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

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

doi:10.1074/jbc.m407380200

Johanna E Cornah, Véronique Germain + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m407380200

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Abstract

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.

Highlights

Introduction of the malate synthase (MLS)cDNA driven by the cauliflower mosaic virus 35 S promoter into the mls-1 knock-out resulted in complementation of the observed phenotypes
We considered the possibility that if instead of blocking the glyoxylate cycle at isocitrate lyase (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)
Isolation of mls Knock-out Mutants—ICL and MLS are both encoded by single genes in Arabidopsis

Summary

Introduction

Introduction of the MLScDNA 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). 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. 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

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