A novel embryo-specific barley cDNA clone encodes a protein with homologies to bacterial glucose and ribitol dehydrogenase

Planta (1994)192:519-525

P l ~ n m 9 Springer-Verlag 1994

A novel embryo-specific barley eDNA clone encodes a protein with homologies to bacterial glucose and ribitol dehydrogenase Roland Alexander, Josefa M. Alamillo, Francesco Salamini, Dorothea Bartels Max-Planck-Institut f/ir Ziichtungsforschung, Carl-von-Linn&Weg 10, D-50829 K61n, Germany Received: 20 September 1993/ Accepted: 8 October 1993

Abstract. In order to analyze the genetic programme expressed during the early stages of embryogenesis a eDNA clone bank was constructed from desiccation-tolerant excised barley embryos 18d after pollination (D. Bartels et al., 1988, Planta 175, 485-492). One of the selected eDNA clones pG31 encodes a transcript of 1300 nudeotides and a protein of 31 kDa, both are specifically expressed in developing embryos and are not detected in other tissues. The expression of the pG31 mRNA is not modulated by the plant hormone eis-abscisic acid but it ceases to be expressed in germinating embryos. The protein sequence deduced from the pG31 transcript shows substantial sequence homologies to bacterial glucose dehydrogenase and ribitol dehydrogenase. Biochemical analysis indicates that glucose dehydrogenase activity is present in protein extracts from embroys 18d after pollination. This glucose dehydrogenase activity is inhibited by antiserum raised against the recombinant pG31 protein. These findings provide evidence for the discovery of a novel pathway in carbohydrate metabolism acting specifically during embryogenesis. Key words: Embryo specific transcript Glucose/ribitol dehydrogenase - Hordeum (embryo development)

Introduction Plant embryogenesis involves the commitment of cells to particular developmental programmes. Different stages of embryogenesis are characterized by the expression of specific sets of genes (Goldberg et al. 1989). Among the regulatory factors coordinating gene expression during embryogenesis the plant hormone eis-abscisic acid Abbreviations : ABA = cis-abscisic acid; DAP = days after pollination; GlcDH=glucose dehydrogenase; LEA=late embryogenesis abundant Correspondence to: D. Bartels; FAX: 49(221)5062413

(ABA) has been identified as one of the potential signals (Quatrano 1986; Black 1991). Many of the isolated embryo-specific genes are inducible by exogenous ABA, and studies of their promoters led to the identification of conserved ABA-responsive elements (Marcotte et al. 1989; Mundy et al. 1990; Lam and Chua 1991). One of the best-studied genes in this group is the Em gene of wheat (Cuming 1984; Litts et al. 1987). A putative transcription factor for this gene represents a leucine zipper protein which binds to the ABA-responsive elements of the Em gene (Guiltinan et al. 1990). Other factors mediating the activation of embryo maturation genes are represented by the maize Vpl gene product and the Arabidopsis homologue Abi3 which encode potential transcription activators (McCarty et al. 1991 ; Giraudat et al. 1992). Evidence that ABA levels are not the only factors regulating the embryogenesis programme comes from a careful examination of cotton embryogenesis which suggests that an unidentified maternal maturation factor and a post-abscission factor may be involved in the regulation of embryogenesis (Hughes and Galau 1991). A specific feature of embryogenesis is the acquisition of desiccation tolerance. We are using immature developing barley embryos as a model system to identify genes leading to desiccation tolerance. Embryos isolated from barley grains 12 d after pollination (DAP) do not germinate after a severe dehydration treatment ( < 10% water content), but desiccated 18-DAP embryos germinate and are defined as desiccation-tolerant embryos (Barrels et al. 1988). This change in the sensitivity to water loss occurs before the embryo starts to loose water and before most of the late-embryogenesis-abundant (LEA) genes are expressed (Galau et al. 1986; Dure et al. 1989; Skriver and Mundy 1990). In barley a subset of new gene products was observed when the protein expression of 18-DAP embryos was compared with that of 12-DAP embryos (Barrels et al. 1988). A cDNA bank was constructed from 18-DAP embryos and eDNA clones abundantly expressed in 18-DAP embryos were selected. One of these clones was modulated by ABA and displayed substantial structural homologies to a gene

520

R. Alexander et al.: A barley cDNA clone encodes a glucose dehydrogenase

family e n c o d i n g N A D P H - d e p e n d e n t aldose reductase (Bartels et al. 1991). In this p a p e r we report the characterization o f a second barley c D N A clone which is expressed in 1 8 - D A P e m b r y o s at similar levels as the aldose reductase m R N A ; in c o n t r a s t to the latter transcript a high expression level was already f o u n d in 1 2 - D A P embryos and exogenous A B A does not appreciably change its level o f transcription. The gene described here for the first time in plants is e m b r y o specific and encodes a protein with structural h o m o l o g y to bacterial glucose and ribitol dehydrogenase. Biochemical evidence is p r o v i d e d that in vivo the gene encodes a protein with glucose d e h y d r o g e n a s e ( G l c D H ) activity.

Materials and methods Plant material. Culture of barley (Hordeum vulgare L. cv. Aura) plants and isolation of stage-specific barley embryos are described by Bartels et al. (1988). If required, 10 -4 M ABA was added to the embryo culture (germination) medium (GM). Isolation of cDNA clones. A cDNA library was constructed using poly(A) § RNA from 18-DAP barley embryos. The library was differentially screened using probes from 12-DAP (not desiccation tolerant) and 18-DAP (desiccation tolerant) embryos (Bartels et al. 1991). For the isolation of full-length cDNA clones a second library was constructed in the lambda zap vector system (Stratagene, Heidelberg, Germany). Recombinant-DNA techniques. Isolation of plasmid DNA, preparation of DNA fragments, ligation and transformation of Eseherichia coli were carried out according to Maniatis et al. (1982). Barley RNA and DNA were isolated and used in Northern and Southern blot experiments as described by Bartels et al. (1991). The DNA probes were radioactively labelled according to Feinberg and Vogelstein (1983). Sequencin9 (?f DNA and computer analysis'. Nucleotide sequences were determined on both strands by subcloning of restriction fragments into pUC19 (Messing and Vieira 1982) followed by dideoxynucleotide sequencing with the T7 polyrnerase kit (Pharmacia LKB, Freiburg, Germany). The program WISGEN of the University of Wisconsin genetic's computer group (Madison, Wis., USA) was used for nucleic-acid and protein-sequence analysis (Devereux et al. 1984), and amino-acid comparisons were done with the TFASTA program (Pearson and Lipman 1988). Construction of E. coli expression clones, purification of recombinant fusion proteins and raisin9 of polyclonal antibodies. The insert of pG31 was ligated into the Smal site of the E. coli plasmid expression vector pGEX-2 (Smith and Johnson 1988) to yield a translational fusion with the glutathione-S-transferase gene (GST). The expression of the fusion protein was induced by the addition of isopropyl-~-D-thiogalactopyranoside (IPTG) to 0.4 mM. Purification of the fusion protein by preparative gel electrophoresis and electroelution, immunization and Western blot analysis were performed as described by Bartels et al. (1991). In-situ analysis'. The insert of pG31 was subcloned into the pBluescript plasmid vector. The recombinant cDNA clone was linearized and transcribed in sense and antisense directions in the presence of ct-[3sS]UTP (Transprobe SP Kit; Pharmacia LKB) using SP6- or T7-RNA-Polymerase. Further treatment of transcripts, preparation of sections, hybridization conditions and photomicroscopy were as described by Schneider et al. (1993). Determination of enzymatic activity. For assay of GlcDH activity, barley embryos were ground to a fine powder under liquid nitrogen.

Proteins were extracted with adequate amounts of 150 mM TrisHC1 buffer (pH 7.5). The suspension was centrifuged (I 1 500 - 9 for 15 rain at 4~C). The supernatent was desalted on a Sephadex G-50 column (medium, Pharmacia) and subsequently the proteins were concentrated using a Centricon microconcentrator. The protein concentration was determined according to Bradford (1976) using the BioRad assay kit. Glucose dehydrogenase (GlcDH) activity was assayed spectrophotometrically in the protein extract following the formation of NAD(P)H at 340 or 366 nm at 30~C. The standard assay mixture contained 150 !umol Tris, 50 lamol D-glucose, 5 p.mol NAD and the appropriate amounts of protein extracts in 1 ml. One unit of GIcDH activity was defined as the amount of enzyme producing 1 lamol NAD(P)H per minute.

Immunoprecipation. Aliquots of protein extracts from 18-DAP embryos were incubated overnight at 4~ with antiserum raised against the pG31 recombinant protein. When indicated the precipitated proteins were isolated with protein A-Sepharose.

Results Isolation o f a c D N A clone with homologies to GlcDH and ribitol dehydrogenase. A c D N A library was constructed f r o m p o l y ( A ) + R N A o f 1 8 - D A P desiccation-tolerant barley e m b r y o s (Bartels et al. 1991); a selected c D N A clone (pG31) encodes a m R N A a b u n d a n t l y expressed at 1 8 D A P . The nucleotide and deduced amino-acid sequence o f this clone are presented in Fig. 1. One possible o p e n reading frame predicts a protein o f 31 kDa. D a t a b a n k searches revealed regions o f homologies spread t h r o u g h o u t the coding region between pG31 and a representative bacterial G l c D H gene f r o m Bacillus megaterium and ribitol dehydrogenase f r o m Klebsiella aerogenes (Fig. 1). Out o f 256 c o m p a r e d a m i n o acids, 32 % and 25 % a m i n o acids, respectively, were identical to the barley pG31. Genomic Southern analysis and expression pattern o f the pG31 transcript and protein. Barley genomic D N A was digested with three different restriction enzymes and p r o b e d with the insert o f pG31. The hybridization pattern suggests that pG31 is represented in one to three copies in the barley g e n o m e (Fig. 2). N o r t h e r n blot experiments were performed to determine the tissue-specific and developmental expression o f the p G 3 1 - e n c o d e d transcript. The R N A was a b u n d a n t l y expressed in e m b r y o s but n o t in leaves, roots or endosperm. In the e m b r y o the pG31 transcript started to accumulate early during embryogenesis and increased during the m a t u r a t i o n phase (Fig. 3); at all stages a single transcript o f 1300 nucleotides was detected. I n c u b a t i o n o f 1 2 - D A P barley e m b r y o s with A B A did not affect the level o f pG31 transcripts, while precocious germination o f 12- and 18-DAP e m b r y o s led to a drastic decrease o f the pG31 m R N A level (Fig. 3). Expression in leaves could not be triggered by osmotic-stress treatments. Polyclonal antibodies raised against the pG31 recombinant protein were used to m o n i t o r the expression o f the pG31 protein during embryogenesis. Protein accumulation followed the R N A pattern and t h r o u g h o u t embryogenesis a protein o f 31 k D a was detected (Fig. 4). The detected molecular mass corresponds to that de-

R. Alexander et al. : A barley cDNA clone encodes a glucose dehydrogenase

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duced from the c D N A clone. Like the m R N A levels the protein disappeared during germination, although the turnover of the protein appears to be slower in older embryos (Fig. 4, compare lanes 2 and 8). When embryo proteins were separated by a two-dimensional electrophoresis the single band o f the pG31 protein could be resolved into two spots with isoelectric points between 7.0 and 6.8 (Fig. 5). The spatial distribution of the pG31 transcript in developing embryos was investigated by R N A in-situ hybridizations (Fig. 6). The transcript was detected in all tissues o f the embryo with a particular high accumulation in the scutellum and a low expression in the coleorhiza. This distribution did not change in 12-DAP and 18-DAP embryos.

Fig. 1. Nucleotide sequence (mRNA strand) and predicted amino-acid sequences of pG31. The nucleotide sequence is shown in the upper line and numbered, the corresponding amino-acid sequence (a) is given below and is aligned with aminoacid sequences from GlcDH of Bacillus megaterium (b) (Heilmann et al. 1988) and ribitol dehydrogenase from Klebsiella aerogenes (c) (Loviny et al. 1985). Identical amino acids are indicated in black. Gaps were introduced to optimize matches

Glucose dehydrogenase activity. The homology of the protein deduced from the barley c D N A clone pG31 to bacterial G l c D H and ribitol dehydrogenases raised the question whether the presence of the barley protein can be linked with related enzymatic activity. The activity of G l c D H was measured in crude protein extracts of 12DAP and 18-DAP embryo and leaf samples. The results from at least three independent experiments are shown in Fig. 7. The highest level of activity was found in 18DAP embryos, while 12-DAP embryos gave a lower activity. N o activity was found in germinated 12- or 18-DAP embryos and in leaves. The enzymatic activity in the 12-DAP and 18-DAP embryos was dependent on glucose as a substrate. Addition of antiserum against the pG31 recombinant fusion protein to the enzyme assays

522

R. Alexander et al. : A barley cDNA clone encodes a glucose dehydrogenase

Fig. 4. Western blot analysis of the pG31 protein during barley embryo development. Protein extracts were subjected to PAGE and analysed via immunoblotting in the presence of the pG31 protein for the following samples : lane 1, 12-DAP embryos; lane 2, 12-DAP embryos on germination medium for 3 d; lane 3, 14-DAP embryos; lane 4, 16-DAP embryos; lane 5, 18-DAP embryos; lane 6, 20-DAP embryos; lane 7, 30-DAP embryos; lane 8, 30-DAP embryos on germination medium for 3 d; lane 9, mature embryos. Reference molecular-size markers are given in kDa

Fig. 2. Southern analysis of genomic DNA from barley. The DNA was cut with Bam HI (1), Eco RI (2) and Hind III (3) and probed with the 32p-labelled insert of pG31; the fragment sizes are given in kilo base-pairs

Fig. 5. Western blot analysis of barley embryo proteins extracted from 18-DAP embryos and separated by two-dimensional electrophoresis. The embryo proteins were first separated by isoelectric focusing (IEF) and then in a 12% SDS-polyacrylamide gel (SDSPAGE) according to Bartels et al. (1988); the proteins were transferred to a nitrocellulose membrane, which was incubated with antibodies raised against the pG31-encoded protein

Fig. 3. A Expression of the pG31 transcript in the barley embryo. RNAs (1 lag poly(A) +) from embryos of different developmental stages were separated on a denaturing agarose gel, blotted to a nylon membrane and hybridized with the 32P-labelled fragment of pG31. RNAs were extracted from the following tissues: lane 1, 12-DAP embryos; lane 2 12-DAP embryos incubated on germination medium for 3 d; lane 3, 15-DAP embryos; lane 4, 18-DAP embryos; lane 5, 18-DAP embryos on germination medium; lane 6, 30 DAP-embryos. B The same blot as in A was hybridized with a ribosomal probe to assure equal loadings of RNA

r e d u c e d the G l c D H activity s u b s t a n t i a l l y ; after r e m o v i n g the p r o t e i n - a n t i g e n c o m p l e x w i t h P r o t e i n A - S e p h a r o s e n o a c t i v i t y w a s f o u n d in the s u p e r n a t a n t ( T a b l e 1). W h e n , in c o n t r o l e x p e r i m e n t s , p r e i m m u n e s e r u m was

used i n s t e a d o f a n t i s e r u m the s u p e r n a t a n t r e t a i n e d the G l c D H activity. T h e ability o f different sugars to reduce N A D ( P ) was tested in e n z y m e assays using b o t h p r o t e i n extracts f r o m 1 8 - D A P e m b r y o s a n d g e r m i n a t e d e m b r y o s . N A D was r e d u c e d b y D-glucose o n l y in 1 8 - D A P e m b r y o extracts b u t n o t in extracts f r o m g e r m i n a t e d e m b r y o s ; this specificity was n o t f o u n d for D-sorbitol, D-xylose, D-fructose a n d D-ribitol for which N A D r e d u c t i o n was o b s e r v e d b o t h with extracts f r o m 1 8 - D A P e m b r y o s a n d with germ i n a t e d e m b r y o s as e n z y m e sources (Fig. 8). W i t h respect to the specificity o f the c o - s u b s t r a t e in the presence o f D-glucose, b o t h N A D a n d N A D P can be reduced, a l t h o u g h N A D P at lower efficiency (40%). T h e a p p a r e n t Km values for G l c D H were d e t e r m i n e d to be 3 m M for glucose a n d 0.3 m M for N A D . T h e activity o f G l c D H was n o t affected by an i n c u b a t i o n u p to 56 ~ C for 10 m i n w h e r e a s the activity using D-ribitol was a b o l i s h e d at this temperature.

R. Alexander et al.: A barley eDNA clone encodes a glucose dehydrogenase

523

Table 1, Glucose dehydrogenase (GlcDH) activity in protein extracts of 18-DAP barley embryos after the addition of antiserum and protein A-Sepharose (Prot. A.S.) Serum added

Prot. A.S. (mg)

GlcDH activity (%)

none 2.5 lal immune serum 5.0 lal immune serum 2.5 gl immune serum 5.0 gl immune serum 2.5 tal preimmune serum 5.0 lal preimmune serum

none none none 2.5 mg 2.5 mg 2.5 mg 2.5 mg

100% 30% 12% 0% 0% 100% 100%

12

9

7 E

6 s 3 < z

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Fig. 6A, B. In-situ RNA hybridizations of the pG31 transcript in developing barley embryos. Cross-sections of 18-DAP barley embryos were hybridized with pG31 3sS-labelled sense (A) and antisense (B) transcripts. Indicated in A are different embryonic tissues : a, embryonic roots; b, shoot apex; c, scutellar tissue; d, coleorhiza; • bar=0.11 mm

Sor

Xyl

Fru

Rib

Fig. 8. Sugar specificity in the enzymatic assay: the reduction of NAD was measured in protein extracts of 18-DAP embryos (shaded bars) and of 18-DAP embryos germinated (open bars) in the presence of D-glucose (Glc), D-sorbitol (Sor), D-xylose (Xyl), D-fructose (Fru) and D-ribitol (Rib). The results represent the average values of several independent experiments

Discussion 4.5 r

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12DAP

12GM

Leaves

Fig. 7. Glucose dehydrogenase activities in protein extracts prepared from different barley tissues: 18-DAP embryos (18-DAP), 18-DAP embryos germinated for 3 d (18GM), 12-DAP embryos (12DAP), 12-DAP embryos germinated for 3 d (12GM) and leaves. The enzyme assays were carried out with glucose (shaded bars) and without glucose (open bars)

In the search for sequences which are expressed early during barley e m b r y o d e v e l o p m e n t a n d possibly associated with the acquisition o f desiccation tolerance, we have identified a novel barley e D N A clone, pG31, with h o m o l o g y to bacterial G l c D H and ribitol dehydrogenase. This pG31 transcript is highly specific for embryogenesis; it ceases to be expressed w h e n i m m a t u r e or m a t u r e e m b r y o s are germinated. M o r e o v e r , in e m b r y o s the transcript could n o t be detected in vegetative tissues or in endosperm, n o r could its expression be triggered by osmotic stress. In c o n t r a s t to several other genes expressed in the e m b r y o the plant h o r m o n e A B A did not appreciably m o d u l a t e the expression level o f pG31 transcripts and therefore its expression m u s t be regulated independently o f A B A . While the A B A - i n d u c i b l e L E A type transcripts a c c u m u l a t e during late stages o f embryogenesis (Dure et al. 1989; Skriver a n d M u n d y 1990) the levels o f pG31 m R N A a n d protein did n o t change appreciably between 12 D A P and maturity.

524

R. Alexander et al.: A barley cDNA clone encodes a glucose dehydrogenase

An interesting feature and a clue to the function of the pG31 gene is the similarity of the encoded protein with GlcDH, as well as ribitol dehydrogenase, from bacteria. Besides the 82 and 65 amino acids identical between the pG31 protein, G l c D H and ribitol dehydrogenases, respectively, all three sequences share a number of similar amino acids. Glucose dehydrogenase catalyses the oxidation of D-glucose using N A D or N A D P as co-substrate and is considered a key enzyme during the early stage of sporogenesis of Bacillus megaterium (Jany et al. 1984). Meanwhile four isoforms of G l c D H with possibly different physiological functions have been identified from B. megaterium (Nagao et al. 1992). Ribitol dehydrogenase catalyses the oxidation of ribitol and the structural gene was isolated from the bacterium Klebsiella aerogenes (Loviny et al. 1985). Both, G l c D H and ribitol dehydrogenase are structurally related and can be grouped into the superfamily of short alcohol-polyolsugar dehydrogenases (J6rnvall et al. 1984). Our finding is the first report that this family of genes extends to plants. The secondary structure of G l c D H has been analysed in detail (J6rnvall et al. 1984; H6nes et al. 1987): the NAD(P)+-binding domain, as well as motifs involved in the dimerisation of subunits, seem conserved in the predicted barley protein. These observations support the conclusion that functionally important D N A sequences have been conserved among plants and bacteria, and similar metabolic pathways are also expected to be active in both types of organism. Besides the sequences discussed above, G l c D H genes have been characterized from other sources such as Acinetobacter calcoaceticus (Cleton-Jansen et al. 1989), Archebacteria (Bright et al. 1993) and Drosophila (Krasney et al, 1990), but they do not display significant structural homology to the barley pG31 protein or the glucose dehydrogenase from Bacillus; possibly these proteins are involved in different metabolic pathways. It is widely accepted that mainly sugar-phosphates are metabolized during plant carbohydrate metabolism (e.g. Turner and Turner 1980). To obtain an indication that the GlcDH-related transcript and protein described here are involved in a novel pathway, not involving phosphorylated intermediates, protein fractions were analysed for their abilities to oxydize D-glucose in the presence of N A D ( P ) +. Specific enzymatic activity was found in extracts from embryos but not from leaves. These biochemical data are in agreement with the expression pattern of the pG31 transcript and proteins (Figs. 3, 4), Although the enzymatic measurements were done with crude protein fractions, the specificity of the assay is largely substantiated by the fact that the GIcDH activity was inhibited by the addition of pG31-specific antiserum. When different sugars were tested as substrates, only D-glucose was oxidized specifically in embryo protein extracts whereas, in the presence of other substrates, N A D was reduced by extracts from embryos and germinated embryos. This observation suggests that the dehydrogenase activity in embryos related to the gene pG31 is restricted to D-glucose as substrate. This is in agreement with the degree of sequence homology found: pG31 seems more homologous to G l c D H than to ribitol

dehydrogenase. Further biochemical characterization (apparent Km values and temperature stability) supports the relatedness between the barley protein and the characterized bacterial G l c D H enzymes (Smith et al. 1989; Nagao et al. 1992). The GlcDH-related c D N A clone is a novel sequence specifically related to embryogenesis and pointing to a metabolic pathway not yet described for higher plants. Previously, an aldose-reductase-related barley embryo transcript was isolated and characterized by our group (Bartels et al. 1991). Although the function of this gene is still not clear, specific enzymatic activity can be attributed to the aldose-reductase-encoded protein (data not shown). Both the GIcDH and the aldose-reductaserelated transcripts are abundantly expressed in developing barley embryos, reaching their maximum steady-state levels before the well-characterized LEA transcripts (Hughes and Galau 1989; Skriver and Mundy 1990). This indicates that the pG31- and aldose-reductaserelated genes contribute to specific functional requirements of early embryo development and suggests the existence in the carbohydrate metabolism specific to embryogenesis of pathways that have not yet been characterized. We thank B. Eilts and M. Feck for excellent technical assistance and M. Pasemann for patiently typing the manuscript. J. Alamillo acknowledges the receipt of a grant from the European Economic Community in the Human Capital and Mobility Program and a Formacion de Personal Investigador-grant from Ministerio de Educacion y Ciencia (Spain). References

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