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Lipid Utilization, Gluconeogenesis, and Seedling Growth in Arabidopsis Mutants Lacking the Glyoxylate Cycle Enzyme Malate Synthase

2004; Elsevier BV; Volume: 279; Issue: 41 Linguagem: Inglês

10.1074/jbc.m407380200

1083-351X

Johanna E. Cornah, Véronique Germain, Jane L. Ward, Michael H. Beale, Steven M. Smith,

Lipid metabolism and biosynthesis

The aim of this research was to test the role of the glyoxylate cycle enzyme malate synthase (MLS) in lipid utilization, gluconeogenesis, and seedling growth in Arabidopsis. We hypothesized that in the absence of MLS, succinate produced by isocitrate lyase (ICL) could still feed into the tricarboxylic acid cycle, whereas glyoxylate could be converted to sugars using enzymes of the photorespiratory pathway. To test this hypothesis we isolated knock-out mls mutants and studied their growth and metabolism in comparison to wild type and icl mutant seedlings. In contrast to icl seedlings, which grow slowly and are unable to convert lipid into sugars (Eastmond, P. J., Germain, V., Lange, P. R., Bryce, J. H., Smith, S. M. & Graham, I. A. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 5669–5674), mls seedlings grow faster, use their lipid more rapidly, and are better able to establish as plantlets. Transcriptome and metabolome analyses show that icl seedlings exhibit many features characteristic of carbohydrate starvation, whereas mls seedlings differ relatively little from wild type. In the light mls seedlings generate more sugars than icl seedlings, and when fed with [14C]acetate, 14C-labeling of sugars is three times greater than in icl seedlings and more than half that in wild type seedlings. The mls seedlings also accumulate more glycine and serine than icl or wild type seedlings, consistent with a diversion of glyoxylate into these intermediates of the photorespiratory pathway. We conclude that, in contrast to bacteria and fungi in which MLS is essential for gluconeogenesis from acetate or fatty acids, MLS is partially dispensable for lipid utilization and gluconeogenesis in Arabidopsis seedlings. The aim of this research was to test the role of the glyoxylate cycle enzyme malate synthase (MLS) in lipid utilization, gluconeogenesis, and seedling growth in Arabidopsis. We hypothesized that in the absence of MLS, succinate produced by isocitrate lyase (ICL) could still feed into the tricarboxylic acid cycle, whereas glyoxylate could be converted to sugars using enzymes of the photorespiratory pathway. To test this hypothesis we isolated knock-out mls mutants and studied their growth and metabolism in comparison to wild type and icl mutant seedlings. In contrast to icl seedlings, which grow slowly and are unable to convert lipid into sugars (Eastmond, P. J., Germain, V., Lange, P. R., Bryce, J. H., Smith, S. M. & Graham, I. A. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 5669–5674), mls seedlings grow faster, use their lipid more rapidly, and are better able to establish as plantlets. Transcriptome and metabolome analyses show that icl seedlings exhibit many features characteristic of carbohydrate starvation, whereas mls seedlings differ relatively little from wild type. In the light mls seedlings generate more sugars than icl seedlings, and when fed with [14C]acetate, 14C-labeling of sugars is three times greater than in icl seedlings and more than half that in wild type seedlings. The mls seedlings also accumulate more glycine and serine than icl or wild type seedlings, consistent with a diversion of glyoxylate into these intermediates of the photorespiratory pathway. We conclude that, in contrast to bacteria and fungi in which MLS is essential for gluconeogenesis from acetate or fatty acids, MLS is partially dispensable for lipid utilization and gluconeogenesis in Arabidopsis seedlings. The glyoxylate cycle catalyzes the conversion of two acetate molecules into succinate, providing the means for microorganisms to grow on ethanol, acetate, or fatty acids (1Kornberg H.L. Krebs H.A. Nature. 1957; 179: 988-991Crossref PubMed Scopus (300) Google Scholar). This function has been confirmed through the analysis of mutants lacking isocitrate lyase (ICL 1The abbreviations used are: ICL, isocitrate lyase; MLS, malate synthase; T-DNA, transfer-DNA; TAG, triacylglycerol; RT, reverse transcription; Suc, sucrose. ; EC 4.1.3.1) and malate synthase (MLS; EC 4.1.3.2), key enzymes of the glyoxylate cycle. Bacterial and fungal mutants do not grow on acetate, ethanol, or fatty acids (2McKinney J.D. zu Bentrup K.H. Munoz-Elias E.J. Miczak A. Chen B. Chan W.T. Swenson D. Sacchettini J.C. Jacobs W.R. Russell D.G. Nature. 2000; 406: 735-738Crossref PubMed Scopus (1116) Google Scholar, 3Lorenz M.C. Fink G.R. Nature. 2001; 412: 83-86Crossref PubMed Scopus (581) Google Scholar, 4Hartig A. Simon M.M. Schuster T. Daugherty J.R. Yoo H.S. Cooper T.G. Nucleic Acid Res. 1992; 20: 5677-5686Crossref PubMed Scopus (78) Google Scholar, 5Pellicer M.T. Fernandez C. Badia J. Aguilar J. Lin E.C.C. Baldoma L. J. Biol. Chem. 1999; 274: 1745-1752Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Loss of ICL or MLS also leads to avirulence in bacterial and fungal pathogens of plants and mammals (2McKinney J.D. zu Bentrup K.H. Munoz-Elias E.J. Miczak A. Chen B. Chan W.T. Swenson D. Sacchettini J.C. Jacobs W.R. Russell D.G. Nature. 2000; 406: 735-738Crossref PubMed Scopus (1116) Google Scholar, 3Lorenz M.C. Fink G.R. Nature. 2001; 412: 83-86Crossref PubMed Scopus (581) Google Scholar, 4Hartig A. Simon M.M. Schuster T. Daugherty J.R. Yoo H.S. Cooper T.G. Nucleic Acid Res. 1992; 20: 5677-5686Crossref PubMed Scopus (78) Google Scholar, 5Pellicer M.T. Fernandez C. Badia J. Aguilar J. Lin E.C.C. Baldoma L. J. Biol. Chem. 1999; 274: 1745-1752Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). For example Mycobacterium tuberculosis lacking ICL cannot grow on fatty acids or acetate and is unable to persist in macrophages, where lipid is the primary carbon source available (2McKinney J.D. zu Bentrup K.H. Munoz-Elias E.J. Miczak A. Chen B. Chan W.T. Swenson D. Sacchettini J.C. Jacobs W.R. Russell D.G. Nature. 2000; 406: 735-738Crossref PubMed Scopus (1116) Google Scholar). Similarly Candida albicans and Saccharomyces cerevisiae lacking ICL cannot grow on acetate, and the Candida mutant is less infectious in mice where macrophage lipid is also the primary carbon source (3Lorenz M.C. Fink G.R. Nature. 2001; 412: 83-86Crossref PubMed Scopus (581) Google Scholar). ICL and MLS have therefore been identified as targets for therapeutic drugs to treat some bacterial and fungal infections since the glyoxylate cycle is absent from vertebrates. In germinating oilseeds the glyoxylate cycle also enables acetate from lipid breakdown to be converted to four-carbon gluconeogenic substrates to support seedling growth (6Beevers H. Nature. 1961; 191: 433-436Crossref PubMed Scopus (95) Google Scholar). In Arabidopsis, seedlings of icl mutants grow poorly because they are unable to convert acetate from fatty acid β-oxidation into sugars (7Eastmond P.J. Germain V. Lange P.R. Bryce J.H. Smith S.M. Graham I.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5669-5674Crossref PubMed Scopus (226) Google Scholar). Instead, the seedlings slowly respire their fatty acids by transferring either acetate or citrate from the peroxisome to the mitochondrion (8Eastmond P.J. Graham I.A. Trends Plant Sci. 2001; 6: 72-77Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 9Cornah J.E. Smith S.M. Baker A. Graham I.A. Plant Peroxisomes: Biochemistry, Cell Biology, and Biotechnological Applications. Kluwer Academic Publishers Group, Dordrecht, Netherlands2002: 57-101Crossref Google Scholar). 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 (10Nishimura M. Yamaguchi J. Mori H. Akazawa T. Yokota S. Plant Physiol. 1983; 80: 313-316Google Scholar, 11Titus D.E. Becker W.M. J. Cell Biol. 1985; 101: 1288-1299Crossref PubMed Scopus (107) Google Scholar, 12Sautter C. Planta. 1986; 167: 491-503Crossref PubMed Scopus (43) Google Scholar). 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 (7Eastmond P.J. Germain V. Lange P.R. Bryce J.H. Smith S.M. Graham I.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5669-5674Crossref PubMed Scopus (226) Google Scholar). Plant Material and Growth Conditions—Seeds of Arabidopsis were surface-sterilized, stratified, and germinated as described by Sherson et al. (13Sherson S.M. Hemmann G. Wallace G. Forbes S. Germain V. Stadler R. Bechtold N. Sauer N. Smith S.M. Plant J. 2000; 24: 849-857Crossref PubMed Google Scholar). Except where stated, seedlings were grown for 48 h, corresponding to principal growth stage 0.6 as defined by Boyes et al. (14Boyes D.C. Zayed A.M. Ascenzi R. Mc Caskill A.J. Hoffman N.E. Davis K.R. Gorlach J. Plant Cell. 2001; 13: 1499-1510Crossref PubMed Scopus (1076) Google Scholar) in continuous light (100 μmol of photons·m-2·s-1) or dark in the presence or absence of 1% (w/v) sucrose. 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 (13Sherson S.M. Hemmann G. Wallace G. Forbes S. Germain V. Stadler R. Bechtold N. Sauer N. Smith S.M. Plant J. 2000; 24: 849-857Crossref PubMed Google Scholar, 15Bechtold N. Ellis J. Pelletier G. C. R. Acad. Sci. Ser. III. 1993; 316 (C. R.): 1194-1199Google Scholar, 16Bouchez D. Camilleri C. Caboche M. C. R. Acad. Sci. Ser. III. 1993; 316: 1188-1193Google Scholar). This screening identified an insertion in a plant in the Wassilewskija ecotype from the Versailles collection (15Bechtold N. Ellis J. Pelletier G. C. R. Acad. Sci. Ser. III. 1993; 316 (C. R.): 1194-1199Google Scholar, 16Bouchez D. Camilleri C. Caboche M. C. R. Acad. Sci. Ser. III. 1993; 316: 1188-1193Google Scholar). 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 (17Alonso J.M. Stepanova A.N. Leisse T.J. Kim C.J. Chen H.M. Shinn P. Stevenson D.K. Zimmerman J. Barajas P. Cheuk R. Gadrinab C. Heller C. Jeske A. Koesema E. Meyers C.C. Parker H. Prednis L. Ansari Y. Choy N. Deen H. Geralt M. Hazari N. Hom E. Karnes M. Mulholland C. Ndubaku R. Schmidt I. Guzman P. Aguilar-Henonin L. Schmid M. Weigel D. Carter D.E. Marchand T. Risseeuw E. Brogden D. Zeko A. Crosby W.L. Berry C.C. Ecker J.R. Science. 2003; 301: 653-657Crossref PubMed Scopus (4153) Google Scholar), 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. RT-PCR Analysis—RNA was isolated from 2-day seedlings using the Qiagen RNAeasy kit and used to generate cDNA with the Qiagen Omniscript RT-PCR kit according to manufacturers' instructions. PCR was carried out with gene-specific primers MS51 and MS30 for MLS and ICL51 and ICL30 for ICL (7Eastmond P.J. Germain V. Lange P.R. Bryce J.H. Smith S.M. Graham I.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5669-5674Crossref PubMed Scopus (226) Google Scholar). Gene-specific primers for the ACT2 gene (At3g18780) were used to normalize the amount of template in each RT-PCR reaction. 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. (7Eastmond P.J. Germain V. Lange P.R. Bryce J.H. Smith S.M. Graham I.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5669-5674Crossref PubMed Scopus (226) Google Scholar). MLS and ICL assays were carried out as described in Cooper and Beevers (18Cooper G. Beevers H. J. Biol. Chem. 1969; 244: 3507-3513Abstract Full Text PDF PubMed Google Scholar). 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 (19Nakamura Y. Tolbert N.E. J. Biol. Chem. 1983; 258: 7631-7638Abstract Full Text PDF PubMed Google Scholar), in which 1 mm glyoxylate was used to start the reaction. Protein content was determined as reported by Bradford (20Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) 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. (21Browse J. Mc Court P.J. Somerville C.R. Anal. Biochem. 1986; 152: 141-145Crossref PubMed Scopus (399) Google Scholar), 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. (22Ward J.L. Harris C. Lewis J. Beale M.H. Phytochemistry. 2003; 62: 949-957Crossref PubMed Scopus (220) Google Scholar) 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. (23Nielsen T.H. Skaerbaek H.C. Karlsen P. Physiol. Plant. 1991; 82: 311-319Crossref Scopus (96) Google Scholar). 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. (7Eastmond P.J. Germain V. Lange P.R. Bryce J.H. Smith S.M. Graham I.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5669-5674Crossref PubMed Scopus (226) Google Scholar), 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). Isolation of mls Knock-out Mutants—ICL and MLS are both encoded by single genes in Arabidopsis. To test the hypothesis that Arabidopsis mls mutant seedlings are capable of lipid utilization and gluconeogenesis from acetate, two independent T-DNA insertion knock-out mutants of the MLS gene (At5g03860) were identified (see “Experimental Procedures”). Homozygous individuals lacking MLS (mls-1 and mls-2;Fig. 2A) and wild type segregants (designated MLS-1and MLS-2) were isolated and analyzed by RT-PCR. This confirmed that neither mls mutant contains a MLS transcript (Fig. 2B and data not shown). RT-PCR using ICL gene-specific primers indicates that the mls mutants have no apparent change in the level of ICL transcripts. Furthermore, although icl-2 mutant seedlings have no ICL transcripts, they have normal levels of MLS transcripts (Fig. 2B). MLS enzyme activity was not detectable in mls seedlings (Fig. 2C and data not shown), whereas the wild type segregants showed a peak of enzyme activity 1.5 days post-imbibition (Fig. 2C). mls mutant seedlings contain ICL enzyme activity at the same level as in wild type seedlings (Fig. 2C and data not shown). The absence of MLS mRNA and MLS enzyme activity confirms that we have isolated two null mutants. 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 (β-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 (7Eastmond P.J. Germain V. Lange P.R. Bryce J.H. Smith S.M. Graham I.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5669-5674Crossref PubMed Scopus (226) Google Scholar). 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. 7Eastmond P.J. Germain V. Lange P.R. Bryce J.H. Smith S.M. Graham I.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5669-5674Crossref PubMed Scopus (226) Google Scholar). 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. 7Eastmond P.J. Germain V. Lange P.R. Bryce J.H. Smith S.M. Graham I.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5669-5674Crossref PubMed Scopus (226) Google Scholar). 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). Introduction of the MLS cDNA driven by the cauliflower mosaic virus 35 S promoter into the mls-1 knock-out resulted in complementation of the observed phenotypes (data not shown). Subsequent experiments were performed only with mls-2 and MLS-2 lines as they are in the same Col-0 ecotype as icl-2. 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. 7Eastmond P.J. Germain V. Lange P.R. Bryce J.H. Smith S.M. Graham I.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5669-5674Crossref PubMed Scopus (226) Google Scholar 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).Table IDifferences in the transcriptomes of 2-day-old icl-2 mutant seedlings compared to ICL-2 seedlings Upper, the top 20 genes underexpressed more than 2-fold in icl-2 compared with ICL-2. Lower, the top 20 genes induced in icl-2 compared with ICL-2. The fold change in gene expression in mls-2 is also shown for comparison. NA, not applicable because the signals for the corresponding genes in MLS-2 and mls-2 are less than 100.Gene numberDescriptionaDescription from the Munich Information Centre for Protein Sequences or NCBI Arabidopsis genome databaseSignalFold inductionICL-2icl-2ICL-2/icl-2MLS-2/mls-2Genes under-expressed in icl-2 seedlingsAt3g21720Isocitrate lyase591949120.50.9At2g16060Class 1 non-symbiotic hemoglobin4682617.7NAAt4g32610Putative protein296268.6NAAt2g17850Senescence-associated protein122145.9NAAt1g77120Alcohol dehydrogenase209355.5NAAt1g53070Protein kinasebIndicates the assignment of function is only putative171315.5NAAt1g48660Hypothetical protein5531105.0NAAt5g46900extA (emb CAA47807.1)153314.91.9At5g44440Berberine bridge enzyme-like protein12982654.7NAAt1g24020Pollen allergen-like protein269574.71.3ndhGNADH dehydrogenase (ND6)23745084.51.6At1g76790O-Methyltransferase family protein162354.51.2At1g78370Glutathione S-transferasebIndicates the assignment of function is only putative199444.41.1At5g08000Glycosyl hydrolase (like β-1,3-glucanase)407914.30.9At5g488803-Keto acyl-CoA thiolase 2133304.21.1At1g06350Hypothetical protein250604.20.9At4g12510pEARLI 1-like protein8142044.01.4At1g11600Putative cytochrome P450109293.8NAAt2g28900Membrane channel proteinbIndicates the assignment of function is only putative3671003.71.1At5g15120Putative protein229643.6NAGenes overexpressed in icl-2 seedlingsAt1g64660Methionine/cystathionine γ lyasebIndicates the assignment of function is only putative312949.6NAAt3g60140β-Glucosidase-like protein2252123.9NAAt5g20230Blue copper-binding protein4468515.5NAAt3g55970Leucoanthocyanidin dioxygenase-like protein171719.8NAAt5g5060011-β-Hydroxysteroid dehydrogenase-like383599.5NAAt1g17810Tonoplast intrinsic proteinbIndicates the assignment of function is only putative242199.0NAAt4g07820Pathogenesis-related proteinbIndicates the assignment of function is only putative817158.81.8At1g08630l-Allothreonine aldolase-like protein18615018.11.7At3g47340Glutamine-dependent asparagine synthetase34326987.92.4At2g43620EndochitinasebIndicates the assignment of function is only putative231697.3NAAt1g10070Branched chain amino acid aminotransferase-like protein1087266.7NAAt5g547402 S storage protein-like1016666.6NAAt1g21400Branched chain α-keto acid dehydrogenase E1 α subunit322066.4NAAt2g05540Glycine-rich proteinbIndicates the assignment of function is only putative66638365.83.5At3g49780Putative protein1015615.6NAAt5g65730Xyloglucan endotransglycosylase-like1578145.21.0At2g32150HydrolasebIndicates the assignment of function is only putative27313835.01.8At2g41100Calmodulin-like protein1698565.01.2At2g41260Late embryogenesis abundant protein361754.8NAAt1g71030Transcription factorbIndicates the assignment of function is only putative964384.5NAAt3g60140β-Glucosidase-like protein2252123.9NAAt5g20230Blue copper-binding protein4468515.5NAa Description from the Munich Information Centre for Protein Sequences or NCBI Arabidopsis genome databaseb Indicates the assignment of function is only putative Open table in a new tab Table IIDifferences in the transcriptomes of mls-2 mutant seedlings compared to MLS-2 seedlings Upper, genes repressed greater than 2-fold in mls-2 compared with MLS-2. Lower, genes induced greater than 2-fold in mls-2 compared with MLS-2. The fold change in gene expression in icl-2 is also shown for comparison. NA, not applicable because the signals for the corresponding genes in MLS-2 and mls-2 are less than 100.Gene numberDescriptionaDescription from the Munich Information Centre for Protein Sequences or NCBI Arabidopsis genome databasesSignalFold inductionMLS-2mls-2MLS-2/mls-2ICL-2/icl-2Genes underexpressed in mls-2seedlingsAt5g03860Malate synthase7675142.92.3At3g14210Myrosinase-associated proteinbIndicates the assignment of function is only putative168247.0NAAt2g45220Putative pectinesterase208583.6NAAt5g09220Amino acid transport protein AAP2138393.5NAAt1g52070Jasmonate-inducible proteinbIndicates the assignment of function is only putative136423.39.3At5g64170Putative protein100342.90.7At1g09240Nicotinamide synthasebIndicates the assignment of function is only putative179642.8NAAt4g04830Putative protein3411402.43.0At1g18980GerminbIndicates the assignment of function is only putative118492.40.4At5g12110Elongation factor 1B α subunit140612.31.4At5g26270Putative protein3871832.11.4At1g52060Jasmonate-inducible proteinbIndicates the assignment of function is only putative106532.0NAGenes overexpressed in mls-2 seedlingsAt2g41850Polygalacturonase-like protein381864.93.5At2g05540Glycine-rich proteinbIndicates the assignment of function is only putative2037083.55.8At5g39580Peroxidase ATP24a2095802.83.9At3g47340Glutamine-dependent asparagine synthetase862072.47.8At1g80160Expressed protein601422.4NAAt3g59930Putative protein591342.33.0At4g18910Major intrinsic protein-like protein1904292.31.9At5g434501-Aminocyclopropane-1-carboxylic acid oxidase581272.23.5At1g75830Expressed protein521112.11.2At1g73260Trypsin inhibitorbIndicates the assignment of function is only putative4068592.13.4a Description from the Munich Information Centre for Protein Sequences or NCBI Arabidopsis genome databasesb Indicates the assignment of function is only putative Open table in a new tab Some of the most highly induced genes in icl-2 seedlings reflect stress responses in this mutant (e.g. pathogenesis-related protein, chitinase, and glycine-rich protein), whereas others reflect carbohydrate limitation (e.g. β-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 (24Brouquisse R. James F. Pradet A. Raymond P. Planta. 1992; 188: 384-395Crossref PubMed Scopus (133) Google Scholar). 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 (22Ward J.L. Harris C. Lewis J. Beale M.H. Phytochemistry. 2003; 62: 949-957Crossref PubMed Scopus (220) Google Scholar). 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 appre