Metabolic and transcriptional responses of glycerolipid pathways to a perturbation of glycerol 3-phosphate metabolism in Arabidopsis

Abstract

Glycerolipid synthesis in plants involves two major metabolic pathways compartmentalized in the chloroplasts and cytosol, respectively. Although these two parallel pathways are regulated with considerable flexibility, the factors mediating this process remain unclear. To investigate the influence of glycerol 3-phosphate (Gly-3-P) on the interactions of the glycerolipid pathways, we generated transgenic Arabidopsis lines with a feedback-resistant Gly-3-P dehydrogenase gene (gpsA(FR)) from Escherichia coli. gpsA(FR) was detected in the cytosol, but augmented Gly-3-P levels were observed in the cytosol as well as in chloroplasts. Glycerolipid composition and fatty acid positional distribution analyses revealed an altered fatty acid flux that affected not only the molar ratios of glycerolipid species but also their fatty acid composition. To decipher this complex pathway, a transgenic line was subjected to lipidomic analysis and a global gene-expression survey. The results revealed that changes in Gly-3-P metabolism caused altered expression of a broad array of genes. When viewed from the perspective of glycerolipid metabolism, coherent networks emerged, revealing that many enzymatic components of the glycerolipid pathways operate in a modular manner under the influence of Gly-3-P. Transcript levels of the enzymes involved in the prokaryotic pathway were mostly induced, whereas genes of the eukaryotic pathway enzymes were largely suppressed. Hence, the gene-expression changes were consistent with the detected biochemical phenotype. Our results suggest that Gly-3-P modulates the balance of the two glycerolipid pathways in Arabidopsis by influencing both metabolic flux and gene transcription.

FIGURE 1.

Immunoblot analysis of gpsA^FR protein and measurement of Gly-3-P levels in the gpsA^FR transgenic lines and wild-type (WT). A, immunoblot analysis of the gpsA^FR protein. Immunoblots of total leaf and chloroplast proteins from both gpsA^FR transgenic and wild-type lines probed with polyclonal antibodies raised against two polypeptides from gpsA^FR. Lane 1, wild-type total leaf protein extract; lane 2, wild-type chloroplast protein extract; lane 3, total leaf protein extract from gpsA^FR transgenic line 84-10; lane 4, chloroplast protein extract from gpsA^FR transgenic line 84-10; lane 5, total leaf protein extract from gpsA^FR transgenic line 102-5; lane 6, chloroplast protein extract from gpsA^FR transgenic line 102-5; lane 7, total leaf protein extract from gpsA^FR transgenic line 22–44; and lane 8, chloroplast protein extract from gpsA^FR transgenic line 22–44. Approximately 10 μg of protein was loaded in each lane. B, Gly-3-P levels in the gpsA^FR transgenic lines and wild-type. Gly-3-P levels were assayed as described under “Experimental Procedures.” Panel a, leaf Gly-3-P levels in the gpsA^FR transgenic lines and wild-type plants; panel b, chloroplast Gly-3-P levels in gpsA^FR transgenic lines and wild-type plants. Error bars represent mean ± S.D. (n = 3). Statistical significance compared with the wild-type was determined by the two-tailed Student's t test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

FIGURE 2.

Fatty acid composition at the sn-2 position of leaf glycerolipids (lyso-derivatives). Individual lipids were purified by two-dimensional TLC on Silica Gel 60, eluted from the gel, and incubated with Rhizopus lipase as described under “Experimental Procedures.” The resulting lyso lipids (representing the sn-2 position of the parent lipid) were purified by TLC and derivatized using 3 n methanolic HCl, and the resulting fatty acid methyl esters were analyzed by gas chromatography. C16 represents the sum of 16:l, 16:2, and 16:3; C18 represents the sum of 18:0, 18:1, 18:2, and 18:3. A, MGDG; B, DGDG; C, PG; D, PC. Error bars represent mean ± S.D. (n = 3). Statistical significance compared with the wild-type (WT) was determined by the two-tailed Student's t test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

FIGURE 3.

Schematic view of glycerolipid biosynthesis pathways integrated with microarray data. To visualize the relative transcript levels between gpsA^FR transgenic plants and the wild-type control, gene-expression data were mapped to the pathway diagrams compiled from the AraCyc data base and Refs. and . Different lipid species are derived either from the ER pathway (sn-1/sn-2, C₁₈:C₁₈ or C₁₆:C₁₈) or from the chloroplast pathway (sn-1/sn-2, C₁₈:C₁₆). Dashed lines represent possible channeling between the ER and chloroplasts. Red diamonds represent induced genes, whereas green diamonds denote repressed genes based on the transcript level ratio. Ph-E, phosphorylethanolamine; Ph-C, phosphorylcholine; CDP-C, CDP-choline; oe, outer envelope; ie, inner envelope; FAS, fatty acid biosynthesis.

FIGURE 4.

Real time quantitative RT-PCR validation of the microarray data. The relative expression levels of representative genes from the microarray data as revealed by real time gene-expression analysis. Real time qRT-PCR results using cDNA from WT (wild-type, gray bar) and 22–44 (gpsA^FR transgenic line, black bar) as templates. For comparison, the results from wild-type control plants were normalized to 1 using StepOne software 2.0 (Applied Biosystems). The values represent the average of three independent replicates. Statistical significance compared with the WT was determined by the two-tailed Student's t test (*, p < 0.05; **, p < 0.01).