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Suspension-cultured
Chenopodium album L. cells are capable of continuous,
long-term growth on a boron-deficient medium. Compared with cultures
grown with boron, these cultures contained more enlarged and detached
cells, had increased turbidity due to the rupture of a small number of
cells, and contained cells with an increased cell wall pore size. These
characteristics were reversed by the addition of boric acid (≥7
μm) to the boron-deficient cells. C. album
cells grown in the presence of 100 μm boric acid entered
the stationary phase when they were not subcultured, and remained
viable for at least 3 weeks. The transition from the growth phase to
the stationary phase was accompanied by a decrease in the wall pore
size. Cells grown without boric acid or with 7 μm boric
acid were not able to reduce their wall pore size at the transition to
the stationary phase. These cells could not be kept viable in the
stationary phase, because they continued to expand and died as a result
of wall rupture. The addition of 100 μm boric acid
prevented wall rupture and the wall pore size was reduced to normal
values. We conclude that boron is required to maintain the normal pore
structure of the wall matrix and to mechanically stabilize the wall at
growth termination.The ultrastructure and physical properties of plant cell walls are
known to be affected by boron deficiency (Kouchi and Kumazawa, 1976;
Hirsch and Torrey, 1980; Fischer and Hecht-Buchholz, 1985; Matoh et
al., 1992; Hu and Brown, 1994; Findeklee and Goldbach, 1996). Moreover,
boron is predominantly localized in the cell wall when plants are grown
with suboptimal boron (Loomis and Durst, 1991; Matoh et al., 1992; Hu
and Brown, 1994; Hu et al., 1996). In radish, >80% of the cell wall
boron is present in the pectic polysaccharide RG-II (Matoh et al.,
1993; Kobayashi et al., 1996), which is now known to exist as a dimer
that is cross-linked by a borate ester between two apiosyl residues
(Kobayashi et al., 1996; O''Neill et al., 1996). Dimeric RG-II is
unusually stable at low pH and is present in a large number of plant
species (Ishii and Matsunaga, 1996; Kobayashi et al., 1996, 1997; Matoh
et al., 1996; O''Neill et al., 1996; Pellerin et al., 1996; Kaneko et
al., 1997). The widespread occurrence and conserved structure of RG-II
(Darvill et al., 1978; O''Neill et al., 1990) have led to the
suggestion that borate ester cross-linked RG-II is required for the
development of a normal cell wall (O''Neill et al., 1996; Matoh, 1997).One approach for determining the function of boron in plant cell walls
is to compare the responses to boron deficiency of growing plant cells
that are dividing and synthesizing primary cell walls with those of
growth-limited plant cells in which the synthesis of primary cell walls
is negligible. Suspension-cultured cells are well suited for this
purpose because they may be reversibly transferred from a growth phase
to a stationary phase. Continuous cell growth phase is maintained by
frequent transfer of the cells into new growth medium (King, 1981;
Kandarakov et al., 1994), whereas a stationary cell population
is obtained by feeding the cells with Suc and by not subculturing them.
Cells in the stationary phase are characterized by mechanically
stabilized primary walls and reduced biosynthetic activity. Here we
describe the responses of suspension-cultured Chenopodium
album L. cells in the growth and stationary phases to boron
deficiency. These cells have a high specific-growth rate, no
significant lag phase, and reproducible changes in their wall pore size
during the transition from the growth phase to the stationary phase
(Titel et al., 1997). 相似文献
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13.
Heme and chlorophyll accumulate to
high levels in legume root nodules and in photosynthetic tissues,
respectively, and they are both derived from the universal tetrapyrrole
precursor δ-aminolevulinic acid (ALA). The first committed step in
ALA and tetrapyrrole synthesis is catalyzed by glutamyl-tRNA reductase
(GTR) in plants. A soybean (Glycine max) root-nodule
cDNA encoding GTR was isolated by complementation of an
Escherichia coli GTR-defective mutant for restoration of
ALA prototrophy. Gtr mRNA was very low in uninfected
roots but accumulated to high levels in root nodules. The induction of
Gtr mRNA in developing nodules was subsequent to that of
the gene Enod2 (early nodule)
and coincided with leghemoglobin mRNA accumulation. Genomic analysis
revealed two Gtr genes, Gtr1 and a 3′
portion of Gtr2, which were isolated from the soybean
genome. RNase-protection analysis using probes specific to
Gtr1 and Gtr2 showed that both genes were
expressed, but Gtr1 mRNA accumulated to significantly
higher levels. In addition, the qualitative patterns of expression of
Gtr1 and Gtr2 were similar to each other
and to total Gtr mRNA in leaves and nodules of mature
plants and etiolated plantlets. The data indicate that
Gtr1 is universal for tetrapyrrole synthesis and that a
Gtr gene specific for a tissue or tetrapyrrole is
unlikely. We suggest that ALA synthesis in specialized root nodules
involves an altered spatial expression of genes that are otherwise
induced strongly only in photosynthetic tissues of uninfected plants.Soybean (Glycine max) and numerous other legumes can
establish a symbiosis with rhizobia, resulting in the formation of root
nodules comprising specialized plant and bacterial cells (for review,
see Mylona et al., 1995). Rhizobia reduce atmospheric nitrogen to
ammonia within nodules, which is assimilated by the plant host to
fulfill its nutritional nitrogen requirement. The high energy
requirement for nitrogen fixation necessitates efficient respiration by
the prokaryote within the microaerobic milieu of the nodule. The plant
host synthesizes a nodule-specific hemoglobin (leghemoglobin) that
serves to facilitate oxygen diffusion to the bacterial endosymbiont and
to buffer the free oxygen concentration at a low
tension (for review, see Appleby, 1992). Both of these functions
require that the hemoglobin concentration be high, and, indeed, it
exceeds 1 mm in soybean nodules (Appleby, 1984)
and is the predominant plant protein in that organ. Once thought to be
confined to legume nodules, hemoglobins are found throughout the plant
kingdom, and leghemoglobin likely represents a specialization of a
general plant phenomenon (for review, see Hardison, 1996). A gene
encoding a nonsymbiotic hemoglobin has been identified in soybean and
other legumes (Andersson et al., 1996); therefore, expression in
nodules involves the specific activation of a subset of genes within a
gene family. Leghemoglobin genes may have arisen from gene duplication,
followed by specialization (Andersson et al., 1996).Hemes and chlorophyll are tetrapyrroles synthesized
from common precursors; chlorophyll is quantitatively the major
tetrapyrrole in plants, with heme and other tetrapyrroles being present
in minor amounts. Legume root nodules represent an exception, in which
heme is synthesized in high quantity in the absence of chlorophyll,
thus requiring the activity of enzymes not normally expressed highly in
nonphotosynthetic tissues. Heme is synthesized from the universal
tetrapyrrole precursor ALA by seven successive enzymatic steps;
chlorophyll formation diverges after the synthesis of protoporphyrin,
the immediate heme precursor (for review, see O''Brian, 1996).
Biochemical and genetic evidence shows that soybean heme biosynthesis
genes are strongly induced in root nodules (Sangwan and O''Brian, 1991,
1992, 1993; Madsen et al., 1993; Kaczor et al., 1994; Frustaci et al.,
1995; Santana et al., 1998), and immunohistochemical studies
demonstrate that induction is concentrated in infected nodule cells
(Santana et al., 1998).ALA is synthesized from Glu in plants by a three-step mechanism called
the C5 pathway (Fig.
(Fig.1);1); the latter two steps are committed to
ALA synthesis and are catalyzed by GTR and GSAT, respectively (for
review, see Beale and Weinstein, 1990; Jahn et al., 1991). Plant cDNA
or genes encoding GTR (Gtr, also called HemA) and
GSAT (Gsa) have been identified in several plant species
(Grimm, 1990; Sangwan and O''Brian, 1993; Hofgen et al., 1994; Ilag et
al., 1994; Frustaci et al., 1995; Wenzlau and Berry-Lowe, 1995; Bougri
and Grimm, 1996; Kumar et al., 1996; Tanaka et al., 1996). Two genes
for each enzyme have been described, and some genes are reported to be
specific to a tissue, tetrapyrrole, or light regimen (Bougri and Grimm,
1996; Kumar et al., 1996; Tanaka et al., 1996). However, soybean
Gsa1 is highly expressed in both leaves and nodules and
contains a cis-acting element in its promoter that binds to
a nuclear factor found in both tissues. (Frustaci et al., 1995). In
this study we isolated soybean Gtr1 and characterized the
genetic basis of GTR expression in root nodules.
Figure 1C5 pathway for ALA synthesis. The
committed steps for ALA synthesis catalyzed by GTR and GSAT are boxed.
Glutamyl-tRNA synthetase (GluRS) and glutamyl-tRNAGlu also
participate in protein synthesis. The gene designations in plants are
shown in parentheses ... 相似文献
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NAD-isocitrate
dehydrogenase (NAD-IDH) from the eukaryotic microalga
Chlamydomonas reinhardtii was purified to
electrophoretic homogeneity by successive chromatography steps on
Phenyl-Sepharose, Blue-Sepharose, diethylaminoethyl-Sephacel, and
Sephacryl S-300 (all Pharmacia Biotech). The 320-kD enzyme was found to
be an octamer composed of 45-kD subunits. The presence of isocitrate
plus Mn2+ protected the enzyme against thermal inactivation
or inhibition by specific reagents for arginine or lysine. NADH was a
competitive inhibitor (Ki, 0.14
mm) and NADPH was a noncompetitive inhibitor
(Ki, 0.42 mm) with respect to
NAD+. Citrate and adenine nucleotides at concentrations
less than 1 mm had no effect on the activity, but 10
mm citrate, ATP, or ADP had an inhibitory effect. In
addition, NAD-IDH was inhibited by inorganic monovalent anions, but
l-amino acids and intermediates of glycolysis and the
tricarboxylic acid cycle had no significant effect. These data support
the idea that NAD-IDH from photosynthetic organisms may be a key
regulatory enzyme within the tricarboxylic acid cycle.IDH catalyzes the oxidative decarboxylation of isocitrate to
produce 2-oxoglutarate. According to the specificity for the electron
acceptor, two enzymes with IDH activity are known, NAD-IDH (EC
1.1.1.41) and NADP-IDH (EC 1.1.1.42) (Chen and Gadal, 1990a).In photosynthetic organisms NADP-IDH has been detected in the cytosol,
chloroplasts, mitochondria, and peroxisomes. Cytosolic NADP-IDH has
been purified from higher plants (Chen et al., 1988) and eukaryotic
algae (Martínez-Rivas et al., 1996), and its cDNA has been
cloned from alfalfa (Shorrosh and Dixon, 1992), soybean (Udvardi et
al., 1993), potato (Fieuw et al., 1995), and tobacco (Gálvez et
al., 1996). This 80-kD isoenzyme is a dimer, and it is likely to be
involved in the synthesis of NADPH for biosynthetic purposes in the
cytosol (Chen et al., 1988), in the synthesis of 2-oxoglutarate for
ammonium assimilation (Chen and Gadal, 1990b), and in the cycling,
redistribution, and export of amino acids (Fieuw et al., 1995).
Chloroplastic NADP-IDH has been studied in higher plants (Gálvez
et al., 1994) and eukaryotic algae (Martínez-Rivas and Vega,
1994). It is a 154-kD dimer that has been proposed to be involved in
the supply of NADPH for biosynthetic reactions in the chloroplast when
photosynthetic NADPH production is low (Gálvez et al., 1994). The
mitochondrial NADP-IDH of higher plants may have a physiological role
in the production of NADPH, which can be converted to NADH by a
transhydrogenase or used to reduce glutathione in the mitochondrial
matrix (Rasmusson and Møller, 1990). NADP-IDH activity has also been
detected in peroxisomes from spinach leaves (Yamazaki and Tolbert,
1970).NAD-IDH is localized exclusively in the mitochondria in association
with the TCA cycle. This enzyme has been purified from several
nonphotosynthetic eukaryotes such as fungi (Keys and McAlister-Henn,
1990; Alvarez-Villafañe et al., 1996) and animals (Giorgio et
al., 1970), in which it appears to be a 300-kD octamer. Its key
regulatory role in the TCA cycle is well documented. The NAD-IDH from
yeast is activated by AMP and citrate (Hathaway and Atkinson, 1963),
whereas the animal enzyme is activated by ADP and citrate (Cohen and
Colman, 1972). In addition, the NAD-IDH cDNAs have been cloned from
yeast (Cupp and McAlister-Henn, 1991, 1992) and animals (Nichols et
al., 1995; Zeng et al., 1995). In these organisms, the enzyme is
composed of two (yeast) or more (animals) different subunits encoded by
different genes.To our knowledge, no NAD-IDH from photosynthetic organisms has yet been
purified to homogeneity, mainly because of the low stability of the
enzyme (Oliver and McIntosh, 1995). However, partial purifications have
been reported from pea (Cox and Davies, 1967; Cox, 1969; McIntosh
and Oliver, 1992), potato (Laties, 1983), spruce (Cornu et al., 1996),
and the eukaryotic microalga Chlamydomonas reinhardtii
(Martínez-Rivas and Vega, 1994). Matrix and membrane forms of
the enzyme have been detected in potato (Tezuka and Laties, 1983) and
pea (McIntosh, 1997). Although it is an allosteric enzyme that exhibits
sigmoidal kinetics with respect to isocitrate (Cox and Davies, 1967;
McIntosh and Oliver, 1992) and is activated in vitro by ABA (Tezuka et
al., 1990), the regulatory importance of NAD-IDH in photosynthetic
organisms is still under debate.To elucidate the regulatory significance of NAD-IDH in photosynthetic
organisms and its apparent contribution to the 2-oxoglutarate
supply for ammonium assimilation, we have purified and characterized
the NAD-IDH from C. reinhardtii. 相似文献
17.
We have achieved, to our knowledge,
the first high-level heterologous expression of the gene encoding
d-ribulose-5-phosphate 3-epimerase from any source, thereby
permitting isolation and characterization of the epimerase as found in
photosynthetic organisms. The extremely labile recombinant spinach
(Spinacia oleracea L.) enzyme was stabilized by
dl-α-glycerophosphate or ethanol and destabilized by
d-ribulose-5-phosphate or 2-mercaptoethanol. Despite this
lability, the unprecedentedly high specific activity of the purified
material indicates that the structural integrity of the enzyme is
maintained throughout isolation. Ethylenediaminetetraacetate and
divalent metal cations did not affect epimerase activity, thereby
excluding a requirement for the latter in catalysis. As deduced from
the sequence of the cloned spinach gene and the electrophoretic
mobility under denaturing conditions of the purified recombinant
enzyme, its 25-kD subunit size was about the same as that of the
corresponding epimerases of yeast and mammals. However, in contrast to
these other species, the recombinant spinach enzyme was octameric
rather than dimeric, as assessed by gel filtration and polyacrylamide
gel electrophoresis under nondenaturing conditions. Western-blot
analyses with antibodies to the purified recombinant enzyme confirmed
that the epimerase extracted from spinach leaves is also octameric.As a participant in the oxidative pentose phosphate pathway, Ru5P
epimerase (EC 5.1.3.1), which catalyzes the interconversion of Ru5P and
Xu5P, is widely distributed throughout nature. Beyond its catabolic
role, the epimerase is also vital anabolically to photosynthetic
organisms in the regenerative phase of the reductive pentose phosphate
pathway (the Calvin cycle). In this capacity, Ru5P epimerase directs
Xu5P, formed in two distinct transketolase reactions of the cycle, to
Ru5P. Phosphorylation of the latter regenerates
d-ribulose-1,5-bisphosphate, the substrate for net
CO2 fixation. Because both the oxidative and
reductive pentose phosphate pathways coexist in chloroplasts
(Schnarrenberger et al., 1995), Ru5P epimerase and R5P isomerase
facilitate partitioning of pentose phosphates between the two pathways,
as dictated by the metabolic needs and redox status of the cell.Scant structural and mechanistic information about Ru5P epimerase is
available despite its inherent importance and dual metabolic roles.
This neglect may in part reflect the low natural abundance of the
enzyme. For example, achievement of electrophoretic homogeneity
required a 2000-fold purification from yeast (Bär et al., 1996)
and spinach (Spinacia oleracea L.) chloroplasts (Teige et
al., 1998) and 9000-fold purification from beef liver (Terada et al.,
1985). Although low overall recoveries (<10%) further limited the
availability of pure material, molecular sieving and denaturing
electrophoresis established that the epimerases from mammals (Wood,
1979; Karmali et al., 1983; Terada et al., 1985) and yeast (Bär
et al., 1996) are homodimers of approximately 23-kD subunits, whereas
the enzyme from spinach chloroplasts may be an octamer of 23-kD
subunits (Teige et al., 1998). DNA-deduced amino acid sequences of Ru5P
epimerases from both photosynthetic and nonphotosynthetic sources,
which confirm this estimated subunit size, show greater than 50%
similarities among the most evolutionarily distant species examined
(Kusian et al., 1992; Blattner et al., 1993; Falcone and Tabita, 1993;
Lyngstadaas et al., 1995; Nowitzki et al., 1995; Teige et al.,
1995).Although Ru5P epimerase has very recently been purified from a
photosynthetic organism (spinach) for the first time (Teige et al.,
1998), the low recovery (100 μg from 3.8 g of soluble
chloroplast protein, representing an overall yield of 5%) imposes
severe constraints on the directions of future experiments.
Furthermore, despite successful cloning of cDNA fragments encoding Ru5P
epimerase of several photosynthetic organisms (Kusian et al., 1992;
Nowitzki et al., 1995; Teige et al., 1995), to our knowledge high-level
heterologous expression and purification of enzymically active
recombinant enzyme have not been achieved. Because of our interest in
the regulation of photosynthetic carbon assimilation and the requisite
need for ample supplies of the participant enzymes for use in
mechanistic studies, we have attempted to optimize the heterologous
expression of the spinach gene for Ru5P epimerase. In this paper we
report cDNA clones that encode the mature chloroplastic enzyme or its
cytoplasmic precursor. We also describe an efficient isolation
procedure for the mature spinach enzyme synthesized in
Escherichia coli and some of the properties of the purified
enzyme. Contrasting features of the plant Ru5P epimerase, relative to
the animal and yeast counterparts, include an octameric rather than a
dimeric structure (also see Teige et al., 1998) and striking
instability under routine laboratory conditions. 相似文献
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19.
Purification and cDNA Cloning of Isochorismate Synthase from
Elicited Cell Cultures of Catharanthus roseus 下载免费PDF全文
Léon J.P. van Tegelen Paolo R.H. Moreno Anton F. Croes Robert Verpoorte George J. Wullems 《Plant physiology》1999,119(2):705-712
Isochorismate is an important
metabolite formed at the end of the shikimate pathway, which is
involved in the synthesis of both primary and secondary metabolites. It
is synthesized from chorismate in a reaction catalyzed by the enzyme
isochorismate synthase (ICS; EC 5.4.99.6). We have purified ICS to
homogeneity from elicited Catharanthus roseus cell
cultures. Two isoforms with an apparent molecular mass of 64 kD were
purified and characterized. The Km values
for chorismate were 558 and 319 μm for isoforms I and II,
respectively. The isoforms were not inhibited by aromatic amino acids
and required Mg2+ for enzyme activity. Polymerase chain
reaction on a cDNA library from elicited C. roseus cells
with a degenerated primer based on the sequence of an internal peptide
from isoform II resulted in an amplification product that was used to
screen the cDNA library. This led to the first isolation, to our
knowledge, of a plant ICS cDNA. The cDNA encodes a protein of 64 kD
with an N-terminal chloroplast-targeting signal. The deduced amino acid
sequence shares homology with bacterial ICS and also with anthranilate
synthases from plants. Southern analysis indicates the existence of
only one ICS gene in C. roseus.The shikimate pathway is a major pathway in primary and secondary
plant metabolism (Herrmann, 1995). It provides chorismate for the
synthesis of the aromatic amino acids Phe, Tyr, and Trp, which are used
in protein biosynthesis, but also serves as a precursor for a wide
variety of aromatic substances (Herrmann, 1995; Weaver and Hermann,
1997; Fig. Fig.1a).1a). Chorismate is also the starting point of a biosynthetic
pathway leading to phylloquinones (vitamin K1)
and anthraquinones (Poulsen and Verpoorte, 1991). The first committed
step in this pathway is the conversion of chorismate into
isochorismate, which is catalyzed by ICS (Poulsen and Verpoorte, 1991;
Fig. Fig.1b).1b). Its substrate, chorismate, plays a pivotal role in the
synthesis of shikimate-pathway-derived compounds, and its distribution
over the various pathways is expected to be tightly regulated. Elicited
cell cultures of Catharanthus roseus provide an example of
the partitioning of chorismate. Concurrently, these cultures produce
both Trp-derived indole alkaloids and DHBA (Moreno et al., 1994). In
bacteria DHBA is synthesized from isochorismate (Young et al.,
1969). Elicitation of C. roseus cell cultures with a fungal
extract induces not only several enzymes of the indole alkaloid
biosynthetic pathway (Pasquali et al., 1992) but also ICS
(Moreno et al., 1994). Information concerning the expression and
biochemical characteristics of the enzymes that compete for available
chorismate (ICS, CM, and AS) may help us to understand the regulation
of the distribution of this precursor over the various pathways. Such
information is already available for CM (Eberhard et al., 1996) and AS
(Poulsen et al., 1993; Bohlmann et al., 1995) but not for ICS.
Figure 1a, Position of ICS in the plant metabolism. SA,
Salicylic acid, OSB, o-succinylbenzoic acid. b, Reaction
catalyzed by ICS.Isochorismate plays an important role in bacterial and plant metabolism
as a precursor of o-succinylbenzoic acid, an intermediate in
the biosynthesis of menaquinones (vitamin K2)
(Weische and Leistner, 1985) and phylloquinones (vitamin
K1; Poulsen and Verpoorte, 1991). In bacteria
isochorismate is also a precursor of siderophores such as
DHBA (Young et al., 1969), enterobactin (Walsh et
al., 1990), amonabactin (Barghouthi et al., 1991), and salicylic acid
(Serino et al., 1995). Although evidence from tobacco would indicate
that salicylic acid in plants is derived from Phe via benzoic acid
(Yalpani et al., 1993; Lee et al., 1995; Coquoz et al., 1998), it
cannot be excluded that it is also synthesized from isochorismate. In
the secondary metabolism of higher plants, isochorismate is a precursor
for the biosynthesis of anthraquinones (Inoue et al., 1984; Sieweke and
Leistner, 1992), naphthoquinones (Müller and Leistner, 1978),
catalpalactone (Inouye et al., 1975), and certain alkaloids in orchids
(Leete and Bodem, 1976).ICS was first extracted and partially purified from crude extracts of
Aerobacter aerogenes (Young and Gibson, 1969). Later, ICS
activity was detected in protein extracts of cell cultures from plants
of the Rubiaceae, Celastraceae, and Apocynaceae families (Ledüc
et al., 1991; Poulsen et al., 1991; Poulsen and Verpoorte, 1992). Genes
encoding ICS have been cloned from bacteria such as Escherichia
coli (Ozenberger et al., 1989), Pseudomonas aeruginosa
(Serino et al., 1995), Aeromonas hydrophila (Barghouthi et
al., 1991), Flavobacterium K3–15
(Schaaf et al., 1993), Hemophilus influenzae
(Fleischmann et al., 1995), and Bacillus subtilis
(Rowland and Taber, 1996). Both E. coli and B.
subtilis have two distinct ICS genes; one is involved in
siderophore biosynthesis and the other is involved in menaquinone
production (Daruwala et al., 1996, 1997; Müller et al., 1996;
Rowland and Taber, 1996). The biochemical properties of the two ICS
enzymes from E. coli are different (Daruwala et al., 1997;
Liu et al., 1990). Sequence analysis has revealed that the bacterial
ICS enzymes share homology with the chorismate-utilizing
enzymes AS and p-aminobenzoate synthase, suggesting that
they share a common evolutionary origin (Ozenberger et al.,
1989).Much biochemical and molecular data concerning the shikimate pathway in
plants have accumulated in recent years (Schmid and Amrhein, 1995;
Weaver and Hermann, 1997), but relatively little work has been done on
ICS from higher plants. The enzyme has been partially purified from
Galium mollugo cell cultures (Ledüc et al., 1991,
1997), but purification of the ICS protein to homogeneity has remained
elusive, probably because of instability of the enzyme.Our interests focus on the role of ICS in the regulation of chorismate
partitioning over the various pathways. Furthermore, we studied ICS in
C. roseus to gain insight into the biosynthesis of DHBA in
higher plants (Moreno et al., 1994). In this paper we report the first
purification, to our knowledge, of ICS to homogeneity from a plant
source and the cloning of the corresponding cDNA. 相似文献