Planta
Planta (1988)173 : 500-508
9 Springer-Verlag 1988
Expression sites and developmental regulation of genes encoding (1--,3,1 4)-fl-glncanases in germinated barley G.I. McFadden t, B. Ahluwalia 2, A.E. Clarke 1 and G.B. Fincher z* 1 Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Parkville, Vic. 3052, and 2 Department of Biochemistry, La Trobe University, Bundoora, Vic. 3083, Australia
Abstract. Expression sites of genes encoding (1 ~3,1-,4)-fl-glucan 4-glucanohydrolase (EC 3.2.1.73) have been mapped in germinated barley grains (Hordeum vulgare L.) by hybridization histochemistry. A 32p-labelled cDNA (copy DNA) probe was hybridized to cryosections of intact barley grains to localize complementary mRNAs. No m R N A encoding (]~3,1---,4)-fl-glucanase is detected in ungerminated grain. Expression of (1--->3,1-,4)-fl-glucanase genes is first detected in the scutellum after 1 d and is confined to the epithelial layer. At this stage, no expression is apparent in the aleurone. After 2 d , levels of (1-->3,1~4)-fi-glucanase m R N A decrease in the scutellar epithelium but increase in the aleurone. In the aleurone layer, induction of (l~3,1--->4)-flglucanase gene expression, as measured by m R N A accumulation, progresses from the proximal to distal end of the grain as a front moving away from, and parallel to, the face of the scutellum. Key words: Aleurone - (1--->3,1--->4)-fl-Glucanase Hordeum (gene regulation) - Hybridization histochemistry - Scutellum Seed germination.
Introduction
An early event in the germination of barley is breakdown of the walls of the starchy endosperm cells (MacLeod et al. 1964; Gibbons 1980). Cellwall degradation is mediated by (1--*3,1--,4)-fl-Oglucan 4-glucanohydrolases (EC 3.2.1.73) (Woodward and Fincher 1982a, b) and other hydrolases, and allows access of c~-amylases and proteases to * To whom correspondence should be addressed Abbreviations: c D N A = copy D N A ; RNase - ribonuclease
their substrates within the endosperm cells. The site of synthesis of hydrolytic enzymes in germinating barley has been debated for almost a century (Brown and Morris 1890; Haberlandt 1890, 1914; MacLeod and Palmer 1966; Palmer 1982; Palmer and Duffus 1986). Various lines of evidence indicate that the hydrolytic enzymes are probably secreted from both the scutellum and the aleurone layer (Dickson and Shands 1941; Briggs 1972; Okamoto et al. 1980; Briggs and MacDonald 1983; Gibbons 1980, 1981; MacGregor etal. 1984; Mundy et al. 1985; Stuart et al. 1986). Degradation of the starchy endosperm first occurs adjacent to the scutellum (Brown and Morris 1890; Dickson and Shands 1941), but this is only circumstantial evidence that the hydrolases originate from the scutellum. Immunocytochemistry shows that ~amylase is located initially near the scutellum (Gibbons 1980, 1981), but because the enzyme was detected after secretion into the starchy endosperm, its cellular origin could not be determined. Both isolated scutella and aleurone layers from barley secrete 0~-amylase and (1 ~3,1 ~4)-fi-glucanases in vitro (MacGregor et al. 1984; Ranki and Sopanen 1984; Stuart et al. 1986). In addition, mRNAs encoding (1 ~3,1 ~4)-fl-glucanase and a-amylase are present in extracts of scutella and aleurone layers excised from germinating barley (Mundy et al. 1985). These observations indicate a role for both tissues in secretion of the hydrolases, but the possibility remains that preparations of isolated scutella are contaminated with aleurone cells (MacLeod and Palmer 1966; Palmer 1982; Palmer and Duffus 1986). Furthermore, it is not clear whether experiments with isolated tissue fragments truly reflect the behaviour of the tissues in vivo. In this study we have used a copy DNA (cDNA) encoding a cell wall-degrading (1 ~3,1-.4)-fl-glucanase (Fincher et al. 1986) to lo-
G.I. McFadden et al. : Developmental regulation of fl-glucanase genes in germinated barley
cate complementary m R N A transcripts in germinated barley grains. We find that the genes encoding (1-,3,1-~4)-fl-glucanase are expressed in both the scutellar epithelium and aIeurone cells, and we t:ave eIucidated the temporal and spatial regulation of the genes during germination.
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Material and methods
hardt's [Maniatis et al. 1982], 50 mM sodium-phosphate buffer, 0.75M NaC1, 5 r a M Naz-ethytenediaminetetraacetic acid [EDTA], 1 mg.ml - I ~onicated herring sperm D N A [type IV~ Sigma Chemical Co,, St. Louis, Mo., USA], 50% deianizcd formamide, pH 7.2) once at room temperature and once at 38 ~ C. Fro-hybridization was for 40 rain at 38 ~ C. Sections were washed briefly in IX SSC (standard saline citrate-0.15 M NaC1, 0.015 M sodium citrale, pH 72) and dehydrated in two ethanol washes. The dehydrated, pre-hybridized sections were stored at 4 ~ C in an ethanolic atmosphere for up to 2 d before application of the probe.
Germination of barley. Barley grams (Hordeum vulgare L. cv.
Hybridization and autoradiography. Aliquots (4 gl, containing
Himalaya) were supplied by the Agronomy Club, Washington State University, Pullman, USA. This grain sample had been used in previous work (Stuart et al. 1986) and the hull-less cultivar was chosen to facilitate section preparation. Grains were surface-sterilized in 0.2% (w/v) silver nitrate for 20 rain, washed thoroughly with 0.5 M NaC1 and sterile distilled water, and imbibed for 24 h in sterile water containing 100 gg. m l - 1 neomycin, 10 gg. ml - ~ chloramphenicol, 100 units- m l - ~ penicillin and 100 units.ml 1 nystatin (Hoy et al. 1981). Grains were blotted dry and incubated on filter paper moistened with the antibiotic solution, in darkness, for up to 4 d at 15~ C (Stuart and Fincher 1983). Seedling development is expressed as the time from spreading imbibed grains on the filter paper. The emergence of roots indicated that germination was complete atld.
approx. 5-105 cpm) of 32p-labelled c D N A probe in hybridization buffer were dispensed onto covershps (10 mm 2) and the pro-hybridized sections inverted onto the coverslips so that the sections were evenly covered, with no bubbles. The sections were hybridized at 42 ~ C in an atmosphere of 50% formamide for at least 20 h. The coverslips were detached by soaking the slides in 4X SSC for approx. 30 rain at room temperature, and the slides with the sections were rinsed m 2X SSC followed by 1X SSC at 38~ for 60 rain, and dehydrated in ethanol. Autoradiographs were prepared by laying X-ray film (Cronex M R F 32; Dupont, Wilmington, Dela., USA) directly on the slides for 3 d. Selected slides were also subjected to liquid emulsion autoradiography (Ilford Nuclear Research Emulsion, I1ford Scientific Products, Cheshire, UK) and exposed for 3 d. Sections were counterstained with 0.025% toluidine blue and mounted in Eukitt (Carl Zeiss, Freiburg, FRG). Whole sections and X-ray film autoradiographs were photographed with a Wild M400 Makroskop (Wild Heerbrugg, Heerbrugg, Switzerland). Liquid emulsion preparations were photographed with an Olympus BH2 microscope (Olympics Optical Co., Tokyo, Japan) and Nikon dark-field condenser (Nippon Kogaku K K . , Tokyo).
Preparation of cDNA probes. The (l ~3~1 -~4)-fl-glucanase c D N A contains 874 nucleotide pairs (excluding d G . d C tails) cloned in the PstI si.te of ptasmid pUC9 (Fincher et aL 1986). Plasmid D N A was prepared (Bimboim and Doly 1979) and a 734 nucIeotide-pair HinFl fragment of the (1 ~ 3 , i -~4)-fl-glucanase c D N A insert was recovered from low-melting-point agarose gels. The Hinfl fragment was used in preference to the large PstI fragmenl (approx. 760 nucleolide pairs; Fincher et al. 1986) because it contained no (dG-dC) tails. The c D N A fragment (25 ng) was labelled to a specific activity of 109-2 -109 c p m . p g - 1 wilh deoxycytidine 5'-[c(-szP]triphosphate using random sequence hexanucleotides (Feinberg and Vogelstein 1983; Amersham International plc, Amersham, Bucks., U K : Multiprime labelling system RPN 1601). Positive and negative hybridization controls of the plasmid bearing the (1--+3,1 ~4)-fl-glucanase insert and plasmid pUC9, respectively, were performed by dot-blot analysis on nitrocellulose filters. In other control hybridizations, a c D N A (776 nucleotide pairs) encoding the self-incompatibility protein $2 from Nicotiana alata (Anderson et al. 1986) was nick-translated (Rigby et al. 1977) to a specific activity of 10 a cpm-gg 1. Sectioning and pre-hybridization, Hybridization histochemistry was performed essentially as described by Cornish et al. (1987). Ten representative grains were selected at intervals of 0, 0.5, 1, 2, 3, and 4 d after the completion of imbibition, immersed in chilled OCT compound (Lab-Tek Products, Naperville, Ill., USA) in an embedding mould (Lab-Tek Products) and frozen in Freon 22 (Dupont, Nashville, Tenn., USA) cooled with liquid nitrogen. Frozen grains were stored for up to four weeks at --70 ~ C. Grains were sectioned (thickness- 8 ~xm) at --18 ~ C on a Reichert Jung Frigocut 2800N (Re/chert Jung G . m b . H . , Nusslich, FRG). The sections were thaw-mounted on stides coated with 1% gelatine hardened with 0.25% formaldehyde, rapidly refl-ozen on solid COz, and left to chill for at least 30 min. Brief fixation (5 l 0 rain in 2% glutaraldehyde, 0,15 M sodium-phosphate buffer, 33% ethylene glycol, pH 7,2, 4 ~ C) was followed by washing m hybridization buffer (5X Den-
Controls. Controls were employed 1o demonslrate lhat the observed labelling represented hybridizalion of the probe to complementary m R N A transcripts in the grains. To check preservation of RNA, pre-hybridized sections were stained for nucleic acids using the metachromatic fluorochrome acridine orange. Following pre-incubation for 5 rain in 0.2 M glycine, pH 2.0, the test sections were stained with acridine orange (1.5 mg. m l - 1 in glycine buffer) for 20 rain, rinsed extensively with glycine buffer and mounted in 0.1% phenylenediamine in PBS :glycerol (2:1, v/v). Acridine-orange-stained sections were viewed using UV epi-illumination with an Olympus CH fluorescence microscope (Olympus Optical Co.). Certain sections were also incubated with ribonuclease (RNase) A (Boehringer, Mannheim, F R G : 1 mg.ml ~ in 0.1 M 2-amino-2-(hydroxymethyl)-l,3propanediol [Tris], 2 mM MgCI2, pH 7.5) for 3 h at 37 ~ C. Digestion was halted by washing with 0.1 M Tris, 5 mM EDTA, pH 7.5. Elimination of R N A was verified by acridine-orange staining and the remaining sections were pre-hybridized and incubated with the probe as described above. As a negative control, sections were incubated with a c D N A encoding the self-incompatibility protein from the styles of Nieotiana alata (Anderson et al. 1986; Cornish eta[. 1987)
Results
Location of nucleic acids. Sections stained with acridine orange and examined by fluorescence microscopy are shown in Fig. 1. Acridine orange produces a yellow-green fluorescence with D N A and
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G.I. McFadden et al. : Developmental regulation of/?-glucanase genes in germinated barley
Fig. 1 a, b. Fluorescence micrographs of the distal aleurone layer in pre-hybridized sections of barley grain (2 d after imbibition) stained with acridine orange, x 450. a Cytoplasm of aleurone cells contains R N A as indicated by orange fluorescence. Nuclei exhibit green/yellow staining, indicating the presence of DNA. The residual walls of the pericarp cross cells exhibit orange fluorescence with acridine orange, while the walls of the aleurone cells autofluoresce blue because of the presence of ferulic acid (Bacic and Stone 1981). b After treatment with RNase, orange fluorescence in the cytoplasm of aleurone and in endosperm cells subtending the aleurone layer disappears. Fluorescence in the walls of the pericarp cross cells and aleurone is not removed by RNase. The green/yellow fluorescence of nuclear D N A is not eliminated by RNase treatment, although the staining is somewhat less intense ( x 300)
an orange fluorescence with R N A and acidic polysaccharides (Pearse 1972). The cytoplasm of aleurone cells and the subtending starchy endosperm cells contains RNA, as indicated by the orange fluorescence (Fig. 1 a). Nuclei are characterized by a bright-yellow fluorescence, and again can be ob-
served both in the aleurone and in cells of the starchy endosperm (Fig. 1 a). After RNase treatment, the orange fluoresence in the cytoplasm is removed, but the yellow fluorescence of the nuclei remains, albeit at lower intensity (Fig. 1 b). These results indicate that the R N A in the sections is P
Fig. 2 a-I. Localization of mRNAs encoding (1 ~ 3,1 ~ 4)-fl-glucanases in germinating barley grains. Four representative germination stages are shown in the top panel. Near median longitudinal sections (middle panel, sections counterstained with toluidine blue) were taken from similar grains and hybridized with a 32p-labelled c D N A probe encoding (1 ~3,1~4)-/?-glucanase. Bound probe was detected by X-ray film autoradiography of the whole section (bottom panel), x 7 ; bar = 1.0 ram. a, b After 0 d germination the embryo (em) is rehydrated. The seutellum (sc) can be detected at the interface of the embryo and the starchy endosperm (en). c The probe binds diffusely over the entire section at low levels, d, e After 1 d germination, the coleoptile (white arrow) has begun to elongate, the roots have emerged (small arrows), and the starchy endosperm (en) is swollen, f A high level of probe is bound in the scutellum (sc). g, h After 2 d, the roots are well developed, the embryo (em) is enlarging, and the coleoptile (white arrow) is almost half the grain length, i High levels of probe bind to the proximal regions of the aleurone layer and binding is more advanced, distally, on the dorsal side (arrows). There is less binding of probe to the scutellum (sc) in 2-d grain than in 1-d-germinated grain (compare i and f). j, k After 4 d, the coleoptile is approximately two thirds the length of the grain (white arrow), and endosperm (en) dissolution is evident in the region adjacent to the embryo, l The probe binds at high levels to the proximal and medial aleurone layer on the ventral side and more distally on the dorsal side (black arrows) and also to the monolayer of aleurone surrounding the scutellum (open arrow). Probe binding in the scutellum (sc) is minimal at 4d
G.I. McFadden et al. : Developmental regulation of/~-glucanase genes in germinated barley
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G.I. McFaddenet al. : Developmentalregulation of fl-glucanasegenes in germinatedbarley
Fig. 3a-d. Control sections, x 12; bar=l mm. a Section from a 4-d grain treated with RNase prior to hybridization with the (1-*3,1~4)-fl-glucanase probe, b X-ray film autoradiograph of the section in a. Concentrations of the probe normally observed in the aleurone at this stage (see Fig. 21) are eliminated by RNase treatment. Only background levels of probe, adsorbed nonspecificallyto the section, remain, e Sectionof 4-d-germinated grain hybridizedwith a cDNA probe encodingthe stylar glycoprotein Sz. d X-ray film autoradiograph of the section in c. The Sz cDNA probe binds over the entire section at levels equivalent to the usual background (compare Figs. 2c and 3b)
not substantially degraded by exogenous or endogenous nucleases during sectioning and pre-hybridization. Similar results were obtained with sections of the scutellum (not illustrated). Acridine-orange staining of grain sections demonstrated the presence of D N A and R N A not only in the aleurone layer, but also in cells of the subaleurone endosperm. Although starchy endosperm cells become metabolically inactive during the final stages of grain maturation, nucleic acids or their partial degradation products remain in these cells in the mature grain (Fig. 1) and are likely to be the substrates for nucleases which are secreted from barley aleurone layers (Chrispeels and Varner 1967; Brown and Ho 1986).
Extent of germination. The rate of germination and development of individual grains in the barley sample used was relatively uniform when assessed by coleoptile length. After incubating the grain for 1 d, seedling roots had emerged and coleoptile extension was detected (Fig. 2d). After 2 d the cole-
optile had extended to almost half the length of the grain (Fig. 2g) and at 4 d to more than half the length of the grain (Fig. 2j). At this stage, endosperm dissolution is visible in the region adjacent to the scutellum (Fig. 2h) and this dissolution is more advanced at 4 d (Fig. 2 k).
Binding pattern of the (l ~3,1~4)-fl-glucanase cDNA probe to germinating grains. To assess the distribution of (1--+3,1~4)-fl-glucanase m R N A during germination and seedling development, a total of 250 sections, from grains incubated for up to 4 d, were probed with the (1 --,3,1 ~4)-fi-glucanase cDNA. Autoradiographs of sections prepared to give an overview of probe binding sites in the whole grain are shown in Fig. 2. In imbibed but ungerminated grain (0 d), lowlevel, diffuse binding of the probe is observed (Fig. 2a~z). After 1 d germination, bound probe is concentrated in the region of the scutellum (Fig. 2d f). After 2 d, levels of probe bound to the scutellum decrease, particularly toward the
G.I. McFadden et al. : Developmental regulation of/?-glucanase genes in germinated barley
505
Fig. 4a-f. Identification of barley grain cells containing mRNAs encoding (1 ~3,1 --*4)-fl-glucanases by hybridization histochemistry and dark-field liquid emulsion autoradiography, a, h, and e, d depict higher magnifications of the boxed areas in Fig. 2e and 2h ( x 7) respectively, a The layer of cells comprising the scutellar epithelium (se) is situated between the scutellar parenchyma (se) and the starchy endosperm (en). x 200; bar = 100 pro. h Dark-field illumination of the area depicted in a shows an accumulation of silver grains over the cells of the scutellar epithelium, indicating hybridization of the probe in these cells, e The two- to three-cell-thick aleurone layer (al) is situated between the layer of empty cells comprising the pericarp testa (pt) and the subtending starchy endosperm (en). x 300; b a r = 30 lain. tl Dark-field illumination of the aleurone layer from e shows hybridization of the probe to aleurone cells, e Proximal end of a 2-d grain sectioned perpendicular to the dorso-ventral plane and hybridized with the (1~3,1 ~4)-,8-glucanase probe. In this orientation, the monolayer of aleurone cells (a/) that attaches to the scutellum (sc) is visible. The scutellar epithelium (se) is situated between the depleted zone (dz) of the starchy endosperm (en) and the scutellar parenchyma (sc). x 20; bar = 200 I~m. f The X-ray film autoradiograph shows that the probe hybridizes to the monolayer of aleurone cells behind the scutellum (arrows), as well as to the scutellar epithelium and the proximal aleurone adjacent to the starchy endosperm
ventral side (Fig. 2 g-i), but high levels of binding to the proximal aleurone layer become apparent (Fig. 2i). Relatively little probe binds to the scutellum after 4 d, while binding in the aleurone layer has advanced toward the distal end (Fig. 2j-l).
Probe-binding in the aleurone layer is consistently more advanced toward the distal end on the dorsal side of the grains (Fig. 2i, 1). By probing consecutive sections from a single grain and sections cut at various orientations, it was found that
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G.I. McFaddenet al. : Developmentalregulationof fl-glucanasegenesin germinatedbarley
this pattern is three-dimensionally consistent (not illustrated). The progression of probe binding from proximal to distal regions of the aleurone is correlated with the extent of elongation of the coleoptile (Fig. 2).
Control sections. In control sections (4-d seedlings) pre-incubated with ribonuclease before hybridization, concentrations of the cDNA probe which normally bind to the aleurone layer at this stage are reduced to background levels (Fig. 3 a, b; compare Fig. 2k, 1). Similarly, a probe prepared from cDNA encoding the $2 self-incompatibility glycoprotein from Nicotiana aIata (Anderson et al. 1986) binds to sections of 4-d seedlings at background levels (Figs. 3c, d; compare Fig. 2k, 1). However, probe associated with folds or other imperfections in the sections is not eliminated by RNase treatment (not illustrated). These controls establish that the cDNA probe is binding to RNA and that the binding is specific for barley (1 -,3,1 -,4)-fi-glucanase mRNA. Identification of cells accumulating (1 -,3,1 -,4 )-figlucanase mRNA. In order to identify the cells in which the (l-,3,1-,4)-fl-glucanase cDNA probe was bound, sections from selected stages of development were subjected to liquid-emulsion autoradiography and examined at higher magnification. When viewed under dark-field illumination, concentrations of bound probe appear as accumulations of white dots. In 1-d germinated grain, the probe binds to the scutellum, but is restricted to the single layer of scutellar epithelial cells which lies at the interface of the scutellum and the endosperm (Fig. 4a, b). In proximal regions of the endosperm of grain incubated for 2 d, cDNA binding is concentrated in the aleurone cells (Fig. 4c, d). Longitudinal sections cut perpendicular to the dorso-ventral plane show that substantial levels of probe bind to the monolayer of aleurone cells that extends past the scutellum (Fig. 4 e, f). Discussion
To identify cells in which (1 43,1 -,4)-fl-glucanase genes are expressed in intact germinated barley grains, sections were probed with 32p-labelled (l-,3,1-,4)-fl-glucanase cDNA (Fincher etal. 1986). For this discussion, we refer to "gene expression" simply as the accumulation of (l-,3,1-,4)-fl-glucanase mRNA, as measured by hybridization histochemistry. The binding pattern of the probe at different stages of germination and seedling development indicates differential expres-
sion of the genes encoding (1-,3,1-,4)-fl-glucanase. No (1-,3,1-,4)-fl-glucanase mRNA is detected in grain immediately after imbibition (Fig. 2c), and no (1-,3,1-,4)-fl-glucanase activity is present at this stage (Stuart and Fincher 1983). This indicates that the (1-,3,1-,4)-fl-glucanase mRNA is transcribed de novo during germination. Expression of the (1 -,3,1 -,4)-fl-glucanase genes is first observed in the scutellum, and at I d germination this tissue is likely to be the major source of (1-,3,1-,4)-fl-glucanase in the grain. Marked expression of the (1 -,3,1 -,4)-fl-glucanase genes in the aleurone layer is not detected until 2 d, and at this stage the level of expression in the scutellum is decreasing. Our observations indicate that induction of (1 -,3,1 -,4)-fl-glucanase gene expression in the aleurone layer progresses from the proximal to distal end of the grain as a front moving away from, and parallel to, the face of the scutellum. This pattern corresponds to the sequence of both endosperm cell-wall breakdown and e-amylase secretion in germinating grains (Briggs 1972; Gibbons 1980, 1981 ; Palmer 1982). Such a pattern indicates that the signal inducing expression of (1 -,3,1 -,4)-fl-glucanase genes is derived from the embryo and diffuses through the germinating grain. This signal is likely to be the phytohormone gibberellin, which is known to enhance both the levels of translatable (1 -,3,1-,4)-fl-glucanase mRNA and the amount of enzyme secreted from isolated aleurone layers (Mundy and Fincher 1986; Stuart et al. 1986). Thus, (l~3,1-,4)-fl-glucanase genes are expressed in both the scutellum and aleurone layer of the germinated barley grain, and also in the monolayer of aleurone cells surrounding the scutellure (Fig. 4; see also Nieuwdorp 1963; Palmer 1982). Gene expression in the scutellum is confined to the single layer of epithelial cells (Fig. 4a, b). This epithelial layer appears to perform a secretory function early in germination, but later becomes predominantly absorptive (see Nieuwdorp and Buys 1964; Gram 1982). Hybridization histochemistry confirms earlier observations that (1-,3,1~4)-fl-glucanase mRNA can be extracted from both tissues in germinated barley samples (Mundy et al. 1985). Stuart et al. (1986) showed that (1-,3,1-,4)-flglucanase isoenzyme I (Woodward and Fincher 1982a, b) is secreted predominantly from isolated scutella, together with a previously undetected protein, designated isoenzyme III. Isoenzyme II is secreted only from aleurone layers (Stuart et al. 1986). Here, we are unable to distinguish mRNAs encoding the (l--,3,1--,4)-fl-glucanase isoenzymes. The
G.I. McFadden et al. : Developmental regulation of fl-glucanase genes in germinated barley
cDNA probe used encodes isoenzyme II (Fincher et al. 1986), and although the sequence homology of the 40 amino acids at the NHz-terminus of isoenzymes I and II is more than 90% (Woodward et al. 1983), the degree of sequence homology at the nucleotide level in the two genes is not yet known. When a cDNA encoding isoenzyme I becomes available and the origin and nature of isoenzyme III are clarified, hybridization histochemistry might be used to define the tissue location and developmental regulation of mRNAs encoding specific (1 ~ 3,1 ~4)-fl-glucanase isoenzymes. In summary, we have identified the expression sites of (1 43,1-,4)-fl-glucanase genes in sections of intact, germinated barley grains by hybridization histochemistry; the study shows that gene expression is subject to temporal and tissue-specific control. We are grateful to Mr. Michael Hansford (School of Botany, University of Melbourne) for assistance in sectioning, and to Ms. Ingrid Bonig (Plant Cell Biology Research Centre) for her expert advice. We thank Dr. John Coghlan and Mrs. Jenny Penschow (Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne) and Professor B.A. Stone (Department of Biochemistry, La Trobe University) for discussion and advice during this study. G.McF. is a Queen Elizabeth II Research Fellow in the Plant Cell Biology Research Centre. This work was supported by grants from the Australian Research Grants Scheme and the Commonwealth Tertiary Education Commission to G.B.F.
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and abscisin in the aleurone layers of barley. Plant Physiol. 42, 1008-1026 Cornish, E.C., Pettitt, J.M., Bonig, I., Clarke, A.E. (1987) Developmentally controlled expression of a gene associated with self-incompatibilityin Nicotiana alata. Nature 326, 99-102 Dickson, J.G., Shands, H.L. (1941) Cellular modification of the barley kernel during malting. Proc. Amer. Soc. Brew. Chem., 1-10 Feinberg, A.P., Vogelstein, B. (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6-13 Fincher, G.B., Lock, P,A., Morgan, M.M., Lingelbach, K., Wettenhall, R.E.H., Mercer, J.F.B., Brandt, A., Thomsen, K.K. (1986) Primary structure of the (1 ~3,1 ~4)-fl-D-glucan 4-glucanohydrolase from barley aleurone. Proc. Natl. Acad. Sci. USA 83, 2081-2085 Gibbons, G.C. (1980) On the sequential determination of c~amylase transport and cell wall breakdown in germinating seeds of Hordeum vulgare. Carlsberg Res. Commun. 45, 172184
Gibbons, G.C. (1981) On the relative role of the scutellum and aleurone in the production of hydrolases during germination of barley. Carlsberg Res. Commun. 46, 215-225 Gram, N.H. (1982) The ultrastructure of germinating barley seeds. I. Changes in the scutellum and aleurone layer in Nordal barley. Carlsberg Res. Commun. 47, 143-162 Haberlandt, G. (1890) Die Kleberschicht des Grasendosperms als Diastase ausscheidendes Drusengewebe. Bet. Dtsch. Bot. Ges. 8, 4048 Haberlandt, G. (1914) Physiological plant anatomy (Engl. transln.). McMillan and Co., London Hoy, J.L., Macauley, B.J., Fincher, G.B. (1981) Cellulases of plant and microbial origin in germinating barley. J. Inst. Brew. 87, 77-80 MacGregor, A.W., MacDonald, F.H., Mayer, C., Daussant, J. (1984) Changes in levels of c~-amylasecomponents in barley tissues during germination and early seedling growth. Plant Physiol. 75, 203-206 MacLeod, A.M., Duffus, J.M., Johnston, C.S. (1964) Development of hydrolytic enzymes in germinating grain. J. Inst. Brew. 70, 521-528 MacLeod, A.M., Palmer, G.H. (1966) The embryo of barley in relation to modification of the endosperm. J. Inst. Brew. 72, 580-589 Maniatis, T., Fritsch, E.F., Sambrook, J. (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., USA Mundy, J., Brandt, A., Fincher, G.B. (1985) Messenger RNAs from the scutellum and aleurone of germinating barley encode (1 ~3,1 ~4)-/~-glucanase, a-amylase and carboxypeptidase. Plant Physiol. 79, 867-871 Mundy, J., Fincher, G.B. (1986) Effects of gibberellic acid and abscisic acid on levels of translatable mRNA for (1--43,1~4)-fl-D-glucanase in barley aleurone. FEBS Lett. 198, 34%352 Nieuwdorp, P.J. (1963) Electron microscopic structure of the epithelial cells of the scutellum of barley. I. The structure of epithelial cells before germination. Acta Bot. Neerl. 12, 295-301 Nieuwdorp, P.J., Buys, M.C. (1964) Electron microscopic structure of the epithelial cells of the scutellum of barley. II. Cytology of the cells during germination. Acta Bot. Neerl. 13, 55%565 Okamoto, K., Kitano, H., Akazawa, T. (1980) Biosynthesis and excretion of hydrolases in germinating cereal grains. Plant Cell Physiol. 21, 201-204 Palmer, G.H. (1982) A reassessment of the pattern of endo-
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sperm hydrolysis (modification) in germinated barley. J. Inst. Brew. 88, 145-153 Palmer, G.H., Duffus, J.H: (1986) Aleurone or scutellar hydrolyric enzymes in malting. J. Inst. Brew. 92, 512-513 Pearse, A.G.E. (1972) Histochemistry: Theoretical and applied, vol. 1. ChurchilI Livingstone, Edinburgh Ranki, H., Sopanen, T. (1984) Secretion of a-amylase by the aleurone layer and the scutellum of germinating barley grain. Plant Physiol. 75, 710-715 Rigby, P.W.J., Dieckmann, M., Rhodes, C., Berg, P. (1977) Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113, 237-251 Stuart, I.M., Fincher, G.B. (1983) Immunological determination of (1-~3,1 ~4)-fi-D-glucan endohydrolase development in germinating barley (Hordeum vulgare). FEBS Lett. 155, 201-204 Stuart, I.M., Loi, L., Fincher, G.B. (1986) Development of
(l~3,1~4)-fl-D-glucan endohydrolase isoenzymes in isolated scutella and aleurone layers of barley (Hordeum vulgare). Plant Physiol. 80, 31(I-314 Woodward, J.R., Fincher, G.B. (1982a) Purification and chemical properties of two (l~3,1~4)-fl-D-gtucan endohydrolases from germinating barley. Eur. J. Biochem. 121, 663-669 Woodward, J.R., Fincher, G.B. (1982b) Substrate specificities and kinetic properties of two (1--,3,1-~4)-fl-D-glucan endohydrolases from germinating barley. Carbohydr. Res. 106, l l I 122 Woodward, J.R., Morgan, F.J., Fincher, G.B. (1982) Amino acid sequence homology in two (1 ~3,1 ~4)-fl-D-glucan endohydrolases from germinating barley (Hordeum vulgare). FEBS Lett. 138, 198-200 Received 6 July; Accepted 28 September 1987
Planta (1988)173 : 500-508
9 Springer-Verlag 1988
Expression sites and developmental regulation of genes encoding (1--,3,1 4)-fl-glncanases in germinated barley G.I. McFadden t, B. Ahluwalia 2, A.E. Clarke 1 and G.B. Fincher z* 1 Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Parkville, Vic. 3052, and 2 Department of Biochemistry, La Trobe University, Bundoora, Vic. 3083, Australia
Abstract. Expression sites of genes encoding (1 ~3,1-,4)-fl-glucan 4-glucanohydrolase (EC 3.2.1.73) have been mapped in germinated barley grains (Hordeum vulgare L.) by hybridization histochemistry. A 32p-labelled cDNA (copy DNA) probe was hybridized to cryosections of intact barley grains to localize complementary mRNAs. No m R N A encoding (]~3,1---,4)-fl-glucanase is detected in ungerminated grain. Expression of (1--->3,1-,4)-fl-glucanase genes is first detected in the scutellum after 1 d and is confined to the epithelial layer. At this stage, no expression is apparent in the aleurone. After 2 d , levels of (1-->3,1~4)-fi-glucanase m R N A decrease in the scutellar epithelium but increase in the aleurone. In the aleurone layer, induction of (l~3,1--->4)-flglucanase gene expression, as measured by m R N A accumulation, progresses from the proximal to distal end of the grain as a front moving away from, and parallel to, the face of the scutellum. Key words: Aleurone - (1--->3,1--->4)-fl-Glucanase Hordeum (gene regulation) - Hybridization histochemistry - Scutellum Seed germination.
Introduction
An early event in the germination of barley is breakdown of the walls of the starchy endosperm cells (MacLeod et al. 1964; Gibbons 1980). Cellwall degradation is mediated by (1--*3,1--,4)-fl-Oglucan 4-glucanohydrolases (EC 3.2.1.73) (Woodward and Fincher 1982a, b) and other hydrolases, and allows access of c~-amylases and proteases to * To whom correspondence should be addressed Abbreviations: c D N A = copy D N A ; RNase - ribonuclease
their substrates within the endosperm cells. The site of synthesis of hydrolytic enzymes in germinating barley has been debated for almost a century (Brown and Morris 1890; Haberlandt 1890, 1914; MacLeod and Palmer 1966; Palmer 1982; Palmer and Duffus 1986). Various lines of evidence indicate that the hydrolytic enzymes are probably secreted from both the scutellum and the aleurone layer (Dickson and Shands 1941; Briggs 1972; Okamoto et al. 1980; Briggs and MacDonald 1983; Gibbons 1980, 1981; MacGregor etal. 1984; Mundy et al. 1985; Stuart et al. 1986). Degradation of the starchy endosperm first occurs adjacent to the scutellum (Brown and Morris 1890; Dickson and Shands 1941), but this is only circumstantial evidence that the hydrolases originate from the scutellum. Immunocytochemistry shows that ~amylase is located initially near the scutellum (Gibbons 1980, 1981), but because the enzyme was detected after secretion into the starchy endosperm, its cellular origin could not be determined. Both isolated scutella and aleurone layers from barley secrete 0~-amylase and (1 ~3,1 ~4)-fi-glucanases in vitro (MacGregor et al. 1984; Ranki and Sopanen 1984; Stuart et al. 1986). In addition, mRNAs encoding (1 ~3,1 ~4)-fl-glucanase and a-amylase are present in extracts of scutella and aleurone layers excised from germinating barley (Mundy et al. 1985). These observations indicate a role for both tissues in secretion of the hydrolases, but the possibility remains that preparations of isolated scutella are contaminated with aleurone cells (MacLeod and Palmer 1966; Palmer 1982; Palmer and Duffus 1986). Furthermore, it is not clear whether experiments with isolated tissue fragments truly reflect the behaviour of the tissues in vivo. In this study we have used a copy DNA (cDNA) encoding a cell wall-degrading (1 ~3,1-.4)-fl-glucanase (Fincher et al. 1986) to lo-
G.I. McFadden et al. : Developmental regulation of fl-glucanase genes in germinated barley
cate complementary m R N A transcripts in germinated barley grains. We find that the genes encoding (1-,3,1-~4)-fl-glucanase are expressed in both the scutellar epithelium and aIeurone cells, and we t:ave eIucidated the temporal and spatial regulation of the genes during germination.
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Material and methods
hardt's [Maniatis et al. 1982], 50 mM sodium-phosphate buffer, 0.75M NaC1, 5 r a M Naz-ethytenediaminetetraacetic acid [EDTA], 1 mg.ml - I ~onicated herring sperm D N A [type IV~ Sigma Chemical Co,, St. Louis, Mo., USA], 50% deianizcd formamide, pH 7.2) once at room temperature and once at 38 ~ C. Fro-hybridization was for 40 rain at 38 ~ C. Sections were washed briefly in IX SSC (standard saline citrate-0.15 M NaC1, 0.015 M sodium citrale, pH 72) and dehydrated in two ethanol washes. The dehydrated, pre-hybridized sections were stored at 4 ~ C in an ethanolic atmosphere for up to 2 d before application of the probe.
Germination of barley. Barley grams (Hordeum vulgare L. cv.
Hybridization and autoradiography. Aliquots (4 gl, containing
Himalaya) were supplied by the Agronomy Club, Washington State University, Pullman, USA. This grain sample had been used in previous work (Stuart et al. 1986) and the hull-less cultivar was chosen to facilitate section preparation. Grains were surface-sterilized in 0.2% (w/v) silver nitrate for 20 rain, washed thoroughly with 0.5 M NaC1 and sterile distilled water, and imbibed for 24 h in sterile water containing 100 gg. m l - 1 neomycin, 10 gg. ml - ~ chloramphenicol, 100 units- m l - ~ penicillin and 100 units.ml 1 nystatin (Hoy et al. 1981). Grains were blotted dry and incubated on filter paper moistened with the antibiotic solution, in darkness, for up to 4 d at 15~ C (Stuart and Fincher 1983). Seedling development is expressed as the time from spreading imbibed grains on the filter paper. The emergence of roots indicated that germination was complete atld.
approx. 5-105 cpm) of 32p-labelled c D N A probe in hybridization buffer were dispensed onto covershps (10 mm 2) and the pro-hybridized sections inverted onto the coverslips so that the sections were evenly covered, with no bubbles. The sections were hybridized at 42 ~ C in an atmosphere of 50% formamide for at least 20 h. The coverslips were detached by soaking the slides in 4X SSC for approx. 30 rain at room temperature, and the slides with the sections were rinsed m 2X SSC followed by 1X SSC at 38~ for 60 rain, and dehydrated in ethanol. Autoradiographs were prepared by laying X-ray film (Cronex M R F 32; Dupont, Wilmington, Dela., USA) directly on the slides for 3 d. Selected slides were also subjected to liquid emulsion autoradiography (Ilford Nuclear Research Emulsion, I1ford Scientific Products, Cheshire, UK) and exposed for 3 d. Sections were counterstained with 0.025% toluidine blue and mounted in Eukitt (Carl Zeiss, Freiburg, FRG). Whole sections and X-ray film autoradiographs were photographed with a Wild M400 Makroskop (Wild Heerbrugg, Heerbrugg, Switzerland). Liquid emulsion preparations were photographed with an Olympus BH2 microscope (Olympics Optical Co., Tokyo, Japan) and Nikon dark-field condenser (Nippon Kogaku K K . , Tokyo).
Preparation of cDNA probes. The (l ~3~1 -~4)-fl-glucanase c D N A contains 874 nucleotide pairs (excluding d G . d C tails) cloned in the PstI si.te of ptasmid pUC9 (Fincher et aL 1986). Plasmid D N A was prepared (Bimboim and Doly 1979) and a 734 nucIeotide-pair HinFl fragment of the (1 ~ 3 , i -~4)-fl-glucanase c D N A insert was recovered from low-melting-point agarose gels. The Hinfl fragment was used in preference to the large PstI fragmenl (approx. 760 nucleolide pairs; Fincher et al. 1986) because it contained no (dG-dC) tails. The c D N A fragment (25 ng) was labelled to a specific activity of 109-2 -109 c p m . p g - 1 wilh deoxycytidine 5'-[c(-szP]triphosphate using random sequence hexanucleotides (Feinberg and Vogelstein 1983; Amersham International plc, Amersham, Bucks., U K : Multiprime labelling system RPN 1601). Positive and negative hybridization controls of the plasmid bearing the (1--+3,1 ~4)-fl-glucanase insert and plasmid pUC9, respectively, were performed by dot-blot analysis on nitrocellulose filters. In other control hybridizations, a c D N A (776 nucleotide pairs) encoding the self-incompatibility protein $2 from Nicotiana alata (Anderson et al. 1986) was nick-translated (Rigby et al. 1977) to a specific activity of 10 a cpm-gg 1. Sectioning and pre-hybridization, Hybridization histochemistry was performed essentially as described by Cornish et al. (1987). Ten representative grains were selected at intervals of 0, 0.5, 1, 2, 3, and 4 d after the completion of imbibition, immersed in chilled OCT compound (Lab-Tek Products, Naperville, Ill., USA) in an embedding mould (Lab-Tek Products) and frozen in Freon 22 (Dupont, Nashville, Tenn., USA) cooled with liquid nitrogen. Frozen grains were stored for up to four weeks at --70 ~ C. Grains were sectioned (thickness- 8 ~xm) at --18 ~ C on a Reichert Jung Frigocut 2800N (Re/chert Jung G . m b . H . , Nusslich, FRG). The sections were thaw-mounted on stides coated with 1% gelatine hardened with 0.25% formaldehyde, rapidly refl-ozen on solid COz, and left to chill for at least 30 min. Brief fixation (5 l 0 rain in 2% glutaraldehyde, 0,15 M sodium-phosphate buffer, 33% ethylene glycol, pH 7,2, 4 ~ C) was followed by washing m hybridization buffer (5X Den-
Controls. Controls were employed 1o demonslrate lhat the observed labelling represented hybridizalion of the probe to complementary m R N A transcripts in the grains. To check preservation of RNA, pre-hybridized sections were stained for nucleic acids using the metachromatic fluorochrome acridine orange. Following pre-incubation for 5 rain in 0.2 M glycine, pH 2.0, the test sections were stained with acridine orange (1.5 mg. m l - 1 in glycine buffer) for 20 rain, rinsed extensively with glycine buffer and mounted in 0.1% phenylenediamine in PBS :glycerol (2:1, v/v). Acridine-orange-stained sections were viewed using UV epi-illumination with an Olympus CH fluorescence microscope (Olympus Optical Co.). Certain sections were also incubated with ribonuclease (RNase) A (Boehringer, Mannheim, F R G : 1 mg.ml ~ in 0.1 M 2-amino-2-(hydroxymethyl)-l,3propanediol [Tris], 2 mM MgCI2, pH 7.5) for 3 h at 37 ~ C. Digestion was halted by washing with 0.1 M Tris, 5 mM EDTA, pH 7.5. Elimination of R N A was verified by acridine-orange staining and the remaining sections were pre-hybridized and incubated with the probe as described above. As a negative control, sections were incubated with a c D N A encoding the self-incompatibility protein from the styles of Nieotiana alata (Anderson et al. 1986; Cornish eta[. 1987)
Results
Location of nucleic acids. Sections stained with acridine orange and examined by fluorescence microscopy are shown in Fig. 1. Acridine orange produces a yellow-green fluorescence with D N A and
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G.I. McFadden et al. : Developmental regulation of/?-glucanase genes in germinated barley
Fig. 1 a, b. Fluorescence micrographs of the distal aleurone layer in pre-hybridized sections of barley grain (2 d after imbibition) stained with acridine orange, x 450. a Cytoplasm of aleurone cells contains R N A as indicated by orange fluorescence. Nuclei exhibit green/yellow staining, indicating the presence of DNA. The residual walls of the pericarp cross cells exhibit orange fluorescence with acridine orange, while the walls of the aleurone cells autofluoresce blue because of the presence of ferulic acid (Bacic and Stone 1981). b After treatment with RNase, orange fluorescence in the cytoplasm of aleurone and in endosperm cells subtending the aleurone layer disappears. Fluorescence in the walls of the pericarp cross cells and aleurone is not removed by RNase. The green/yellow fluorescence of nuclear D N A is not eliminated by RNase treatment, although the staining is somewhat less intense ( x 300)
an orange fluorescence with R N A and acidic polysaccharides (Pearse 1972). The cytoplasm of aleurone cells and the subtending starchy endosperm cells contains RNA, as indicated by the orange fluorescence (Fig. 1 a). Nuclei are characterized by a bright-yellow fluorescence, and again can be ob-
served both in the aleurone and in cells of the starchy endosperm (Fig. 1 a). After RNase treatment, the orange fluoresence in the cytoplasm is removed, but the yellow fluorescence of the nuclei remains, albeit at lower intensity (Fig. 1 b). These results indicate that the R N A in the sections is P
Fig. 2 a-I. Localization of mRNAs encoding (1 ~ 3,1 ~ 4)-fl-glucanases in germinating barley grains. Four representative germination stages are shown in the top panel. Near median longitudinal sections (middle panel, sections counterstained with toluidine blue) were taken from similar grains and hybridized with a 32p-labelled c D N A probe encoding (1 ~3,1~4)-/?-glucanase. Bound probe was detected by X-ray film autoradiography of the whole section (bottom panel), x 7 ; bar = 1.0 ram. a, b After 0 d germination the embryo (em) is rehydrated. The seutellum (sc) can be detected at the interface of the embryo and the starchy endosperm (en). c The probe binds diffusely over the entire section at low levels, d, e After 1 d germination, the coleoptile (white arrow) has begun to elongate, the roots have emerged (small arrows), and the starchy endosperm (en) is swollen, f A high level of probe is bound in the scutellum (sc). g, h After 2 d, the roots are well developed, the embryo (em) is enlarging, and the coleoptile (white arrow) is almost half the grain length, i High levels of probe bind to the proximal regions of the aleurone layer and binding is more advanced, distally, on the dorsal side (arrows). There is less binding of probe to the scutellum (sc) in 2-d grain than in 1-d-germinated grain (compare i and f). j, k After 4 d, the coleoptile is approximately two thirds the length of the grain (white arrow), and endosperm (en) dissolution is evident in the region adjacent to the embryo, l The probe binds at high levels to the proximal and medial aleurone layer on the ventral side and more distally on the dorsal side (black arrows) and also to the monolayer of aleurone surrounding the scutellum (open arrow). Probe binding in the scutellum (sc) is minimal at 4d
G.I. McFadden et al. : Developmental regulation of/~-glucanase genes in germinated barley
503
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G.I. McFaddenet al. : Developmentalregulation of fl-glucanasegenes in germinatedbarley
Fig. 3a-d. Control sections, x 12; bar=l mm. a Section from a 4-d grain treated with RNase prior to hybridization with the (1-*3,1~4)-fl-glucanase probe, b X-ray film autoradiograph of the section in a. Concentrations of the probe normally observed in the aleurone at this stage (see Fig. 21) are eliminated by RNase treatment. Only background levels of probe, adsorbed nonspecificallyto the section, remain, e Sectionof 4-d-germinated grain hybridizedwith a cDNA probe encodingthe stylar glycoprotein Sz. d X-ray film autoradiograph of the section in c. The Sz cDNA probe binds over the entire section at levels equivalent to the usual background (compare Figs. 2c and 3b)
not substantially degraded by exogenous or endogenous nucleases during sectioning and pre-hybridization. Similar results were obtained with sections of the scutellum (not illustrated). Acridine-orange staining of grain sections demonstrated the presence of D N A and R N A not only in the aleurone layer, but also in cells of the subaleurone endosperm. Although starchy endosperm cells become metabolically inactive during the final stages of grain maturation, nucleic acids or their partial degradation products remain in these cells in the mature grain (Fig. 1) and are likely to be the substrates for nucleases which are secreted from barley aleurone layers (Chrispeels and Varner 1967; Brown and Ho 1986).
Extent of germination. The rate of germination and development of individual grains in the barley sample used was relatively uniform when assessed by coleoptile length. After incubating the grain for 1 d, seedling roots had emerged and coleoptile extension was detected (Fig. 2d). After 2 d the cole-
optile had extended to almost half the length of the grain (Fig. 2g) and at 4 d to more than half the length of the grain (Fig. 2j). At this stage, endosperm dissolution is visible in the region adjacent to the scutellum (Fig. 2h) and this dissolution is more advanced at 4 d (Fig. 2 k).
Binding pattern of the (l ~3,1~4)-fl-glucanase cDNA probe to germinating grains. To assess the distribution of (1--+3,1~4)-fl-glucanase m R N A during germination and seedling development, a total of 250 sections, from grains incubated for up to 4 d, were probed with the (1 --,3,1 ~4)-fi-glucanase cDNA. Autoradiographs of sections prepared to give an overview of probe binding sites in the whole grain are shown in Fig. 2. In imbibed but ungerminated grain (0 d), lowlevel, diffuse binding of the probe is observed (Fig. 2a~z). After 1 d germination, bound probe is concentrated in the region of the scutellum (Fig. 2d f). After 2 d, levels of probe bound to the scutellum decrease, particularly toward the
G.I. McFadden et al. : Developmental regulation of/?-glucanase genes in germinated barley
505
Fig. 4a-f. Identification of barley grain cells containing mRNAs encoding (1 ~3,1 --*4)-fl-glucanases by hybridization histochemistry and dark-field liquid emulsion autoradiography, a, h, and e, d depict higher magnifications of the boxed areas in Fig. 2e and 2h ( x 7) respectively, a The layer of cells comprising the scutellar epithelium (se) is situated between the scutellar parenchyma (se) and the starchy endosperm (en). x 200; bar = 100 pro. h Dark-field illumination of the area depicted in a shows an accumulation of silver grains over the cells of the scutellar epithelium, indicating hybridization of the probe in these cells, e The two- to three-cell-thick aleurone layer (al) is situated between the layer of empty cells comprising the pericarp testa (pt) and the subtending starchy endosperm (en). x 300; b a r = 30 lain. tl Dark-field illumination of the aleurone layer from e shows hybridization of the probe to aleurone cells, e Proximal end of a 2-d grain sectioned perpendicular to the dorso-ventral plane and hybridized with the (1~3,1 ~4)-,8-glucanase probe. In this orientation, the monolayer of aleurone cells (a/) that attaches to the scutellum (sc) is visible. The scutellar epithelium (se) is situated between the depleted zone (dz) of the starchy endosperm (en) and the scutellar parenchyma (sc). x 20; bar = 200 I~m. f The X-ray film autoradiograph shows that the probe hybridizes to the monolayer of aleurone cells behind the scutellum (arrows), as well as to the scutellar epithelium and the proximal aleurone adjacent to the starchy endosperm
ventral side (Fig. 2 g-i), but high levels of binding to the proximal aleurone layer become apparent (Fig. 2i). Relatively little probe binds to the scutellum after 4 d, while binding in the aleurone layer has advanced toward the distal end (Fig. 2j-l).
Probe-binding in the aleurone layer is consistently more advanced toward the distal end on the dorsal side of the grains (Fig. 2i, 1). By probing consecutive sections from a single grain and sections cut at various orientations, it was found that
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G.I. McFaddenet al. : Developmentalregulationof fl-glucanasegenesin germinatedbarley
this pattern is three-dimensionally consistent (not illustrated). The progression of probe binding from proximal to distal regions of the aleurone is correlated with the extent of elongation of the coleoptile (Fig. 2).
Control sections. In control sections (4-d seedlings) pre-incubated with ribonuclease before hybridization, concentrations of the cDNA probe which normally bind to the aleurone layer at this stage are reduced to background levels (Fig. 3 a, b; compare Fig. 2k, 1). Similarly, a probe prepared from cDNA encoding the $2 self-incompatibility glycoprotein from Nicotiana aIata (Anderson et al. 1986) binds to sections of 4-d seedlings at background levels (Figs. 3c, d; compare Fig. 2k, 1). However, probe associated with folds or other imperfections in the sections is not eliminated by RNase treatment (not illustrated). These controls establish that the cDNA probe is binding to RNA and that the binding is specific for barley (1 -,3,1 -,4)-fi-glucanase mRNA. Identification of cells accumulating (1 -,3,1 -,4 )-figlucanase mRNA. In order to identify the cells in which the (l-,3,1-,4)-fl-glucanase cDNA probe was bound, sections from selected stages of development were subjected to liquid-emulsion autoradiography and examined at higher magnification. When viewed under dark-field illumination, concentrations of bound probe appear as accumulations of white dots. In 1-d germinated grain, the probe binds to the scutellum, but is restricted to the single layer of scutellar epithelial cells which lies at the interface of the scutellum and the endosperm (Fig. 4a, b). In proximal regions of the endosperm of grain incubated for 2 d, cDNA binding is concentrated in the aleurone cells (Fig. 4c, d). Longitudinal sections cut perpendicular to the dorso-ventral plane show that substantial levels of probe bind to the monolayer of aleurone cells that extends past the scutellum (Fig. 4 e, f). Discussion
To identify cells in which (1 43,1 -,4)-fl-glucanase genes are expressed in intact germinated barley grains, sections were probed with 32p-labelled (l-,3,1-,4)-fl-glucanase cDNA (Fincher etal. 1986). For this discussion, we refer to "gene expression" simply as the accumulation of (l-,3,1-,4)-fl-glucanase mRNA, as measured by hybridization histochemistry. The binding pattern of the probe at different stages of germination and seedling development indicates differential expres-
sion of the genes encoding (1-,3,1-,4)-fl-glucanase. No (1-,3,1-,4)-fl-glucanase mRNA is detected in grain immediately after imbibition (Fig. 2c), and no (1-,3,1-,4)-fl-glucanase activity is present at this stage (Stuart and Fincher 1983). This indicates that the (1-,3,1-,4)-fl-glucanase mRNA is transcribed de novo during germination. Expression of the (1 -,3,1 -,4)-fl-glucanase genes is first observed in the scutellum, and at I d germination this tissue is likely to be the major source of (1-,3,1-,4)-fl-glucanase in the grain. Marked expression of the (1 -,3,1 -,4)-fl-glucanase genes in the aleurone layer is not detected until 2 d, and at this stage the level of expression in the scutellum is decreasing. Our observations indicate that induction of (1 -,3,1 -,4)-fl-glucanase gene expression in the aleurone layer progresses from the proximal to distal end of the grain as a front moving away from, and parallel to, the face of the scutellum. This pattern corresponds to the sequence of both endosperm cell-wall breakdown and e-amylase secretion in germinating grains (Briggs 1972; Gibbons 1980, 1981 ; Palmer 1982). Such a pattern indicates that the signal inducing expression of (1 -,3,1 -,4)-fl-glucanase genes is derived from the embryo and diffuses through the germinating grain. This signal is likely to be the phytohormone gibberellin, which is known to enhance both the levels of translatable (1 -,3,1-,4)-fl-glucanase mRNA and the amount of enzyme secreted from isolated aleurone layers (Mundy and Fincher 1986; Stuart et al. 1986). Thus, (l~3,1-,4)-fl-glucanase genes are expressed in both the scutellum and aleurone layer of the germinated barley grain, and also in the monolayer of aleurone cells surrounding the scutellure (Fig. 4; see also Nieuwdorp 1963; Palmer 1982). Gene expression in the scutellum is confined to the single layer of epithelial cells (Fig. 4a, b). This epithelial layer appears to perform a secretory function early in germination, but later becomes predominantly absorptive (see Nieuwdorp and Buys 1964; Gram 1982). Hybridization histochemistry confirms earlier observations that (1-,3,1~4)-fl-glucanase mRNA can be extracted from both tissues in germinated barley samples (Mundy et al. 1985). Stuart et al. (1986) showed that (1-,3,1-,4)-flglucanase isoenzyme I (Woodward and Fincher 1982a, b) is secreted predominantly from isolated scutella, together with a previously undetected protein, designated isoenzyme III. Isoenzyme II is secreted only from aleurone layers (Stuart et al. 1986). Here, we are unable to distinguish mRNAs encoding the (l--,3,1--,4)-fl-glucanase isoenzymes. The
G.I. McFadden et al. : Developmental regulation of fl-glucanase genes in germinated barley
cDNA probe used encodes isoenzyme II (Fincher et al. 1986), and although the sequence homology of the 40 amino acids at the NHz-terminus of isoenzymes I and II is more than 90% (Woodward et al. 1983), the degree of sequence homology at the nucleotide level in the two genes is not yet known. When a cDNA encoding isoenzyme I becomes available and the origin and nature of isoenzyme III are clarified, hybridization histochemistry might be used to define the tissue location and developmental regulation of mRNAs encoding specific (1 ~ 3,1 ~4)-fl-glucanase isoenzymes. In summary, we have identified the expression sites of (1 43,1-,4)-fl-glucanase genes in sections of intact, germinated barley grains by hybridization histochemistry; the study shows that gene expression is subject to temporal and tissue-specific control. We are grateful to Mr. Michael Hansford (School of Botany, University of Melbourne) for assistance in sectioning, and to Ms. Ingrid Bonig (Plant Cell Biology Research Centre) for her expert advice. We thank Dr. John Coghlan and Mrs. Jenny Penschow (Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne) and Professor B.A. Stone (Department of Biochemistry, La Trobe University) for discussion and advice during this study. G.McF. is a Queen Elizabeth II Research Fellow in the Plant Cell Biology Research Centre. This work was supported by grants from the Australian Research Grants Scheme and the Commonwealth Tertiary Education Commission to G.B.F.
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