Quantitative Analysis of ER Body Morphology in an Arabidopsis Mutant

Abstract

Although fluorescence microscopy screening has proven useful in the identification of genes involved in plant organelle biogenesis and integrity, the quantitative and statistical study of the geometric phenotype is highly limited. This situation could generate unconscious bias in the understanding and presentation of a mutant phenotype. Therefore, we have developed an automated quantification system for green fluorescent protein (GFP) images, which enabled us to easily obtain quantitative data on ER bodies (an endoplasmic reticulum-derived organelle). We isolated an ER body morphology mutant of Arabidopsis thaliana, leb-1 (long ER body). The leb-1 mutant had significantly fewer and larger ER bodies than the wild-type. An amino acid substitution of Cys29 with tyrosine (C29Y) on PYK10, a major component protein of ER bodies, was found in leb-1. Non-reducing SDS–PAGE revealed that the electrophoretic mobility of PYK10 in the leb-1 mutant was clearly different from that in the wild type. This difference suggests that the C29Y amino acid substitution caused a tertiary structural change of the PYK10 protein. While the bglu21-1 and pyk10-1 single mutations slightly affected the number and morphology of the ER bodies, a bglu21-1 pyk10-1 double mutant had fewer and larger ER bodies than the wild type. The quantitative ER body phenotypes of leb-1 were similar to those of bglu21-1 pyk10-1 and bglu21-1 leb-1, suggesting that the leb-1 mutation allele acts dominantly to the BGLU21 wild-type allele. The leb-1 type PYK10 protein, which has an abnormal structure, may competitively inhibit interactions between the wild-type BGLU21/PYK10 protein and an unknown partner.