The Plant Cell, Vol. 4, 99-1 10, January 1992 O 1992 American Society of Plant Physiologists

Patterns of Soybean Proline-Rich Protein Gene Expression

Robert E. Wyatt, Ron T. Nagao, and Joe L. Key' Department of Botany, University of Georgia, Athens, Georgia 30602

The expression patterns of three members of a gene family that encodes proline-rich proteins in soybean (SbPRPs) were examined using in situ hybridization experiments. In most instances, the expression of SbPRP genes was intense in a limited number of cell types of a particular organ. SbPRPl RNA was localized in several cell types of soybean hypocotyls, including cells within the phloem and xylem. SbPRPl expression increased within epidermal cells in the elongating and mature regions of the hypocotyl; expression was detected also in lignified cells surrounding the hilum of mature seeds. SbPRP2 RNA was present in cortical cells and in the vascular tissue of the hypocotyl, especially cells of the phloem. This gene was expressed also in the inner integuments of the mature seed coat. SbPRP3 RNA was localized specifically to the endodermoid layer of cells surrounding the stele in the elongating region of the hypocotyl, as well as in the epider- mal cells of leaves and cotyledons. These data show that members of this gene family exhibit cell-specific expression. The members of the SbPRP gene family are expressed in different types of cells and in some cell types that also express the glycine-rich protein or hydroxyproline-rich glycoprotein classes of genes.

INTRODUCTION

Plant cells are unique among eukaryotes in that they are sur- rounded by a cell wall. Mature plant cells are surrounded by a rigid cell wall that varies in composition and structure among the many cell types. It is therefore important that the design and components of plant cell walls be under strict spa- tia1 and developmental controls. The cell walls of plants are composed of 4 0 % protein (Lamport, 1965). As several re- cent studies have demonstrated, these proteins have their own unique distribution patterns among the various organs, tissues, and cells of plants (Keller et al., 1988; Hong et al., 1989; Stiefel et al., 1990; Ye and Varner, 1991). To date, three primary classes of cell wall structural proteins have been characterized; all are encoded by small gene families (for reviews of cell wall proteins, see Cassab and Varner, 1988; Showalter and Rumeau, 1990). The hydroxyproline-rich gly- coproteins (HRGPs) include the extensins. The extensins contain a characteristic repeat of the peptide Ser-Pro4. Ex- tensins localize to the palisade epidermal and hourglass cells of soybean seed coat (Cassab and Varner, 1987) and the cambial cells and parenchymous cells of soybean stems (Ye and Varner, 1991). The glycine-rich proteins (GRPs) contain characteristic (Gly-X], motifs, where X often is glycine. The GRPs localize primarily to lignified cells of the xylem (Condit and Meagher, 1986; Keller et al., 1989; Ye and Varner, 1991). Their occurrence in cells with highly specialized secondary walls probably reflects some unique aspect of their function. The third class of cell wall proteins, the proline-rich proteins (PRPs), are composed of a number of Pro-Pro-Val-X-Lys repeats, where X is usually glutamic acid or tyrosine (Hong et al., 1987, 1990; Datta et al., 1989). Hong et al. (1989), using RNA gel blot and dot blot hybridization analyses, demon-

To whom correspondence should be addressed.

strated the developmental regulation of the soybean PRP (SbPRP) gene family by analyzing various organs at different stages of development. Collectively, the distinct patterns of regulation and localization of cell wall proteins and their mRNAs indicate that they may play important roles in the de- velopment, structure, and function of the cell walls of particu- lar cells within the body of the plant.

In the studies reported here, the primary objective was to determine the cellular distribution patterns of the SbPRP mRNAs and to compare those patterns with the known local- izations of the HRGP and GRP cell wall proteins or their respective mRNAs. Gene-specific probes were used to inves- tigate the in situ localization of the SbPRP mRNAs. The results of this study demonstrated that SbPRP gene expres- Sion is regulated in a cell-specific as well as in a developmen- tal fashion, that the SbPRP mRNAs are highly localized in several different cell types of young soybean seedlings and mature plants, and that members of the SbPRP gene family are differentially expressed throughout the plant body. The different SbPRP genes may be expressed individually in sev- era1 different cell types or collectively at different times during the development of a particular tissue or cell type.

RESULTS

Spatial Expression of the SbPRP Gene Family in Soybean Hypocotyls

The developmental regulation of the SbPRP gene family has been determined by analyzing various organs at different stages of development (Hong et al., 1989). The data showed

100 The Plant Cell

that the overall organ expression pattern of the SbPRPs differed markedly from the extensins. The specific localiza- tions of the HRGP and GRP families of cell wall proteins and mRNAs (Cassab and Varner, 1987; Keller et al., 1989; Stiefel et al., 1990; Ye and Varner, 1991) are better known than those of the PRP family of genes. In situ hybridization experiments using gene-specific probes permitted observation of the ex- pression patterns of the individual members of the SbPRP gene family. Fixed, frozen sections of 4-day-old soybean seedlings were hybridized with single-stranded 35S-RNA gene-specific probes transcribed from the 3’ untranslated re- gion of the various SbPRP genes.

SbPRPl mRNA Localization

As seen in Figure 1, the probe recognizing SbPRPl mRNA localized primarily to the vascular and epidermal regions of hypocotyl sections. Cross-sections of the hypocotyl (stem) of young soybean seedlings were taken from the apical region (near the hook), the elongating region (area of cell expan- sion), and the mature region (near the base of the hypocotyl) and were hybridized in situ and examined. Bright-field micro- graphs show that hybridization grains (seen as dark areas or grains over unstained sections) are evident over cells in both xylem and phloem tissues of the stele (Figures lA, 16, lD, and 1F). Most of the cells in the vascular tissue had SbPRP1 mRNA, whereas RNase-treated or sections hybridized with SbPRPl sense-strand probes showed only background sig- na1 grains (data not shown). Hybridization signals became evident over epidermal cells of sections from the elongation region (Figure 1C) and over epidermal as well as a few layers of adjacent cortical cells of sections from the mature hypo- cotyl region (Figure 1E). The timing and localization of SbPRPl gene expression in seedling hypocotyls also cor- related with the transition of hypocotyl cells from elongating growth to more rigid, maturing tissues.

SbPRP2 mRNA Localization

Figure 2 shows the distribution of SbPRP2 mRNAs in cross- sections taken from the apical, elongating, and mature regions of soybean seedling hypocotyls. The hybridization patterns obtained from anti-SbPRP2 RNA probes were dis- tinct from those of anti-SbPRP1 probes. Figures 28, 2D, and 2F show that within the vascular cylinder, SbPRP2 mRNAs were present only in small discrete regions. Figures 2A, 2C, and 2E show corresponding hypocotyl cross-sections stained only with toluidine blue. The localization of SbPRP2 to phloem tissue is more evident in the higher magnification and fluorescent micrographs shown in Figure 3 (comparing SbPRP gene expression). Figures 3A and 3C detail the clusters of cells expressing SbPRP2, whereas Figure 3D re- veals that the wall structure of the Same cells is apparently different from that of the immediately surrounding cells. In the

same sections from soybean hypocotyls, SbPRP2 mRNAs were also detected in pith and cortical parenchyma cells, al- though signals in the innermost layer of cortical cells (see large arrow, Figure 3A) and the outermost two or three layers of pith cells were barely detectable or absent. In the hypo- cotyl, both the SbPRPl and SbPRP2 genes were expressed in phloem and epidermal tissues.

SbPRP3 mRNA Localization

Figure 4 shows the localization of SbPRP3 mRNA. SbPRP3 mRNA was not detected in sections from the apical region of the hypocotyl (Figure 4A). SbPRP3 gene expression was first evident in the elongating region of the hypocotyl (Figure 4C). In this region, hybridization grains were evident primarily over the innermost layer of cortical cells, termed the “en- dodermoid layer” or “starch sheath (Esau, 1976). The walls of these cells in soybean are not suberized and lack a Casparian strip (Cumbie, 1960), distinguishing this tissue from the true endodermis tissue of the root. It is, however, morphologically distinct, and the walls of endodermal (en- dodermoidal) cells of other species may contain suberin or other modifications (Esau, 1976).

The expression pattern of the SbPRP3 gene in elongating hypocotyls was especially interesting when directly com- pared to SbPRP2 mRNA distribution, as shown in Figures 3A and 3B. SbPRP2 and SbPRP3 gene expression patterns were complementary within the elongating hypocotyl cortex. SbPRP2 was expressed in the majority of cortical cells, but not the innermost layer, whereas SbPRP3 mRNA was found primarily in this endodermoid layer. SbPRP3 expression in sections from the mature region increased in the remainder of the cortex and pith (Figure 4E), and, of the SbPRP genes, had a pattern of expression that most closely resembled the pattern of HRGP (extensin) gene family expression in seed- lings of comparable age, as shown by Ye and Varner (1991). Expression of SbPRP3 in the vascular region sections from the mature region was compared with SbPRPl expression in comparable tissue in Figures 3E and 3F.

SbPRP Gene Expression in Mature Organs

Figure 5 shows the in situ hybridizations of SbPRP1, SbPRP2, and SbPRP3 in severa1 tissues other than the hypocotyl. Two of the three SbPRP genes studied have been shown to be expressed in soybean seed coat (Hong et al., 1989). The in situ localization of SbPRPl mRNAs in maturing soybean seed coats revealed hybridization within a compact group of sclerid cells in the chalazal end of the seed near the hilum (Figure 5C). The walls of these cells are lignified (Fig- ure 5D) and contribute to the strength of the seed coat (Esau, 1976. RNA dot blot hybridization analyses showed that SbPRPl mRNA was expressed at a very high level in the seed coat when compared to either the level of SbPRP2

Figure 1. Localization of SbPRPI mRNAs in the Hypocotyl and Seed Coat of Soybean Seedlings.

(A) and (B) Bright-field micrographs of in situ hybridization of an anti-SbPRP1 mRNA probe with apical hypocotyl cross-sections of 4-day-oldseedlings.(C) and (D) Bright-field micrographs of in situ hybridization of an anti-SbPRP1 mRNA probe with elongating hypocotyl cross-sections of 4-day-oldseedlings.(E) and (F) Bright-field micrographs of in situ hybridization of an anti-SbPRP1 mRNA probe with mature hypocotyl cross-sections of 4-day-oldseedlings.(A), (C), and (E) Whole hypocotyl cross-sections. Bar in (A) = 400 \iM for (A), (C), and (E).(B), (D), and (F) The vascular region of hypocotyls. Bar in (B) = 800 nM for (B), (D), and (F).p, phloem; x, xylem.

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Figure 2. Localization of SbPRP2 mRNAs in Hypocotyls.

(A) Toluidine blue-stained, unhybridized cross-section of a soybean hypocotyl from the apical region.(B) In situ hybridization of an anti-SbPRP2 mRNA probe with the apical cross-section of a soybean hypocotyl.(C) Toluidine blue-stained, unhybridized cross-section of a soybean hypocotyl from the elongating region.(D) In situ hybridization of an anti-SbPRP2 mRNA probe with the elongating cross-section of a soybean hypocotyl.(E) Toluidine blue-stained, unhybridized cross-section of a soybean hypocotyl from the mature region.(F) In situ hybridization of an anti-SbPRP2 mRNA probe with the mature cross-section of a soybean hypocotyl.Bar in (A) = 400 nM for all sections.co, cortex; p, phloem; pp, pith parenchyma; x, xylem.

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Figure 3. Comparisons of SbPRPt, SbPRP2, and SbPRP3 mRNA Localizations in the Soybean Hypocotyl Vascular Region.

(A) Anti-SbPRP2 mRNA probe hybridizing over vascular tissue of a hypocotyl cross-section from the elongating region. Arrowhead indicatesthe endodermoid layer of cells. Arrows show hybridization over the phloem. Bar = 1000 nM.(B) Anti-SbPRP3 mRNA probe hybridizing with the endodermoid layer of cells of a hypocotyl cross-section from the elongating region. Arrowheadindicates the endodermoid layer of cells. Bar = 1000 nM.(C) Detail from (A). Arrowheads indicate hybridization grains over the phloem. Bar = 1000 nM.(D) UV fluorescent micrograph showing the structure of cells hybridizing with the anti-SbPRP2 mRNA probe (arrowheads). Bar = 1000 nM.(E) Anti-SbPRP3 mRNA probe hybridizing with vascular tissue from the mature region. Bar = 800 nM.(F) Anti-SbPRP1 mRNA probe hybridizing with vascular tissue from the mature region. Bar = 800 nM.co, cortex; v, vascular cylinder (stele); x, xylem.

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*/ •VFigure 4. Localization of SbPRP3 mRNAs in Soybean Hypocotyl and SbPRP2 and SbPRP3 mRNAs in Germinating Soybean SeedlingCotyledon.

(A) In situ hybridization of an anti-SbPRP3 mRNA probe with the apical cross-section of a soybean hypocotyl. Bar = 400 nM.(B) In situ hybridization of an anti-SbPRP3 mRNA probe with the longitudinal section of a soybean cotyledon. Bar = 500 nM.(C) In situ hybridization of an anti-SbPRP3 mRNA probe with the elongating cross-section of a soybean hypocotyl. Bar = 400 nM.(D) Higher magnification of the cotyledon. Bar = 500 |iM.(E) In situ hybridization of an anti-SbPRP3 mRNA probe with the mature cross-section of a soybean hypocotyl. Bar = 400 ^M.(F) In situ hybridization of an anti-SbPRP2 mRNA probe with the germinating soybean seedling cotyledon. Bar = 500 nM.ct, cotyledon; en, endodermoid layer; ep, epidermis; p, phloem; x, xylem.

Figure 5. Localization of SbPRPI, SbPRP2, and SbPRP3 mRNAs in Soybean Seeds and Mature Leaves.

(A) Cross-section through a mature soybean leaflet hybridized with an anti-SbPRP3 mRNA probe. Bar = 200 nM.(B) Higher magnification of (A). Bar = 200 |iM.(C) Hilum end of the mature soybean seed hybridized with an anti-SbPRP1 probe. Bar = 600 nM.(D) In situ hybridization of an anti-SbPRP3 mRNA probe with the seed coat showing no hybridization. The arrowhead indicates the lignified tra-cheids that hybridize with the anti-SbPRP1 probe. Bar = 600 nM.(E) In situ hybridization of an anti-SbPRP2 mRNA probe with the soybean seed coat. Bar = 600 nM.(F) High magnification of the seed coat hybridized with the anti-SbPRP2 mRNA probe. Bar = 600 |iM.a, aleurone; ct, cotyledon; ep, epidermal cells; h, hilum; hg, hourglass cells; sp, storage parenchyma; x, xylem.

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Figure 6. Hybridization of Anti-SbPRP2 mRNA Probe with Mature Soybean Leaflet.(A), (B), and (C) Cross-section of mid-veins; signal is evident over the vein in the cap of sclerenchyma cells.(D) Cross-section of smaller vein; arrowheads indicate intense hybridization over cytoplasm of cells,ep, epidermis; sc, sclerenchyma cap; x, xylem (tracheids). Bars = 500 nM.

mRNA in the same tissue or the level of SbPRPI mRNA inother tissues (Hong et al., 1989).

SbPRP2 mRNA localized to the inner integuments of theseed coat, primarily the aleurone layer and at a lower levelwithin the parenchymous cells of the degenerating en-dosperm (Figures 5E and 5F). No hybridization of any SbPRPmRNA was detected in the embryo. SbPRP2 mRNA waspresent at low levels, however, in mesophyll parenchymacells of germinating soybean seedling axis and cotyledons(Figure 4F). SbPRP3 expression was also detected in the

cotyledons of germinating soybean seedlings, primarily inthe upper epidermis (Figures 4B and 4D).

The leaves of 3-week-old soybean plants were also exam-ined for SbPRP gene expression. SbPRP3 mRNA was de-tected in the adaxial epidermal cells of the leaves (Figures 5Aand 5B) but not in the abaxial epidermis. As shown in Figure6, the SbPRP2 gene was strongly expressed in a cap of ligni-fied schlerenchyma cells that covers the vascular bundles ofsoybean leaflets (Figure 6D); expression was especially evi-dent over the midvein (Figures 6A, 6B, and 6C).

Proline-Rich Protein Gene Expression 107

DISCUSSION

PRP Gene Family Members Are Expressed in Diverse Cell Types

The complex patterns of localization of the SbPRP mRNAs in soybean tissues are summarized in Table 1. The cell- specific expression of the SbPRP genes in young soybean seedlings can be described as follows. The gene encoding SbPRPl is expressed in hypocotyl vascular tissue, xylem, and phloem. SbPRPl mRNA becomes highly abundant in epidermal cells of the elongating and mature regions of the seedling hypocotyl. SbPRPl mRNA also localizes to tra- cheids cells near the hilum of the seed coat.

SbPRPP mRNA is present in most, but not all, of the paren- chyma cells in the pith and cortex of seedling hypocotyls. In

Table 1. In Situ Expression of SbPRP Genes in Soybean

Gene 4-Day-Old Seedling Hypocotyla

SbPRPl SbPRP2 SbPRP3

Apical

eP co en x p pp

Elongating

SbPRPl SbPRPP SbPRP3

+ - - + + - + - - + + + - - - - +/- + Mature

SbPRPl SbPRP2 SbPRP3

Gene Other Tissuesb

Mature Leaf

ep(ab) ep(ad) me X P

SbPRPl - - - - - SbPRP2 - - +/- + - SbPRP3 - + - - -

Mature Seed

hg al SP co hi

- + SbPRPl - - - SbPRPP - + SbPRP3 -

+ I - - - - - - -

the seed coat, SbPRP2 localizes to cells of the aleurone layer of seeds. SbPRP2 mRNA is also present in the parenchyma cells of germinating seedling cotyledons. This is especially interesting because SbPRP2 expression occurs when the re- serve materials of these cells are being mobilized and further functioning of the cells of the aleurone or cotyledon is limited. The SbPRP3 gene is expressed primarily in the endoder- moid layer of cells surrounding the stele in the elongating re- gion of the hypocotyl and in parenchymous cells as well as the endodermoid layer of the mature region. The expression of SbPRP3 (and absence of SbPRPl and SbPRPP mRNAs) in the endodermoid layer of cells provides a distinct marker for this cell type. These cells mark the interface between the cortex and the vascular region, creating a physiological bar- rier at the stele (Esau, 1976).

At least one other gene, a phenylalanine-ammonia lyase (PAL) gene of bean, also exhibits strong and exclusive ex- pression in cells of the endodermis similar to SbPRP3 ex- pression. When attached to the P-glucuronidase reporter gene, the 5’ upstream region of the bean PAL gene conferred stem endodermis expression in tobacco (Bevan et al., 1989). The PAL gene product catalyzes the formation of a phenyl- propanoid precursor that has been correlated with the syn- thesis of lignin (Hammerschmidt, 1984) and suberin (Smith and Rubery, 1981). The cell walls of the hypocotyl endo- dermis of soybean are not thought to be suberized but may have other modifications. SbPRP3 mRNA also is present in the adaxial epidermis of soybean leaves; both of these der- mal cell types that express SbPRP3 may act as protective barriers. SbPRP3 mRNA was not detected in the abaxial epidermal cells. The lower leaf epidermal cells of soybean are smaller and have more convolutions or infoldings of the radial cell walls than the adaxial cells (Dzikowski, 1936) and are distinguishable from those of the upper epidermis.

Some of the cells in which SbPRP gene expression is found have only primary cell walls and are not lignified. The exceptions are SbPRP1 expression in the xylem and the tra- cheal bar at the micropylar area of the hilum and SbPRP2 ex- pression in the sclerenchyma of the leaf midrib and lignified cells within the phloem. These lignified cells express the respective SbPRP genes at a very high level. These data, as well as the complex expression patterns observed among the members of the SbPRP gene family, preclude a simple expla- nation of SbPRP function that correlates with cell wall forma- tion. The expression patterns of PRP genes in mature plant tissues (e.g., leaves or seed coats) and the absence of any detectable SbPRP expression in soybean embryos further complicate simple attempts to associate SbPRP expression patterns with known cell wall structure.

~~ ~

aep, epidermis; co, cortex; en, endodermis; x, xylem; p, phloem; pp, pithparenchyma. Cell Wall Proteins ep(ab), abaxial epidermis; ep(ad), adaxial epidermis; me, meso-

phyll; hg, hourglass cells; al, aleurone; SP, storage parenchyma; hi, hilum sclerids.

SbPRP mRNA Localiration 1s Distinct from Other

One of the initial observations on the cellular localization of structural cell wall proteins was the presence of HRGPs

108 The Plant Cell

(extensins) in soybean seed coats. The extensins were found primarily in the sclerified cells of the outer integuments, called “hourglass cells,” and in the palisade-like epidermal cells of the seed coat (Cassab and Varner, 1987). SbPRP2 mRNA localizes to the inner integuments, specifically the aleurone layer of cells that have thin primary cell walls. The cellular localization of the proteins and/or mRNAs of the HRGP and GRP genes has also been demonstrated by Keller et al. (1989), Stiefel et ai. (1990), Ye and Varner (1991), and others. Thus far, the occurrence of HRGPs and GRPs has shown the same developmental and tissue-specific patterns of distribution as their respective mRNAs. HGRP proteins and mRNAs are found in the meristematic cells of soybean stem cambium. Thus, HGRPs might be important in the early formation or assembly of the cell walls of expanding or malle- able cells. However, HRGPs also occur within the thick- walled, distinctive, hourglass-shaped cells of soybean seed coats. It is possible, therefore, that HRGPs could play differ- ent roles in these different cell types. The SbPRP genes are also expressed in cells surrounded by both thin primary and thickened secondary cell walls. In the elongating and mature regions, soluble HRGPs are present only in a few layers of the cortex around vascular bundles (Ye and Varner, 1991). The extensins also localize to some of the same cell types as the SbPRPs (e.g., pith-parenchyma and cortical) in the apical region of 2-day-old soybean hypocotyls (Ye and Varner, 1991). In more mature seedlings, extensins are present in a few layers of cortical cells surrounding the vascular cylinder. This same area of the cortex shows reduced expression of the SbPRP2 gene relative to expression in the remainder of the surrounding cortical parenchyma.

The GRPs are primarily associated with lignified cells in xy- lem tissue, e.g., the protoxylem of young soybean seedlings (Keller et al., 1989; Ye and Varner, 1991). Observations on the intramural location of the GRPs provide some understanding of the roles of cell wall structural proteins in cell wall form and function. For instance, the GRPs have a close association with the lignin portions of these cell walls (Keller et al., 1989). This pattern suggests that GRPs may influence the polymer- ization of lignin or direct the sites of lignin deposition by serv- ing as a scaffold (Condit and Keller, 1990). SbPRP mRNAs also are found in cells with lignified walls, but the intracellular localization of the proteins in these lignified cells is not yet known.

The proteins of the SbPRP gene family are under investi- gation. Averyhart-Fullard et al. (1988) isolated a PRP from soybean cell walls that has an amino acid composition identi- cal to SbPRPP and reported that 50% of the prolines are hydroxylated. Lindstrom and Vodkin (1991) recently isolated SbPRPl and showed that -50% of the proline residues are hydroxylated and that the second residue in the repeat motif is probably the modified proline. This SbPRP1 protein was isolated from soybean seed coat, and its abundance was shown to be correlated with an anthocyanin gene affecting seed coat color.

The presence of SbPRP1 and SbPRP2 mRNAs in cells of the hypocotyl, other than those of the vascular tissue (e.g., cortical, pith, and epidermal cells), indicated that the en- coded proteins may have a somewhat different role in cell wall structure than that of the GRPs, which are found primar- ily in lignified cells (Keller et al., 1989; Ye and Varner, 1991).

All of the cell wall structural protein gene families studied thus far show tissue- and/or cell-specific expression. The in- dividual members of the HRGP gene families studied are ex- pressed in similar cell types, as is also true of the GRP gene family. Ye and Varner (1991), using a combination of tissue printing and RNA gel blot analyses, showed that the HRGP genes are developmentally regulated within cambial cells; each gene within the family is expressed during a different period in a cell’s development, but it is expressed basically in the same cells. Expression of the GRP gene family seems to occur almost exclusively within vascular tissue, primarily in cells of the xylem, but this expression can also be found in the phloem (Ye and Varner, 1991). The SbPRP gene family members exhibit differential expression among the various types of cells. One member of the gene family may be ex- pressed in a certain cell type, whereas the other family mem- bers may not be expressed. One example is the expression of SbPRP3 in the endodermoid layer and the exclusion of SbPRP2 expression in the same cells. The SbPRPs vary in size and repeat organization/composition, and it seems rea- sonable to assume that this variation may affect the function of these proteins within the cell wall.

The results presented here show that the SbPRP genes are expressed in specific tissues, as are the HRGP and GRP gene families. The SbPRP genes are expressed in some of the same cell types as HRGPs and GRPs. We have shown that the different members of the SbPRP gene family are ex- pressed in different cell types and, therefore, may have differ- ent roles in cell wall formation and function than the other members of the SbPRP gene family and the other cell wall protein gene families. Further studies on the cellular and ul- trastructural localization of structural cell wall proteins may reveal the role they play in cell wall formation and structure.

METHODS

Plant Material

Soybean (Glycine max cv Wayne) seeds were germinated and grown in moist vermiculite at 28 to 3OoC in the dark. After 4 days, seedlings were harvested for localization studies. To obtain tissues from more mature plants, soybean seeds were planted and grown in the green- house. Hypocotyls of 4-day-old etiolated seedlings were divided into apical, elongating, and mature regions, where “apical” refers to the uppermost 0.5 cm of the seedling hypocotyl, including part of the api- cal hook, “elongating” refers to a 1-cm section of hypocotyl immedi- ately below the apical hook, and “mature” refers to sections obtained by taking 1 cm of tissue starting 2.5 cm below the apical hook. Other samples were obtained from the second set of trifolate leaves of

Proline-Rich Protein Gene Expression 109

3-week-old greenhouse-grown soybean plants. Developing seeds with attached seed coats were collected from older plants and staged. Fully mature green seeds (24 to 26 days postanthesis) were used for this study.

Fixation and Sectioning

Soybean seedlings were cut into 1-cm sections and fixed for 2 hr at room temperature as follows. A 4% paraformaldehyde, 0.25% glutaraldehyde, 50 mM Pipes buffer, pH 7.3, solution was phase parti- tioned into heptane (McFadden et al., 1988), and the heptanel glutaraldehyde phase was used for this initial fixation. Fixation was continued for 6 hr at room temperature under vacuum in aqueous 4% paraformaldehyde, 0.25% glutaraldehyde, 50 mM Pipes, pH 7.3. The fixed samples were washed three times for 15 min each with Pipes, pH 7.3, placed into Tissue Tek (Fisher Scientific, Pittsburgh, PA) freez- ing compound, and frozen in liquid nitrogen. Frozen embedded tis- sue was cut into 8-pm sections using a Frigocut cryostat (model2280; The Reichert Co., Vienna, Austria). The frozen sections were picked up on gelatin subbed Probe-on (Fisher Scientific) slides and attached to the slides by a 5-min incubation in fixative. Severa1 sections from different tissue sources were placed on each slide to more directly compare hybridization signals.

RNA Transcriptions

RNAs to be used as riboprobes were transcribed as recommended by Stratagene cloning systems. The DNA templates were gene- specific sequences obtained from the 3’ untranslated regions of the SbPRP genes, as described by Hong et al. (1989). The transcription reactions were carried out in 25 pL of 40 mM Tris-HCI, pH 8.0, 8 mM MgC12, 2 mM Spermidine, 50 mM NaCI, 400 pM ATP, 400 pM CTP, 400 pM GTP, 40 pM UTP, 30 mM Dithiothreitol, 2.5 pg DNA template, 50 pCi 35S-UTP, 10 units RNasin, and 10 units T3 or T7 polymsrase; the reaction was incubated at 37% for 30 min. The reaction mixtures were diluted 10-fold with 40 mM Tris-HCI, pH 7.5, 6 mM MgClp, 10 mM NaCI, and digested with 1 unit of RQI DNase (Promega Biotech) per microgram of DNAfor 30 minto remove the template. The control probes used included SbPRPl sense strand or transcribed vector. Sense strands transcribed from SbPRP2 and SbPRP3 gene-specific templates appeared to hybridize in situ to ribosomal RNA.

In Situ Hybridization

Before hybridization, selected control slides were treated with RNase to remove any hybridization signal by a 2-hr, 37°C incubation in 500 mM NaCI, 10 mM Tris-HCI, pH 8.0, 1 mM EDTA, 1 mg/mL RNase A. The remaining slides were incubated in the same buffer without the RNase. All of the slides were then extensively rinsed in 50 mM Tris- HCI, pH 8.0, 1 mM EDTA. The prehybridization and hybridization solution contained 50% deionized formamide, 300 mM NaCI, 10 mM Tris-HCI, pH 8.0, 1 mM EDTA, 10% dextran sulfate, 1 x Denhardt’s solution (0.02% Ficoll, 0.02% PVP, 0.02% BSA), 20 mM Dithiothreitol, and 500 pglmL yeast RNA; 150 pL of hybridization solution was placed into each slide chamber containing the sections and in- cubated in a moist slide container at 42OC. The slides were prehybrid- ized overnight, the prehybridization solution was then replaced with

hybridization solution containing 2.5 x 107 cpmM5O pL, and the slides were hybridized for 6 hr at 42OC.

In Situ Posthybridization Tmatments

After hybridization, the slides were washed for 2 hr at 42OC in 2 x SSC (1 x SSC is 0.15 M NaCI, 0.015 M sodium citrate). The unhybrid- ized RNA probes were removed by a 2-hr, 37% incubation in 500 mM NaCI, 10 mM Tris-HCI, pH 8.0, 1 mM EDTA, 0.2 mglmL RNase A. The slides were then washed for severa1 hours in 50% formamide, 10 mM Tris-HCI, pH 7.4, rinsed once in 2 x SSC, dehydrated through etha- nol, and air dried. The slides were emulsed in Kodak NTB-2 nuclear tract emulsion diluted 1:l and kept desiccated in the dark at room temperature for 2 weeks before development.

Microscopic Evaluation

After development of the emulsion, some slides were stained with 0.5% Toluidine blue or 4:6-diamidino-2-phenylindole to visualize nuclei. They were then dried and examined on a Zeiss microscope using bright-field, differential interference contrast, or epifluores- cence optics. Photomicrographs were taken with an MClOO camera (Carl Zeiss, Inc., Thornwood, NY) using Tech-Pan 4615 film (Kodak).

ACKNOWLEDGMENTS

We thank Dr. Barry Palevitz and Joyce Kochert for a critical reading of this manuscript. This research was supported in part by National lnstitutes of Health Grant GM30317 (to J.L.K.) and a McKnight Foun- dation Award for interdisciplinary research in plant biology.

Received July 10, 1991; accepted November 21, 1991.

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R E Wyatt, R T Nagao and J L KeyPatterns of soybean proline-rich protein gene expression.

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