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1.
Borate-Rhamnogalacturonan II Bonding Reinforced by
Ca2+ Retains Pectic Polysaccharides in Higher-Plant Cell
Walls
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The extent of in vitro formation of
the borate-dimeric-rhamnogalacturonan II (RG-II) complex was stimulated
by Ca2+. The complex formed in the presence of
Ca2+ was more stable than that without Ca2+. A
naturally occurring boron (B)-RG-II complex isolated from radish
(Raphanus sativus L. cv Aokubi-daikon) root contained
equimolar amounts of Ca2+ and B. Removal of the
Ca2+ by
trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic
acid induced cleavage of the complex into monomeric RG-II. These data
suggest that Ca2+ is a normal component of the B-RG-II
complex. Washing the crude cell walls of radish roots with a 1.5%
(w/v) sodium dodecyl sulfate solution, pH 6.5, released 98% of the
tissue Ca2+ but only 13% of the B and 22% of the pectic
polysaccharides. The remaining Ca2+ was associated with
RG-II. Extraction of the sodium dodecyl sulfate-washed cell walls with
50 mm
trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic
acid, pH 6.5, removed the remaining Ca2+, 78%
of B, and 49% of pectic polysaccharides. These results suggest that
not only Ca2+ but also borate and Ca2+
cross-linking in the RG-II region retain so-called chelator-soluble
pectic polysaccharides in cell walls.Boron (B) is an essential element for higher plant growth,
although its primary function is not known (Loomis and Durst, 1992).
Determining the sites of B in cells is required to identify its
function. In cultured tobacco cells more than 80% of cellular B is in
the cell wall (Matoh et al., 1993), whereas the membrane fraction
(Kobayashi et al., 1997) and protoplasts (Matoh et al., 1992) do not
contain a significant amount of B. In radish (Raphanus
sativus L. cv Aokubi-daikon) root cell walls, B cross-links two
RG-II regions of pectic polysaccharides through a borate-diol ester
(Kobayashi et al., 1995, 1996). The association of B with RG-II has
been confirmed in sugar beet (Ishii and Matsunaga, 1996), bamboo
(Kaneko et al., 1997), sycamore and pea (O''Neill et al., 1996), and
red wine (Pellerin et al., 1996). In cultured tobacco cells the B
associated with RG-II accounts for about 80% of the cell wall B
(Kobayashi et al., 1997) and RG-II may be the exclusive carrier of B in
higher plant cell walls (Matoh et al., 1996). Germanic acid, which
partly substitutes for B in the growth of the B-deprived plants (Skok,
1957), also cross-links two RG-II chains (Kobayashi et al., 1997).
These results suggest that the physiological role of B is to cross-link
cell wall pectic polysaccharides in the RG-II region and thereby form a
pectic network.It is believed that in the cell wall pectic polysaccharides are
cross-linked with Ca2+, which binds to carboxyl
groups of the polygalacturonic acid regions (Jarvis, 1984). Thus, the
ability of B and Ca2+ to cross-link cell wall
pectic polysaccharides needs to be evaluated. In this report we
describe the B-RG-II complex of radish root and the role of B-RG-II and
Ca2+ in the formation of a pectic
network. 相似文献
2.
3.
The effect of copper on
photoinhibition of photosystem II in vivo was studied in
bean (Phaseolus vulgaris L. cv Dufrix). The plants were
grown hydroponically in the presence of various concentrations of
Cu2+ ranging from the optimum 0.3 μm
(control) to 15 μm. The copper concentration of leaves
varied according to the nutrient medium from a control value of 13 mg
kg−1 dry weight to 76 mg kg−1 dry weight.
Leaf samples were illuminated in the presence and absence of lincomycin
at different light intensities (500–1500 μmol photons
m−2 s−1). Lincomycin prevents the concurrent
repair of photoinhibitory damage by blocking chloroplast protein
synthesis. The photoinhibitory decrease in the light-saturated rate of
O2 evolution measured from thylakoids isolated from treated
leaves correlated well with the decrease in the ratio of variable to
maximum fluorescence measured from the leaf discs; therefore, the
fluorescence ratio was used as a routine measurement of photoinhibition
in vivo. Excess copper was found to affect the
equilibrium between photoinhibition and repair, resulting in a decrease
in the steady-state concentration of active photosystem II centers of
illuminated leaves. This shift in equilibrium apparently resulted from
an increase in the quantum yield of photoinhibition (ΦPI)
induced by excess copper. The kinetic pattern of photoinhibition and
the independence of ΦPI on photon flux density were not
affected by excess copper. An increase in ΦPI may
contribute substantially to Cu2+ toxicity in certain plant
species.Cu2+ is an essential micronutrient but in
excess is toxic for plants. It is a redox-active metal that functions
as an enzyme activator and is an important part of prosthetic groups of
many enzymes (for review, see Sandmann and Böger, 1983). Copper
concentrations in healthy plant tissues range from 5 to 20 mg
kg−1 dry weight. In
Cu2+-rich environments, accumulation of
Cu2+ in plant tissues depends on the species and
cultivar. Cu2+ seems to have several sites of
action, which vary among plant species. Toxic concentrations of
Cu2+ inhibit metabolic activity, which leads to
suppressed growth and slow development. Most Cu2+
ions are immobilized to the cell walls of roots or of mycorrhizal fungi
(Kahle, 1993).When the tolerance mechanisms in the root zone become overloaded,
Cu2+ is translocated by both the xylem and phloem
up to the leaves. Excess Cu2+ may replace other
metals in metalloproteins or may interact directly with SH groups of
proteins (Uribe and Stark, 1982). Cu2+-induced
free-radical formation may also cause protein damage (for review, see
Fernandes and Henriques, 1991; Weckx and Clijsters, 1996). High
concentrations of Cu2+ may catalyze the formation
of the hydroxyl radical from O2 and
H2O2. This
Cu2+-catalyzed Fenton-type reaction takes place
mainly in chloroplasts (Sandmann and Böger, 1980). The hydroxyl
radical may start the peroxidation of unsaturated membrane lipids and
chlorophyll (Sandmann and Böger, 1980), and these inhibitory
mechanisms might contribute to the observed inhibition of
photosynthetic electron transport by excess Cu2+
(Clijsters and Van Assche, 1985).The role of Cu2+ as an inhibitor of
photosynthetic electron transport has been studied in vitro. Both the
donor (Cedeno-Maldonado and Swader, 1972; Samuelsson and Öquist,
1980; Schröder et al., 1994) and acceptor (Mohanty et al., 1989;
Yruela et al., 1992, 1993, 1996a, 1996b; Jegerschöld et al.,
1995) sides of PSII have been proposed to be the most sensitive site
for Cu2+ action. On the donor side,
Cu2+ is thought to inhibit electron transport to
P680, the primary donor of PSII (Schröder et al., 1994). On the
acceptor side, Cu2+ interactions with the
pheophytin-QA-Fe2+-domain
or Cu2+-induced modifications in the amino acid
or lipid structure close to the QA- and
QB-binding sites have been suggested to cause the
inhibition of electron transport (Jegerschöld et al., 1995;
Yruela et al., 1996a, 1996b).Celeno-Maldonado and Swader (1972) noticed that preincubation of
chloroplasts in the light enhanced the
Cu2+-induced inhibition of electron transport,
and that PSII was more susceptible to this kind of inhibition than was
PSI. The hypothetical acceptor- and donor-side mechanisms of the
light-induced inhibition of electron transport, photoinhibition,
involve the same domains of attack as Cu2+. Both
acceptor- and donor-side photoinhibition trigger the D1 polypeptide of
the PSII reaction center for degradation (for review, see Aro et al.,
1993). The damaged D1 protein is degraded, and the recovery of PSII
activity needs de novo synthesis of D1 protein. Photoinhibition occurs
at all light intensities (Tyystjärvi and Aro, 1996); therefore,
the cycle of PSII photoinhibition, which is followed by degradation,
and, finally, resynthesis of the D1 protein, runs constantly in plant
cells in the light. If the photoinhibition-repair cycle is allowed to
run for some time at a constant light intensity, equilibrium is
reached. At equilibrium (steady state), all three reaction rates
(photoinhibition, D1 degradation, and D1 synthesis) are equal. Healthy
plants are often able to maintain a high steady-state concentration of
active PSII under widely varying light intensities. Even if the
concentration of active PSII is lowered by high light, the
concentration of D1 protein tends to stay fairly constant (Cleland et
al., 1990; Kettunen et al., 1991). In the bean (Phaseolus
vulgaris L.) plants used in this study the steady-state D1 protein
content remained almost constant even in the presence of excess
Cu2+.The effect of Cu2+ on photoinhibition in
vivo has been studied very little. Vavilin et al. (1995) suggest that
excess Cu2+ may slow the PSII repair cycle in the
green alga Chlorella pyrenoidosa, and Ouzounidou et al.
(1997) suggest that Cu2+ inhibits adaptation to
light in maize. In the current study we show that excess
Cu2+ induces a large increase in the rate
constant of photoinhibition in vivo in a higher plant. 相似文献
4.
5.
Lidia Osuna Jean-N?el Pierre María-Cruz González Rosario Alvarez Francisco J. Cejudo Cristina Echevarría Jean Vidal 《Plant physiology》1999,119(2):511-520
Phosphoenolpyruvate
carboxylase (PEPC) activity was detected in aleurone-endosperm extracts
of barley (Hordeum vulgare) seeds during germination,
and specific anti-sorghum (Sorghum bicolor)
C4 PEPC polyclonal antibodies immunodecorated constitutive
103-kD and inducible 108-kD PEPC polypeptides in western analysis. The
103- and 108-kD polypeptides were radiolabeled in situ after imbibition
for up to 1.5 d in 32P-labeled inorganic phosphate. In
vitro phosphorylation by a Ca2+-independent PEPC protein
kinase (PK) in crude extracts enhanced the enzyme''s velocity and
decreased its sensitivity to l-malate at suboptimal pH and
[PEP]. Isolated aleurone cell protoplasts contained both
phosphorylated PEPC and a Ca2+-independent PEPC-PK that was
partially purified by affinity chromatography on blue dextran-agarose.
This PK activity was present in dry seeds, and PEPC phosphorylation in
situ during imbibition was not affected by the cytosolic
protein-synthesis inhibitor cycloheximide, by weak acids, or by various
pharmacological reagents that had proven to be effective blockers of
the light signal transduction chain and PEPC phosphorylation in
C4 mesophyll protoplasts. These collective data support the
hypothesis that this Ca2+-independent PEPC-PK was formed
during maturation of barley seeds and that its presumed underlying
signaling elements were no longer operative during germination.Higher-plant PEPC (EC 4.1.1.31) is subject to in vivo
phosphorylation of a regulatory Ser located in the N-terminal domain of
the protein. In vitro phosphorylation by a
Ca2+-independent, low-molecular-mass (30–39 kD)
PEPC-PK modulates PEPC regulation interactively by opposing metabolite
effectors (e.g. allosteric activation by Glc-6-P and feedback
inhibition by l-malate; Andreo et al., 1987), decreasing
significantly the extent of malate inhibition of the leaf enzyme
(Carter et al., 1991; Chollet et al., 1996; Vidal et al., 1996; Vidal
and Chollet, 1997). These metabolites control the rate of
phosphorylation of PEPC via an indirect target-protein effect (Wang and
Chollet, 1993; Echevarría et al., 1994; Vidal and Chollet,
1997).Several lines of evidence support the view that this protein-Ser/Thr
kinase is the physiologically relevant PEPC-PK (Li and Chollet, 1993;
Chollet et al., 1996; Vidal et al., 1996; Vidal and Chollet, 1997). The
presence and inducible nature of leaf PEPC-PK have been established
further in various C3, C4,
and CAM plant species (Chollet et al., 1996). In all cases, CHX proved
to be a potent inhibitor of this up-regulation process so that apparent
changes in the turnover rate of PEPC-PK itself or another, as yet
unknown, protein factor were invoked to account for this observation
(Carter et al., 1991; Jiao et al., 1991; Chollet et al., 1996).
Consistent with this proposal are recent findings about PEPC-PK from
leaves of C3, C4, and CAM
plants that determined activity levels of the enzyme to depend on
changes in the level of the corresponding translatable mRNA (Hartwell
et al., 1996).Using a cellular approach we previously showed in
sorghum (Sorghum bicolor) and hairy crabgrass
(Digitaria sanguinalis) that PEPC-PK is
up-regulated in C4 mesophyll cell protoplasts
following illumination in the presence of a weak base
(NH4Cl or methylamine; Pierre et al., 1992;
Giglioli-Guivarc''h et al., 1996), with a time course (1–2 h) similar
to that of the intact, illuminated sorghum (Bakrim et al., 1992) or
maize leaf (Echevarría et al., 1990). This light- and
weak-base-dependent process via a complex transduction chain is likely
to involve sequentially an increase in pHc, inositol
trisphosphate-gated Ca2+ channels of the
tonoplast, an increase in cytosolic Ca2+, a
Ca2+-dependent PK, and PEPC-PK.Considerably less is known about the up-regulation of PEPC-PK and
PEPC phosphorylation in nongreen tissues. A sorghum root PEPC-PK
purified on BDA was shown to phosphorylate in vitro both recombinant
C4 PEPC and the root
C3-like isoform, thereby decreasing the enzyme''s
malate sensitivity (Pacquit et al., 1993). PEPC from soybean root
nodules was phosphorylated in vitro and in vivo by an endogenous PK
(Schuller and Werner, 1993; Zhang et al., 1995; Zhang and Chollet,
1997). A Ca2+-independent nodule PEPC-PK
containing two active polypeptides (32–37 kD) catalyzed the
incorporation of phosphate on a Ser residue of the target enzyme and
was modulated by photosynthate transported from the shoots (Zhang and
Chollet, 1997). Regulatory seryl phosphorylation of a heterotetrameric
(α2β2) banana fruit
PEPC by a copurifying, Ca2+-independent PEPC-PK
was shown to occur in vitro (Law and Plaxton, 1997). Although
phosphorylation was also detected in vivo and found to concern
primarily the α-subunit, PEPC exists mainly in the dephosphorylated
form in preclimacteric, climacteric, and postclimacteric fruit.In a previous study we showed that PEPC undergoes regulatory
phosphorylation in aleurone-endosperm tissue during germination of
wheat seeds (Osuna et al., 1996). Here we report on PEPC and the
requisite PEPC-PK in germinating barley (Hordeum vulgare)
seeds. PEPC was highly phosphorylated by a
Ca2+-independent Ser/Thr PEPC-PK similar to that
found in other plant systems studied previously (Chollet et al., 1996);
however, the PK was already present in the dry seed and its activity
did not require protein synthesis during imbibition. 相似文献
6.
7.
8.
Characterization of Zinc Uptake, Binding, and Translocation in
Intact Seedlings of Bread and Durum Wheat Cultivars 总被引:1,自引:0,他引:1
Jonathan J. Hart Wendell A. Norvell Ross M. Welch Lori A. Sullivan Leon V. Kochian 《Plant physiology》1998,118(1):219-226
Durum wheat (Triticum
turgidum L. var durum) cultivars exhibit lower
Zn efficiency than comparable bread wheat (Triticum
aestivum L.) cultivars. To understand the physiological
mechanism(s) that confers Zn efficiency, this study used
65Zn to investigate ionic Zn2+ root uptake,
binding, and translocation to shoots in seedlings of bread and durum
wheat cultivars. Time-dependent Zn2+ accumulation during 90
min was greater in roots of the bread wheat cultivar. Zn2+
cell wall binding was not different in the two cultivars. In each
cultivar, concentration-dependent Zn2+ influx was
characterized by a smooth, saturating curve, suggesting a
carrier-mediated uptake system. At very low solution Zn2+
activities, Zn2+ uptake rates were higher in the bread
wheat cultivar. As a result, the Michaelis constant for
Zn2+ uptake was lower in the bread wheat cultivar (2.3
μm) than in the durum wheat cultivar (3.9
μm). Low temperature decreased the rate of
Zn2+ influx, suggesting that metabolism plays a role in
Zn2+ uptake. Ca inhibited Zn2+ uptake equally
in both cultivars. Translocation of Zn to shoots was greater in the
bread wheat cultivar, reflecting the higher root uptake rates. The
study suggests that lower root Zn2+ uptake rates may
contribute to reduced Zn efficiency in durum wheat varieties under
Zn-limiting conditions.Soils that contain insufficient levels of the essential plant
micronutrient Zn are common throughout the world. As a result, Zn
deficiency is a widespread problem in crop plants, especially cereals
(Graham et al., 1992). The importance of plant foods as sources of Zn,
particularly in the marginal diets of developing countries, is well
established (Welch, 1993). The development of crop plants that are
efficient Zn accumulators is therefore a potentially important
endeavor. In addition to its effects on nutrition, Zn deficiency in
crops is relevant to other areas of human health. Another consequence
of Zn-deficient soils is the tendency for plants grown in such soils to
accumulate heavy metals. For example, in the Great Plains region of
North America, where soil Zn levels are low and naturally occurring Cd
is present, durum wheat (Triticum turgidum L. var
durum) grains accumulate Cd to relatively high
concentrations (Wolnik et al., 1983). The presence of Cd in food
represents a potential human health hazard and, in response,
international trade standards have been proposed to limit the levels of
Cd in exported grain (Codex Alimentarius Commission, 1993).
Thus, there is a need to understand the physiological processes that
control acquisition of Zn from soil solution by roots and mobilization
of Zn within plants.It has been demonstrated in recent years that crop plants vary in their
ability to take up Zn, particularly when its availability to roots is
limited. Zn efficiency, defined as the ability of a plant to grow and
yield well in Zn-deficient soils, varies among wheat cultivars (Graham
and Rengel, 1993). In field trials, durum wheat cultivars have been
shown to be consistently less Zn efficient than bread wheat
(Triticum aestivum L.) cultivars (Graham et al., 1992).
Similarly, durum wheat varieties were reported to be less Zn efficient
than bread wheat varieties when grown in chelate-buffered hydroponic
nutrient culture (Rengel and Graham, 1995a).The physiological mechanism(s) that confers Zn efficiency has not been
identified. Processes that could influence the ability of a plant to
tolerate limited amounts of available Zn include higher root uptake,
more efficient utilization of Zn, and enhanced Zn translocation within
the plant. Cakmak et al. (1994) showed that a Zn-inefficient durum
wheat cultivar exhibited Zn-deficiency symptoms earlier and more
intensely than a Zn-efficient bread wheat cultivar even though the Zn
tissue concentrations were similar in both lines, suggesting
differential utilization of Zn in the two cultivars. Rates of Zn
translocation to shoots were shown to vary among sorghum cultivars,
although correlations with Zn efficiency were not established (Ramani
and Kannan, 1985). Root uptake kinetics have been reported to vary
between rice cultivars having different Zn requirements, with
high-Zn-requiring cultivars exhibiting consistently higher root uptake
rates (Bowen, 1986). In contrast, a correlation between Zn efficiency
and rates of root Zn uptake in bread and durum wheat cultivars could
not be demonstrated (Rengel and Graham, 1995b).In grasses Zn influx into the root symplasm has been hypothesized to
occur as the free Zn2+ ion (Halvorson and
Lindsay, 1977), as well as in the form of Zn complexes with nonprotein
amino acids known as phytosiderophores (Tagaki et al., 1984) or
phytometallophores (Welch, 1993). Concentration-dependent uptake of
free Zn2+ ions has been shown to be saturable in
several species, including maize (Mullins and Sommers, 1986), barley
(Veltrup, 1978), and wheat (Chaudhry and Loneragan, 1972), suggesting
that ionic uptake in grasses occurs via a carrier-mediated system.
However, several of these studies have been criticized on the basis
that excessively high (and physiologically unrealistic)
Zn2+ concentrations were used (Kochian, 1993).This study was undertaken to examine unidirectional
Zn2+ influx and translocation to shoots in
Zn-efficient bread wheat lines and Zn-inefficient durum wheat lines.
Experiments were performed in the absence of added phytometallophores
and results are presumed to represent influx of ionic
Zn2+. Zn activities in the nanomolar range were
used to more closely mimic free Zn2+ levels
occurring naturally in soil solution. The results presented here
indicate that a Zn-efficient bread wheat cultivar maintained higher
rates of Zn uptake than a Zn-inefficient durum wheat cultivar,
particularly at low (and physiologically relevant) solution
Zn2+ activities. 相似文献
9.
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
10.
11.
A Steep Dependence of Inward-Rectifying Potassium Channels on
Cytosolic Free Calcium Concentration Increase Evoked by
Hyperpolarization in Guard Cells
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Inactivation of inward-rectifying
K+ channels (IK,in) by a rise in
cytosolic free [Ca2+] ([Ca2+]i)
is a key event leading to solute loss from guard cells and stomatal
closure. However, [Ca2+]i action on
IK,in has never been quantified, nor are its
origins well understood. We used membrane voltage to manipulate
[Ca2+]i (A. Grabov and M.R. Blatt [1998]
Proc Natl Acad Sci USA 95: 4778–4783) while recording
IK,in under a voltage clamp and
[Ca2+]i by Fura-2 fluorescence
ratiophotometry. IK,in inactivation
correlated positively with [Ca2+]i and
indicated a Ki of 329 ± 31
nm with cooperative binding of four Ca2+ ions
per channel. IK,in was promoted by the
Ca2+ channel antagonists Gd3+ and calcicludine,
both of which suppressed the [Ca2+]i rise,
but the [Ca2+]i rise was unaffected by the
K+ channel blocker Cs+. We also found that
ryanodine, an antagonist of intracellular Ca2+ channels
that mediate Ca2+-induced Ca2+ release, blocked
the [Ca2+]i rise, and Mn2+
quenching of Fura-2 fluorescence showed that membrane hyperpolarization
triggered divalent release from intracellular stores. These and
additional results point to a high signal gain in
[Ca2+]i control of
IK,in and to roles for discrete
Ca2+ flux pathways in feedback control of the
K+ channels by membrane voltage.Ca2+ underlies many fundamental regulatory processes
in plants, including adaptive responses to abiotic environmental stress
(Knight et al., 1996; Russell et al., 1996; McAinsh et al., 1997) and
programmed cell death evoked by pathogen attack (Low and Merida, 1996;
Hammondkosack and Jones, 1997). Coordination of changes in
[Ca2+]i and its
integration with downstream response elements are central in coupling
stimulus input to cellular response in these processes.In stomatal guard cells, the best characterized higher-plant cell
model, major downstream targets of
[Ca2+]i and their roles
in stomatal function have been identified. Increasing
[Ca2+]i is known to
inactivate IK,in and to activate
Cl− channels, events that bias plasma membrane
transport for net efflux of osmotically active solute and a loss of
turgor, which drives stomatal closure (Blatt and Grabov, 1997).
Furthermore, changes in
[Ca2+]i are associated
with ABA, CO2, and the growth hormone auxin
(Blatt and Grabov, 1997; McAinsh et al., 1997). These
[Ca2+]i signals have been
observed to oscillate (McAinsh et al., 1995; Webb et al., 1996),
characteristics that may constitute “Ca2+
signatures” to encode specific downstream responses (Berridge, 1996).
Yet, despite the evidence for
[Ca2+]i signaling in
guard cells, surprisingly little detail is known about the link between
[Ca2+]i changes and ion
channel activity at the plasma membrane or about the mechanisms
mediating such [Ca2+]i
changes. To our knowledge, in no instance have the characteristics of
ion channel regulation by Ca2+ been quantified
directly in any higher-plant cell.We recently described the coupling of membrane voltage to
[Ca2+]i, demonstrating
that hyperpolarization, whether under a voltage clamp or in the
presence of low [K+]o,
evoked [Ca2+]i increases
in guard cells, and that the voltage threshold for
[Ca2+]i rise was
profoundly altered by ABA (Grabov and Blatt, 1998). Our observations
indicated a link to Ca2+ influx across the plasma
membrane and raised questions about the efficacy of
[Ca2+]i in inactivating
IK,in and about the contributions of
intracellular Ca2+ release to the
[Ca2+]i signal. We have
used membrane voltage to experimentally manipulate
[Ca2+]i and report that
IK,in is strongly dependent on
[Ca2+]i, consistent with
a cooperative binding of four Ca2+ ions to effect
inactivation. Additional experiments indicate that voltage-evoked
[Ca2+]i increases depend
both on Ca2+ influx and on release of
Ca2+ from intracellular stores. These results
underscore the role of
[Ca2+]i as a high-gain
“switch” in the control of IK,in, and
implicate [Ca2+]i in
feedback control linking membrane voltage to the activity of the
K+ channels. 相似文献
12.
Cloning and Characterization of the Pseudomonas aeruginosa zwf Gene Encoding Glucose-6-Phosphate Dehydrogenase, an Enzyme Important in Resistance to Methyl Viologen (Paraquat) 总被引:1,自引:0,他引:1
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Ju-Fang Ma Paul W. Hager Michael L. Howell Paul V. Phibbs Daniel J. Hassett 《Journal of bacteriology》1998,180(7):1741-1749
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NAD+ (nicotinamide adenine dinucleotide) is an essential cofactor involved in various biological processes including calorie restriction-mediated life span extension. Administration of nicotinamide riboside (NmR) has been shown to ameliorate deficiencies related to aberrant NAD+ metabolism in both yeast and mammalian cells. However, the biological role of endogenous NmR remains unclear. Here we demonstrate that salvaging endogenous NmR is an integral part of NAD+ metabolism. A balanced NmR salvage cycle is essential for calorie restriction-induced life span extension and stress resistance in yeast. Our results also suggest that partitioning of the pyridine nucleotide flux between the classical salvage cycle and the NmR salvage branch might be modulated by the NAD+-dependent Sir2 deacetylase. Furthermore, two novel deamidation steps leading to nicotinic acid mononucleotide and nicotinic acid riboside production are also uncovered that further underscore the complexity and flexibility of NAD+ metabolism. In addition, utilization of extracellular nicotinamide mononucleotide requires prior conversion to NmR mediated by a periplasmic phosphatase Pho5. Conversion to NmR may thus represent a strategy for the transport and assimilation of large nonpermeable NAD+ precursors. Together, our studies provide a molecular basis for how NAD+ homeostasis factors confer metabolic flexibility.The pyridine nucleotide NAD+ and its reduced form NADH are primary redox carriers involved in metabolism. In addition to serving as a coenzyme in redox reactions, NAD+ also acts as a cosubstrate in protein modification reactions including deacetylation and ADP-ribosylation (1, 2). NAD+ also plays an important role in calorie restriction (CR)2-mediated life span extension via regulating NAD+-dependent longevity factors (3, 4). CR is the most effective regimen known to extend life span in various species (5, 6). CR also ameliorates many age-related diseases such as cancer and diabetes (5). The Sir2 family proteins are NAD+-dependent protein deacetylases, which have been shown to play important roles in several CR models in yeast (3, 7) and higher eukaryotes (8, 9). By coupling the cleavage of NAD+ and deacetylation of target proteins, the Sir2 family proteins serve as a molecular link relaying the cellular energy state to the machinery of life span regulation. Mammalian Sir2 family proteins (SIRT1–7) have also been implicated in stress response, cell survival, and insulin and fat metabolism (8–10), supporting a role for SIRT proteins in age-related metabolic diseases and perhaps human aging.In eukaryotes, NAD+ is generated by de novo synthesis and by salvaging various intermediary precursors (see Fig. 1A). In yeast, the de novo pathway is mediated by Bna1–5 and Qpt1 (Bna6), which produces nicotinic acid mononucleotide (NaMN) from tryptophan (11). Because the de novo pathway requires molecular oxygen as a substrate, cells grown under anaerobic growth conditions would rely on exogenous NAD+ precursors for the nicotinamide (Nam) moiety (11). Yeast cells also salvage Nam from NAD+ consuming reactions or nicotinic acid (NA) from environment via Tna1, Pnc1, and Npt1, leading to NaMN production. NaMN is then converted to NAD+ via Nma1/2 and Qns1 (see Fig. 1A). Nma1/2 are adenylyltransferases with dual specificity toward NMN and NaMN (12, 13), and Qns1 is a glutamine-dependent NAD+ synthetase. Recent studies also showed that supplementing nicotinamide riboside (NmR) and nicotinic acid riboside (NaR) to growth medium rescued the lethality of NAD+ auxotrophic mutants (14–16). Assimilations of exogenous NmR and NaR are mainly mediated by a conserved NmR kinase (Nrk1) and three nucleosidases (Urh1, Pnp1, and Meu1). Nrk1 phosphorylates NmR and NaR to produce nicotinamide mononucleotide (NMN) and NaMN, respectively (14, 16). Urh1, Pnp1, and Meu1 catabolize NmR and NaR to generate Nam and NA (15, 16).Open in a separate windowFIGURE 1.Nicotinamide riboside (NmR) is an endogenous metabolite in yeast. A, the current model of the NAD+ biosynthesis pathways. Extracellular NmR enters the salvage cycle through Nrk1, Urh1, Pnp1, and Meu1. B, NAD+ prototrophic cells release metabolites into growth medium to cross-feed NAD+ auxotrophic cells (the npt1Δqpt1Δ and qns1Δ mutants). Micro-colonies of the NAD+ auxotrophic mutants become visible after 2-day incubation at 30 °C, which show “gradient” growth patterns descending from the side adjacent to WT. C, Nrk1 is required for NAD+ auxotrophic cells to utilize NmR. Anaerobic growth conditions (−O2) are utilized to block de novo NAD+ biosynthesis in the npt1Δ and npt1Δnrk1Δ mutants. D, Nrk1 is required to utilize cross-feeding metabolites. E, cross-feeding activity is modulated by factors in NmR metabolism. Cells defective in NmR utilization (left panel) or transport (middle panel) show increased cross-feeding in spot assays. Overexpressing Nrk1 decreases cross-feeding activity (right panel). The results show growth of the npt1Δqpt1Δ recipient (plated on YPD at a density of ∼9000 cells/cm2) supported by feeder cells (∼2 × 104 cells spotted directly onto the recipient lawn). oe, overexpression.NmR supplementation has recently been shown to be a promising strategy for prevention and treatment of certain diseases (17). For example, NmR protected neurons from axonal degeneration via functioning as a NAD+ precursor (18, 19). Given that several NmR assimilating enzymes and NmR transporters have been characterized and many are conserved from fungi to mammals (14, 15, 20–22), NmR has been speculated to be an endogenous NAD+ precursor (17, 23). Here, we provided direct evidence for endogenous NmR as an integral part of NAD+ metabolism in yeast. We also determined the biological significance of salvaging endogenous NmR and studied its role in CR-induced life span extension. Moreover, we demonstrated that the NmR salvage machinery was also required for utilizing exogenous NMN, which has recently been shown to increase NAD+ levels in mammalian cells (24). Finally, we discussed the role of Sir2 in modulating the flux of pyridine nucleotides between alternate routes. 相似文献
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Utilization of an NF-ATp Binding Promoter Element for EGR3 Expression in T Cells but Not Fibroblasts Provides a Molecular Model for the Lymphoid Cell-Specific Effect of Cyclosporin A 总被引:2,自引:1,他引:1
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Hans W. Mages Rima Baag Birgit Steiner Richard A. Kroczek 《Molecular and cellular biology》1998,18(12):7157-7165