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Plant Physiol. (1998) 117: 997-1006
Molecular and Biochemical Characterization of Cytosolic
Phosphoglucomutase in Maize1
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Phosphoglucomutase (PGM) catalyzes the interconversion of glucose (Glc)-1- and Glc-6-phosphate in the synthesis and consumption of sucrose. We isolated two maize (Zea mays L.) cDNAs that encode PGM with 98.5% identity in their deduced amino acid sequence. Southern-blot analysis with genomic DNA from lines with different Pgm1 and Pgm2 genotypes suggested that the cDNAs encode the two known cytosolic PGM isozymes, PGM1 and PGM2. The cytosolic PGMs of maize are distinct from a plastidic PGM of spinach (Spinacia oleracea). The deduced amino acid sequences of the cytosolic PGMs contain the conserved phosphate-transfer catalytic center and the metal-ion-binding site of known prokaryotic and eukaryotic PGMs. PGM mRNA was detectable by RNA-blot analysis in all tissues and organs examined except silk. A reduction in PGM mRNA accumulation was detected in roots deprived of O2 for 24 h, along with reduced synthesis of a PGM identified as a 67-kD phosphoprotein on two-dimensional gels. Therefore, PGM is not one of the so-called "anaerobic polypeptides." Nevertheless, the specific activity of PGM was not significantly affected in roots deprived of O2 for 24 h. We propose that PGM is a stable protein and that existing levels are sufficient to maintain the flux of Glc-1-phosphate into glycolysis under O2 deprivation.
In cells of prokaryotic and eukaryotic organisms, PGM (EC
5.4.2.2), a phosphoenzyme, catalyzes an important trafficking point in
carbohydrate metabolism. In one direction, Glc-1-P produced from Suc
catabolism is converted to Glc-6-P, the first intermediate in
glycolysis. In the other direction, conversion of Glc-6-P to Glc-1-P
provides a substrate for synthesis of UDP-Glc, which is required for
synthesis of a variety of cellular constituents, including cell wall
polymers and glycoproteins. An obligatory step in the PGM reaction
mechanism is the transfer of phosphate from the Ser of the
phosphoenzyme to the substrate (Glc-6-P or Glc-1-P) to form the
dephosphorylated enzyme and Glc-1,6-P2, a catalytic intermediate and cofactor.
Two isoforms of PGM are present in plants, one located in the cytosol
and the other in the chloroplast stroma (Mühlbach and Schnarrenberger, 1978 Gene expression in plants is regulated both developmentally and by the
environment. Environmental stresses such as flooding (O2 deprivation/hypoxia and anoxia) affect Glc-P
metabolism in many agronomically important plants (for review, see
Drew, 1997 Many of the proteins synthesized in O2-deprived
roots participate in Suc breakdown, Glc-P metabolism, or ethanolic
fermentation (for review, see Sachs et al., 1996 As a first step toward a molecular characterization of plant cytosolic
PGM, we describe the isolation and characterization of full-length
cDNAs that encode cytosolic PGMs of maize. Maize lines with distinct
pgm1 and pgm2 genotypes were used to determine if
the cDNAs encode the known cytosolic isozymes of PGM. Since enzymes
involved in Glc-P metabolism are expressed during seed development and
in response to O2 deprivation, RNA analysis was performed to examine the developmental and environmental regulation of
PGM mRNA accumulation. Molecular and biochemical techniques were used
to confirm that PGM is a phosphoprotein in extracts from maize roots
and to examine the synthesis of this phosphoprotein in
O2-deprived roots.
Reagent-grade chemicals were purchased from United States
Biochemical, Sigma, or Boehringer Mannheim.
[ Plant Material
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
). In maize (Zea mays L.) cytosolic
isozymes of PGM are encoded by pgm1 and pgm2
(Goodman et al., 1980; Stuber and Goodman, 1983), which map to
duplicated chromosomal regions on 1L and 5S, respectively (Helentjaris
et al., 1988). The products of these loci are monomeric and are
detected in cell extracts from various tissues, including roots,
coleoptiles, leaves, scutella, and pollen (Stuber and Goodman, 1983).
Despite the extensive use of cytosolic PGM isozymes as genetic markers,
very little is known about pgm gene structure or expression
in plants.
). During the first few hours of O2
deprivation in maize, the synthesis of proteins made under aerobic
conditions is rapidly suppressed, whereas the synthesis of a small
group of proteins called ANPs is enhanced (Sachs et al., 1980). The
increased synthesis of the ANPs is not only due to increased
transcription of specific genes that encode them but also to selective
mRNA translation (Bailey-Serres and Freeling, 1990; Russell and Sachs,
1992; Fennoy and Bailey-Serres, 1995; Sachs et al., 1996; Manjunath and
Sachs, 1997; S. Fennoy, T. Nong, and J. Bailey-Serres, unpublished
data).
). The ANP Suc synthase
catalyzes the formation of UDP-Glc, which is subsequently converted to
Glc-1-P, the substrate of PGM. Enzymes involved in Glc-P metabolism are also up-regulated during the development of the kernel embryo and
endosperm (Chourey, 1981). This up-regulation is due to transcriptional induction of these genes, as evidenced by transient expression analysis
of alcohol dehydrogenase1 (adh1) promoter
constructs (Klein et al., 1989) and increased transcription of
shrunken1 (encoding Suc synthase) in nuclei isolated from
developing kernels (Kodrzycki et al., 1989).
MATERIALS AND METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
-32P]ATP (3000 Ci
mmol
1) and [35S]Met
(1200 Ci mmol1) were purchased from DuPont-New
England Nuclear and [
-32P]dCTP was
purchased from Andotek (Tustin, CA).
. Primary roots (3-5 cm)
and coleoptiles were cut directly into liquid N2
and stored at 
70°C until further use.
DNA Probes
We identified a rice EST cDNA (gift of Rice Genome Research Program NIAR/STAFF, Ibaraki, Japan; GenBank accession no. D24288) as a putative PGM clone on the basis of protein coding sequence similarity to human PGM (GenBank accession no. M83088). Rice and maize pgm cDNA inserts were obtained by digesting DNA clones with EcoRI and XhoI and electrophoresis on a low-melt agarose gel. cDNAs were labeled with [-32P]dCTP using a random-prime labeling kit
(Promega). For adh1, a 893-bp PstI fragment was
isolated by restriction digestion of the recombinant plasmid pZmL793
(Dennis et al., 1984). The 18S rDNA probe was a 210-bp fragment from a
tomato clone DB292 (D. Bird, personal communication).
cDNA Library Screening and DNA Sequencing
A cDNA library constructed in the Uni-ZAP-XR vector from poly(A+) mRNA of 6-h O2-deprived roots (3-d-old seedlings) of maize inbred B73 Ht was generously provided by Dr. M.M. Sachs, U.S. Department of Agriculture/Agricultural Research Station (Urbana, IL). Approximately 300,000 plaque-forming units were screened according to the manufacturer's protocol using a cDNA-synthesis kit (
-ZAPII, Stratagene), with the rice EST as the
probe at moderate stringency (hybridization in 7% SDS [w/v] and 50 mM NaPO4, pH 7.0, at 55°C; washed
with 0.1% SDS, 0.1 × SSC at 55°C). Both strands of cDNA were
sequenced using the fmol DNA cycle-sequencing system
(Promega) with commercially available and custom primers (Heligen
Laboratory, Huntington Beach, CA). A portion of the sequencing was
performed by the DNA Sequencing Core Laboratory (University of Florida,
Gainsville). Nucleotide and amino acid sequence analyses were performed
with the computer programs ClustalW (Thompson et al., 1994) and
Alscript (Barton, 1993) from the Genetics Computer Group (Madison, WI).
Southern Analysis
Genomic DNA was isolated from 5-d-old maize roots using the mixed alkyltrimethylammonium bromide (M-7635, Sigma) extraction procedure (Boyce et al., 1989). Fifty micrograms of DNA was digested with
HindIII (2.5 units µg1 DNA)
overnight in a reaction volume of 200 µL. The DNA was concentrated by
ethanol precipitation, fractionated on a 1% agarose gel, and transferred to a nylon membrane (MagnaGraph, MSI, Westbourgh, MA)
(Sambrook et al., 1989). Prehybridization was carried out in 50% (v/v)
formamide, 5× SSC, 20 mM NaPO4, pH
6.8, 1% (w/v) SDS, Denhardt's solution (0.02% [w/v] PVP, 0.02%
[w/v] Ficoll, and 0.02% [w/v] BSA), 5% dextran sulfate
(w/v), and 100 µg mL1 of sheared and
heat-denatured salmon-sperm DNA (Sambrook et al., 1989) for 4 h at
42°C. Hybridization to a [32P]-labeled
pgm2 cDNA probe was carried out with fresh prehybridization solution for 16 to 18 h at 42°C. Blots were washed and exposed to Hyperfilm (Amersham), as described by Manjunath and Sachs (1997).
Seed Germination, O2-Deprivation Treatment, and Labeling in Vivo with [35S]Met
O2-deprivation treatment was by submergence of intact seedlings in induction buffer (0.5 mM Tris-HCl, pH 8.0, 7.5 µg mL1 chloramphenicol) sparged
with argon in a closed Mason jar, as described by Fennoy and
Bailey-Serres (1995). Dissolved O2 concentration was measured with a Clark-type electrode (no. 97-08-00, Orion, Cambridge, MA) and meter (no. 611, Orion). For labeling proteins in
vivo, the apical 1 cm of the primary root of intact seedlings was
immersed in 150 µCi [35S]Met
mL1 induction buffer in a 1-mL plastic syringe
with the plunger removed and the tip sealed with Parafilm. Seedlings
were taken from open trays (aerobic) or after submergence in induction
buffer sparged with argon for 22 h (22 h O2
deprived). Labeling was carried out for the final 2 h of the
treatment in a humidified Mason jar that was open and maintained in air
(aerobic) or closed and sparged with argon (24 h
O2 deprived).
RNA Isolation and Northern Hybridization
Total RNA was extracted from various maize organs and tissues following the CsCl-gradient method described by Cone et al. (1986). RNA
(20 µg) was electrophoresed on a 2.2 M formaldehyde/1.3% agarose gel and transferred onto a nylon membrane (MSI) by capillary action with 10× SSC. Hybridizations were performed as described for
Southern blots. Blots were exposed to a phosphor imager screen and
quantified using ImageQuant software (Molecular Dynamics, Sunnyvale,
CA). To correct for differences in loading, 18S rDNA was used as a
hybridization probe. When needed, blots were regenerated by soaking in
0.1× SSC, 0.5% SDS for 15 min at 90°C.
Root Protein Extraction and Phosphorylation in Vitro
All procedures were carried out at 4°C unless otherwise indicated. Roots were homogenized in 2 mL of extraction buffer (20 mM Tris-HCl, pH 8.0, 4 mM DTT, unless indicated otherwise in figure legends) per gram of fresh weight with a pestle (Kontes, Vineland, NJ) in a microcentrifuge tube. The extract was centrifuged for 5 min at 16,000g, and the crude supernatant fraction was collected for enzyme assays, for in vitro phosphorylation, or for estimation of soluble-protein concentration. Protein extracts (10-30 µg) were phosphorylated for 6 min at room temperature in a 30-µL reaction mixture containing root extract, 5 µCi [-32P]ATP, and one or more of the following:
0.1 mM Glc-1,6-P2, 5 mM
MgCl2, 2 mM Glc, and 2 units
mL1 hexokinase (H-5375, Sigma), as indicated in
the figure legends.
Assay of Enzyme-Specific Activity
Specific activity of enzymes was assayed using crude cell extracts in 20 mM Tris-HCl, pH 8.0, 4 mM DTT, and 5 mM MgCl2. An assay coupled with Glc-6-PDH and the reduction of NADP+ was used to determine PGM activity. This assay was carried out in a 250-µL reaction mixture containing 20 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM Na2-Glc-1-P, 0.25 mM NADP+, and 0.1 mM Glc-1,6-P2. The reaction was initiated by the addition of 10 µL of extract (20-30 µg of protein) and 1 unit of Glc-6-PDH (G-5760, Sigma). The reduction of NADP+ was measured every 0.5 s for 1 min at 340 nm and at room temperature with a spectrophotometer (Lambda 3B, Perkin-Elmer Cetus). The specific activity of alcohol dehydrogenase in the direction of ethanol oxidation was determined as described by Kelley and Freeling (1984b). Specific activity was determined from reactions that were
linear for at least 2 min. One unit of enzyme activity is defined as 1 µmol of NADP+ or NAD+
reduced min1. Specific activity is expressed in
units per milligram of protein. Bradford reagent (United States
Biochemical) was used to determine protein concentration using BSA as
the standard.
Protein Gel Electrophoresis
Soluble root proteins (10-30 µg of extract per lane) were fractionated by native (nondenaturing)-PAGE at 4°C, as described by Bailey-Serres et al. (1992). Gels were stained for PGM activity immediately after electrophoresis by incubation in a fresh solution of
0.13 mM NADP+, 1 mM nitro
blue tetrazolium, 0.4 mM phenazine methosulfate, 100 mM Tris, pH 7.5, 5 mM
MgCl2, 0.3 mM EDTA, 7 mM
Na2-Glc-1-P, and 0.4 unit
mL1 Glc-6-PDH for 30 to 45 min at room
temperature. Gel staining was stopped by immersion in 10% (v/v)
glacial acetic acid for 5 min, and gels were stored in water prior to
drying. Two-dimensional native/SDS-PAGE was carried out as described
previously (Sachs et al., 1980). Total protein was visualized by
staining with Coomassie blue R-250. Labeled proteins were visualized by
exposure of dry gels to Hyperfilm at 70°C with an intensifying
screen for [-32P]ATP and at room temperature
for [35S]Met.
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Maize Cytosolic PGM Is Encoded by Two Nearly Identical Genes
Screening of a maize root cDNA library with a rice EST cDNA that has deduced polypeptide similarity to mammalian PGMs resulted in the isolation of two nearly identical cDNA clones (GenBank accession nos. U89341 and U89342). Both of the putative PGM cDNAs encode an open reading frame of 1752 bp. The overall DNA sequence identity between the coding regions of the two cDNAs was 96.7%. DNA sequence identity was also high in the 5
(87.0%) and 3 (88.5%) UTRs, but sequence
divergence increased in the 3 UTRs distal to the stop codon.
Analysis of PGM Transcript Accumulation during Development
PGM Transcript Accumulation Is Reduced during O2
Deprivation
Confirmation of Maize PGM as a Phosphoprotein
Identification of a 67-kD Phosphoprotein as PGM in Aerobic and
O2-Deprived Roots
PGM Specific Activity in Roots Is Not Significantly Affected by
O2 Deprivation
Plant cells possess cytosolic and plastidic PGM activity. We
identified cytosolic PGM as a phosphoprotein with an apparent molecular
mass of 67 kD in roots of maize. We isolated two cDNAs of maize that
encode PGMs of 98.5% deduced amino acid sequence identity and a
calculated molecular mass of 63 kD. The pgm cDNAs most
likely encode cytosolic PGM isozymes because: (a) Southern hybridization with a pgm cDNA probe identified RFLPs that
correspond to pgm1 and pgm2 genotype; (b) the
deduced amino acid sequences of maize PGM1 and PGM2 are 56% identical
to PGM of humans (Whitehouse et al., 1992 Received December 23, 1997;
accepted March 30, 1998.
Abbreviations:
ANP, anaerobic polypeptide.
DAP, days after
pollination.
EST, expressed sequence tag.
Glc-1,6-P2, Glc-1,6-bisphosphate.
Glc-6-PDH, Glc-6-phosphate dehydrogenase.
PGM, phosphoglucomutase.
RFLP, restriction fragment-length polymorphism.
UTR, untranslated region.
We thank Thoa Nong and Shaune Senter for technical assistance,
and members of the Bailey-Serres laboratory for their comments on the
manuscript. Alan Williams is thanked for his help with the multiple
protein alignment figure.
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UTRs to confidently
determine by Southern-blot hybridization which cDNA encodes PGM1 or
PGM2. Nevertheless, the detection of RFLPs corresponding to the
pgm1 and pgm2 genotype supports the conclusion
that the cDNAs we characterized encode the cytosolic isozymes PGM1
and/or PGM2.
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Figure 1.
Southern analysis of RFLPs identified by
hybridization of a maize pgm cDNA probe to genomic DNA
from maize lines with different pgm1 and
pgm2 genotypes. Fifty micrograms of genomic DNA from B73
(Pgm1-9; Pgm2-4),
2-null (Pgm1-5;
Pgm2-null), and
1-null (Pgm1-null; Pgm2-3) was digested with
HindIII, fractionated by electrophoresis on a 1%
agarose-TBE gel, transferred to a nylon membrane, probed with a
full-length pgm2 cDNA under high-stringency conditions, and exposed to radiographic film. M, The position of migration of
molecular mass markers (in kb). RFLPs characteristic of the pgm1 and pgm2 genotype are indicated to
the right. Hybridizing fragments present in all genotypes are not
labeled.
). On the basis of this information we tentatively
identify the cDNA that encodes the PGM of lower pI (5.47) as
pgm1 and the cDNA that encodes the PGM of higher pI (5.48)
as pgm2.
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Figure 2.
Alignment and comparison of the deduced amino acid
sequence of PGMs. Z. mays (Zmpgm1 and Zmpgm2),
Mesembryanthemum crystallinum (Mcpgm; GenBank accession
no. U84888), Homo sapiens (Hspgm; M83088),
Saccharomyces cerevisiae (Scpgm; P33401), A. tumefaciens (Atpgm; P39671), D. discoideum
(Ddpgm; U61984), E. coli (Ecpgm1: M77127; Ecpgm2:
U08369), and plastidic S. oleracea (Sopgm: X75898). The
numbers above the alignment correspond to residues of the maize PGMs.
Amino acid residues that are conserved in 7 of the 10 sequences are
shaded. The predicted catalytic center and the metal-ion-binding site
are boxed and labeled. The nine deduced amino acid sequence differences
between the two maize cytosolic PGMs are boxed and indicated with an
asterisk.
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Figure 5.
In vitro phosphorylation of PGM in crude extracts
of soluble proteins from aerobic roots. Root extracts were incubated
with [ -32P]ATP for 6 min at room temperature and
fractionated by native-PAGE. Lane 1, Activity gel stained in situ for
PGM activity; lanes 2 to 4, proteins visualized by autoradiography.
Lane 2, In vitro phosphorylation of crude root extract in extraction
buffer. Lane 3, Same as lane 2, with the addition of 0.1 mM
Glc-1,6-P2. Lane 4, Same as lane 2, with the addition of 5 mM MgCl2, 0.1 mM
Glc-1,6-P2, and 2 units mL1 hexokinase. The
position of PGM in the activity-stained gel and the direction of
electrophoresis are indicated.
). The catalytic reaction center and metal-ion- binding
loop of known animal and fungal PGMs are 100% conserved. On the basis
of this identity, the phosphorylation site of PGM1 and PGM2 is most
likely Ser-124. The hydrophobic-rich region at residues 300 to 304 in the multiple alignment shows very high identity to the metal-ion- binding loop Asp-Gly-Asp-Gly-Asp identified in rabbit muscle PGM (Dai
et al., 1992). Many additional regions of unknown function are highly
conserved between PGMs. However the PGMs of maize, ice plant, humans,
yeast, Dictyostelium discoideum, and Agrobacterium tumefaciens had considerably lower homology to the PGMs of
Escherichia coli and spinach plastids (see ``Discussion'')
(Fig. 2).
). To investigate the developmental expression of
these genes at the mRNA level, northern analysis was performed with
total RNA from roots, coleoptiles, leaves, pollen, silk, and developing
seed by hybridization to the full-length pgm2 cDNA. This
probe hybridizes to both pgm1 and pgm2; it was
not possible to generate gene-specific probes due to the high sequence
identity between the two cDNAs. The RNA blot shown in Figure
3 reveals that an approximately 2-kb PGM
transcript accumulated to detectable levels in all samples
except silk. Among the organs and tissues studied, PGM transcript
accumulation was highest in roots and coleoptiles relative to 18S rRNA
levels. PGM mRNA levels remained relatively unchanged throughout embryo
development, decreased slightly during endosperm development, and
decreased significantly during aleurone development.
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[in a new window]
Figure 3.
Developmental regulation of PGM mRNA
accumulation. A, Total RNA was isolated from various tissues, and 20 µg was fractionated by electrophoresis and hybridized at high
stringency to the coding region of a maize cytosolic
pgm2 cDNA and 18S rRNA, as described in ``Materials and Methods''. B, The hybridization signal in each lane was quantified with a phosphor imager, and the signal in each lane was normalized to
the level of 18S rRNA in the same sample. RNA level in roots was given
a value of 1.0, and fold increase/decrease over root levels is
presented as the relative mRNA level.
; Rowland and Strommer, 1986; Russell and Sachs, 1989; Fennoy and
Bailey-Serres, 1995; Manjunath and Sachs, 1997). Since PGM catalyzes
the formation of Glc-6-P, the first glycolytic intermediate, we
analyzed mRNA accumulation in O2-deprived roots
using the full-length pgm2 cDNA as a probe (Fig.
4, A and B). As mentioned earlier, this
probe hybridizes to both pgm1 and pgm2. A
1.5-fold increase in PGM mRNA accumulation was observed in the first
2 h of O2 deprivation, followed by a 4-fold
reduction over 24 h relative to aerobic levels. By contrast,
adh1 transcript accumulation increased over 40-fold relative
to aerobic levels after 24 h of O2
deprivation in the same samples.
View larger version (47K):
[in a new window]
Figure 4.
PGM mRNA accumulation in roots of seedlings
deprived of O2. A, Four- to five-day-old maize seedlings
were deprived of O2 for 0 to 24 h, total RNA was
isolated, and PGM transcript levels were analyzed with use of the
full-length pgm2 cDNA as described in Figure 3. The blot
was sequentially stripped and re-probed with an adh1
cDNA and an 18S rRNA probe. Transcript levels were quantified with a
phosphor imager. The corresponding aerobic values were given the value
of 1.0, and the increase/decrease over aerobic control is expressed as
the relative mRNA level.
; Salvucci et
al., 1990). An in vitro phosphorylation reaction and native-PAGE system
was developed to identify autophosphorylated PGM in crude extracts from
maize roots. Incubation of the buffered extract with
[-32P]ATP resulted in the efficient
phosphorylation of two proteins that were resolved by native PAGE. One
of the phosphoproteins comigrated with cytosolic PGM activity in the
native gel (Fig. 5, compare lanes 1 and
2). The reaction mixture was modified to improve the specificity of the
phosphorylation assay. The addition of
Glc-1,6-P2, an activator of PGM activity (Ray and
Peck, 1972), resulted in a marginal improvement in the specificity of
phosphorylation of the polypeptide that comigrated with PGM activity
(Fig. 5, lane 3). The addition of hexokinase and
Mg2+ to stimulate conversion of Glc to
Glc-6-[32P] significantly increased the
specificity of phosphorylation of the protein that comigrated with PGM
activity (Fig. 5, lane 4) and reduced the labeling of the more slowly
migrating phosphoprotein. These results confirm that cytosolic PGM of
maize can be phosphorylated in vitro in crude extracts from roots.
described the synthesis of 10 major and 10 minor soluble ANPs in anaerobically stressed roots of maize seedlings.
The same two-dimensional gel system was used to examine whether PGM is
synthesized in roots under aerobic and
O2-deprivation conditions. Seedling roots were
labeled in vivo with [35S]Met to examine de
novo protein synthesis. PGM was autophosphorylated by incubation of
crude root extracts with [-32P]ATP in the
presence of MgCl2,
Glc-1,6-P2, Glc, and hexokinase. Figure
6 shows a comparison of proteins of
aerobic and 24-h O2-deprived roots stained for
PGM activity (Fig. 6A), labeled in vivo with [35S]Met (Fig. 6B) and in vitro with
[-32P]ATP (Fig. 6, C and D).
View larger version (61K):
[in a new window]
Figure 6.
Two-dimensional gel analysis of soluble root
protein from aerobic (control) and O2-deprived seedling
roots. Twenty-five micrograms of protein was fractionated on a
native-polyacrylamide gel in the first dimension (A and C) and a 9%
(w/v) polyacrylamide-SDS gel in the second dimension (B and D). A, Gel
stained in situ for PGM activity. B, Proteins of seedling roots labeled
for 2 h in vivo with [35S]Met under aerobic
conditions or after 22 h of O2 deprivation. C and D,
Proteins phosphorylated in vitro with [ -32P]ATP in the
presence of 5 mM MgCl2, 0.1 mM
Glc-1,6-P2, 2 mM Glc, and 2 units/mL hexokinase
for 6 min at room temperature. Radiolabeled proteins were visualized by
autoradiography. Arrows indicate the positions of PGM, the 67-kD
polypeptide, and specific ANPs mentioned in ``Results''.
). A [35S]Met-labeled protein that
comigrated with PGM activity was observed in the samples from both
aerobic and 24-h O2-deprived roots (Fig. 6B,
unlabeled arrow) and corresponded to the 67-kD phosphoprotein (Fig.
6D). There were ANPs of 64 or 65 kD, neither of which had the same
electrophoretic mobility of PGM (Fig. 6B; ANP64 and ANP65). The
synthesis of the 67-kD polypeptide was reduced over the time course of
the O2 deprivation relative to the synthesis of
the Suc-synthase isozymes (Fig. 6B; ANP87). Since the synthesis of PGM
in maize roots is reduced in response to O2
deprivation, it is not an ANP.
View this table:
Table I.
Dissolved O2 concentration in induction
buffer and PGM- and ADH-specific activities in control and
O2-deprived roots
Dissolved O2 concentration data are from one representative
experiment. Specific activity data are means and SD from
three experiments. Values followed by a different letter are
significantly different (P 
0.01) as determined by the
Student's t test.
DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References
) and only 19% identical to
the putative plastidic PGM of spinach (Penger et al., 1994); (c)
pgm1 and pgm2 do not encode an N-terminal transit
peptide typically found on chloroplast-targeted proteins, including the
plastidic PGM of spinach (Heijne and Nishikawa, 1991; Penger et al.,
1994); and (d) pgm transcripts and PGM1 and PGM2 isozymes
accumulate in pollen (Stuber and Goodman, 1983), a cell type that in
maize does not contain plastids.
) and is located at residues 122 to 126 of maize PGM. The hydroxyl O2 of Ser-116 of human PGM (most likely Ser-124
of maize PGM) serves as the phosphate acceptor/donor in the catalytic
process (Ray and Peck, 1972). A metal-ion-binding loop of PGM,
Asp-Gly-Asp-Gly-Asp (Dai et al., 1992), located at residues 300 to 304 of maize PGM, is also highly conserved among these PGMs, except that
the second Gly residue is replaced by Ala in plant PGMs. Figure 2 also
shows the alignment of maize cytosolic PGMs with two PGMs of E. coli and a putative plastidic PGM of spinach.
). This PGM has limited sequence
identity to the PGMs of maize (19%) and E. coli (19%
identity to pgm2 [Lu and Klechner, 1994] and 27% identity to pgm1 [GenBank accession no. M77127]). Despite the low
level of overall identity, the multiple alignment analysis shows
conservation of the phosphorylated catalytic center and
metal-ion-binding site in the spinach plastid and E. coli
PGMs, suggesting a related enzymatic activity. Mutation of E. coli pgm2 led to partial blocking of Glc-1-P metabolism (Lu and
Klechner, 1994), indicating the presence of an alternative
Glc-1-P-utilization pathway. Subsequent analysis revealed that the
algC gene encoding the phosphomannomutase of
Pseudomonas aeruginosa can complement the pgm2
mutation in E. coli (Lu and Klechner, 1994). On the basis of
these observations, pgm1 of E. coli may encode an
alternative form of PGM or phosphomannomutase.
) and mRNA accumulation were detected in many plant organs
and tissues (Fig. 3). Northern analysis revealed significant PGM mRNA
accumulation in roots and coleoptiles. Transcript levels were lower per
microgram of rRNA in mature green leaves, pollen, and immature ears.
PGM transcripts were undetectable in silk, although a number of
transcripts encoding translation factors and ribosomal proteins (S. Manjunath and J. Bailey-Serres, unpublished data; K. Szick and J. Bailey-Serres, unpublished data) have been detected in the same mRNA
sample. The reason for the absence or low abundance of PGM mRNA in silk
tissue is not known.
). After about 15 DAP the kernel-fill period
begins (Ingel et al., 1965), and the activities of many enzymes
involved in starch and storage-protein synthesis increase rapidly
(Prioul et al., 1990). Doehlert et al. (1994) established that
shrunken1, -zein, aldolase, waxy,
shrunken2, and brittle2 transcripts peak at 15 or
30 DAP, then decrease to virtually undetectable levels by 55 DAP. We
observed that pgm transcripts were high in embryo and
endosperm from 15 to 25 DAP. Consistent with the expression of
pgm transcripts in these tissues, Tsai et al. (1970)
detected increased PGM activity throughout maize endosperm development.
; Drew, 1997). Therefore, we considered that PGM may also be synthesized in roots deprived of
O2. Northern analyses demonstrated that
accumulation of PGM mRNA was transiently induced by 2 h of
O2 deprivation, then decreased over 24 h of flooding. Concomitant with the decrease in PGM mRNA levels, in vivo
labeling of PGM with [35S]Met was also lower in
24-h O2-deprived roots than in aerobic roots.
Since PGM synthesis is reduced during O2
deprivation, we conclude that it is not an ANP. However, despite
reduced de novo synthesis, the specific activity of PGM in crude root
extracts was not significantly altered by 24 h of
O2 deprivation.
),
Fru-1,6-P2-aldolase (Kelley and Freeling, 1984b),
and enolase (Lal et al., 1991) are synthesized at or above aerobic
levels (i.e. are ANPs), but show no significant increase in specific activity and/or steady-state levels in
O2-deprived maize roots. However, cytosolic
glyceraldehyde-3-P-dehydrogenase activity is maintained during
O2 deprivation due to the induced expression of
two of four of the genes that encode this enzyme (Russell and Sachs,
1992; Manjunath and Sachs, 1997). Our results suggest that PGM may be a
stable protein in flooded roots. It is possible that the synthesis of
PGM is not up-regulated in response to O2
deprivation because of sufficient enzyme activity to maintain the flux
of Glc-6-P into glycolysis. This hypothesis could be tested by
measurement of carbon flux through Glc-6-P into glycolysis in maize
lines with varying levels of cytosolic PGM.
; Helentjaris et al., 1988), which are transcribed in
a number of tissues and organs and in response to
O2 deprivation. The failure to detect
tissue-/organ-specific differences in the accumulation of PGM1 and PGM2
isozymes (Stuber and Goodman, 1983) indicates that pgm1 and
pgm2 may be functionally redundant. High sequence identity
throughout the pgm cDNAs made it impossible for us to
determine if accumulation of mRNA encoding the two gene products was
differentially regulated. Cytosolic PGM was identified as a 67-kD
phosphoprotein that is synthesized at reduced levels in
O2-deprived roots. Nonetheless, PGM-specific activity was not affected by 24 h of O2
deprivation. Our analysis indicates that the cytosolic PGMs of higher
plants are distinct from the reported plastidic PGMs; however, the PGMs
of the two subcellular compartments possess the phosphate-transfer
catalytic center and metal-ion-binding site that are highly conserved
in all known prokaryotic and eukaryotic PGMs and hexose mutases.
1
This project was funded by the U.S. Department
of Agriculture/National Research Initiative Competitive Grants Program
(nos. 92-02016 and 95-00866) and by a University of
California-Riverside Academic Senate Research Award.
FOOTNOTES
*
Corresponding author; e-mail serres{at}mail.ucr.edu; fax
1-909-787-3738.
The GenBank accession numbers for the sequences reported in this
article are U89341 and U89342.
ABBREVIATIONS
ACKNOWLEDGMENTS
LITERATURE CITED
Top
Abstract
Introduction
Methods
Results
Discussion
References
Copyright Clearance Center: 0032-0889/98/117/0997/10
© 1998 American Society of Plant Physiologists
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