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Expansins are a family of proteins capable of inducing stress relaxation of isolated cell walls. In earlier studies, we showed that the expression of expansin genes in deepwater rice (Oryza sativaL.) is regulated by developmental, hormonal and environmental stimuli. Here, we describe the spatial distribution pattern of expansin transcripts and proteins in tissues and organs of deepwater rice usingin situmRNA hybridization and immunohistochemical analysis. Expansin transcripts and proteins are present at high levels in the growing internodal epidermis, which has thick cell walls and acts, therefore, as a growth-limiting cell layer. Expansins are also concentrated in the differentiating vascular bundles of internodes. In the primary root, expansins are predominantly expressed in the tip region, particularly in the epidermis, differentiating vascular cylinder, and around the pericyle. Developing adventitious roots and lateral root primordia also contain high levels of expansin mRNA. In the shoot apex, expansin transcripts are abundant in the emerging leaf primordia. Our results indicate that expansins play an important role in the expansion and differentiation of plant tissues and organs.
Plant cell expansion is based on stress relaxation of the cell wall ( Cosgrove 1993). A family of cell wall proteins, the expansins, have been identified as wall-loosening factors (reviewed in Cosgrove 1996;McQueen-Mason 1995), and evidence has been provided that expansins act by disrupting hydrogen bonds between cellulose microfibrils and matrix polymers ( McQueen-Mason & Cosgrove 1994). Although the role of expansins in mediating stress relaxation of isolated cell walls has been clearly demonstrated, less is known about the in vivo function of expansins.
In previous work, we have isolated two expansin proteins from deepwater rice internodes ( Cho & Kende 1997a), and four expansin genes have been identified from rice ( Cho & Kende 1997c;Shcherban et al. 1995 ). We have shown that cell walls from the growing region of the internode are more susceptible to expansin and also contain more expansin than the walls from the nongrowing region ( Cho & Kende 1997b). We have further shown that rice expansin genes are differentially expressed in response to hormonal and environmental stimuli and at different developmental stages ( Cho & Kende 1997c).
In our present study, we aimed at determining the spatial distribution of expansin transcripts and proteins in various rice tissues and organs. We hoped, thereby, to gain further insight into the possible role of expansin in growth and organ differentiation. Our results show that expansin transcripts and proteins are predominantly localized in growth-limiting tissues and in organ initials of deepwater rice.
Pattern of expansin expression in the internode
The structure of the uppermost, elongating rice internode is shown in Fig. 1. It is a hollow cylinder, consisting of the epidermis, vascular bundles, and parenchymal tissue. Above the second highest node, there is a ring of non-growing, white tissue, followed by the intercalary meristem, the elongation zone, and the differentiation zone. Axillary buds and adventitious roots originate from the white tissue and the node.
The spatial distribution patterns of expansin mRNA and protein in deepwater rice internodes were analyzed by in situ mRNA hybridization and immunohistochemistry. Antisense probes for expansin mRNAs showed strong hybridization in the epidermis and in the tissue surrounding the vascular bundles ( Fig. 2a,c). The ground parenchyma contained low levels of expansin transcripts, and sense probes did not show any detectable signal in those tissues ( Fig. 2b,d,f). In the vascular bundles, the expansin transcripts were concentrated in the fiber sheath cells that surround the conducting tissues ( Fig. 2c). Figure 2(a–d) represents the hybridization patterns in the elongation zone of the internode, and Fig. 2(e–g) in the differentiation zone. The distribution of expansin mRNA in the intercalary meristem was similar to that in the elongation zone (data not shown). In contrast, the differentiation zone contained low levels of expansin transcripts, both in the epidermis and in the vascular bundles ( Fig. 2e). The longitudinal distribution of expansin transcripts was consistent with previous RNA gel blot analyses, which showed that Os-EXP1 and Os-EXP4 mRNAs are most abundant in the intercalary meristem and elongation zone ( Cho & Kende 1997c). The in situ hybridizations in Fig. 2(a–f) were performed with the Os-EXP4 gene-specific probe. Results with the Os-EXP1 and Os-EXP2 gene-specific probes were similar (results not shown), except that expression of Os-EXP2 was maintained in the differentiation zone ( Fig. 2g), again confirming the results of gel blot analysis ( Cho & Kende 1997c). The level of expansin transcripts in the epidermis of the white tissue ( Fig. 1) was low, whereas the epidermis of the young axillary bud, whose basal regions keep a spatial continuum with the white tissue, contained high levels of expansin mRNAs ( Fig. 2h). In the region of the axillary bud, the expansin transcripts were abundant in the developing vasculature ( Fig. 2h).
Immunohistochemistry was performed with antibody raised against cucumber expansin (Cs-EXP1;Li et al. 1993 ). Using immunogold labeling with cucumber antibodies, we showed earlier at the electron microscopic level that expansin proteins are almost exclusively localized in the cell walls of corn coleoptiles and deepwater rice internodes ( Hoffmann-Benning et al. 1994 ;Hoffmann-Benning 1993). Cs-EXP1 antibody is expected to recognize preferentially Os-EXP2, Os-EXP4, and any close homologs that may be present ( Cho & Kende 1997a, c). Results obtained by immunohistochemistry confirmed those of the in situ mRNA hybridization experiments. Expansin proteins were found to be most abundant in the epidermis and around the vascular bundles ( Fig. 2i). The localization of expansin in the inner epidermal layer shown by tissue-print immunoblotting ( Cho & Kende 1997b) could not be reproduced by immunohistochemistry in the present study, most likely because of technical differences between the two methods. The inner cell layer of the internode consists of a loose network of broken cells. It is possible that parts of this loose cell network remained on the nylon membrane and caused the binding of antibody.
Pattern of expansin expression in the root
Figure 3(a–g) shows in situ hybridizations in primary roots performed with the gene-specific probe for Os-EXP3. We found earlier that the Os-EXP3 gene is specifically expressed in the rice root and in no other organ examined ( Cho & Kende 1997c). The spatial pattern of expansin gene expression in the root was, with minor quantitative variations, similar among all four known rice expansin genes (data not shown). In agreement with our previous results using RNA gel blot analysis ( Cho & Kende 1997c), in situ hybridization showed that expansin transcripts are abundant in the root tip ( Fig. 3a); they are particularly high in the epidermis and the vascular cylinder ( Fig. 3a,c,e). The root cap initial contained a low level of expansin mRNA, whereas the root cap itself contained little detectable expansin transcript ( Fig. 3a,c,d). The quiescent center had low levels of expansin mRNA ( Fig. 3c). The high transcript abundance around the pericycle was maintained in the upper regions of the root ( Fig. 3a,g,h).
Pattern of expansin expression in organ primordia
Because the formation of organ initials requires cell expansion and therefore cell wall modifications, we investigated the expression pattern of expansin genes in three organ primordia, namely in lateral root primordia, adventitious root primordia formed in the internode, and in leaf primordia at the shoot apex. The pericycle in the root-hair zone of the primary root, from which lateral roots are initiated, retained a considerable amount of expansin transcripts. In this region, lateral root primordia with 2–3 cell layers begin to appear. Figure 3(h) shows the in situ hybridization of the Os-EXP2 gene-specific probe to a lateral root primordium. Gene-specific probes of Os-EXP3 and Os-EXP4 also showed considerable levels of hybridization (data not shown). In contrast, hybridization with the gene-specific probe for Os-EXP1 was as weak as that seen with the sense probe ( Fig. 3i,j).
The in situ hybridizations of Fig. 4(a,b) were performed on cross-sections from the white tissue located between the intercalary meristem and node below it ( Fig. 1). This region contains several adventitious root initials, an axillary bud, and a number of procambial strands ( Kaufman 1959). Figure 4(a) shows in situ hybridization using the gene-specific probe for Os-EXP2. The transcript for Os-EXP2 was present at high levels in adventitious root primordia ( Fig. 4a), as were the other three rice expansin transcripts (data not shown). Expansin genes were also expressed along the procambial strands from which the adventitious root primordia emerge.
In the shoot apex, expansins were highly expressed in the leaf initials and emerging leaf primordia ( Fig. 4c–e). Os-EXP1, Os-EXP2, and Os-EXP4 showed a similar expression pattern with one minor difference: Os-EXP1 was mainly expressed in the first leaf primordial bulge ( Fig. 4d). Gene-specific probes for Os-EXP2 and Os-EXP4 showed hybridization signals in both the emerging leaf primordium and in the very young leaves ( Fig. 4c,e). Our results are similar to those of Fleming et al. (1997) who showed by in situ hybridization that the expansin transcript level in the shoot apex of tomatoes was highest in the leaf primordium.
Growing plant stems consist of tissues whose cell walls have different thicknesses. The outer epidermal cell walls of stems are considerably thicker than the inner epidermal cell walls and the cell walls of the inner tissues ( Kutschera 1992). This is also true for the internodes of deepwater rice ( Hoffmann-Benning & Kende 1994). Therefore, elongation of the stem is limited by the thick-walled epidermis, and loosening of the epidermal cell wall is essential for elongation of the stem. Our results from both in situ mRNA hybridization and immunohistochemistry demonstrate that expansin mRNA and protein are highly concentrated in the epidermis of the elongating internodal region ( Fig. 2a,c,i). The low content of expansin transcripts in the epidermis of non-growing tissues, such as the differentiation zone ( Fig. 2e), and the white tissue ( Fig. 2h) of the internode indicates a correlation between the requirement for expansins and the capacity for internodal cell elongation. We propose therefore that the high level of expansin in the epidermis contributes to loosening of the thick cell wall and elongation of the stem. The native epidermal cell wall of rice extends in response to acid buffer, and the heat-inactivated epidermal cell wall in response to exogenous expansin (H.-T. Cho and H. Kende, unpublished data), indicating that the epidermal cell wall contains expansin and is also susceptible to it.
Expansin is also abundant in the vascular bundles of the internode ( Fig. 2a,c). In the vascular system of grass stems, the conducting tissues are surrounded by small fiber sheath cells ( Esau 1965; Chapter 10). The walls of fully differentiated fiber sheath cells are thickened and lignified to provide mechanical support to the conducting elements. Rice expansins are highly expressed in the fiber sheath cells of the undifferentiated, as well as differentiating, tissues. In the intercalary meristem of rice internodes, the length of immature fiber cells is approximately 10 μm, whereas the mature fiber sheath cells are several hundred micrometers long ( Bleecker et al. 1986 ). It is likely that expansins are involved in mediating this cell elongation process, as well as the cell wall modifications that accompany it. It is also conceivable that the fiber sheath cells supply expansins to the metabolically inactive conducting tissues to help mediate modifications of their cell walls. The notion that expansins may play a role in the differentiation of the conducting tissues is supported by the fact that the differentiating vascular cylinder in the root tip ( Fig. 3a,c) and the developing vasculature in the white tissue of the internode ( Fig. 2h) also contain high levels of expansin transcripts.
The epidermal and fiber cells are smaller and less vacuolated than the parenchymal cells. Because of this, it is likely that they are enriched in metabolites and that the strong in situ hybridization signal of expansin results from a high concentration of RNA in these small, cytoplasm-rich cells. Indeed, we found that the total extractable RNA content of the parenchymal cells is, on a fresh-weight basis, only 6% of that isolated from epidermal tissue. We do not think, however, that this invalidates the proposition that the accumulation of expansin transcripts in the epidermal and fiber tissues is of physiological significance. To overcome the growth-limiting constraints of thickened cell walls, the concentration of expansin protein in the wall is expected to be elevated. It is very likely that this would be the result of increased levels of expansin mRNA in the underlying cytoplasm. The similarity in the patterns of the in situ hybridization ( Fig. 2a) and immunohistochemistry ( Fig. 2i) strongly support the physiological relevance of our observations.
Organ formation in plants is accomplished by a combination of oriented cell divisions and cell expansion. Recently, Fleming et al. (1997) reported that expansin applied to the shoot meristem of tomato plants induced the formation of leaf primordia. This result showed that expansin-mediated cell wall loosening and the ensuing cell expansion can control organogenesis. Our present study strongly supports this hypothesis. Expansin transcripts are abundant in emerging leaf primordia of the shoot apex ( Fig. 4c–e), in developing adventitious roots of the internode ( Figs 2h and 4a), and in lateral root primordia ( Fig. 3h). During organ formation, expansin may function both as a factor triggering organogenesis and as a factor sustaining growth of the respective organ. The exact biological function of expansins in rice is being tested by overexpressing expansin genes or repressing their expression.
Rice (Oryza sativa L., cv. Pin Gaew 56) seeds were germinated on two sheets of moist Whatman no. 1 filter paper in a Petri dish in darkness at 30°C for 3 days. Sections 3–4 mm long containing the root-tip region, and 5 mm sections 10–15 mm above the tip containing the root-hair region, were excised for fixation. For experiments with internodes, rice plants were grown as described previously ( Stünzi & Kende 1989). Twenty cm long stem sections containing the uppermost internode were excised from 11- to 13-week-old plants and submerged in distilled water for 6 h ( Kutschera & Kende 1988). Approximately 2 mm long tissue sections were excised from the intercalary meristem, the elongation zone, the differentiation zone, and the white tissue just below the intercalary meristem ( Fig. 1). Shoot apices were also isolated from 11- to 13-week-old plants.
In situ mRNA hybridization
In situ hybridization was performed essentially as described by Cox et al. (1984) . Plant tissue was fixed in 50% ethanol, 5% acetic acid, and 3.7% formaldehyde at room temperature for 16 h. The tissue was dehydrated in a graded series of ethanol and xylene and embedded in Paraplast Plus (Oxford Labware, St. Louis, MO, USA). Sections, 8 μm thick, were attached to Superfrost Plus microscope slides (Fisher Scientific, Pittsburgh, PA). Sections were deparaffinized with xylene and rehydrated through a graded ethanol series. They were subsequently pretreated with 1 μg ml–1 proteinase K in 100 m m Tris–HCl, pH 7.5, and 50 m m EDTA at 37°C for 30 min, dehydrated in graded ethanol, and dried under vacuum for 2 h. Sections were hybridized with sense or antisense probes in 50% (v/v) formamide, 300 m m NaCl, 10 m m Tris–HCl, pH 7.5, 1 m m EDTA, 5% (w/v) dextran sulfate, 1% (w/v) blocking reagent (Boehringer Mannheim), 150 μg ml–1 tRNA (Sigma, catalog no. R8759), and 500 μg ml–1 Poly A (Sigma, catalog no. P9403) at 37°C for 16 h. After washing in 2× SSC (1× SSC is 150 m m NaCl, 15 m m sodium citrate) and 0.2× SSC for 20 min each at room temperature, sections were treated with 2 μg ml–1 RNase A in 10 m m Tris–HCl, pH 7.5, 1 m m EDTA, and 500 m m NaCl at 37°C for 30 min, and washed three times in 0.2× SSC for 15 min each at room temperature. For immunological detection, the sections were subsequently treated with 1% (w/v) blocking reagent in 10 m m maleic acid-NaOH, pH 7.5, 150 m m NaCl, and 1% (w/v) BSA (Sigma, catalog no. A7030) in TNT (100 m m Tris–HCl, pH 7.5, 150 m m NaCl, and 0.3% (v/v) Triton-X 100) for 45 min at room temperature. Following the blocking reaction, sections were incubated with antidigoxygenin-alkaline phosphatase conjugate (Boehringer Mannheim, 1:600 dilution) in 1% BSA solution at room temperature for 2 h, washed three times in TNT for 45 min, and rinsed with alkaline phosphatase buffer (100 m m Tris–HCl, pH 9.5, 100 m m NaCl, and 50 m m MgCl2). The color reaction was performed in alkaline phosphatase buffer containing nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate and stopped by placing the slides into 10 m m Tris–HCl, pH 8.0, and 1 m m EDTA. For slide mounting, sections were dehydrated in graded ethanol, dipped in xylene, and mounted with Permount (Fisher Scientific). Sections were photographed with a Zeiss Axiophot microscope.
Preparation of riboprobes
Gene-specific 3′ untranslated regions were prepared either by restriction digestion or by PCR from the respective cDNA and cloned into pBluescript II SK-(Stratagene). For the Os-EXP1-specific probe, a 329-bp fragment was generated by restriction digestion with PstI and EagI after PCR with the primers 5′-AACTGCAGGACGTTCGGCCAGACCTT-3′ and 5′-CAGGAAACAGCTATGAC-3′. A 443-bp fragment between the PstI and EagI restriction sites was used for the Os-EXP2-specific probe. For the Os-EXP3-specific probe, a 330-bp fragment was generated by restriction digestion with PstI and SacI after PCR with the primers 5′-AACTGCAGCCCGTCCAACTG-3′ and 5′-CAGGAAACAGCTATGAC-3′. A 350-bp fragment between the EagI and HindIII restriction sites was used for the Os-EXP4-specific probe. Antisense RNA probes were generated in a reaction mixture containing digoxigenin-UTP using T3 or T7 polymerase (Boehringer Mannheim), depending on the orientation of the inserts. A sense probe was prepared from Os-EXP4. The probes were partially hydrolyzed to a length of about 100 nucleotides by incubation in 40 m m NaHCO3/60 m m Na2CO3.
DNA dot blot analysis was performed to confirm the specificity of the probes. Ten or 100 ng of purified plasmid DNA containing rice expansin cDNAs was blotted onto a nylon membrane using a dot blotting manifold. The membrane was blocked, hybridized with each probe, washed, and stained as in the in situ mRNA hybridization experiments, except that the hybridization was performed at room temperature. This DNA dot blot analysis showed that each gene-specific probe only bound to its respective cDNA (data not shown).
Fixation, paraffin embedding, sectioning and proteinase K treatment of the tissues were performed as in the in situ mRNA hybridizations. Following proteinase K treatment, sections were washed three times in TNT for 45 min, treated with 3% BSA in TNT, and incubated for 16 h with rabbit polyclonal antiserum (1:250 dilution in 3% BSA) prepared against cucumber expansin (Cu-EXP1, Li et al. 1993 ). After three washes in TNT, sections were incubated with antirabbit immunoglobulin G alkaline phosphatase conjugate (Sigma) for 2 h and washed three times in TNT and once in alkaline phosphatase buffer for 1 h. The color reaction, mounting and photography were performed as in the in situ mRNA hybridization experiments.
We thank Dr Daniel J. Cosgrove (Pennsylvania State University) for the generous gift of the cucumber expansin antibody. This research was supported by grant no. IBN 9722915 from the National Science Foundation and grant no. DE-FG02–91ER20021 from the U.S. Department of Energy.