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Plant Physiol. (1999) 119: 255-266
Purification and Molecular Genetic Characterization of ZPU1, a
Pullulanase-Type Starch-Debranching
Enzyme from Maize1
Mary K. Beatty2, 3,
Afroza Rahman2,
Heping Cao,
Wendy Woodman,
Michael Lee,
Alan M. Myers, and
Martha G. James*
Department of Biochemistry, Biophysics, and Molecular Biology
(M.K.B., A.R., H.C., A.M.M., M.G.J.), and Department of Agronomy (W.W.,
M.L.), Iowa State University, Ames, Iowa 50011
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ABSTRACT |
This
study identified and purified specific isoamylase- and pullulanase-type
starch-debranching enzymes (DBEs) present in developing maize
(Zea mays L.) endosperm. The cDNA clone Zpu1 was
isolated based on its homology with a rice (Oryza sativa
L.) cDNA coding for a pullulanase-type DBE. Comparison of the protein product, ZPU1, with 18 other DBEs identified motifs common to both
isoamylase- and pullulanase-type enzymes, as well as class-specific sequence blocks. Hybridization of Zpu1 to genomic DNA defined a
single-copy gene, zpu1, located on chromosome 2. Zpu1
mRNA was abundant in endosperm throughout starch biosynthesis, but was not detected in the leaf or the root. Anti-ZPU1 antiserum specifically recognized the approximately 100-kD ZPU1 protein in developing endosperm, but not in leaves. Pullulanase- and isoamylase-type DBEs
were purified from extracts of developing maize kernels. The
pullulanase-type activity was identified as ZPU1 and the
isoamylase-type activity as SU1. Mutations of the
sugary1 (su1) gene are known to cause
deficiencies of SU1 isoamylase and a pullulanase-type DBE. ZPU1
activity, protein level, and electrophoretic mobility were altered in
su1-mutant kernels, indicating that it is the affected
pullulanase-type DBE. The Zpu1 transcript levels were equivalent in
nonmutant and su1-mutant kernels, suggesting that coordinated regulation of ZPU1 and SU1 occurs posttranscriptionally.
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INTRODUCTION |
Amylopectin is a branched Glc polymer that is a major constituent
of plant starch granules and is the primary determinant of their
structural and physical properties. The spatial positioning of
 (1 6) glycosidic bonds, i.e. branch linkages, is a critical aspect
of the three-dimensional structure of amylopectin (Gallant et al.,
1997 ). Branch linkages are introduced by the actions of starch
branching enzymes and are hydrolyzed by the actions of DBEs (for recent
reviews, see Preiss and Sivak, 1996; Smith et al., 1997).
Mutations that result in DBE deficiencies, such as the
sugary1 (su1) mutations of maize (Zea
mays L.) and rice (Pan and Nelson, 1984; James et al., 1995;
Nakamura et al., 1996b; Rahman et al., 1998), alter the number and
spatial distribution of branches in amylopectin. Therefore, DBEs are
believed to be involved in branch-pattern determination, possibly
providing an editing function (Ball et al., 1996).
Two classes of DBEs have been identified in plants that are
distinguishable by their substrate specificities (Lee and Whelan, 1971;
Doehlert and Knutson, 1991). "Isoamylase-type" DBEs cleave (16) branch linkages in amylopectin and glycogen, but do not hydrolyze the chemically identical bonds in pullulan, an
(16)-linked maltotriose polymer. In contrast,
"pullulanase-type" DBEs, also referred to as R-enzymes or
limit-dextrinases (Manners, 1997), readily hydrolyze (16)
linkages of pullulan or amylopectin, but have little activity toward
glycogen. Biochemical fractionation experiments identified both
isoamylase- and pullulanase-type DBE activities in developing maize
kernels during the starch biosynthetic period (Pan and Nelson, 1984;
Doehlert and Knutson, 1991), but the genetic identities and specific
functions of these two enzymes have not yet been established.
The primary sequences of a pullulanase-type DBE from rice endosperm and
an isoamylase-type enzyme from maize endosperm were discovered from
cloned cDNAs. Rice RE was purified and characterized as a
pullulanase-type DBE, and the cDNA coding for RE was cloned (Toguri,
1991; Nakamura et al., 1996a). A maize cDNA identified from a cloned
fragment of the su1 gene codes for a protein similar to
bacterial isoamylases (James et al., 1995). The su1 gene
product, SU1, functions as an isoamylase-type DBE and is present in
amyloplasts of developing maize endosperm during the time that starch
is synthesized (Rahman et al., 1998; Yu et al.,
1998).
Expression of the isoamylase- and pullulanase-type DBEs of maize
seemingly is coordinately controlled. Although the su1 locus codes for an isoamylase-type enzyme (Rahman et al., 1998), previous studies have demonstrated a reduction in the activity of a
pullulanase-type DBE in su1-mutant endosperms (Pan and
Nelson, 1984). Consistent with these data, a protein related
immunologically to rice RE is present in nonmutant maize kernels at 20 DAP, but is deficient in su1-mutant kernels of the same age
(Rahman et al., 1998). Thus, su1 mutations apparently result
in the deficiency of two distinct DBEs. A similar situation is likely
to occur in rice, in which the su1 mutation controlling RE
expression maps to a chromosomal location that is distinct from the
gene that codes for RE (Nakamura et al., 1996a).
To determine how DBEs affect starch structure, we are seeking to
identify and characterize completely these enzymes in maize endosperm.
Here we describe a full-length cDNA, designated Zpu1 (for
Zea mays pullulanase-type DBE),
which codes for a protein similar in sequence to known pullulanase-type
DBEs. Zpu1 transcript accumulation was characterized, and the
corresponding gene, zpu1, was mapped. The gene product ZPU1
was purified from developing endosperm and shown to be a
pullulanase-type enzyme. Analyses of ZPU1 in a su1-Ref
mutant indicated that its expression, electrophoretic mobility, and
enzymatic activity are dependent on the presence of a functional
Su1 gene.
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MATERIALS AND METHODS |
Plant Materials and Nomenclature
Nonmutant plants in the maize (Zea mays L.) inbred
lines W64A and Oh43 and plants homozygous for the reference mutation
su1-Ref (Correns, 1901), introgressed in these same genetic
backgrounds, were used for gel-blot and protein analyses. Standard
genetic nomenclature for maize is used as described by Beavis et al.
(1995). In addition, nonitalicized gene symbols are used to designate cDNAs and transcripts.
Characterization of Zpu1 cDNA
A random-primed maize endosperm cDNA library in  gt11 (K. Cone,
University of Missouri, Columbia) was screened using a 1.2-kb HindIII fragment from the rice RE cDNA as a hybridization
probe (Nakamura et al., 1996a). Standard procedures were followed for preparation of phage lifts, phage amplification, and single-plaque purification, plasmid construction, and growth of Escherichia coli cells (Ausubel et al., 1989; Sambrook et al., 1989). DNA was
isolated from purified phage by the Wizard DNA Purification Kit
(Promega). cDNA inserts were characterized with regard to their length
by gel electrophoresis after digestion with EcoRI. The
longest, in-clone 14-1 was 2.3 kb in length and was subcloned into
pBluescript KS+ to create plasmid pMB12. Subsequent screens of the
endosperm cDNA library with the entire 2.3-kb insert from pMB12 (probe
EE2.3) identified clones overlapping the 3 end (6A, 10A, 16A,
and 17C), and screens using the 680-bp
EcoRI/HindIII fragment as a probe (EH.68)
identified clones overlapping the 5 end (2A, 3A, 3C, and
3E). Nucleotide sequences were determined by standard procedures
(Ausubel et al., 1989) for both strands of the cDNA inserts in pMB12
and phage clones 17C, 3C, and 3E. Universal and synthetic
primers specific to portions of the cDNA inserts or to DNA were
used for sequence analysis.
Production of Anti-ZPU1 Polyclonal Antisera
To express part of ZPU1 as a fusion protein, the 2.3-kb
EcoRI insert from pMB12 (see Fig. 1) was subcloned into
plasmid pGEX-4T-2 (Pharmacia), creating pML1. E. coli DH5
cells containing pML1 were grown in 50 mL of LBA medium (Luria broth
supplemented with 40 µg/mL ampicillin) at 37°C for 7 h, and
the entire culture was then transferred to 1 L of LBA medium
supplemented with 2.5 mM betaine and 1 M sorbitol and grown at 30°C for 24 h.
Fusion protein expression was induced by the addition of
isopropyl- -D-thiogalactopyranoside to 0.1 mM, and incubation was continued at 37°C for 3 h.
Cell lysis and affinity purification of glutathione
S-transferase-ZPU1 using glutathione-agarose beads was
performed as described previously (Rahman et al., 1998). The fusion
protein was eluted in 100 mM Tris-HCl, pH 8.0, 120 mM NaCl, and 20 mM
glutathione.
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| Figure 1.
A, Overlapping bacteriophage clones provide
the full-length Zpu1 cDNA. The 3261-bp nucleotide sequence of the Zpu1
cDNA was determined from overlapping sequences of three clones. The
Zpu1 cDNA sequence is available as accession no. AF080567. B, Primary
sequence alignment of ZPU1 with rice RE. Amino acid residues deduced
from the Zpu1 cDNA (Zm) and rice RE cDNA (Os; accession no. D50602) are
shown in alignment. Boxed residues are the same in both polypeptides.
The asterisk designates the transit peptide cleavage site known in
RE.
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To produce polyclonal anti-ZPU1 serum, 0.5 mL (approximately 300 µg)
of purified glutathione S-transferase-ZPU1 in 1× PBS was
mixed with 0.5 mL of Freund's complete adjuvant (Sigma) and injected
into each of two New Zealand White rabbits, according to standard
procedures (Harlow and Lane, 1988). Inoculations were repeated four
times at 3-week intervals using approximately 200 µg of fusion
protein emulsified in Freund's incomplete adjuvant (Sigma). Immune
serum was collected 6 weeks after the final inoculation, assayed for
antibody titer, and stored at  80°C in the presence of 0.02% sodium
azide.
DNA and RNA Gel-Blot Analyses
DNA Gel Blots
Genomic DNAs were isolated from line W64A seedling leaves
according to the procedure of Dellaporta et al. (1983), digested with
restriction enzymes, and electrophoresed and transferred to nylon
membrane, as described previously (James et al., 1995). DNA gel blots
were hybridized with probe EE2.3 or EH.68 labeled with
32P by the random-primer method (Ausubel et al.,
1989).
RNA Gel Blots
Total RNAs were isolated from maize tissues and subjected to
gel-blot analysis, as described previously (Gao et al., 1998). RNA gel
blots were hybridized with probe EE2.3.
Mapping of zpu1
The zpu1 gene locus was mapped to a specific maize
chromosome by analysis of restriction fragment-length polymorphisms in the T232×CM37 and CO159×Tx303 RI populations, consisting of 48 and 41 individuals, respectively (Burr et al., 1988). Segregation data
produced from both populations were used (Burr et al., 1993; Matz et
al., 1994). Genomic DNAs were isolated from immature leaves of parental
inbreds and RI plants according to the method of Saghai-Maroof et
al. (1984), digested with EcoRI, and subjected to gel
electrophoresis. Gel blots were hybridized with Zpu1 probe EE2.3.
Maximum-likelihood estimates of linkage and map distances were
determined using the Mapmaker program (Lander et al., 1987). Genetic
linkage was determined with a recombination value of 50 and a LOD
threshold of 4.0.
Fractionation, Enzymatic Assay, and Immunoblot Analysis of DBEs
Cell Extract Preparation and Ammonium Sulfate Precipitation
Kernels were harvested 20 DAP, quickly frozen in liquid nitrogen,
and stored at 80°C. Approximately 15 g of frozen kernels or
endosperm tissue was pulverized in liquid nitrogen, then stirred overnight at 4°C in 40 mL of extraction buffer (50 mM
Hepes-NaOH, pH 7.5, 10 mM EDTA, 5 mM DTT, 1 mM PMSF, and 0.5 mL of protease inhibitor cocktail per gram
of tissue [no. P2714, Sigma]). The suspension was centrifuged at
39,000g for 20 min. The supernatant was filtered through
four layers of Miracloth (Calbiochem) and centrifuged again under the
same conditions. The supernatant was then passed through a 0.45-µm
syringe filter to yield the crude kernel extract. This solution was
made up to 40% ammonium sulfate and stirred for 1 h at 4°C.
Precipitated proteins were collected by centrifugation at
16,000g for 20 min, suspended in 20 mL of buffer A (50 mM Hepes-NaOH, pH 7.5, 10 mM EDTA, 5 mM DTT, and 5%
glycerol), and dialyzed overnight at 4°C in 1 L of the same buffer.
Anion-Exchange Chromatography
The dialyzed protein solution was centrifuged at
10,000g for 15 min, and the supernatant was passed through a
0.45-µm syringe filter. The solution was then applied to a
preequilibrated Q-Sepharose Fast-Flow column (1.5 cm × 46 cm
column, 80 mL bed volume; approximately 1.3 mg of protein loaded per
milliliter of bed volume; Pharmacia). After washing the column with 850 mL of buffer A, bound proteins were eluted with a linear, 600-mL
gradient of 0 to 1 M NaCl in buffer A. Fractions
(8 mL) assayed for DBE activity were concentrated approximately 80-fold
using an Ultrafree-4 centrifugal filter-unit concentrator (model
NMWL-10K, Millipore), pooled, made up to 50% in glycerol, quickly
frozen in liquid nitrogen, and stored at 80°C.
Gel-Permeation Chromatography
Proteins from the 40% ammonium sulfate precipitate or, for
purification A (see ``Results''), pooled fractions from the
Q-Sepharose column that exhibited pullulanase-type DBE activity, were
applied to a Sephacryl S-200 superfine gel-permeation column (2.5 cm × 90 cm column, 440 mL bed volume; Pharmacia) and eluted with
the equilibration buffer (10 mM Hepes-NaOH, pH 7.5, 5 mM DTT, and 5 mM MgCl2)
at a flow rate of 0.5 mL min 1. Aliquots from
7.5-mL fractions were checked for pullulanase-type DBE activity.
Fractions containing the enzyme were pooled and concentrated as
described.
Fast-Protein Liquid Chromatography
Pullulanase-type DBE fractions from the Sephacryl S-200 column
containing 3 mg of protein were diluted to 10 mL in buffer A, then
loaded onto a Mono-Q column (1 mL bed volume; Pharmacia) equilibrated
with buffer A. The column was washed with 10 mL of buffer A, then
eluted with a linear, 50-mL gradient of 0 to 0.5 M NaCl in
the buffer. Fractions were assayed again for pullulanase-type DBE
activity, concentrated, and stored as described above.
Affinity Chromatography
Pullulanase-type DBE fractions pooled after anion-exchange
chromatography with Q-Sepharose were dialyzed in citrate buffer (50 mM sodium citrate, pH 5.5, and 5 mM DTT) and
concentrated as described. Approximately 0.4 mg of protein was applied
to a column containing epoxy-activated Sepharose (Sigma) conjugated with cyclohexa-amylose (Sigma) (Vretblad, 1974) and equilibrated with
the citrate buffer (0.7- × 8-cm column, 3 mL bed volume). Bound
pullulanase-type DBE was eluted with 1 mg/mL -cyclodextrin (cyclohepta-amylose) (Sigma) in citrate buffer. The fraction
exhibiting pullulanase-type DBE activity was desalted and concentrated
approximately 200-fold.
Immunoblot Analysis
Proteins in concentrated fractions were separated by SDS-PAGE in
6% gels and transferred to nitrocellulose membranes according to
standard procedures (Ausubel et al., 1989; Sambrook et al., 1989).
Immunodetection was modified from the ECL protocol (Amersham) as
described previously (Rahman et al., 1998) using affinity-purified anti-SU1 diluted 1:200 or crude anti-ZPU1 antiserum diluted 1:25,000. Alternatively, the chromatogenic 5-bromo-4-chloro-3-indolyl
phosphate/nitroblue tetrazolium reagent system (Bio-Rad) was employed
using crude anti-ZPU1 diluted 1:2,000. For comparative analysis of
protein fractions from nonmutant and su1-Ref mutant kernels,
equivalent amounts of protein were analyzed identically.
DBE Assays
For isoamylase assays, 100 µL of each column fraction was
incubated in a total volume of 0.2 mL containing 5 mg of amylopectin (Sigma) and 50 mM Hepes-NaOH, pH 7.0, for 2 h at
30°C. A 50-µL aliquot of each reaction was mixed with 700 µL of
water and 250 µL of 0.01 M I2/0.5
M KI solution. The change in
A550 was measured relative to a blank
amylopectin reaction lacking protein as the reference. To measure
reducing equivalent formation, the reactions were inactivated by mixing
a 50-µL aliquot of each reaction with 25 µL of 1 M
Na2CO3. Reducing
equivalents were determined as described by Fox and Robyt (1991) using
maltose as the standard. For pullulanase-type DBE assays, 50 µL of
each column fraction was incubated in a total volume of 0.1 mL
containing 5 mg of pullulan (Sigma) and 50 mM citrate, pH 5.5, for 2 h at 37°C. Reducing equivalents were
determined as described above using maltotriose as the standard.
Determinations of specific activities were made on pooled fractions
from each purification step. Aliquots were assayed after reaction times of 30 min, 1 h, and 2 h to demonstrate a linear increase in
activity.
Computational Analyses
For sequence analyses we used the Genetics Computer Group Sequence
Analysis Software Package (Madison, WI), and for multiple sequence
alignment we used the program PILEUP. Conserved sequence motifs were
assigned based on the presence of at least one invariant residue in the
19 polypeptides analyzed, as well as conservative substitutions of
several nearby residues in aligned positions. Consensus sequences were
determined by votes, according to the method of Posfai et al. (1989).
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RESULTS |
Characterization of a Maize cDNA Coding for a Predicted
Pullulanase-Type DBE
The near-full-length Zpu1 cDNA was cloned from a random-primed
maize endosperm cDNA library using a fragment of the rice RE cDNA as a
hybridization probe. The complete Zpu1 cDNA nucleotide sequence of 3261 bp was determined from three overlapping bacteriophage clones
(designated 3C, 14-1, and 17C) (Fig.
1A) (accession no. AF080567). Zpu1
contains an ATG-initiated, continuous open reading frame of 2886 bp
that predicts a 962-amino acid polypeptide, ZPU1, of approximately 106 kD. The ZPU1 protein is highly similar in sequence to rice RE,
exhibiting 78% identity among 880 aligned residues with three gaps in
the alignment (Fig. 1B). The mature rice RE begins with the Ala-Val
sequence located at predicted residues 75 to 76 (Nakamura et al.,
1996a). This sequence is conserved in ZPU1 at predicted residues 75 to
76 as well (Fig. 1B), suggesting that the preceding 74 residues
constitute a transit peptide for protein targeting. ZPU1 also shows
extensive similarity to a characterized pullulanase-type DBE from
spinach leaves (accession no. X83969); these two proteins are 59%
identical among 882 aligned residues with no gaps (data not shown).
There are genomic and cDNA sequences from barley that predict another
pullulanase-type DBE (accession no. AF022725); ZPU1 is identical to
this deduced amino acid sequence at 79% of 832 aligned residues, with
one gap of a single amino acid.
Sequence Motifs Conserved in Pullulanase- and Isoamylase-Type
Enzymes
Further sequence comparisons among plant and bacterial -(16)
glucan hydrolases indicate that the pullulanase- and isoamylase-type DBEs have been conserved separately in evolution. The high degree of
conservation in plants among pullulanase-type DBEs also occurs among
the plant isoamylases: the maize isoamylase-type DBE SU1 is 71%
identical over 690 aligned residues to an Arabidopsis protein predicted
from genomic sequence data (accession no. AF002109; data not shown).
However, each plant DBE is more similar to the bacterial enzymes of the
same class than to the plant enzyme of the other class. For example, in
the 200-residue span of ZPU1 and SU1 that is most similar, 32% of the
amino acids are identical. Within the same 200 aligned residues,
however, ZPU1 is 46% identical to pullulanase from Klebsiella
aerogenes and SU1 is 47% identical to isoamylase from
Pseudomonas amyloderamosa (data not shown). These
observations suggest that isoamylase- and pullulanase-type DBEs
diverged before establishment of the plant kingdom, and that the
function of each type of DBE has been selected independently during the
evolution of plants.
Comparison of the entire ZPU1 sequence to 18 other known or predicted
isoamylase- and pullulanase-type enzymes from plants and prokaryotes
supported the preceding conclusion. As noted previously, both types of
DBEs contain all four regions (motifs I-IV) conserved within the
-amylase superfamily of starch hydrolytic enzymes (Jesperson et al.,
1993; James et al., 1995; Nakamura et al., 1996a, 1997). Two additional
conserved sequence blocks, designated motifs V and VI, are identified
here that occur in all of the DBEs that we examined, whether they fall
within the isoamylase- or the pullulanase-type class (Fig.
2). Among these six common motifs, 20 residues are conserved in each of the 19 DBEs analyzed. Class-specific,
conserved sequence blocks were also identified (Fig. 2). Enzymes
grouped in the pullulanase-type class contain five conserved regions
that are not found in the isoamylase-type enzymes. Similarly, eight
motifs conserved among the isoamylase-type enzymes do not occur in the
pullulanase-type enzymes.
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| Figure 2.
Multiple sequence alignment of pullulanase- and
isoamylase-type DBEs from higher plants and prokaryotes. DBEs are
grouped based on characterized enzymatic activity and/or sequence
similarity; any polypeptide within a class is significantly more
similar to others within that group than to those of the other class.
Conservative substitutions in the consensus sequences are noted when
they fall into the functional groups defined by Dayhoff and Orcutt
(1979), which are AGPST, ILMV, HKR, DENQ, RWY, and C. Residues
invariant in all 19 sequences are noted by asterisks. Rare exceptions
to the consensus sequence are underlined. Numerals refer to amino acid
position beginning at the first ATG codon of the open reading frame.
The number of nonconserved amino acids adjacent to each conserved motif
is indicated. Conserved motifs in boxes are present in both the
pullulanase- and isoamylase-type classes, whereas conserved motifs
without boxes are specific to one of the classes as indicated. Motifs I
to IV are those defined previously that occur in all members of the
-amylase superfamily (Jesperson et al., 1993), and are numbered
accordingly. Abbreviations and accession numbers are provided for the
following DBES. Pullulanases and pullulanase-type DBEs: Bth
(Bacteroides thetaiotaomicron, U67061); Csa
(Caldicellulosiruptor saccharolyticus, L39876); Kae
(Klebsiella aerogenes, M16187); Hvu (Hordeum
vulgare, AF022725); Kpn (Klebsiella pneumoniae,
X52181); Osa (Oryza sativa, D50602); Sol
(Spinacia oleracea, X83969); Tma (Thermotoga
maritima, AJ001087); Tsp (Thermus sp. IM6501,
AF060205); Zma (Z. mays, AF080567, this study).
Isoamylases and isoamylase-type DBEs: Art (artificial gene, A10906);
Ath (Arabidopsis, AF002109); Eco (E. coli, U18997); Fla
(Flavobacterium sp., U90120); Psu
(Pseudomonas sp., A28109, A37035); Sac
(Sulfolobus acidocaldarius, D83245); Sso
(Sulfolobus solfataricus, Y08256); Syn1
(Synechocystis sp., U44761); Syn2
(Synechocystis sp., D90908); and Zma (Z. mays, U18908).
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Mapping of zpu1 within the Maize Genome
DNA gel-blot analysis of genomic DNA from maize inbred line W64A
revealed that the zpu1 locus is unique within the maize
genome. Restriction enzymes that do not cleave the Zpu1 cDNA were used, and the 2.3-kb EcoRI fragment of the cDNA containing codons
160 to 930 (Fig. 1) was used as a hybridization probe (designated probe
EE2.3). The probe hybridized to a unique KpnI genomic
fragment (Fig. 3), indicating that
zpu1 is a single-copy gene. To support this conclusion, the
blot was stripped of the probe and rehybridized with a smaller portion
of the Zpu1 cDNA, the 680-bp EcoRI/HindIII fragment comprising codons 160 to 387 (designated probe EH.68; Fig. 1).
In this analysis unique genomic fragments were identified using six
different restriction enzymes (Fig. 3).
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| Figure 3.
The copy number of the zpu1 locus
was determined by gel-blot analysis of genomic DNA. DNA from maize
inbred W64A was digested with the indicated restriction enzymes (B,
BamHI; K, KpnI; N, NotI;
P, PstI; S, SstI; X, XbaI;
and Xh, XhoI). The gel blot was hybridized at high
stringency with probe EE2.3 (left panel), then stripped of probe and
hybridized with probe EH.68 (right panel) of the Zpu1 cDNA.
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The zpu1 locus was mapped to chromosome 2 (Burr et al.,
1994). Probe EE2.3 (Fig. 1) was used to identify restriction
fragment-length polymorphisms in two populations of RIs. In the two
sets of parental inbreds polymorphisms were detected by digestion with
EcoRI, which produced Zpu1-homologous fragments of 4.3 and
3.0 kb in line CM37, 5.2 kb in line T232, 5.4 and 4.3 kb in line Tx303,
and 5.0 and 3.9 kb in line CO159 (data not shown). The detection of
more than one band in three lines is most likely attributable to the
presence of an internal EcoRI site in the zpu1
locus, given that each pair of bands was inherited as a single allele.
Identification of the parental allele in individual plants of the
CM37×T232 and Tx303×CO159 RI populations allowed us to determine the
genetic linkage to previously mapped physical markers using the program
Mapmaker. These linkage data placed zpu1 approximately 2.7 centimorgans from marker accA and 1.2 centimorgans from marker pps15 in
the Tx303×CO159 RI population, with a LOD score of 9.4. Similar
results were obtained with the CM37×T232 population, with
zpu1 mapping approximately 2.5 centimorgans from marker accA
and 1.1 centimorgans from marker isu142, with a LOD score 11.4. Thus,
zpu1 was localized to the region of Bin 2.05 to 2.06 (Gardiner et al., 1993), although specific placement to either the
short or the long arm of the chromosome could not be made.
Tissue and Developmental Expression of Zpu1 mRNA
The tissues in which Zpu1 mRNA accumulates were identified by RNA
gel-blot analysis. Total RNAs isolated from maize embryos, developing
endosperm, leaves, roots, and tassels were separated by gel
electrophoresis and hybridized with probe EE2.3. A transcript of
approximately 3.2 kb was abundant in endosperm from kernels harvested
20 DAP, and was weakly expressed in both the embryo and tassel tissues
(Fig. 4A). Transcript was not detected in
leaf or root tissue, indicating that Zpu1 expression is
specific to the reproductive tissues of the plant. The 3.2-kb size of
the transcript matches the length of the cloned cDNA, providing further confirmation that the clone is nearly full length. Probe EE2.3 also
identified a smaller-sized transcript of approximately 1.4 kb that
corresponded on all RNA blots with accumulation of the larger
transcript. The identity of the 1.4-kb transcript is not known at this
time. Although the RNAs detected by the Zpu1 probe migrate
at nearly the same rate as the rRNAs, the signal does not result from
nonspecific binding because the rRNAs are equally abundant in all of
the samples, whereas the Zpu1 transcript is tissue specific.
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| Figure 4.
Total RNAs from various sources were hybridized
with probe EE2.3. The RNAs as they appeared in the ethidium bromide
(EtBr)-stained gel before transfer are shown to indicate RNA integrity
and loading differences. A, RNAs from embryo (Em) and endosperm (En)
harvested 20 DAP, seedling leaves (L), immature root (R), and immature
tassel (T). B, RNAs from maize endosperm harvested at various times
after pollination. C, RNAs from nonmutant (Su1) and
mutant (su1-Ref ) kernels harvested 20 DAP.
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Zpu1 mRNA levels over the course of endosperm development were
determined as well. Total RNAs isolated from wild-type kernels at 7, 12, 14, 18, 20, 26, and 30 DAP were analyzed. Zpu1 mRNA was shown to be
weakly expressed at 12 and 14 DAP, but strongly and uniformly expressed
from 18 to at least 32 DAP (Fig. 4B).
Immunological Detection and Purification of DBE Activities from
Developing Maize Kernels
ZPU1 was detected in soluble kernel extracts by immunological
methods. The polyclonal antiserum anti-ZPU1 was raised in rabbits against the 770 residues of ZPU1 derived from codons 160 to 930. Anti-ZPU1 detected a major polypeptide of approximately 100 kD among
proteins from crude extracts of developing wild-type kernels (data not
shown) and in specific fractions (Fig.
5A). The apparent size of this protein
corresponds with that predicted by the Zpu1 cDNA. Anti-ZPU1 failed to
detect ZPU1 in protein extracts from seedling leaves harvested during
the light or dark cycle (data not shown). This observation, together
with the transcript-accumulation data, demonstrated that
zpu1 is not expressed in leaves during either phase of the
photosynthetic period.
View larger version (36K):
[in this window]
[in a new window]
| Figure 5.
A, Q-Sepharose chromatography. Fractions eluted
from the column were assayed for DBE activity using pullulan ( ) or
amylopectin ( ) as a substrate. Products of the amylopectin reaction
were complexed with iodine, and the change in
A550 value relative to untreated substrate
was plotted ( ). Activity units for the amylopectin digestion are
microgram maltose equivalents produced after a 2-h incubation of
substrate with 100 µL of protein fraction. Activity units for the
pullulan digestion are microgram maltotriose equivalents produced after
a 2-h incubation of the substrate with 50 µL of the protein fraction.
Fractions with DBE activity were subjected to immunoblot analysis with
anti-ZPU1 or anti-SU1 antiserum, as indicated (right-hand panels). B,
Gel-filtration chromatography. The peak fractions of pullulanase-type
activity from Q-Sepharose columns were pooled, concentrated, and
applied to a Sephacryl S-200 superfine gel-permeation column. DBE
activity in the fractions eluted from this column was assayed using
pullulan as the substrate; activity units are as described for A. Fractions were also assayed for the presence of ZPU1 by immunoblot
analysis (right-hand panel). C, Mono-Q chromatography. The peak
fractions (7-11) of pullulanase-type activity from the Sephacryl S-200
column were pooled, concentrated, and applied to a Pharmacia
fast-protein liquid chromatography Mono-Q column. DBE activity in
fractions eluted from this column was assayed using pullulan as the
substrate; activity units are as described for A. Fractions were
assayed for the presence of ZPU1 in immunoblots (right-hand panels). D,
Affinity chromatography. The peak fractions of pullulanase-type
activity from Q-Sepharose columns were pooled, concentrated, and
applied to a column containing epoxy-activated Sepharose conjugated
with cyclohexa-amylose. DBE activity in the four fractions eluted from
this column was assayed using pullulan as the substrate; activity units
are as described for A. Proteins from two of the fractions were
separated by SDS-PAGE and the gel was silver stained; a duplicate gel
was subjected to immunoblot analysis with anti-ZPU1 (right-hand
panels).
|
|
The product of the Zpu1 cDNA cofractionated with a pullulanase-type DBE
activity purified from extracts of developing maize kernels, thereby
confirming the identity of ZPU1 as a pullulanase-type enzyme. The DBE
activities present in the 40% ammonium sulfate precipitate
from extracts of nonmutant kernels harvested 20 DAP were
separated by anion-exchange chromatography on Q-Sepharose (Fig. 5A).
Pullulanase-type DBE activity was assayed by measuring increases in
reducing sugar concentrations after incubation of the substrate
pullulan with protein fractions. DBE activity was also determined by
increased reducing value measurements using amylopectin as the
substrate, and by changes in the A550 value of the glucan-iodine complexes formed after incubating amylopectin with
the protein fractions. Owing to substrate specificity, the assays using
pullulan were expected to detect only pullulanases, whereas the
amylopectin assays could identify either isoamylase- or
pullulanase-type DBEs.
One peak of DBE activity was observed using pullulan as the substrate,
and two peaks were observed with amylopectin as the substrate, one of
which coincided with the pullulanase-type DBE peak (Fig. 5A).
Immunoblot analysis using anti-ZPU1 or anti-SU1 (Rahman et al., 1998)
was used to determine whether ZPU1 or SU1 could be correlated with
either activity. ZPU1 was identified only in the fractions exhibiting
pullulanase-type DBE activity, whereas SU1 was present only in those
DBE fractions that constituted the second peak of activity toward
amylopectin (Fig. 5A), i.e. each activity peak yielded a positive
immunoblot signal with only one of the two antisera. This analysis
provided a clear distinction between the pullulanase- and
isoamylase-type activities in developing maize kernels, and identified
the particular DBE responsible for each activity peak. Thus, the
su1 gene product was identified specifically as an
isoamylase-type DBE active in developing kernels, and the
zpu1 gene product was identified specifically as an active pullulanase-type DBE in the same tissue. The increased
A550 of the glucan-iodine complex ("blue
value") obtained after amylopectin digestion (Fig. 5A) indicates that
a DBE, as opposed to contaminating -amylase activity, is responsible
for the increased reducing value in the peak assigned as isoamylase
(fractions 42-49). Contaminating -amylase activity can also be
excluded as the cause of the peak assigned as pullulanase-type DBE
(fractions 25-32), because the former enzyme does not hydrolyze
pullulan.
Pullulanase-type DBE activity was further purified by gel-filtration
chromatography followed by another anion-exchange chromatography step.
Q-Sepharose fractions that displayed pullulanase-type DBE activity were
pooled and separated on the basis of size using Sephacryl S-200
chromatography. Assays of these fractions identified a pullulanase-type
DBE activity, and immunoblot analysis revealed that ZPU1 again
cofractionated with the activity peak (Fig. 5B). The enzyme was further
purified by anion-exchange chromatography on a Mono-Q column; once
again, ZPU1 cofractionated with the purified pullulanase-type DBE (Fig.
5C).
Measurements of the pullulanase-type DBE activity after the
anion-exchange and gel-permeation chromatography steps (purification A)
are presented in Table I. Specific
activity increased with each round of purification, resulting in a
100-fold purification of the enzyme from the ammonium sulfate
precipitate. This purification stage was used as the baseline because
contaminating hydrolases have been shown by others to artificially
elevate the apparent pullulanase-type DBE activity in crude extracts
(Lee et al., 1971; Maeda et al., 1978).
A further purification of the pullulanase-type DBE was achieved by
means of affinity chromatography (Fig. 5D). Q-Sepharose fractions that
exhibited pullulanase-type DBE activity (Fig. 5A) were pooled and the
proteins separated on the basis of their affinity for cyclohexa-amylose
Sepharose (purification B). Pullulanase-type DBE activity was detected
in only one of the four fractions eluted from the affinity column.
SDS-PAGE and silver staining of the proteins in these fractions
revealed one band of approximately 100 kD, which coeluted with
pullulanase-type DBE activity. This protein was identified as ZPU1 by
immunoblot analysis (Fig. 5D). The Q-Sepharose and affinity
chromatography steps resulted in a 200-fold purification of ZPU1 (Table
I), again using the ammonium sulfate precipitate as the baseline. The
fact that ZPU1 was the only protein present in the most pure enzyme
preparation provides definitive evidence that ZPU1 and the purified
pullulanase-type DBE are one and the same.
Accumulation of ZPU1 Protein and Zpu1 mRNA in Nonmutant and
su1-Mutant Kernels
The previous finding that pullulanase-type DBE activity is greatly
reduced in su1 mutants (Pan and Nelson, 1984) prompted further characterization of the DBEs in su1-Ref kernels.
Proteins from nonmutant and mutant kernels harvested 20 DAP were
separated on the basis of size using Sephacryl S-200 chromatography.
Pullulanase-type DBE activity was assayed by measuring hydrolysis of
pullulan, and isoamylase-type DBE activity was assayed by blue-value
determinations after hydrolysis of amylopectin. As expected from the
Q-Sepharose fractionation (Fig. 5A), distinct peaks of activity were
observed for each DBE in the nonmutant extracts (Fig.
6A). Immunoblot analyses again confirmed
that the pullulanase-type DBE activity corresponded with ZPU1 and the
isoamylase-type DBE activity corresponded with SU1. Both peaks of DBE
activity were reduced in the su1 mutant (Fig. 6B).
Immunoblot analyses of the protein fractions from both genotypes were
performed under identical conditions. The pullulanase- and
isoamylase-type DBE activities affected by su1-Ref
corresponded with reduced accumulation of the ZPU1 and SU1 proteins,
respectively (Fig. 6B). However, a shift was also detected in the
electrophoretic mobility of ZPU1 in certain su1-Ref
fractions. Direct comparison of corresponding nonmutant and
su1-Ref fractions revealed that the anti-ZPU1 antiserum
identifies a polypeptide doublet of approximately 100 and 105 kD,
respectively, and that the form with the apparent lower molecular mass
predominates in the nonmutant kernels (Fig. 6C). This polypeptide was
greatly reduced in the su1-Ref mutant, but the form with the
apparent higher molecular mass was increased (Fig. 6C). Similar results
were observed with the independent allele
su1-R4582::Mu1 (James et al., 1995; data not
shown).
View larger version (39K):
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| Figure 6.
Fractionation of DBEs from nonmutant and
su1-Ref kernels by gel-permeation chromatography. A,
DBEs in nonmutant kernels. Proteins from nonmutant kernels harvested 20 DAP were applied to a Sephacryl S-200 superfine gel-permeation column.
Fractions were assayed for pullulanase-type DBE activity by measuring
formation of new reducing ends after incubation with pullulan (activity
units, ), and for isoamylase-type DBE activity by determination of
iodine-complex absorbance maxima after incubation with amylopectin
(A550,  ). Equivalent amounts of protein
from fractions exhibiting DBE activity were analyzed for the presence
of ZPU1 or SU1 on immunoblots with the indicated antisera. B, DBEs in
su1-Ref kernels. Proteins from su1-Ref
kernels harvested 20 DAP were fractionated and assayed for DBE
activity, and immunoblot analysis was performed, as described for A. C,
Comparative immunoblot analysis. Nonmutant proteins in fractions 9 to
12 from A (lanes +) and su1-Ref proteins in fractions 9 to 12 from B (lanes m) were subjected to immunoblot analysis with the
anti-ZPU1 antibody. Equivalent amounts of protein were loaded, and each
lane contained twice as much protein as the immunoblots shown in A and
B.
|
|
To determine whether the effect of su1 mutations on ZPU1
expression occurs at the level of transcription, the steady-state level
of Zpu1 mRNA was compared in nonmutant and su1-Ref-mutant kernels harvested 20 DAP. Full-length Zpu1 transcripts were
approximately equal in both size and abundance in the nonmutant and
mutant kernels (Fig. 4C). Thus, no obvious changes were detected in the
transcription of Zpu1 in the su1 mutants compared with
nonmutant kernels.
| |
DISCUSSION |
This study identified the specific genetic elements responsible
for each of two distinct DBE activities in developing maize endosperm
tissue, extending the analysis of DBE activities described previously
by Doehlert and Knutson (1991). Activities of both isoamylase- and
pullulanase-type DBEs were purified from developing maize kernels. The
pullulanase-type enzyme activity corresponds with the product of the
gene zpu1 identified in this report, and the isoamylase-type
enzyme activity corresponds to the product of the su1 gene.
In a previous study recombinant expression of su1 produced
an active isoamylase-type DBE (Rahman et al., 1998). Taken together,
these data clarify the cast of DBEs present in maize endosperm cells:
zpu1 codes for a pullulanase-type DBE, su1 codes
for an isoamylase-type DBE, and both enzymes are present in amyloplasts
of endosperm tissue during the time that starch granules are being
produced.
Two lines of evidence support the conclusion that ZPU1 is a
pullulanase-type DBE. First, the polypeptide predicted by the Zpu1 cDNA
is highly similar in sequence to all known bacterial pullulanases and
plant pullulanase-type DBEs (Figs. 1 and 2). Second, antibodies raised
against the Zpu1 product detected an endosperm protein (or
protein doublet) that cofractionated with pullulanase-type DBE activity
in four different chromatography purification steps (Figs. 5 and 6).
After a nearly 200-fold purification of the pullulanase-type enzyme
activity, the 100-kD protein that reacts with anti-ZPU1 appeared to be
the only polypeptide present in the fraction.
The zpu1 gene is expressed predominantly in endosperm. The
small amount of transcript observed in the embryo could indicate a role
for the pullulanase-type DBE in embryo starch metabolism or,
alternatively, may result from endosperm contamination of the tissue
sample. Small amounts of Zpu1 transcript were detected in the tassel,
possibly indicating a role in pollen starch metabolism. The fact that
Zpu1 transcript was not detected in leaves is significant because
pullulanase-type DBEs have been characterized in photosynthetic tissue
from several species (Okita and Preiss, 1980; Li et al., 1992; Ghiena
et al., 1993). Presuming that one or more pullulanase-type DBEs are
present in maize leaves, they must be coded for by genes other than
zpu1.
The presence of both isoamylase and pullulanase types of DBEs may be a
general feature of tissues that produce storage starch. Both enzymes
have been reported in developing maize kernels (Doehlert and Knutson,
1991) and potato tubers (Drummond et al., 1970; Ishizaki et al., 1983).
In a recent study pea embryos were found to possess two distinct
pullulanase-type DBEs in addition to an isoamylase (Zhu et al., 1998).
From the fact that both zpu1 and su1 are highly conserved within the plant kingdom, we speculate that most
starch-producing sink tissues contain functional homologs of the DBEs
coded for by these two maize genes.
Two possibilities can be envisioned for the function of ZPU1. The
simplest explanation is that this DBE hydrolyzes storage starch during
seed germination. Even though ZPU1 is expressed during starch
biosynthesis, it may accumulate in an inactive form and be restricted
from action until after germination. Such restriction, however, would
have to occur even though the enzyme is catalytically active in
endosperm cell extracts. Furthermore, ZPU1 was shown by immunoblot
analysis to be enriched in the amyloplast stromal fraction of
developing endosperm (H. Mu, B. Wasserman, personal communication).
Thus, ZPU1 is present during the time that starch biosynthesis occurs
and in the same subcellular compartment. For these reasons we favor the
explanation that ZPU1 functions directly in starch biosynthesis as
opposed to starch utilization. A starch biosynthetic function has been
proposed for SU1 isoamylase (Ball et al., 1996), based on the fact that
su1 mutations result in the production of an overly
branched polysaccharide (Sumner and Somers, 1944). Mutations of
zpu1 are not known; however, because the cDNA sequence is
currently available, reverse genetic strategies can be used to
identify a mutant allele. Such a mutation could be used to determine
whether ZPU1 is needed for normal starch biosynthesis.
Previous studies showed that a pullulanase-type enzyme activity is
deficient in maize endosperm homozygous for the su1-Ref mutation (Pan and Nelson, 1984). The current study, however, together with the characterization of recombinant SU1 (Rahman et al., 1998), demonstrates unequivocally that Su1 does not code for a
pullulanase-type enzyme but instead specifies an isoamylase-type DBE.
Therefore, the reduction in pullulanase-type DBE activity in
su1 mutants must be explained by a pleiotropic effect. This
report shows that some or all of the pullulanase-type DBE affected
pleiotropically by su1 mutations is ZPU1. Pan and Nelson
(1984) identified three peaks of pullulanase-type DBE activity in
hydroxyapatite columns, all of which were affected to some extent by
the su1-Ref mutation. Further analysis is required to
determine whether all three peaks are attributable to zpu1
or, alternatively, if additional pullulanase-type DBEs exist in maize
endosperm.
Previously, our laboratory reported that su1-Ref mutant
kernels are deficient in an approximately 100-kD polypeptide identified by antisera to rice RE (Rahman et al., 1998). Anti-rice RE identified only a single polypeptide in nonmutant kernels in the region of 100 kD.
The current study shows that antiserum raised against the maize protein
ZPU1 identifies a polypeptide doublet of approximately 100 and 105 kD.
The lower band of this doublet is strongly reduced in su1
mutants. This is consistent with the previous report, because anti-rice
RE specifically identifies the protein of apparent lower molecular mass
(data not shown). Identification of the larger form of ZPU1, which is
increased in su1 mutants, suggests that the overall decrease
in ZPU1 protein resulting from su1 mutations is less than
previously thought. Rather, the data imply that the relative
accumulation of the two ZPU1 polypeptide forms is dependent on SU1
isoamylase, and that the form with the apparent lower molecular mass is
enzymatically active, whereas the larger form is functionally inactive.
The pleiotropic effect of su1 mutations on zpu1
gene expression could occur at either the transcriptional or the
posttranscriptional level. Transcriptional mechanisms regulating starch
biosynthetic gene expression have been demonstrated previously for
sugar-accumulating mutants of maize (Giroux et al., 1994). Zpu1
transcription is normal in su1-mutant kernels as far as can
be resolved by RNA gel-blot analysis, which suggests that changes in
transcription initiation are not responsible for the effects of
su1 mutations on ZPU1.
Several potential posttranscriptional mechanisms could account for the
changes in ZPU1 in su1 mutants. The possibility that altered
splicing of the Zpu1 pre-mRNA occurs as the result of su1
mutations has not been ruled out. At the level of protein-protein interaction, the possibility exists that SU1 and ZPU1 participate in an
enzyme complex. According to this model, loss of SU1 could lead to
destabilization and/or altered modification of ZPU1. Observation of SU1
and ZPU1 in distinct chromatographic fractions makes this model less
plausible, although the possibility remains that the complex
dissociates upon cell lysis or during fractionation. An explanation
that does not require direct SU1-ZPU1 interaction is that loss of the
isoamylase-type DBE results in an altered concentration or form of the
preferred substrate for the pullulanase-type DBE. Substrate binding in
turn might affect the stability of ZPU1 and/or alter its ability to
undergo further posttranslational modification. Finally, Su1
activity may be directly required to achieve or maintain an active form
of ZPU1. For example, SU1 might remove covalently linked glucan from
ZPU1, analogous to the known ability of Pseudomonas sp.
isoamylase to cleave the glucosyl-tyrosyl linkage in glycogenin (Lomako
et al., 1992). In any event, coordinate regulation of the two types of
DBE in maize endosperm cells suggests a cooperative function. Both the
timing of gene expression and the effects of su1 mutations
on starch structure suggest that SU1 and ZPU1 cooperate to play a
direct role in the biosynthesis of amylopectin.
| |
FOOTNOTES |
1
This work was supported by grants from the U.S.
Department of Agriculture (no. 96-35301-3159 to M.G.J. and A.M.M.) and
from the National Science Foundation (no. DIR-9113593 to the Iowa State University Signal Transduction Training Group). This is journal paper
no. J-18035 of project no. 3418 of the Iowa Agriculture and Home
Economics Experiment Station, Ames.
2
These authors contributed equally to this
work.
3
Present address: Pioneer HiBred International,
Inc., Johnston, IA 50131.
*
Corresponding author; e-mail mgjames{at}iastate.edu; fax
1-515- 294-0453.
Received July 31, 1998;
accepted October 12, 1998.
| |
ABBREVIATIONS |
Abbreviations:
DAP, days after pollination.
DBE, starch-debranching enzyme.
LOD, log of the odds.
RE, R-enzyme.
RI, recombinant inbred.
| |
ACKNOWLEDGMENTS |
We thank Dr. Yasunori Nakamura for providing the rice RE cDNA,
Dr. Karen Cone for providing the maize endosperm cDNA library, and Ming
Li for technical assistance. DNA sequence analysis was performed by the
Iowa State University DNA Synthesis and Sequencing Facility.
| |
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