Summary To check for the presence of Em-like proteins in seeds of the leguminous tree Robiniapseudoacacia L. (black locust), a cDNA library constructed from mRNA isolated from developing seeds was screened using radiolabeled cDNA en-coding the Em protein from Vignaradiata as a probe. Sequence analysis of the identified cDNA clones revealed two classes of Em proteins in Robinia. The nucleotide sequence data for each class have been submitted to Genbank/EMBL Data library and are available under the accession numbers U40820 and U40821. Northern blot analysis demonstrated that the Robinia Em proteins are translated from mRNAs of approximately 800 nucleotides, which are abundant in the seed but are not ex-pressed in the bark. Southern blot analysis indicated that the Em proteins in Robinia are encoded by at least two genes. The Robinia Em proteins have a high degree of sequence homology with previously characterized Em-like proteins from both monocots and dicots.
Keywords: cDNA, embryogenesis, Northern blot, Southern blot.
Introduction
Maturing seeds progressively desiccate during late embryo-genesis. This developmental stage is characterized by a loss of water and an accumulation of abscisic acid. In addition, the late stages of embryogenesis are associated with the expres-sion of specific genes that encode mRNAs that survive desic-cation and accumulate in the dry seeds. These mRNAs disappear soon after the onset of germination; therefore, the synthesis of the corresponding proteins is confined to late embryogenesis and to the early phase of germination (Galau et al. 1987, Espelund et al. 1992). Because of the time of their expression, the proteins encoded by these mRNAs are usually referred to as Lea (Late embryogenesis abundant) proteins.
The first Lea protein was discovered in wheat embryos when it appeared as the most conspicuous spot on a fluorogram after separation of the proteins in a total extract from 35 S-methion-ine-labeled germinating wheat embryos. Accordingly, the pro-tein was originally described as an Early-methionine-labeled (Em) polypeptide (Cuming and Lane 1979). The wheat Em polypeptide accumulates during the late maturation stages of
developing grains. Both abscisic acid and osmotic stress en-hance the expression of the Em genes in developing and germinating wheat embryos (Williamson et al. 1985, Marcotte et al. 1989, Morris et al. 1990). Molecular cloning and analysis of the cDNA that encodes the wheat Em polypeptide indicate that the protein represents a unique type of albumin (Litts et al. 1987, Futers et al. 1990).
Since the discovery of the wheat Em polypeptide and the molecular cloning of the corresponding gene, Em-like cDNAs or genomic clones have been isolated and characterized from numerous plants, including the monocots maize (Williams and Tsang 1991), barley (Espelund et al. 1992) and rice (Litts et al. 1992), and the dicots cotton (Baker et al. 1988, Galau et al. 1992), radish (Raynal et al. 1989), carrot (Ulrich et al. 1990), sunflower (Almoguera and Jordano 1992) and Arabidopsis (Gaubier et al. 1993). Based on the common positive response of Em genes to abscisic acid and the high sequence homology among Em-like proteins, it is assumed that Em-like proteins have similar functions in different plant species. Although the exact function of Em-like proteins is not fully understood, their hydrophilicity and other biophysical properties (McCubbin et al. 1985) suggest that they play a role in water-stress tolerance during seed desiccation (Grzelczak et al. 1982, McCubbin et al. 1985, Galau et al. 1987) and early germination (Morris et al. 1990).
No Em-like proteins have been reported in seeds of tree species. Because seeds of the legume Vignaradiata (mung bean) contain proteins and genes that have a high sequence homology with previously described Em polypeptides (Gen-bank/EMBL Data Library), we used the cDNA of two mung bean Em proteins to probe a cDNA library constructed from mRNA isolated from developing seeds of the leguminous tree Robinia pseudoacacia L. (black locust). We present evidence that the black locust tree expresses Em-like proteins in its seeds.
Materials and methods
Construction of cDNA library
Developing seeds were harvested from a single black locust (Robinia pseudoacacia L.) tree in August. After removal of the
Molecular cloning of two classes of Em-like proteins from the seeds of
the leguminous tree
Robinia
pseudoacacia
ELS J. M. VAN DAMME and WILLY J. PEUMANS
Catholic University of Leuven, Laboratory for Phytopathology and Plant Protection, Willem de Croylaan 42, 3001 Leuven, Belgium
Received December 19, 1995
seed coat, the seeds were frozen in liquid nitrogen and stored at −70 °C until used for the isolation of mRNA, according to the procedure described previously (Van Damme and Peumans 1993). A cDNA library was constructed from isolated polyA+ RNA with the cDNA synthesis kit from Pharmacia (Uppsala, Sweden). The cDNA fragments were inserted in the EcoRI site of the multifunctional phagemid pT7T3 18U (Pharmacia, Uppsala, Sweden). The library was propagated in Escherichia coli XL1 Blue (Stratagene, La Jolla, CA).
Isolation and sequence analysis of cDNA clones
The Robinia seed cDNA library was screened using random-primer-32P-labeled cDNA clones encoding the Em proteins from mung bean, EMVIGNARA1 and EMVIGNARA2 (avail-able in the Genbank/EMBL Data Library under the accession numbers U31210 and U31211), as probes. Colony hybridiza-tion was carried out at 55 °C, as described previously (Van Damme et al. 1992a). In later experiments, the cDNA clones EMRPS19 and EMRPS24, which encode Em-like proteins from Robinia, were used as probes to identify more Em-pro-tein cDNA clones in the Robinia seed cDNA library. Colonies that produced positive signals were selected and rescreened at low density under the same hybridization conditions. Plasmids were isolated from purified single colonies on a miniprep scale using the alkaline lysis method as described by Mierendorf and Pfeffer (1987) and sequenced by the dideoxy method (Sanger et al. 1977). The DNA sequences were analyzed using pro-grams from PC Gene (Intelligenetics, Mountain View, CA).
Northern blot analysis
Electrophoresis of RNA was performed according to Maniatis et al. (1982). Approximately 3 µg of polyA+ RNA was dena-tured in glyoxal and DMSO and separated in a 1.2% (w/v) agarose gel. Following electrophoresis, the RNA was trans-ferred to an Immobilon N membrane (Millipore, Bedford, USA). The 32P-labeled EMRPS19 cDNA was used to probe the membrane, according to the hybridization protocol reported by Van Damme et al. (1992b). We used RNA markers (0.16--1.77 kb) to estimate the transcript size.
Genomic DNA analysis
Total DNA from Robinia bark was isolated according to the procedures described by Dellaporta et al. (1983) and de
Ko-chko and Hamon (1990). To remove RNA, the isolated DNA was treated with RNAse. Approximately 50 µg of genomic DNA was singly digested with the restriction endonucleases BamHI, EcoRI, HindIII and PstI and subjected to electropho-resis in a 0.8% (w/v) agarose gel. The DNA was transferred to Immobilon N membranes (Millipore, Bedford, USA) and hy-bridized at 68 °C with 32P-labeled EMRPS19 and EMRPS24 cDNA.
Results
Molecular cloning of Em-like cDNA clones from Robinia
Screening of the Robinia seed cDNA library with the cDNA clones EMVIGNARA1 and EMVIGNARA2, which encode Em proteins from mung bean (Manickam et al. 1996), and later with the cDNA clones EMRPS19 and EMRPS24, which en-code Em-like proteins from Robinia, led to the isolation of several full length cDNA clones. Sequence analysis of eight Robinia clones revealed two distinct classes of Em-like genes. The two classes had an overall sequence homology of about 54% at the nucleotide level and 85% at the amino acid level, but differed significantly from each other at some positions in their sequences. The complete nucleotide sequence and de-duced amino acid sequence (Figure 1) of a representative of each class were determined. The first class, represented by the cDNA clone EMRPS19, had 674 bp with an open-reading-frame encoding a 112-amino acid polypeptide with a calcu-lated molecular mass of 12.2 kDa. The second class, represented by the clone EMRPS24, encoded a 99-amino acid protein with a calculated molecular mass of 10.8 kDa. A comparison of the amino acid composition of the proteins encoded by EMRPS19 and EMRPS24 revealed a small differ-ence in the percentage of charged amino acids in each protein. This difference was reflected in the estimated isoelectric points for the proteins: 6.23 for EMRPS19 and 5.64 for EMRPS24.
The Em-like proteins encoded by EMRPS19 and EMRPS24 showed a high degree of sequence homology (84.8% sequence identity; 86.8% sequence similarity). Alignment of the de-duced amino acid sequences of the two Em-like proteins re-vealed that the EMRPS19 protein contains a repeated segment 20 amino acids in length (Figure 1), whereas the EMRPS24 protein contains this segment only once. The EMRPS19
tein lacks seven amino acids at the C-terminal, compared to the EMRPS24 protein (Figure 1).
Sequence homology between cDNA clones from Robinia pseudoacacia and other Em-like proteins
Alignment of the Robinia Em-protein amino acid sequences with sequences of other Em-like proteins from dicot and monocot species revealed a high degree of homology (Fig-ure 2). The Em sequences from Robinia were most homolo-gous to the sequences of the Em-like proteins from mung bean. The proteins encoded by EMRPS19 and EMVIGNARA1 had 86% sequence identity and those encoded by EMRPS24 and EMVIGNARA2 were 80% homologous.
Northern blot analysis
To determine the size of the mRNAs encoding the Em-like proteins from Robinia pseudoacacia, the cDNA clones EMRPS19 and EMRPS24 were hybridized to polyA+ RNA isolated from Robinia seeds and Robinia bark. Both probes yielded the same result, revealing that the Em-like proteins are encoded by mRNAs with an approximate length of 800 nu-cleotides (Figure 3) isolated from seeds. There was no hybridi-zation to mRNA isolated from bark.
Southern blot analysis
Southern blot analysis was performed to confirm the hypothe-sis that two genes for Em-like proteins are expressed in Ro-biniapseudoacacia, as indicated by cDNA sequence analysis. Genomic DNA was digested with restriction enzymes that do not cut the EMRPS19 or EMRPS24 cDNA clones. To visualize the restriction fragments containing Em-like sequences, the blots were hybridized with labeled EMRPS19 and EMRPS24 cDNA. Figure 4 shows that the labeled cDNA hybridized to two EcoRI and two PstI restriction fragments.
Discussion
In this report, we have described the isolation of two classes of Em-like genes from a cDNA library constructed from mRNA of developing Robiniapseudoacacia seeds. Our results pro-vide epro-vidence that seeds of an angiosperm tree species express Em-like proteins. All other Em and Lea proteins and genes reported previously were isolated from seeds of herbaceous plant species (namely wheat, barley, rice, maize, cotton, carrot, radish, Arabidopsis and mung bean). The occurrence of Em-like genes in trees had been indirectly inferred for the conifer white spruce (Piceaglauca (Moench) Voss) (Leal and Misra
Figure 2. Alignment of the deduced amino acid sequences of the cDNA clones encoding the Em-like proteins from Robiniapseudoacacia
1993), which contains seed mRNAs that cross-hybridize with a cDNA encoding a radish Em-like protein.
Northern blot analysis demonstrated that Em genes are highly expressed in Robinia pseudoacacia seeds and that there is no detectable expression in bark. In this respect, the Em proteins differ from the black locust lectins, which are abun-dant in both seeds and bark (Van Damme et al. 1995a, 1995b). Southern blot analysis and cDNA cloning indicated that the black locust Em proteins are each encoded by a single gene, or by a few tandemly arrayed genes. Similar results have been described for all reported Em and Lea proteins, suggesting that there are no major differences in the number of genes encoding Em proteins in trees and herbs.
Amino acid sequence analysis revealed that both classes of black locust Em proteins have a high degree of homology to previously described Em-like proteins. The Robinia Em pro-teins are most homologous to the Em-like propro-teins of mung bean, which is the only other legume known to express Em-like genes in its seeds (Manickam et al. 1996). Like mung bean Em proteins, one of the classes of Em proteins in Robinia contains a repeat of a 20-amino acid segment, whereas the other class contains this segment only once. The sequence similarity be-tween the Em proteins of Robinia and Em proteins from other plant species demonstrates that Em proteins are highly con-served and thus likely have similar functions in all plant spe-cies in which they have been identified.
Acknowledgments
This work was supported in part by grants from the Catholic Univer-sity of Leuven (OT/94/17) and the National Fund for Scientific Re-search (Belgium). W. Peumans is a ReRe-search Director and E. Van Damme is a Postdoctoral Fellow of this fund.
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