A Temperature-sensitive Mutation in the Arabidopsis thaliana Phosphomannomutase Gene Disrupts Protein Glycosylation and Triggers Cell Death

Abstract

Eukaryotic phosphomannomutases (PMMs) catalyze the interconversion of mannose 6-phosphate to mannose 1-phosphate and are essential to the biosynthesis of GDP-mannose. As such, plant PMMs are involved in ascorbic acid (AsA) biosynthesis and N-glycosylation. We report on the conditional phenotype of the temperature-sensitive Arabidopsis thaliana pmm-12 mutant. Mutant seedlings were phenotypically similar to wild type seedlings when grown at 16 -18°C but died within several days after transfer to 28°C. This phenotype was observed throughout both vegetative and reproductive development. Protein extracts derived from pmm-12 plants had lower PMM protein and enzyme activity levels. In vitro biochemical analysis of recombinant proteins showed that the mutant PMM protein was compromised in its catalytic efficiency (K cat /K m ). Despite significantly decreased AsA levels in pmm-12 plants, AsA deficiency could not account for the observed phenotype. Since, at restrictive temperature, total glycoprotein patterns were altered and glycosylation of protein-disulfide isomerase was perturbed, we propose that a deficiency in protein glycosylation is responsible for the observed cell death phenotype.

Arabidopsis Phosphomannose Isomerase 1, but Not Phosphomannose Isomerase 2, Is Essential for Ascorbic Acid Biosynthesis

“…GDP-Man is essential for post-translational modifications such as protein glycosylation and glycosylphosphatidylinositol anchoring in eukaryotes, because mannose, derived from GDP-Man, is a crucial building block of the core glycan chain attached to the modified proteins (33,54,55). It has been reported recently that the Arabidopsis pmm-12 mutants had an impairment of protein glycosylation as well as a reduction in AsA content and, interestingly, showed lethal phenotypes because of the impaired glycosylation rather than the AsA depletion under mild heat stress (33). These findings lead us to ponder whether PMI1 would have consequences for not only AsA biosynthesis, but also glycosylation, cell wall biogenesis, and/or environmental stress response.…”

“…Accordingly, we concluded that PMI2 does not contribute to the biosynthesis. As described above, mannose is one of the major hexose components of plant cell constituents such as cell wall polysaccharides and many types of glycoproteins (33,(53)(54)(55). Under sugar-starved conditions, the degradation of cell walls and proteins was increased in plant cells (57)(58)(59).…”

“…After bleedings, the antisera were separated from the blood (37). Western blot analysis was carried out as described previously (33). The protein bands were detected by using specific antibodies as the primary antibody, and anti-mouse IgG-horseradish peroxidase conjugate (Bio-Rad) as the secondary antibody.…”

“…The Arabidopsis vtc1 mutant had reduced GMP activity, and a null mutation of this gene resulted in death by arresting embryonic development at a very early stage (32). Moreover, pmm-12 mutant with markedly decreased activity of PMM had reduced levels of AsA in Arabidopsis (33). Although these biochemical and genetic findings suggest that the pathway from hexose phosphates to GDP-Man involved in PMI, GMP, and PMM is essential for the biosynthesis of AsA in higher plants, very little is known about PMI in higher plants.…”

Cellular redox regulation, signaling, and stress response in plants

“…9,20,21) In this pathway, GDP-D-Man is synthesized from D-fructose-6-phosphate (Fru-6P) in three steps, which are catalyzed by phosphomannose isomerase (PMI), phosphomannomutase, and GDP-D-Man pyrophosphorylase (GMP). [30][31][32] Arabidopsis has two PMI genes, PMI1 and PMI2. Expressional and reverse genetic analyses revealed that only PMI1 was involved in the biosynthesis of AsA.…”