (w/v) milk protein (PBS/MP, Marvel, Premier Beverages, Knighton Adbaston, UK) and a 10-fold dilution of LM7 for 1 h. Samples were then washed in PBS at least three times and incubated with a 100-fold dilution of anti-rat IgG (whole molecule) linked to fluorescein isothiocyanate (Sigma) in PBS/MP for 1 h in darkness. The samples were washed at least three times and mounted in a glycerol-based antifade solution (Citifluor AF1, Agar Scientific, Stansted, UK) and observed on an Olympus (Tokyo) BH-2 microscope equipped with epifluorescence irradiation. In the case of PAM1scFv, the sections were incubated in a 20-fold dilution of PAM1scFv in PBS/MP for 1 h. After washing, the sections were incubated in a 100-fold dilution of mouse anti-polyHistidine monoclonal antibody (Sigma) in PBS/MP for 1 h followed by a 100-fold dilution of anti-mouse IgG (whole molecule) linked to fluorescein isothiocyanate in PBS/MP for 1 h in darkness. The sections were then washed and mounted as described for LM7. For light microscopy, fresh stem segments were embedded in 4% agar and cut into 100- to 150-mm sections using a vibratome. Sections were stained with tolouidine blue 0.05%. Photomicrographs were taken in a Zeiss Axiophot microscope (Jena, Germany) using a Canon Powershot G3 photocamera (Tokyo). For scanning electron microscopy, fully expanded leaf explants were attached to the sample holder with a thin layer of clay and immediately transferred to the vacuum chamber of the electron microscope to be directly examined.

ACKNOWLEDGMENTS We thank Daniela Pontiggia and Giuseppe Caruso for their valuable technical assistance. Received March 16, 2004; returned for revision April 5, 2004; accepted April 5, 2004.

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