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Plant Physiol, May 2001, Vol. 126, pp. 267-277 Induction of Lipid Metabolic Enzymes during the Endoplasmic Reticulum Stress Response in Plants1Departments of Crop Science (K.J.S., R.E.D.) and Botany (K.J.S., P.S., I.B., W.F.B., R.S.B), Boxes 7620 and 7612, North Carolina State University, Raleigh, North Carolina 27695
The endoplasmic reticulum (ER) stress response is a signal transduction pathway activated by the perturbation of normal ER metabolism. We used the maize (Zea mays) floury-2 (fl2) mutant and soybean (Glycine max) suspension cultures treated with tunicamycin (Tm) to investigate the ER stress response as it relates to phospholipid metabolism in plants. Four key phospholipid biosynthetic enzymes, including DG kinase and phosphatidylinositol (PI) 4-phosphate 5-kinase were up-regulated in the fl2 mutant, specifically in protein body fractions where the mutation has its greatest effect. The third up-regulated enzyme, choline-phosphate cytidylyltransferase, was regulated by fl2 gene dosage and developmental signals. Elevated accumulation of the fourth enzyme, PI 4-kinase, was observed in the fl2 endosperm and soybean cells treated with Tm. The activation of these phospholipid biosynthetic enzymes was accompanied by alterations in membrane lipid synthesis and accumulation. The fl2 mutant exhibited increased PI content in protein body membranes at 18 d after pollination and more than 3-fold higher triacylglycerol accumulation in the endosperm by 36 d after pollination. Incorporation of radiolabeled acetate into phospholipids in soybean culture cells increased by about 30% with Tm treatment. The coordinated regulation of ER stress related proteins and multiple components of phospholipid biosynthesis is consistent with signaling through a common pathway. We postulate that the plant ER stress response has an important role in general plant metabolism, and more specifically in integrating the synthesis of protein and lipid reserves to allow proper seed formation.
Seed development requires coordination between formation of a pre-emergent embryo and synthesis of the major starch, lipid, and protein storage reserves. Regulation of storage reserve synthesis is complex because of a need to keep the individual components in their proper ratios for the development of mature, viable seeds that will desiccate and subsequently germinate properly. Little is known about how these processes are regulated or how seeds adapt to perturbations in the metabolic pathways essential for their growth and maturation. Because the endoplasmic reticulum (ER) is the site for processing of secretory proteins and formation of membrane and storage lipids, it is likely to play a key role in coordinating the metabolism of proteins and lipids during seed development. One of the simplest systems for exploring intracellular communication
between protein and phospholipid biosynthesis is the formation of
protein bodies in maize (Zea mays). These membrane-bound organelles arise directly from the ER as ordered aggregates of storage
proteins within the ER lumen. Much attention has been given to the
packaging of storage proteins within protein bodies (Okita and Rogers,
1996 A more dramatic alteration in ER membranes is seen in the endosperm of
the maize mutant, floury-2 (fl2).
Protein bodies derived from the ER are deeply convoluted in this mutant
and remain clustered near nuclei rather than dispersed throughout the
endosperm (Zhang and Boston, 1992 The chaperone induction and changes in membrane phenotype previously
observed in the fl2 endosperm are similar to characteristics of mammalian and yeast cells during ER stress. Accumulation of unfolded
proteins in the ER triggers a well-defined signal transduction pathway
in yeast called the unfolded protein response (UPR; for review, see
Chapman et al., 1998 Several lines of evidence suggest a connection between phospholipid
biosynthesis and UPR signaling. Overexpression of integral membrane
proteins is correlated with an increase in phospholipid biosynthesis,
proliferation of ER, and induction of the UPR (Chapman et al., 1998 The fl2-mediated induction of ER stress occurs in a tissue
devoted primarily to synthesis of storage reserves and gives us the
means to determine whether or not protein and lipid metabolism are
coordinated during protein body formation in seeds. Cell cultures provide an additional system to investigate the ER stress response in
plants without the pleiotropic effects of the fl2 mutation. A number of pharmacological agents, including tunicamycin (Tm), an
inhibitor N-linked glycosylation of proteins, can be used to induce an
ER stress response in cell cultures. Tm is a potent inducer of the ER
stress response (Watowich and Morimoto, 1988 In this study we report that four key phospholipid biosynthetic enzymes are up-regulated in fl2 protein bodies during early kernel development. Levels of phosphatidylinositol (PI) in fl2 protein body membranes and total triacylglycerols in the fl2 endosperm are increased when compared with their normal counterparts. In addition, expression levels of PI 4-kinase and overall phospholipid synthesis are elevated in soybean (Glycine max) cells in which an ER stress response is chemically induced. The data presented here provide new insights into coordination of the ER stress response and phospholipid metabolism in plants.
Enzymes Assayed We assayed protein bodies of normal and fl2 maize for
activities of enzymes that are believed to be involved at regulatory or
rate-limiting steps in phospholipid biosynthesis (Fig.
1). Choline-phosphate
cytidylyltransferase (CCT, enzyme 1), is considered to be the
rate-limiting step in the biosynthesis of phosphatidylcholine (PC), the
major phospholipid component of most eukaryotic membranes (for a review
of plant phospholipid metabolism, see Ohlrogge and Browse, 1995
CCT Activity Increases with fl2 Gene Dosage To determine if CCT activity was affected by the fl2
mutation we analyzed enzyme activity associated with protein bodies of normal and fl2 kernels. Because of the triploid nature of
maize endosperm, a gene dosage series ranging from zero to three copies of a given gene can be generated by reciprocal crosses between homozygous parental lines. Figure 2 shows
the effects of fl2 gene dosage on CCT activity at 18 d
after pollination (DAP). Although a single copy of the fl2
gene had no significant effect on the enzyme activity associated with
protein bodies, two and three copies resulted in increases of about
1.2- and 1.5-fold, respectively. This dosage effect correlates well
with our previous findings that visible morphological effects of
fl2 on protein bodies increase minimally with one copy of
the mutant gene, and become progressively more severe with the
introduction of two and three copies (Zhang and Boston, 1992
CCT Activity Changes during Seed Development To obtain a profile of the differences in CCT activity associated with normal and fl2 protein bodies during seed development we assayed endosperm at developmental stages from 10 to 36 DAP. During early seed fill, CCT activity was high in normal and fl2 maize (Fig. 3). As the seed matured, CCT activity remained high in fl2 protein bodies, but dropped in normal ones, with the largest differences appearing at 14 to 18 DAP. At later stages of endosperm maturation, CCT activity dropped overall and differences between normal and fl2 protein bodies decreased.
Lipid Kinase Activities Increase in fl2 Protein Bodies The activities of enzymes previously implicated in vesicle formation, DG kinase, PI 4-kinase, and PIP 5-kinase, were assayed in protein bodies from kernels harvested 18 and 28 DAP. An increase in each of these activities in protein body fractions was observed in fl2 relative to normal maize. Table I shows the averages of duplicate values obtained from a single experiment. Because considerable variation was observed in the total amount of radioactivity incorporated among independently isolated protein body fractions, the results of five separate experiments were analyzed as the relative increase in enzyme activity in fl2 versus normal for each independent experiment. These results are shown in Table I as mean ratios ± SE. To determine if differences in lipid kinase activities at 18 DAP were specific to protein bodies, ER-enriched fractions from the Suc gradients were assayed. No significant differences were observed (data not shown). By 28 DAP, the mean lipid kinase activities tended to be higher in fl2 protein bodies than in normal protein bodies at the same developmental stage, but the increases were not significant.
PI 4-Kinase Expression Increases in fl2 Protein Bodies To determine if up-regulation of lipid kinases was due, at least
in part, to an increase in enzyme amounts, we investigated the
accumulation of PI 4-kinase by immunoblot analysis. Protein body
fractions from normal and fl2 endosperm and a microsomal membrane fraction from carrot cells grown in suspension culture were
subjected to SDS-PAGE as described in "Materials and Methods." A
band of approximately 65 kD was detected in maize and carrot (Fig.
4). This band most likely corresponds to
the low-Mr PI 4-kinase that has been
previously described by Westergren et al. (1999)
PI 4-Kinase Expression Increases with Chemical Induction of the ER Stress Response The common feature between the fl2 mutation and chemical treatment of cell cultures with Tm is induction of an ER stress response. We investigated the expression of PI4-kinase in response to chemical induction of an ER stress response as a way of verifying that the effect on lipid metabolism is directly due to the ER stress response and not due to pleiotropic effects of the mutation. Soybean suspension cultures incubated with Tm showed a strong induction of the molecular chaperones BiP and PDI compared with control cultures, as judged by immunoblot analysis of whole-cell extracts (Fig. 5, A and B). PI4K immunoblots of the same samples also showed increases in response to chemical induction (Fig. 5C). Modest increases in molecular chaperone and PI4-kinase accumulation were observed over the time course of these experiments in control cells treated only with the N, N-dimethylformamide solvent. Similar results were observed with untreated soybean cultures, however, suggesting that the abundance of these proteins also changes with culture age (data not shown).
One limitation to immunoblot analysis is that the observed signal represents the combined accumulation of protein produced before and after the application of the stress-inducing compound. To overcome this limitation we performed short-term radiolabeling and immunoprecipitation of treated soybean cells to determine the effects of the Tm treatment on protein synthesis. Immunoprecipitation from whole-cell extracts revealed a strong induction of the ER stress response by Tm as shown by increased synthesis of the chaperones BiP, PDI, and calreticulin (Fig. 6A). Immunoprecipitation of newly synthesized proteins from ER-enriched fractions showed induction of BiP and PI4K expression in as little as 6 h after Tm treatment (Fig. 6, B and C). The multiple cross-reacting proteins (known active forms are designated by the arrowheads to the right of the figure) all increase in response to chemical treatment.
Normal and fl2 Protein Body Membranes Differ in Composition The differences in activities of lipid metabolic enzymes suggested that the phospholipid composition of fl2 protein bodies may differ from that of normal protein bodies. To investigate whether or not membranes were affected by the fl2 mutation we quantified the amounts of the major phospholipids from protein body and ER fractions. Protein body membranes from the fl2 mutant contained much lower levels of PA and higher levels of PI than normal protein body membranes (Fig. 7A). Amounts of PA, PI, PC, phosphatidylethanolamine, and phosphatidylglycerol in ER did not differ significantly between normal and fl2 samples (Fig. 7B). PIP and PIP2 were not detected in this assay.
Triaglycerol Accumulation Increases in fl2 Endosperm Martiniello et al. (1978)
Acetate Incorporation into Lipid Species Increases in Treated Soybean Cells If induction of ER stress does indeed lead to increased phospholipid biosynthesis, we might expect to see this reflected as an increase in acetate incorporation into these species in induced cell cultures. Investigation of lipid synthesis in soybean cells revealed that overall incorporation of acetate into various lipid species was up-regulated with induction of the ER stress response by Tm (Table III). An increase in acetate incorporation of about 30% was seen in response to treatment with Tm for 12 and 24 h. TLC separation of the radiolabeled compounds revealed a general overall increase in the synthesis of each of the major phospholipids (data not shown) as opposed to specific increases in any one phospholipid species.
The ER mediates synthesis and storage of proteins, phospholipids,
and triacylglycerols in the developing seed. Our findings suggest that phospholipid metabolism and protein production in protein
bodies are coordinately regulated through a common ER pathway that
shares many characteristics of the ER stress response. Examination of
phospholipid metabolism in the fl2 mutant revealed alterations in enzyme activities and the phospholipid composition of
protein body membranes. Given that PC is a major structural phospholipid of ER and protein body membranes, it is not surprising that signals eliciting the need for increased membrane biogenesis would
result in enhanced activity of CCT, the rate-limiting enzyme in PC
synthesis. The greatest differences in CCT activity were seen from 14 to 18 DAP. It has been previously shown that at 14 and 18 DAP, mRNA and
protein of the major zeins decrease in the fl2 mutant when
compared with normal maize (Jones, 1978 Because the products of the DG, PI, and PIP kinase reactions represent
relatively minor membrane components, we suggest that the increases in
their activities are less likely to be needed for membrane biogenesis
and instead are related to the roles these phospholipids play in
secretory vesicle formation. PA and polyphosphoinositides are involved
in secretory vesicle formation in mammalian and yeast cells and are
essential for vesicle trafficking (Roth, 1999 It is likely that protein body formation in maize is controlled in a
manner similar to vesicle budding and that the fl2 mutant zein may interfere with this process. Investigation of fl2
endosperm morphology reveals deeply invaginated protein bodies that are clustered near nuclei rather than being dispersed throughout the cell
(Zhang and Boston, 1992 Previous studies have identified a number of specialized ER subdomains
with differing morphological and biochemical properties such as
non-random localization of mRNAs, unequal distribution of molecular
chaperones, and localized assembly and packaging of proteins into
protein bodies (Okita and Rogers, 1996 In addition to mediating the synthesis of phospholipids and seed
storage proteins, the ER controls production of triacylglycerols that
are stored as oil bodies. With the exception of DG acyltransferase, the
final enzyme in triacylglycerol synthesis, all other steps of the
triacylglycerol pathway are shared with phospholipid biosynthetic pathways (Ohlrogge and Browse, 1995 Taken together, the results presented here demonstrate that the fl2 mutation affects lipid biosynthesis, as well as the storage protein composition of maize seeds. Chemical induction of the ER stress response in soybean cell cultures showed similar results. In both cases the ER stress response in plants lead to increases in molecular chaperone levels and lipid biosynthesis. In maize kernels, ER stress specifically leads to alterations in protein body membrane composition and morphology and increases in phospholipid biosynthetic enzyme activities. Future studies will be directed toward understanding the underlying mechanisms that coordinate the integrated regulation of protein and lipid metabolism in plants.
Plant Materials Maize (Zea mays) inbred W64A (normal) and its
near isogenic mutant, W64Afloury-2 (fl2),
were grown at the North Carolina Central Crops Research Station
(Clayton) in the summers of 1998 and 1999. Maize kernels were harvested
at specific DAP, frozen in liquid nitrogen, and stored at
Soybean (Glycine max) cell cultures were grown according
to Abusteit et al. (1985) Fractionation of ER and Protein Bodies All centrifugation and extraction steps were performed at 0°C
to 4°C. Buffer A [100 mM {Tricine
N-[2-hydroxy-1,1-Bis(hydroxymethyl)ethyl]glycine}-NaOH, pH 7.5, 1 mM EDTA, and 10 mM KCl] was used for
enzyme activity assays. Buffer B (10 mM Tris-HCl [pH 8.5 at 25°C], 10 mM KCl, and 5 mM
MgCl2) was used for immunoblot and lipid analyses.
Homogenization was carried out in buffer A made 20% (w/v) Suc and 10 mM dithiothreitol or buffer B made 7.2% (w/v) Suc and 10 mM dithiothreitol as noted below for specific procedures.
For maize protein extractions, endosperm was removed from kernels and
was ground in buffer (1:2, w/v) with a mortar and pestle. Homogenates
were subjected to centrifugation at 300g for 10 min to
remove cell debris. Supernatants were applied to discontinuous Suc
gradients (Larkins and Hurkman, 1978 An alternative method of fractionation was used to obtain protein
for the CCT activity assay. After centrifugation at 300g to remove cell debris, supernatant was subjected to a
5,000g centrifugation for 10 min to separate protein
bodies (5,000g pellet) from ER and other membranes
(5,000g supernatant). Supernatant was subjected to
centrifugation at 100,000g for 15 min in a fixed angle
rotor. Resulting protein body and ER pellets from either method were resuspended in homogenization buffer and were quantified for protein using the Bio-Rad Protein Assay Kit I, with a bovine CCT Activity Assay Protein bodies prepared in buffer A (50 µg in 25 µL of
buffer) were added to an equal volume of CCT reaction buffer (Kinney et
al., 1987 Lipid Kinase Activity Assay Protein body and ER fractions from Suc gradients made with
buffer A were resuspended in 30 mM Tris-HCl (pH 7.2 at
25°C) and were assayed for endogenous lipid kinase activities.
Reactions of 20 µg of protein from protein body fractions or 2 µg
of protein from ER fractions in 30 mM Tris-HCl (pH 7.2),
7.5 mM MgCl2, 1 mM NaMolybdate,
0.01% (v/v) Triton X-100, and 0.9 mM
[ Immunoblotting Protein bodies from Suc gradients made with buffer B were washed
twice by dilution in buffer B followed by a 5,000g
centrifugation at 4°C for 10 min. The final pellet was resuspended in
buffer B containing 0.15 M NaCl, made 1% (v/v) Triton
X-100, and were allowed to mix on a Nutator (Innovative Medical
Systems, Ivyland, PA) for 1 h at 4°C. Prior to
fractionation through 8% (w/v; PI 4-kinase) or 10% (w/v; BiP)
SDS-polyacrylamide gels, one volume SDS-PAGE sample buffer (Laemmli,
1970 The PI 4-kinase immunoblots were probed with antibodies raised
against a recombinant protein encoding the C-terminal one-third of
AtPI4K Protein Labeling At the designated times after treatment, 1-mL aliquots of
soybean suspension culture cells were transferred to a 24-well tissue culture plate (Becton-Dickinson, Lincoln Park, NJ). Cells were incubated with Easytag Expre35S35S -protein
labeling mix (NEN Life Science Products) for 30 min and were washed
according to the protocol described by Malik et al. (1999) ER-enriched fractions for immunoprecipitation were prepared by grinding the frozen cells in buffer A made 20% (w/v) Suc using a mortar and pestle with a small amount of glass beads. Cellular debris was removed by a 2,000g centrifugation for 2 min and the supernatant was applied to a discontinuous Suc gradient prepared as a 0.6-mL step of 1.5 M Suc and a 1.0-mL step of 1.0 M Suc, both in buffer A. The remaining fractionation steps were identical to those described above for fractionation of ER from maize. Immunoprecipitation All immunoprecipitation steps were conducted at 0°C to 4°C.
Radiolabeled cells were resuspended at a ratio of 2 mL g As a clearing step, 25 µL of a 50% (v/v) slurry of Protein-A agarose
(Life Technologies, Rockville, MD) in TBS was added to each sample (40 µg of protein per incubation diluted to 200 µL with lysis buffer).
After clearing, proteins were incubated with antibodies against spinach
BiP (1D9, StressGen Biotechnologies; 1 µL), calreticulin (Coughlan et
al., 1997 Lipid Analysis Endosperm was homogenized in buffer B, left undisturbed for 15 min on ice to allow starch to settle, and then decanted prior to a
low-speed centrifugation at 80g for 5 min and
fractionation through Suc gradients as described above for separation
of ER and protein bodies. Fractions from the gradients were diluted to
the refractive index of the homogenization buffer and were subjected to
centrifugation at 5,000g in a tabletop centrifuge (protein bodies) or 100,000g in a fixed angle rotor
(ER). ER and protein body pellets were resuspended in buffer B prior to
extraction of the lipids into the organic phase by overnight incubation
in chloroform:methanol (2:1, v/v) at For total PL and triacylglycerol analysis, five to seven kernels of
each phenotype were dissected. Endosperm and embryos were separated
from each other and were placed in a drying oven (80°C) for 2 d
prior to measuring of dry weights. Dried samples were pulverized with a
mallet and lipids were extracted in chloroform:methanol (2:1, v/v)
overnight at Lipid extracts were evaporated under nitrogen at 50°C, resuspended in
100 µL of chloroform:methanol (2:1), and applied to a 60-Å silica
gel plate (Whatman). Lipid species were resolved by TLC with a
developing solvent of petroleum ether:diethyl ether:acetic acid
(80:20:1, v/v) to separate total PL and triacylglycerol or chloroform:methanol:concentrated NH4OH (60:30:1.5,
v/v) to separate individual phospholipid species. All lipids
separated by TLC were visualized with 2,7-dichlorofluorescein in 95%
(w/v) ethanol and were identified by comigration with known standards.
The regions of the TLC plate corresponding to individual lipid species
were extracted with hexane and quantified by gas chromatography (Browse et al., 1986 [14C]Acetate Incorporation An aliquot of cells was removed at the designated time after
treatment with Tm and was incubated with shaking at room temperature for 2 h with [14C]-acetic acid (NEN Life Science
Products) at a concentration of 0.5 µCi mL Labeled cells were thawed on ice and rinsed with ice-cold 5% (w/v) TCA. Lipids were extracted, dried under nitrogen, resuspended, and resolved by TLC as described above. Bands were visualized and quantified with an imaging scanner (500, Bioscan System, Washington, DC).
We are grateful to Nozomu Koizumi and Maarten J. Chrispeels for sharing unpublished data. Special thanks to Bill Novitzky for technical assistance with the TLC and GC analyses, Bonnie Sheldon for sharing her soybean cell cultures, Weibing Xing for implementing the CCT enzyme activity assay, Jeff Gillikin for sharing his protein fractionation expertise, and members of the Boston, Boss, and Dewey laboratories for helpful discussions.
Received October 13, 2000; returned for revision January 8, 2001; accepted February 7, 2001. 1 This work was supported by the U.S. Department of Energy (grant no. DE-FG02-00ER150065 to R.S.B., R.E.D., and W.F.B.), by the National Science Foundation (grant nos. MCB96-04285 [to W.F.B.], IBN-9513582 [to R.E.D.], and MCB93-17303 [to R.S.B.]), by the North Carolina Agricultural Research Service (to W.F.B., R.S.B., and R.E.D.), and by the National Science Foundation for Interdisciplinary Research Training Group on Transgenic Plant Technology for Laboratory and Field Applications (fellowship no. BIR-9420689 to K.J.S.).
2 Present address: BASF Plant Sciences, Research Triangle Park, NC 27709.
3 Present address: Department of Biochemistry, Box 7622, North Carolina State University, Raleigh, NC 27695.
* Corresponding author; email ralph_dewey{at}ncsu.edu; fax 919-515-7959.
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TOP
ABSTRACT
INTRODUCTION
-zein
storage protein that prevents the signal peptide of the zein from being
cleaved (Coleman et al., 1995
1 mg
80°C.
-globulin protein standard (Bio-Rad, Hercules, CA).
