A Role for Arabinogalactan-Proteins in Root Epidermal Cell Expansion

A Role for Arabinogalactan-Proteins in Root Epidermal Cell Expansion

Planta (1997) 203: 289±294 A role for arabinogalactan-proteins in root epidermal cell expansion Lei Ding, Jian-Kang Zhu Department of Plant Sciences, University of Arizona, Tucson, AZ 85721, USA Received: 13 February 1997 / Accepted: 1 April 1997 Abstract. Arabinogalactan-proteins (AGPs) are abund- membrane, cell wall and intercellular spaces (Fincher ant plant proteoglycans that react with (b-D-Glc)3 but et al. 1983; Komalavilas et al. 1991; Serpe and Nothnagel not (b-D-Man)3 Yariv reagent. We report here that 1995). The carbohydrate moiety of AGPs consists of treatment with (b-D-Glc)3 Yariv reagent caused inhibi- mainly arabinose and galactose with minor amounts of tion of root growth of Arabidopsis thaliana (L.) Heynh. other sugars including uronic acids (Fincher et al. 1983; seedlings. Moreover, the treated roots exhibited numer- Komalavilas et al. 1991). The protein moieties of AGPs ous bulging epidermal cells. Treatment with (b-D-Man)3 are typically rich in hydroxyproline, serine, alanine, Yariv reagent did not have any such eects. These threonine and glycine (Fincher et al. 1983; Showalter results indicate a role for AGPs in root growth and and Varner 1989). The primary structures of several control of epidermal cell expansion. Because treatment AGP core proteins have recently been elucidated via with (b-D-Glc)3 Yariv reagent phenocopies the reb1 (root gene cloning (Chen et al. 1994; Du et al. 1994; Cheung epidermal cell bulging) mutant of Arabidopsis, AGPs et al. 1995; Mau et al. 1995; Du et al. 1996). were extracted from the reb1-1 mutant and compared The expression of AGPs is highly regulated during with those of the wild type. The reb1-1 roots contained plant development (Knox et al. 1989; Pennell and an approximately 30% lower level of AGPs than the Roberts 1990; Stacey et al. 1990; Schindler et al. 1995). wild type. More importantly, while the pro®le of AGPs Experiments with monoclonal antibodies to particular from wild-type roots showed two major peaks upon AGP epitopes have demonstrated that the expression of crossed electrophoresis, the pro®le of AGPs from reb1-1 AGPs correlates with cell dierentiation (Pennell and roots exhibited only one of the major peaks. Therefore, Roberts 1990; Knox et al. 1991). For example, the AGP the reb1 phenotype appears to be a result of defective or epitope recognized by the monoclonal antibody missing root AGPs. Taken together, this pharmacolog- MAC207 is present in all cells of vegetative meristems, ical and genetic evidence strongly indicates a function of primordia and organs, and throughout undeveloped AGPs in the control of root epidermal cell expansion. ¯ower buds, but not in speci®c cells of developing stamens and carpels (Pennell and Roberts 1990). There- Key words: Arabidopsis (reb1 mutant) ± Arabinogalactan- fore, AGPs have been proposed to serve as speci®c cell protein ± Cell expansion ± Mutant (reb1) ± root epidermal surface markers. cell bulging ± Root growth Several functions of AGPs have been established recently. Speci®c AGPs have been found to be important for somatic embryogenesis of Daucus carota L., because addition of AGPs extracted from carrot seeds to a two- year-old, non-embryogenic cell line resulted in the re- Introduction induction of embryogenic potential (Kreuger and van Holst 1993). Serpe and Nothnagel (1994) observed that Arabinogalactan-proteins (AGPs) are a family of treatment of rose cell suspension with ( -D-Glc) Yariv proteoglycans that are widely distributed throughout b 3 reagent, a chromophoric molecule that selectively binds the plant kingdom (Fincher et al. 1983; Basile and Basile AGPs, caused inhibition of cell proliferation. More 1987). They are found predominantly in the plasma recently, secreted AGPs in the tobacco style have been demonstrated to stimulate and guide pollen tube growth Abbreviations: AGP = arabinogalactan protein; Gal = galactose; (Cheung et al. 1995; Wu et al. 1995). Glc = glucose; Man = mannose It has long been known that AGPs bind to Yariv Correspondence to: J.K. Zhu; E-mail: [email protected]; reagents, synthetic molecules containing phenyl-b-gly- Fax: 1 (520) 621 7186 cosides (Yariv et al. 1962; Clarke et al. 1978). This 290 L. Ding and J.-K. Zhu: Arabinogalactan-proteins and root cell expansion property has been used extensively in the detection, 90 mM boric acid and 2 mM EDTA, pH 8.3). The running buer quanti®cation and isolation of AGPs from plant tissues was the same as the gel buer. The second dimension was run with and cultured cells (Clarke et al. 1978; van Holst and 1% agarose gel containing 15 lM(b-D-Glc)3 Yariv reagent. The gel and running buer consisted of 25 mM Tris and 200 mM glycine, Clarke 1986; Komalavilas et al. 1991; Zhu et al. 1993). pH 8.4. After completion of electrophoresis, gels were washed Because the glycosidic linkage in Yariv reagents is overnight with 2% (w/v) NaCl and dried onto ®lter paper. required to be in the b-anomeric conjugation to react with AGPs, AGPs sometimes have also been referred to as b-lectins (Jermyn and Yeow 1975; Clarke et al. 1978). Results Yariv phenylglycosides having sugars with cis-hydroxyl or other substitutions on C2 of the b-D-glycopyranosyl b-glucosyl Yariv reagent treatment of Arabidopsis pheno- determinant also do not bind to AGPs (Nothnagel and copies the reb1 root epidermal cell bulging mutant. Lyon 1986). For example, while (b-D-Glc)3 Yariv Because (b-D-Glc)3 Yariv reagent can inhibit cell growth reagent can bind AGPs, (b-D-Man)3 or (a-D-Gal)3 in rose suspension cultures (Serpe and Nothnagel 1994), Yariv reagents do not react with AGPs. These latter we were interested in determining whether it may also Yariv reagents can serve as valuable controls to distin- alter the growth of Arabidopsis thaliana seedlings, with guish AGP-related eects from non-speci®c eects when the aim of exploring a molecular genetic approach to the using Yariv reagents in biological systems. study of AGP function. When Arabidopsis seeds were In the course of initiating a genetic screen for possible sown onto media containing up to 50 lM b-glucosyl Arabidopsis thaliana mutants with an altered response to Yariv reagent, their germination was not aected. Yariv reagent, we made a fortuitous observation that However, subsequent root growth was substantially exposure to (b-D-Glc)3 Yariv reagent causes the bulging inhibited by as low as 10 lM b-glucosyl Yariv reagent. of root epidermal cells. The morphological alterations Figure 1A illustrates this inhibition on roots grown on resemble the phenotype of the reb (root epidermal cell an agar surface when the plates were placed vertically. bulging) mutant of Arabidopsis isolated by Baskin et al. For comparison, no root inhibition was observed on (1992). We then found that the root of the reb1-1 mutant seedlings grown on media containing (b-D-Man)3 Yariv contains AGPs that are electrophoretically dierent reagent, which does not bind to AGPs (Fig. 1A). To from those of the wild type. These results strongly evaluate how fast the root inhibiton could occur, 4-d-old indicate a role of AGPs in controlling root epidermal cell seedlings grown on vertical agar plates without Yariv expansion. reagent were transferred onto a medium containing 10 lM(b-D-Glc)3 Yariv reagent, or as controls onto medium containing (b-D-Man)3 Yariv reagent (Fig. 1B) Materials and methods or to medium without Yariv reagent (not shown). While root growth was visible less than 1 h after the transfer on Treatment with Yariv reagents. Wild-type Arabidopsis thaliana (L.) seedlings transferred to control medium without Yariv Heynh. (ecotype Columbia) seeds were surface-sterilized and sown reagent, or with (b-D-Man)3 Yariv reagent, seedlings onto agar media containing Murashige and Skoog (Murashige and transferred to (b-D-Glc)3 Yariv reagent failed to exhibit Skoog 1962) salts, 3% sucrose, 1.2% agar, pH 5.7 and supple- appreciable root growth. Figure 1B shows that 2 d after mented with various amounts of Yariv reagents. Yariv reagents the seedling transfer, the roots on ( -D-Glc) Yariv were prepared according to the method of Yariv et al. (1962). b 3 Plants were grown at 23 2 °C under constant white light reagent had only a slight growth compared with 2 1 (approx. 70 lmol photons á m) á s) ). Roots were observed and substantial growth on (b-D-Man)3 Yariv reagent. The photographed directly inside the agar media using an inverted roots transplanted on (b-D-Glc)3 Yariv reagent also microscope. showed a series of sharp twists (Fig. 1B). Shoot growth of the seedlings on (b-D-Glc)3 Yariv reagent was not Extraction and electrophoretic analysis of AGPs. Wild type and inhibited until much later, which was probably an reb1-1 mutant seedlings were grown for 10 d on vertical agar plates. indirect consequence of the reduced root growth. Roots were separated from the shoots at the base of hypocotyl with A close scrutiny of roots grown inside agar medium a razor blade and collected and frozen immediately in liquid nitrogen. To extract AGPs, 5 g of frozen root or shoot samples was containing 10 lM(b-D-Glc)3 Yariv reagent revealed ground in 5 ml of extraction buer containing 50 mM Tris-HCl surprising morphological changes. While root epidermal (pH 8.0), 10 mM EDTA, 2 mM Na2SO5, and 1% (v/v) Triton X- cells including root hairs and non-hair cells are elongat- 100. The extraction procedure is essentially as described in van ed in untreated plants, we found that many of them Holst and Clarke (1986) except that the desalting step was omitted.

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