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Plant Physiol. (1999) 119: 1535-1546 Molecular Analysis of (R)-(+)-Mandelonitrile Lyase Microheterogeneity in Black Cherry1
Department of Biological Sciences, The University of Iowa, Iowa City, Iowa 52242
The flavoprotein (R)-(+)-mandelonitrile lyase (MDL; EC 4.1.2.10), which plays a key role in cyanogenesis in rosaceous stone fruits, occurs in black cherry (Prunus serotina Ehrh.) homogenates as several closely related isoforms. Biochemical and molecular biological methods were used to investigate MDL microheterogeneity and function in this species. Three novel MDL cDNAs of high sequence identity (designated MDL2, MDL4, and MDL5) were isolated. Like MDL1 and MDL3 cDNAs (Z. Hu, J.E. Poulton [1997] Plant Physiol 115: 1359-1369), they had open reading frames that predicted a flavin adenine dinucleotide-binding site, multiple N-glycosylation sites, and an N-terminal signal sequence. The N terminus of an MDL isoform purified from seedlings matched the derived amino acid sequence of the MDL4 cDNA. Genomic sequences corresponding to the MDL1, MDL2, and MDL4 cDNAs were obtained by polymerase chain reaction amplification of genomic DNA. Like the previously reported mdl3 gene, these genes are interrupted at identical positions by three short, conserved introns. Given their overall similarity, we conclude that the genes mdl1, mdl2, mdl3, mdl4, and mdl5 are derived from a common ancestral gene and constitute members of a gene family. Genomic Southern-blot analysis showed that this family has approximately eight members. Northern-blot analysis using gene-specific probes revealed differential expression of the genes mdl1, mdl2, mdl3, mdl4, and mdl5.
Cyanogenesis, the release of the respiratory inhibitor HCN, is but
one of many chemical defense systems used by higher plants against
herbivory (Nahrstedt, 1985 During the past several years, we have used molecular biological
approaches to improve our understanding of MDL microheterogeneity and
function in black cherry. This work has yielded two full-length MDL cDNA clones, designated MDL1 and MDL3, with ORFs that share 81%
amino acid identity (Cheng and Poulton, 1993 Plant Material
Isolation and Sequencing of MDL2 cDNA An unamplified ZAP II cDNA library (2 × 105 plaque-forming units), constructed from
poly(A+) RNA isolated from immature black cherry
seeds (Zheng and Poulton, 1995 ), was screened by standard
methods (Sambrook et al., 1989) using the MDL1 cDNA labeled with
[ -32P]dCTP by random priming. After four
rounds of plaque hybridization, phage DNA was isolated from positive
clones using a liquid-culture method (Sambrook et al., 1989). Insert
sizes were determined by PCR, and cDNA inserts greater than 1.7 kb in
length were subcloned into pBluescript SK by in
vivo excision according to the manufacturer's instructions (Stratagene). Plasmids were isolated using the Wizard mini kit (Promega) and subjected to restriction analysis, followed in some cases
by partial sequencing. One clone (pMDL2), whose insert differed from
the MDL1 and MDL3 cDNAs in its restriction map and nucleotide sequence,
was serially deleted by exonuclease III and S1 nuclease digestion
(Sambrook et al., 1989) and sequenced in both directions by the dideoxy
chain-termination method (Sanger et al., 1977) using Sequenase version
2.0 (United States Biochemical).
Isolation and Sequencing of MDL4 and MDL5 cDNAs Isolation of Probe for Screening of the Leaf cDNA Library Total RNA was isolated from seedling tops (uppermost 6 cm of 10-cm-tall seedlings) as described by Logemann et al. (1987). Reverse
transcription was carried out at 37°C for 45 min in a 20-µL
reaction volume containing 2 µg of heat-denatured (70°C for 10 min)
RNA, 1 µg of oligo(dT)20 primer, 200 µM of each dNTP, 40 units of RNasin
(Promega), and 200 units of SuperScript II RNase
H RT (GIBCO-BRL). After the reaction was
stopped by heating at 70°C for 10 min, first-strand cDNA
amplification was carried out using the primers
5 -TGGGTTGAAGACACTATTGTGT-3 (sense) and 5-AATGAAATTACGAGGATTGTCAT-3 (antisense), which correspond to highly conserved regions
of the mdl1, mdl2, and mdl3 genes (Hu
and Poulton, 1997). Each reaction (100 µL) contained 10 µL of RT
reaction product, 0.4 µM of each primer, 200 µM of each dNTP, 2 units of Taq DNA
polymerase, and 1× PCR buffer. Reactions were cycled 34 times at
94°C for 1 min, 50°C for 1 min, and 72°C for 1 min (10 min for
the final cycle). The resulting PCR product (0.5 kb) was purified by
the GENECLEAN II kit (Bio 101, Inc., Vista, CA), blunt ended with
Klenow enzyme (New England Biolabs), and ligated into the
SmaI site of pBluescript SK for
transformation into Escherichia coli XL1-Blue competent
cells. The insert was sequenced from both ends by primer walking using a dye-termination sequencing protocol run on the fluorescent automated sequencer at the University of Iowa DNA Facility (Iowa City).
Construction and Screening of the Leaf cDNA Library Total RNA (1 µg) from seedling tops was used to construct a leaf cDNA library ingt11 by the CapFinder PCR cDNA library construction method according to the manufacturer's suggestions (Clontech, Palo
Alto, CA). Approximately 5 × 104 phage
plaques were screened with the 0.5-kb PCR fragment described above,
which had been labeled with [32P]dCTP by random
priming (Boehringer Mannheim). After three successive rounds of plaque
hybridization, phage DNA was isolated from positive clones by a
plate-lysate method (Ausubel et al., 1995). After restriction analysis
had identified two classes of recombinant phages corresponding to MDL4
and MDL5, cDNA inserts were recovered by NotI digestion,
purified by the GENECLEAN II kit, and subcloned into the
NotI site of pBluescript SK for
automated double-strand sequencing.
Isolation and Sequencing of mdl1, mdl2, and mdl4 Genomic Sequences Genomic DNA was isolated from arborescent leaves, as described by Dellaporta et al. (1983). Genomic sequences corresponding to the MDL1,
MDL2, and MDL4 cDNAs were obtained by PCR amplification using gene-specific primers. For mdl1 amplification, five
parallel reactions were undertaken; each reaction contained up to 2 µg of genomic DNA, 0.1 µM each of the sense
(5- TTGAGTTATCAAAAAACATGGA-3) and antisense
(5-GATAGTCTCGATATTACAAG-3) primers, 200 µM of each deoxyribonucleotide triphosphate, 2 units of Pfu DNA
polymerase, and 1× PCR buffer (Stratagene) in a total volume of 100 µL. Reactions were cycled 25 times at 94°C for 1 min,
53°C for 1 min, and 72°C for 3 min (10 min for the final cycle).
All five reactions generated a major PCR product of approximately 2.3 kb, which was purified using the QIAquick gel- extraction kit (Qiagen,
Dusseldorf, Germany) and individually ligated into the SmaI
site of pBluescript SK. After transformation
into E. coli XL1-Blue competent cells, one clone resulting
from each ligation was subjected to partial sequencing by the dideoxy
chain-termination method. After confirmation that these PCR
products had identical sequences in the regions examined, a
single clone was selected for complete sequencing in both directions by
primer walking. For comparative purposes, two other clones that
originated from parallel amplification reactions were sequenced
in specific regions as required. Genomic sequences corresponding
to the MDL2 and MDL4 cDNAs were obtained by identical means using
the following oligonucleotides as primers: MDL2,
5-GCACGAGATTCAGAAACAT-3 (sense) and
5-AGAGAACTAGAACATCACAAAG-3 (antisense); and MDL4, 5-TGAAAGTGTGAAAGAAATTTTAAGAA-3 (sense) and
5-GAGTAAATAGAAAGCACAAG-G-3 (antisense).
Isolation and N-Terminal Sequencing of a Major MDL Isoform from Young Seedlings Enzyme Purification All procedures were performed at 4°C. Seedling tops (180 g; uppermost 6 cm of 10-cm-tall seedlings) were homogenized using a blender with 500 mL of buffer A (0.1 M histidine-HCl, pH 6.0) containing 10 g of polyvinylpolypyrrolidone and 12 g of quartz sand. The homogenate was filtered through four layers of cheesecloth and centrifuged for 30 min at 20,000g. The resulting supernatant was dialyzed overnight against 4 L of buffer B (10 mM His-HCl, pH 6.0, containing 0.17 M NaCl) and applied to a Con A-Sepharose 4B column (1.6 × 10 cm) preequilibrated with buffer B. After the column was washed extensively with buffer B, bound proteins were eluted with 100 mL of 0.5 M-methyl-D-glucoside in buffer B. Active MDL
fractions derived from Con A-Sepharose chromatography were pooled and
dialyzed overnight against 4 L of buffer C (20 mM
sodium acetate, pH 5.0). The dialyzed pool was applied to a DEAE-cellulose column (1.6 × 12 cm) preequilibrated with buffer C. After unbound proteins were removed by washing with 500 mL of buffer
C, MDL was eluted with a linear gradient (0-350
mM NaCl; total volume, 150 mL) in the same
buffer. MDL fractions (7.5 mL each) from DEAE-cellulose chromatography
were pooled, dialyzed overnight against 4 L of buffer C, and applied to
a Reactive Red 120-agarose column (1.6 × 12 cm) preequilibrated
with buffer C. The column was washed with 10 column volumes of buffer C
to remove unbound proteins. Bound proteins were eluted with a linear
gradient (0-0.5 M NaCl; total volume, 400 mL) in
buffer C.
Enzyme Assay MDL activity was measured as described previously (Yemm and Poulton, 1986).
SDS-PAGE and N-Terminal Sequence Analysis To assess protein homogeneity, SDS-PAGE was performed on 10% resolving gels, as described by Sambrook et al. (1989), before staining
with Coomassie brilliant blue. After electrophoresis, polypeptides were
transferred to PVDF membranes (Bio-Rad) in
3-(cyclohexylamino)-1-propane-sulfonic acid transfer buffer and
visualized by brief Coomassie brilliant blue staining (LeGendre et al.,
1993). The MDL band was excised and subjected to sequence analysis by
Edman degradation on a protein sequencer (model 475A, Applied
Biosystems) at the University of Iowa Protein Structure Facility.
Sequence Analyses Computer analyses of DNA and amino acid sequences were performed with the University of Wisconsin Genetics Computer Group sequence-analysis software package (Devereux et al., 1984). Comparison of coding regions was performed using the BestFit program, and intron
sequences were compared by the GAP program (for both programs, gap
creation penalty = 50 and gap extension penalty = 3). Deduced amino acid sequences were aligned by the Megalign Clustal program (DNASTAR, Inc., Madison, WI). For phylogenetic tree construction, the
derived amino acid sequences of the black cherry and almond MDLs were
aligned using the PileUp program (gap creation penalty = 12 and
gap extension penalty = 4). Based on the corresponding amino acid
sequence alignment, distance matrices were constructed using the Kimura
(1980) protein-distance method, and phylogenetic reconstruction was
performed using a neighbor-joining method (Saitou and Nei, 1987).
Bootstrap analysis (100 replications; Felsenstein, 1985) was performed
using the same tree reconstruction method to assess the stability of
tree elements.
Northern-Blot Hybridization Total RNA was extracted from immature seeds, inflorescences, and the leaves, roots, and cotyledons of young seedlings, as described by Logemann et al. (1987). After separation by electrophoresis (10 µg
per lane) on denaturing 1.2% agarose gels containing 2.2 M
formaldehyde, RNA was blotted onto membranes (Hybond N, Amersham) by
standard methods (Sambrook et al., 1989). After cross-linking for
2 h at 80°C in a vacuum oven, blots were prehybridized for 1 h at 50°C in 20 mM
Na2HPO4, pH 6.8, containing
6× SSC, 0.4% SDS, 5× Denhardt's solution, and 0.5 mg
mL1 denatured salmon sperm DNA. Overnight
hybridization was carried out in this medium (without Denhardt's
solution) with [ -32P]ATP end-labeled,
gene-specific oligonucleotide probes generated from the MDL1 (positions
489-466), MDL2 (positions 413-389), or MDL4 (positions
1839-1818) cDNAs. Blots were washed twice for 15 min in 6× SSC
and 0.1% SDS at room temperature and twice for 15 min at 55°C before
autoradiography. To study MDL3 and MDL5 expression, membranes were
probed as described for Southern-blot analysis using either an
MDL3 cDNA fragment (positions 1663-1878; Hu and Poulton, 1997) or an
MDL5 cDNA fragment (positions 1962-2232) previously
32P labeled by random priming. Equal loading of
RNA samples was assessed by hybridization with a 1-kb DNA fragment
encoding soybean 28S rRNA labeled by random priming. Autoradiography
was subsequently undertaken with intensifying screens at  70°C from
overnight to up to 5 d.
Southern-Blot Hybridization Genomic DNA was isolated from leaves of a single black cherry tree as described above, digested overnight with BamHI, HincII, or EcoRI, and separated by electrophoresis (10 µg per lane) on a 0.8% agarose gel. After blotting onto membranes (Hybond-N+, Amersham), the samples were UV cross-linked. Six different probes were generated for Southern-blot analysis. Highly conserved among all of the known MDL genes, nucleotides 453 to 534 of the mdl1 gene served as an 82-bp consensus probe that was labeled with [-32P]dCTP by PCR (Mertz and Rashtchian,
1994). In addition, five gene-specific probes were generated by PCR
amplification; these probes spanned the second introns of the
mdl1, mdl2, and mdl4 genes, the first
intron of the mdl3 gene, and the 3-UTR of the MDL5 cDNA.
These probes were separated by agarose-gel electrophoresis, purified
using the GENECLEAN II kit, and labeled with
[-32P]dATP by random priming. Specificity of
the gene-specific probes was confirmed by Southern-blot analysis
against full-length fragments of the five mdl genes.
In cyanophoric plants, Microheterogeneity of Rosaceous Stone-Fruit MDLs One puzzling aspect of the biochemistry of rosaceous stone-fruit MDLs is their microheterogeneity. With only one known exception (Xu et al., 1986), all MDLs isolated from seeds of members of the Prunoideae
and Maloideae subfamilies exist as multiple forms (Hickel et al.,
1996). Within a given species, these isoforms display slight
differences in molecular mass, pI, electrophoretic mobility, and
specific activity, but they have identical antigenic properties.
Further microheterogeneity is observed when these seed MDLs are
compared with their counterparts in postembryonic tissues. For example,
in black cherry the hypocotyl and epicotyl MDLs (70 kD) are much larger
than the seed isoforms (57-59 kD). To date, few studies have focused
on the structural differences between the MDL isoforms within a species
or addressed the possibility that individual isoforms may have unique
functions (Gerstner et al., 1971; Yemm and Poulton, 1986).
Likely sources of MDL microheterogeneity include allelic differences at
the structural locus for the polypeptide (i.e. allozymes), alteration
of the translated polypeptide (e.g. N- and C-terminal processing,
glycosylation, and phosphorylation), and gene duplication leading to
multigene families (Weeden, 1983). In higher plants, polyploidy is
perhaps the most conspicuous mechanism for gene duplication. Several
rosaceous stone fruits are polyploid (Darlington, 1928), including
black cherry, which is a tetraploid species (2n = 4x = 32) of ancient origin whose chromosomes pair as
bivalents during meiosis (Maynard et al., 1991). Thus, it is possible
that allozymic forms could contribute significantly to the observed MDL
multiplicity of some Prunus species.
Isolation and Characterization of MDL2 cDNA In the present study, a novel MDL cDNA, designated MDL2, was obtained by probing a new seed cDNA library (Zheng and Poulton, 1995)
with the radiolabeled MDL1 cDNA under low-stringency conditions. The
MDL2 cDNA was sequenced in both directions, and its nucleotide sequence
was assigned accession number AF040078. As shown in Table
I, the cDNA is 1932 nucleotides in length
and consists of 17 nucleotides of 5-UTR, a 1731-nucleotide ORF, and a
3-UTR of 184 nucleotides terminated by a 9-nucleotide
poly(A+) tail. The ORF encodes a polypeptide of
576 amino acids with a predicted molecular mass of 62.7 kD and a pI of
4.39. Five putative polyadenylation (AATAAA) signals are located 3, 108, 144, 148, and 152 nucleotides downstream of the TAA stop codon.
Isolation and Characterization of MDL4 and MDL5 cDNAs Isolation of the MDL4 cDNA Fragment by RT-PCR To assess rapidly which MDL genes are expressed in black cherry leaves, RT-PCR was used with total RNA from this source acting as the template. Using two primers based on highly conserved regions of the MDL1, MDL2, and MDL3 cDNAs, a 0.5-kb fragment was obtained that was subcloned into the pBluescript vector for double-strand sequencing. This partial-length cDNA (495 bp) shared 84% to 86% similarity with the MDL1, MDL2, and MDL3 cDNAs. In view of the uniqueness of this MDL sequence, a seedling-leaf cDNA library was constructed with the goal of obtaining full-length clones.Isolation of MDL4 and MDL5 cDNAs by Screening of a Leaf cDNA Library Using the CapFinder PCR cDNA library construction system, a leaf cDNA library was constructed in thegt11 vector using mRNA isolated
from leaves of 3- to 5-week-old seedlings. Despite its small size
(5 × 104 plaque-forming units), this library yielded
26 putative MDL clones when screened with the 495-bp MDL fragment
described above. Restriction analysis using SalI,
EcoRI, and HindIII revealed that these
clones fell into two groups, one with 25 members and one with 1 member. DNA inserts of two clones from the first group and of the sole member
of the second group were obtained by NotI digestion and subcloned into the pBluescript vector. Partial sequencing revealed that
the two clones from the first group were identical and matched the
495-bp fragment obtained by RT-PCR; they were designated MDL4. The
single clone from the second group was clearly dissimilar from
clones MDL1 to MDL4 and was designated MDL5. The cDNA inserts of
one MDL4 clone and the single MDL5 clone were fully sequenced and
subsequently assigned accession nos. AF053384 and AF053386, respectively. The major features of these cDNAs and of the predicted proteins that they encode are summarized in Table I.
Comparison of the Six Known MDL cDNA Sequences The three novel black cherry MDL cDNAs reported here were compared with the previously reported black cherry MDL1 and MDL3 cDNAs (Cheng and Poulton, 1993; Hu and Poulton, 1997) and the almond MDL cDNA
(Suelves and Puigdomènech, 1998) by the BestFit algorithm of the
Genetics Computer Group sequence-analysis software package. Table
II shows the high similarity between
these sequences. Among black cherry MDLs, the amino acid identity
ranged from 75.3% to 87.6%. This degree of similarity suggests that
they represent members of a multigene family rather than multiple
alleles. This notion is also strongly supported by the intron data and
differential expression patterns presented below. Even greater
sequence identity was observed between MDL5 and almond MDL (93.7%
identity and 97% similarity). Because both were expressed in
postembryonic tissues, this pair probably represents orthologs. The
lowest similarity was observed with MDL2 and almond MDL, yet they still
shared 75.1% identity and 84.6% similarity. When amino acid alignment
was performed using the Megalign Clustal program, the most obvious
differences between the six deduced sequences were (a) the presence in
MDL2 and MDL4 of an additional amino acid at positions 11 and 34, respectively, (b) a single amino acid deletion in MDL1 at position 251, and (c) an additional six to nine amino acids at or near the C termini of MDL2, MDL3, and MDL4 (Fig. 1).
N-Terminal Signal Sequence
FAD-Binding Site As mentioned above, a hallmark of rosaceous HNLs is their FAD content, although the enzymatic reaction that they catalyze does not involve a net oxidation-reduction (Jorns, 1979). Available evidence
suggests that this dinucleotide binds to a hydrophobic region near the
enzyme's catalytic site (Baerwald and Jaenicke, 1978; Jorns, 1979).
Sequence analysis now shows that a highly conserved (>90%)
 -fold, which probably serves as the FAD-binding site, lies
close to the predicted N termini of all six mature MDL proteins (Fig.
1). This fold exhibits most of the consensus residues recommended by
Wierenga et al. (1986) as a diagnostic "fingerprint" for
binding of the ADP moieties of FAD and NAD+,
including the sequence G-X-G-X-X-G.
N-Glycosylation Sites As noted above, all black cherry MDL isoforms bind to Con A-Sepharose, indicative of their glycosylation by-D-Man
and/or -D-Glc moieties (Yemm and Poulton, 1986).
Consistent with this observation, the five black cherry MDL cDNA
sequences predicted several potential N-glycosylation sites
(N-X-S/T), although the number varied widely (Fig. 1). Although the
MDL2, MDL3, MDL4, and MDL5 sequences include 15, 13, 10, and 15 potential sites, respectively, the MDL1 sequence has only 5. This
striking difference may underlie the differential glycosylation
detected by Con A-Sepharose chromatography (Yemm and Poulton, 1986) and
will be pursued further. It is noteworthy that the five sites shown by
MDL1 are conserved in at least four of the six known MDL sequences.
Catalytically Active Cys Residue In 1984, Jaenicke and Preun reported that bitter almond HNL was inhibited in 1:1 stoichiometry by the active-site-directed, irreversible inhibitor 3-OPP. This pseudo-first-order inactivation process was inhibited by substrate and competitive inhibitors. Complete hydrolysis of the boronate-reduced, 3-OPP-modified apoprotein yielded L-2-amino-4-thia-DL-7-hydroxy-7-phenylheptanoic acid, the reduced linear addition product of 3-OPP to a Cys-SH group. This suggests that a catalytically important Cys is involved in the active site of this enzyme. In support of this idea, the almond and black cherry MDLs were inhibited by iodoacetamide and iodoacetic acid, respectively (Yemm and Poulton, 1986; Wajant et al., 1995).
Isolation and Characterization of the mdl1, mdl2, and mdl4 Genomic Sequences and Comparison with the mdl3 Gene In an earlier publication (Hu and Poulton, 1997), we reported the
sequences of the mdl3 gene and its respective cDNA (MDL3). Representing the first information about the gene organization of any
plant HNL, we showed that the 2156-bp coding region of this gene is
interrupted by three short introns (217, 107, and 110 bp in length). To
determine whether other black cherry MDL genes are similarly organized,
genomic sequences corresponding to the MDL1, MDL2, and MDL4 cDNAs were
obtained by PCR amplification using black cherry genomic DNA as the
template. Designated mdl1, mdl2, and
mdl4, respectively, these nucleotide sequences were assigned
accession nos. U78814, AF040079, and AF053385. The major features of
the mdl1, mdl2, mdl3, and
mdl4 genes are summarized in Table
III. Comparison of these four genomic
sequences, both among themselves and with their respective cDNAs,
revealed that the ORF of each gene is interrupted at identical
positions by three AT-rich introns (Fig.
3). This constitutes strong, additional evidence that these genes were derived from a common ancestral gene. In
contrast to the conservation of intron locations, the lengths of the
introns varied considerably among the four genes, presumably as a
result of insertions and/or deletions (Table III). For example, intron
I varied in length from 116 to 216 bp. Nevertheless, within matched
regions individual introns showed high homology (45%-85% identity)
across the four genes. With the exception of intron II of
mdl3, which is flanked by the dinucleotides GC and AG, all
12 exon-intron junction sequences conform to the GT-AG rule for RNA
splicing (Mount, 1982). A GC-AG junction sequence has been reported for
several other plant genes, including seven myrosinase genes (Xue and
Rask, 1995).
N-Terminal Sequencing of Seedling MDL To ascertain whether either of the MDL4 and MDL5 cDNAs encodes the 70-kD MDL polypeptide detected in young seedlings by immunoblotting studies (Swain and Poulton, 1994b), MDL protein was extensively purified from this source to obtain N-terminal sequence information. The purification protocol used here mirrored that of Wu and Poulton (1991) for the purification of MDL from black cherry seeds. When the
dialyzed crude extract from seedlings was applied to a Con A-Sepharose
4B column, MDL bound to this matrix, thereby demonstrating its
glycoprotein character. This step provided an effective means of MDL
purification, because the majority of seedling proteins failed to bind
to this affinity resin (Fig. 4). After
its elution by -methyl-D-glucoside, the leaf MDL was
subjected to DEAE-cellulose and Reactive Red 120-agarose
chromatography. Unlike seed MDL (Li et al., 1992), the seedling MDL did
not bind to the Reactive Red 120-agarose column. Nevertheless, this
column was retained in the purification protocol because it removed
many contaminating proteins. Subsequent analysis of the highly purified
seedling MDL by SDS-PAGE revealed two polypeptides with molecular
masses of 70 and 63 kD (Fig. 4). These are believed to represent the seedling MDL and PH, respectively. After these polypeptides were electroblotted onto PVDF membranes, the 70-kD band was excised and
subjected to 15 cycles of N-terminal sequencing. Its N terminus was
LAXTSSEHDFGYLKF. Excluding the third residue, for which an assignment
could not be unequivocally made, this sequence matches exactly the
derived amino acid sequence of the MDL4 cDNA beginning at residue 28 and differs significantly from those of the other known black cherry
MDL isoforms. Failure to assign the third residue may be the result of
glycosylation, because in the MDL4 cDNA this residue is an Asp
that lies within the context of a putative
N-glycosylation site (N-X-S/T).
Black Cherry MDL Is Encoded by a Small Gene Family The size of the putative MDL gene family was assessed by Southern-blot analysis. Genomic DNA from a single tree was digested with BamHI, HincII, or EcoRI and probed with an 82-bp fragment corresponding to nucleotides 453 to 534 of the mdl1 gene (hereafter designated the consensus probe). This region was selected because comparison of the MDL1 to MDL5 cDNAs showed that it is highly conserved (approximately 91% identity) and lacks restriction sites for these endonucleases. Moreover, the short length of this probe would minimize the possibility of other target sequences in the genome containing these sites. As a result, the number of bands hybridizing to the consensus probe would more likely reflect the actual size of the MDL gene family. After the blot was washed at low stringency (0.5× SSC and 0.1% SDS at 50°C), approximately eight bands were detected on the membrane in BamHI-, HincII-, or EcoRI-digested DNA (data not shown). These patterns remained unchanged after the blot was washed twice at high stringency (0.1× SSC and 0.1% SDS at 65°C) (Fig. 5). These data suggest that black cherry MDL is encoded by a multigene family of approximately eight members.
Northern-Blot Analysis Reveals Differential Expression of Known MDL Genes In previous work, the temporal expression of MDL in developing black cherry seeds was analyzed by northern-blot analysis using the full-length MDL1 cDNA as a probe (Zheng and Poulton, 1995). MDL
transcripts were first detectable approximately 40 DAF, when embryos
became visible macroscopically, and peaked around 49 DAF before
declining to negligible levels by fruit maturity (82 DAF). This time
course is consistent with the levels of MDL proteins measured during
fruit ripening by direct enzyme assay of seed homogenates (Zheng and
Poulton, 1995). In agreement with previously published data (Swain et
al., 1992a), MDL activities were undetectable until 44 DAF. They then
increased rapidly during mid-phase II, essentially reaching a plateau
at full fruit maturity. Despite this excellent correlation, the high
degree of sequence identity now demonstrated for mdl1 to
mdl5 makes it highly unlikely that the MDL1 cDNA probe was
specific for any one MDL gene. Thus, it remains unclear how many MDL
genes are being expressed during fruit maturation.
Physiological Significance of the MDL Gene Family
* Corresponding author; e-mail jonathan-poulton{at}uiowa.edu; fax 1-319-335-3620. Received October 21, 1998;
accepted January 7, 1999.
Abbreviations:
3-OPP, 3-oxo-3-phenylpropene.
Con A, concanavalin A.
DAF, days after flowering.
HNL,
We thank Drs. Debashish Bhattacharya, Chi-Lien Cheng, and Ming-Che Shih for their helpful suggestions and for carefully reviewing the manuscript. We are also grateful to Dr. Bhattacharya for assistance with sequence analysis.
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Abstract
Introduction
