A Temperature-sensitive Mechanism That Regulates Post-translational Stability of a Plastidial ω-3 Fatty Acid Desaturase (FAD8) in Arabidopsis Leaf Tissues

Osamu Matsuda,Hikaru Sakamoto,Tadafumi Hashimoto,Koh Iba

doi:10.1074/jbc.m407226200

Abstract

Trienoic fatty acids (TAs) are the major constituents in plant membrane lipids. In Arabidopsis, two plastidial isozymes of omega-3 fatty acid desaturase, FAD7 and FAD8, are the major contributors for TA production in leaf tissues. Despite a high degree of structural relatedness, activities of these two isozymes are regulated differentially in response to temperature. Elevated temperatures lead to decreases in leaf TA level due to temperature sensitivity of FAD8 activity. A series of FAD7-FAD8 chimeric genes, each encoding a functional plastidial omega-3 desaturase, were introduced into the Arabidopsis fad7fad8 double mutant. Constructs with or without a c-Myc epitope tag were tested. Functionality of each chimeric gene in response to temperature was assayed by Northern and Western analyses and by examining the fatty acid composition. All transformants harboring a chimeric gene containing the FAD8-derived C-terminal coding region (44 amino acids) showed a marked decrease in TA level when exposed to high temperature, similarly as transgenic lines complemented with the native form of FAD8. The reduction of TA level was accompanied by a decrease in the amount of omega-3 desaturase protein but not necessarily by a decrease in its transcript level. Analysis of the decay of c-Myc-tagged products after inhibiting protein synthesis revealed that the FAD8-derived C-terminal region acts in an autoregulatory fashion to destabilize the protein at high temperature. This suggests that the regulation of post-translational stability of FAD8 provides an important regulatory mechanism for modifying its activity in response to temperature, mediating a decrease in TA level at elevated temperatures.

Highlights

Trienoic fatty acids (TAs) are the major constituents in plant membrane lipids
It was expected that expression of FAD8 in this genetic background would result in acquisition of an fad7 mutant-like phenotype, which is discernible by its fatty acid composition
In the fad8 mutant, the decrease in leaf TA level resulting from the same treatment was 5.5 Ϯ 0.7% (Table I, ⌬TA), an amount significantly lower than the value obtained in the fad7 mutant (p Ͻ 0.01)

Summary

A Temperature-sensitive Mechanism That Regulates

Post-translational Stability of a Plastidial ␻-3 Fatty Acid Desaturase (FAD8) in Arabidopsis Leaf Tissues*□S. Analysis of the decay of c-Myc-tagged products after inhibiting protein synthesis revealed that the FAD8-derived C-terminal region acts in an autoregulatory fashion to destabilize the protein at high temperature This suggests that the regulation of post-translational stability of FAD8 provides an important regulatory mechanism for modifying its activity in response to temperature, mediating a decrease in TA level at elevated temperatures. A major hindrance preventing thorough investigation into the regulation of FAD8 expression has been the lack of an effective way to quantify the amount and activity of ␻-3 desaturase To overcome these difficulties, a series of FAD7-FAD8 chimeric genes, each encoding a functional plastidial ␻-3 desaturase, either with or without a c-Myc epitope tag, was introduced into the Arabidopsis fad7fad double mutant. Net changes in leaf TA and DA levels attributable to transgene (or endogenous FAD7 or FAD8) expression relative to the fad7fad double mutant (⌬⌬TA[X]22327 and ⌬⌬DA[X]22327) were calculated as follows

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION