| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 279, Issue 41, 42916-42923, October 8, 2004
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
**
From the
Institute of Cell and Molecular Biology, University of Edinburgh, King's Bldgs., Mayfield Rd., Edinburgh, EH9 3JH, United Kingdom, ¶UMR Physiologie et Biotechnologie Végétales, Instítut de Biologie Végétale Moléculaire-Centre de Recherches Institut National de la Recherche Agronomique de Bordeaux, 71, Avenue Edouard Bourleaux-BP 81, 33883 Villenave D'Ornon Cedex, France, and ||Crop Performance and Improvement Division, Rothamsted Research, Harpenden, AL5 2JQ, United Kingdom
Received for publication, July 1, 2004 , and in revised form, July 21, 2004.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
In germinating oilseeds the glyoxylate cycle also enables acetate from lipid breakdown to be converted to four-carbon gluconeogenic substrates to support seedling growth (6). In Arabidopsis, seedlings of icl mutants grow poorly because they are unable to convert acetate from fatty acid 
-oxidation into sugars (7). Instead, the seedlings slowly respire their fatty acids by transferring either acetate or citrate from the peroxisome to the mitochondrion (8, 9). Without ICL, the tricarboxylic acid cycle cannot be supplied with succinate or with malate produced from glyoxylate by MLS. Thus, oxaloacetate cannot be generated for gluconeogenesis. We considered the possibility that if instead of blocking the glyoxylate cycle at ICL, it is blocked downstream at MLS, the succinate would still feed into the tricarboxylic acid cycle to regenerate oxaloacetate, and the glyoxylate could potentially feed into the photorespiratory pathway for conversion to sugar (Fig. 1). Although seed germination (radicle emergence) is fueled by a limited reserve of sugars in Arabidopsis, the subsequent growth of the seedling is fueled largely by oil breakdown, which occurs concurrently with the acquisition of photosynthetic competence. It has been clearly established by three independent studies that glyoxylate cycle and photorespiratory enzymes co-exist in the same peroxisomes during seedling growth in oilseed species (10–12). Thus, in mls mutants, glyoxylate produced by ICL could serve as a substrate for serine-glyoxylate aminotransferase, leading to glycine and subsequently to serine, hydroxypyruvate, glycerate, and ultimately sugars (Fig. 1). We, therefore, hypothesized that mls mutant seedlings would be capable of gluconeogenesis from lipid and would grow better than icl seedlings. To test this hypothesis we isolated two independent mls mutants and studied their growth and metabolism relative to icl mutants. Consistent with our hypothesis, mls mutant seedlings grow much better, break down their lipid more rapidly, and accumulate more sugars than icl mutant seedlings. In addition they are capable of gluconeogenesis from acetate, unlike icl seedlings (7).
|
|
EXPERIMENTAL PROCEDURES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
Screening of T-DNA Insertion Lines and Isolation of mls Knock-out Mutants—PCR screening was used to identify T-DNA insertions the Arabidopsis MLS gene (At5g03860). The gene-specific primers MS50 (5'-ATG GAG CTC GAG ACC TCA GTT TAT C-3'), MS51 (5'-GCT GCT TTC GAG GAC GCT TTG TCT C-3'), MS30 (5'- GAG CCT TGA GAC ATT GAT AGG GTA G-3'), and MS31 (5'-ACA AGT ACG GAT GAG AAG ATC AGA G-3') were used in combination with T-DNA left border primers (13, 15, 16). This screening identified an insertion in a plant in the Wassilewskija ecotype from the Versailles collection (15, 16). DNA sequence analysis revealed two copies of the T-DNA in the first intron of the MLS gene (mls-1; Fig. 2A). A second mutant was identified in the SALK SIGnAL T-DNA collection, which was generated in the Col-0 ecotype (17), containing a T-DNA insertion in the third intron of the MLS gene (mls-2, SALK_060987; Fig. 2A). In both knock-out lines kanamycin resistance conferred by the T-DNA co-segregated with the interrupted MLS gene (data not shown). A wild type segregant was isolated concurrently with each mutant and named MLS-1 and MLS-2 to indicate the mutant to which each corresponds.
|
Transcriptome Analysis—Microarray analysis using Affymetrix ATH1 genome arrays was carried out by the Nottingham Arabidopsis Stock Centre (nasc.nott.ac.uk) under the auspices of the Genomic Arabidopsis Resource Network (www.york.ac.uk/res/garnet/garnet.htm). The procedures are Minimum Information about a Microarray Experiment compliant, and data are available on the NASC website.
Biochemical Analysis—For enzyme assays, tissue extracts were prepared from Arabidopsis seedlings as described in Eastmond et al. (7). MLS and ICL assays were carried out as described in Cooper and Beevers (18). A coupled assay for serine-glyoxylate aminotransferase and hydroxypyruvate reductase activity was carried out using a modified version of the method described by Nakamura and Tolbert (19), in which 1 mM glyoxylate was used to start the reaction. Protein content was determined as reported by Bradford (20) using bovine serum albumin as the standard. Total fatty acids were extracted and quantified by gas chromatography-mass spectroscopy using the method described by Browse et al. (21), with C20:1 as a marker for triacylglycerol (TAG). 1H NMR was used to analyze major polar metabolites in 2-day-old seedlings as reported by Ward et al. (22) under the auspices of the Genomic Arabidopsis Resource Network (as above). The levels of glucose (Glc), fructose (Fru), and sucrose (Suc) were quantified in ethanol-soluble extracts as described in Nielsen et al. (23).
14C-Labeling Experiments and Paper Chromatography—The metabolism of sodium [2-14C]acetate, [U-14C]glycine, and L-[U-14C]serine by triplicate batches of 100 2-day-old Arabidopsis seedlings grown on 1% (w/v) Suc in continuous light was analyzed according to the method described previously by Eastmond et al. (7), with the following modifications. Seedlings were incubated in the dark (to prevent re-fixation of 14CO2) for 4 h, during which time released 14CO2 was trapped in a well containing 200 µl of 5 N KOH. The soluble components of the seedlings were then extracted in three 1-ml aliquots of 80% (v/v) ethanol at 80 °C followed by 1 ml of water at 40 °C. The total soluble extracts were combined, and the hydrophobic components were extracted with 1.5 ml of chloroform. The ethanol-soluble components were dried, resuspended in water, and then further separated into neutral, basic, and acidic fractions by ion exchange chromatography. The amount of 14C present in each fraction was quantified by scintillation counting. The presence of mainly [14C]Suc, [14C]Glc, and [14C]Fru in the neutral fraction was confirmed by paper chromatography of aliquots of the neutral fraction and subsequent scintillation counting of the spots corresponding to sugar markers on the chromatogram. Between 80 and 90% of the radioactivity in the neutral fraction was found on the chromatogram in the regions corresponding to these sugar makers (data not shown).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
The expression patterns for genes encoding a number of key enzymes of lipid metabolism were analyzed in mls and icl-2 mutant lines: 3-keto-acyl CoA thiolase (-oxidation), two peroxisomal isoforms each of citrate synthase and malate dehydrogenase (glyoxylate cycle), and two isoforms of phosphoenolpyruvate carboxykinase (gluconeogenesis) showed no apparent changes in the patterns of the transcripts for these genes in the mutant lines (data not shown).
The mls Mutants Have a Less Severe Phenotype than the icl Mutant—The mls seedling phenotypes were examined along-side the icl-2 mutant line isolated previously together with wild type revertant ICL-2 (7). The icl-2 mutant has a stunted phenotype during post-germinative growth, which can be rescued by the addition of exogenous Suc or by growth in high light conditions (Fig. 3A; Ref. 7). However, mls-1 and mls-2 mutants have only a slightly stunted phenotype when grown in the absence of exogenous sugar in the light (Fig. 3A). The mls phenotype is more obvious in the dark where hypocotyl elongation is inhibited, and little root development takes place (Fig. 3A). mls mutant seedlings can be rescued by the addition of exogenous Suc in all conditions (Fig. 3A). Furthermore, lower concentrations of Suc are able to restore the mls-2 mutant seedlings to wild type growth in the light than are required to rescue icl-2 seedlings (data not shown). A further phenotype observed in icl-2 mutants was the failure of seedlings to establish into plantlets with true leaves under conditions of limited light (Fig. 3B; Ref. 7). In contrast, the establishment frequency of mls-2 seedlings is similar to wild type levels and only becomes compromised when seedlings are grown in short days (Fig. 3B).
|
Transcriptome Analysis Reveals Major Differences in the Two Glyoxylate Cycle Mutants—To investigate differences in gene expression in the two glyoxylate cycle mutants, RNA was isolated from triplicate batches of 2-day-old, light-grown mutant and wild type seedlings. This time point represents the onset of the period of most rapid lipid breakdown (Ref. 7 and see below), so that any differences in gene expression may be most marked. RNAs were each hybridized to Affymetrix ATH1 genome chips. The average hybridization signals detected in each mutant line were compared with the signal strengths in the corresponding wild types (Fig. 4). The data reveal that there are many more differences in gene expression in icl-2 (Fig. 4A) than in mls-2 (Fig. 4B) relative to their wild type controls. In icl-2, 161 genes showed a more than 2-fold increase in expression, and 236 genes showed a more than 2-fold decrease relative to wild type (Table I indicates the 20 genes with the most altered gene expression in each class). In contrast, in mls-2 only 10 genes showed increased expression, and 12 genes decreased relative to wild type (Table II).
|
|
|
-glucosidase, asparagine synthetase, and enzymes of branched chain amino acid catabolism). Other induced genes include those encoding enzymes of protein degradation (e.g. proteases, aminotransferases, amino acid transporters) and numerous glycohydrolases and senescence-associated proteins. The transcript data from mls-2 indicates that these seedlings are not carbohydrate-limited. Gene transcripts expressed at low levels in the mutant include two jasmonate-inducible genes but otherwise do not fall into any obvious functional groupings, and most of them are not underexpressed in icl-2 (Table II). However, the 10 genes overexpressed in mls-2 relative to wild type include several that are also overexpressed in icl-2 (Table II). Among these is that encoding Gln-dependent asparagine synthetase, which is the only induced transcript coding for an enzyme known to be associated with carbohydrate limitation (24).
Metabolome Analysis Shows That mls Mutant Seedlings Are Not Carbohydrate-limited and Accumulate Glycine and Serine—To establish the effects of the mutations on levels of sugar, organic acids, and amino acids in mutant seedlings, the metabolomes of icl-2 and mls-2 seedlings were investigated and compared with wild types using non-biased 1H NMR (22). Principal component analysis of the data was carried out, and a six-component model explained 90% of the variance. The first two principal components reveal that icl-2 forms a distinct cluster separate from wild types and mls-2 (Fig. 5A). This clear separation is due mainly to decreased levels of glutamine (Gln) and glucose (Glc) in icl-2 compared with all other lines (Fig. 5, B and C). There were also small increases in valine, isoleucine, and lysine in icl-2. In contrast, the glucose levels in mls-2 did not differ appreciably from MLS-2, although the Gln level in mls-2 was also appreciably reduced (Fig. 5, B and C). A further significant finding in this analysis was that mls-2 seedlings have appreciably increased levels of serine (Ser) and glycine (Gly) relative to all other lines (Fig. 5C). There were no changes in organic acids detectable using this method in any genotypes (data not shown).
|
|
-oxidation, whereas icl-2 seedlings are not (7). mls Seedlings Carry Out Gluconeogenesis from [14C]Acetate, whereas icl Seedlings Do Not—To directly test the hypothesis that mls-2 seedlings are capable of gluconeogenesis from acetate, 2-day-old light-grown seedlings were fed with [2-14C]acetate and fractionated, and the amount of 14C was incorporated into each fraction determined (Fig. 7). mls-2 and wild type seedlings incorporated the same total amount of 14C during the experiment (123 ± 6 and 122 ± 4 x 104 dpm, respectively); the pattern of incorporation in the two genotypes was similar in all fractions except that mls-2 seedlings incorporate 60% of the amount of 14C into sugars and release twice as much 14CO2 compared with wild type seedlings. In contrast, icl-2 seedlings incorporated less total 14C during the experiment (75 ± 5 x 104 dpm), and significantly less 14C was detected in all ethanol-soluble fractions than in wild type seedlings. Only 20% of 14Cis detected in the sugar fraction of icl-2 seedlings compared with wild type relative to total incorporation (5% in absolute terms). icl-2 seedlings release more than three times the percentage of 14CO2 compared with wild type seedlings (Fig. 7). These data confirm that mls-2 seedlings are capable of gluconeogenesis from acetate, whereas icl-2 seedlings are not.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
NMR analysis of polar solutes showed that icl-2 seedlings have low levels of Glc, whereas mls-2 seedlings contain Glc at wild type levels (Fig. 5B). Both icl-2 and mls-2 seedlings have low Gln (Fig. 5C), which is consistent with both having elevated expression of a Gln-dependent asparagine synthetase gene, but Asn levels were not elevated. The elevated levels of valine, isoleucine, and lysine in icl-2 seedlings may result from increased protein catabolism, as suggested by transcriptome data. Changes in amino acids and increased expression of genes encoding enzymes of branched-chain amino acid catabolism in icl-2 are likely to be causally related.
The greater rate of breakdown of TAG in mls relative to icl seedlings (Fig. 6A) and the accumulation of sugars in mls seedlings as they grow (Fig. 6, B and C) imply that mls seedlings are able to convert fatty acids from TAG into sugars. This was confirmed by the transfer of 14C from acetate to sugars in mls-2 at a level of 60% that of wild type seedlings (Fig. 7). In contrast to icl-2 seedlings, the pattern of 14C labeling in mls-2 seedlings differed from wild types only in the small increase in level of radioactivity released as CO2 and the small decrease incorporated into sugars (Fig. 7). This is entirely consistent with gluconeogenesis from acetate in mls-2 seedlings, which is less efficient than in wild type, such that more of the [14C]acetate is respired in mls-2 but is far more efficient than in icl-2 seedlings. The observation that seedlings lacking MLS are capable of gluconeogenesis from acetate is in marked contrast to the observation that ICL is required for such gluconeogenesis in Arabidopsis seedlings (7) and fulfils our original hypothesis. This observation also contrasts with the observation that both MLS and ICL are essential for gluconeogenesis and growth of microorganisms on ethanol, acetate, or fatty acids (2–5).
Because icl mutant seedlings are essentially incapable of gluconeogenesis from lipid (Fig. 7; Ref. 7), we deduce that ICL is required for gluconeogenesis in the mls mutants. The succinate produced by ICL will be oxidized in the mitochondria and oxaloacetate regenerated to maintain the cycle. ICL can only continue to produce succinate as long as the glyoxylate is metabolized. Furthermore, conversion of this glyoxylate to sugar is required for the net conversion of TAG to sugar to support plant growth; its respiration or degradation would achieve no more than is achieved by the respiration of TAG in the icl mutant. We hypothesized that this glyoxylate would enter the photorespiratory gluconeogenic pathway. Some bacteria can achieve gluconeogenesis from glyoxylate using the "glycerate pathway" employing glyoxylate carboligase, tartronic semialdehyde reductase, and glycerate kinase (26). To our knowledge, glyoxylate carboligase and tartronic semialdehyde reductase have not been detected in plants, and no genes are predicted in the Arabidopsis genome. Micrococcus denitrificans converts glyoxylate to oxaloacetate via erythro--hydroxyaspartate (27), but again, we can find no evidence for the appropriate enzymes in plants.
Final confirmation of our proposed pathway of MLS-independent gluconeogenesis from acetate will depend upon detailed genetic and molecular characterization in the future. However, our hypothesis that glyoxylate will feed into the photorespiratory pathway is supported by elevated levels of Gly and Ser in mls seedlings and by the transfer of 14C from Gly and Ser into sugars. Microarray data show that the genes encoding the enzymes of the photorespiratory pathway are expressed as highly in 2-day-old seedlings as in 7-day-old seedlings, and mature leaves (information obtained from nasc.nott.ac.uk). We have also determined that serine-glyoxylate aminotransferase activity coupled to hydroxypyruvate reductase activity in mls-2 seedlings was 20.8 ± 2.1 and 39 ± 4.3 µmol·min-1·mg of protein-1 at days 2 and 5, respectively. This is similar to enzyme activity measured in MLS-2 seedlings of 17.7 ± 1.2 and 32.4 ± 1.6 µmol·min-1·mg of protein-1 at days 2 and 5, respectively, and is sufficient to account for the observed flux of carbon from fatty acids to sugar.
We propose that at the transition from germinated seed to seedling, the mls mutant will be compromised by the absence of the glyoxylate cycle and inability to assimilate acetate into sugars. This is reflected in the low hexose content of such seedlings after 2 days of growth (Fig. 6C) and an accumulation of Ser and Gly (Fig. 5C). We further propose that around day 2, when the seedlings start to turn green, they acquire the ability to metabolize glyoxylate through the photorespiratory pathway, and the block caused by the lack of MLS is alleviated. At this point TAG breakdown starts to accelerate, and sugars begin to accumulate.
The observation that seeds of icl-2 accumulate less lipid than wild type while mls-2 seeds accumulate normal amounts (Fig. 6A; Ref. 7) provides evidence that ICL has an important function at other stages of the life cycle for which MLS is not required. There are reports of ICL activity in the absence of MLS in green leaves (28, 29, 30), which may thus function in such a pathway.
The phenotypes of glyoxylate cycle mutants clearly indicate that there is a cost to Arabidopsis seedlings lacking ICL and MLS. However, although the absence of ICL and thus gluconeogenesis from lipid results in severely compromised seedling growth (Fig. 3; Ref. 7), the absence of MLS results in a much less severe phenotype because the seedlings are able to employ an alternative gluconeogenic mechanism. This novel metabolic pathway from acetate to sugar, potentially employing photorespiratory enzymes in conjunction with glyoxylate cycle enzymes, in mls mutant seedlings is an example of the remarkable metabolic flexibility of Arabidopsis.
|
FOOTNOTES |
|---|
Funded by the Biotechnology and Biological Sciences Research Council, Swindon, United Kingdom.
** To whom correspondence should be addressed. Tel.: 44-131-6505318; Fax: 44-131-6505392; E-mail: s.smith{at}ed.ac.uk.
1 The abbreviations used are: ICL, isocitrate lyase; MLS, malate synthase; T-DNA, transfer-DNA; TAG, triacylglycerol; RT, reverse transcription; Suc, sucrose.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  B. S. Zheng, E. Ronnberg, L. Viitanen, T. A. Salminen, K. Lundgren, T. Moritz, and J. Edqvist Arabidopsis sterol carrier protein-2 is required for normal development of seeds and seedlings J. Exp. Bot., September 1, 2008; 59(12): 3485 - 3499. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Calderon-Vazquez, E. Ibarra-Laclette, J. Caballero-Perez, and L. Herrera-Estrella Transcript profiling of Zea mays roots reveals gene responses to phosphate deficiency at the plant- and species-specific levels J. Exp. Bot., June 6, 2008; (2008) ern115v2. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Mazzella, M. I. Zanor, A. R. Fernie, and J. J. Casal Metabolic responses to red/far-red ratio and ontogeny show poor correlation with the growth rate of sunflower stems J. Exp. Bot., June 1, 2008; 59(9): 2469 - 2477. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. G. van Doorn and E. J. Woltering Physiology and molecular biology of petal senescence J. Exp. Bot., March 3, 2008; (2008) erm356v2. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Andre and C. Benning Arabidopsis Seedlings Deficient in a Plastidic Pyruvate Kinase Are Unable to Utilize Seed Storage Compounds for Germination and Establishment Plant Physiology, December 1, 2007; 145(4): 1670 - 1680. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Ramirez-Trujillo, S. Encarnacion, E. Salazar, A. G. de los Santos, M. F. Dunn, D. W. Emerich, E. Calva, and I. Hernandez-Lucas Functional Characterization of the Sinorhizobium meliloti Acetate Metabolism Genes aceA, SMc00767, and glcB J. Bacteriol., August 15, 2007; 189(16): 5875 - 5884. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Joshi, K. M. Laubengayer, N. Schauer, A. R. Fernie, and G. Jander Two Arabidopsis Threonine Aldolases Are Nonredundant and Compete with Threonine Deaminase for a Common Substrate Pool PLANT CELL, December 1, 2006; 18(12): 3564 - 3575. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Footitt, J. Marquez, H. Schmuths, A. Baker, F. L. Theodoulou, and M. Holdsworth Analysis of the role of COMATOSE and peroxisomal beta-oxidation in the determination of germination potential in Arabidopsis J. Exp. Bot., August 1, 2006; 57(11): 2805 - 2814. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Cernac, C. Andre, S. Hoffmann-Benning, and C. Benning WRI1 Is Required for Seed Germination and Seedling Establishment Plant Physiology, June 1, 2006; 141(2): 745 - 757. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. J. Eastmond SUGAR-DEPENDENT1 Encodes a Patatin Domain Triacylglycerol Lipase That Initiates Storage Oil Breakdown in Germinating Arabidopsis Seeds PLANT CELL, March 1, 2006; 18(3): 665 - 675. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Rubio, T. R. Larson, M. Gonzalez-Guzman, S. Alejandro, I. A. Graham, R. Serrano, and P. L. Rodriguez An Arabidopsis Mutant Impaired in Coenzyme A Biosynthesis Is Sugar Dependent for Seedling Establishment Plant Physiology, March 1, 2006; 140(3): 830 - 843. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Pracharoenwattana, J. E. Cornah, and S. M. Smith Arabidopsis Peroxisomal Citrate Synthase Is Required for Fatty Acid Respiration and Seed Germination PLANT CELL, July 1, 2005; 17(7): 2037 - 2048. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||
) and MLS-1 (
) seedlings. Data plotted are the mean ± S.D. for measurements on three batches of seedlings. For both experiments seedlings were grown on half-strength Murashige and Skoog (
icl-2; 
, ICL-2. B, typical NMR traces for the characteristic peaks of Glc in mls-2 (solid line) and MLS-2 (dashed line) seedlings (i) and icl-2 (solid line) and ICL-2 (dashed line) seedlings (ii). C, typical NMR traces for the characteristic peaks of Gly, Ser, and Gln in mls-2 (solid line) and MLS-2 (dashed line) seedlings (i) and icl-2 (solid line) and ICL-2 (dashed line) seedlings (ii).
