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

Abstract

Trienoic fatty acids (TAs) are the major constituents in plant membrane lipids. In Arabidopsis, two plastidial isozymes of ω-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 ω-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 ω-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. Trienoic fatty acids (TAs) are the major constituents in plant membrane lipids. In Arabidopsis, two plastidial isozymes of ω-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 ω-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 ω-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. Membrane lipids of plant cells are characterized by a high content of polyunsaturated fatty acids. Typically, dienoic and trienoic fatty acids (DAs and TAs, respectively), 1The abbreviations used are: DA, dienoic fatty acid; TA, trienoic fatty acid; ER, endoplasmic reticulum; MS, Murashige-Skoog; FAD (fad), fatty acid desaturase; 18:3, α-linolenic acid; 16:3, hexadecatrienoic acid; 18:2, α-linoleic acid; 16:2, hexadecadienoic acid; CHX, cycloheximide. account for as much as 70% of total fatty acids in leaf and root lipids (1Harwood J.L. Stumpf P.K. Conn E.E. The Biochemistry of Plants: A Comprehensive Treatise. 4. Academic Press, New York1980: 1-55Google Scholar). The abundance of TAs relative to DAs changes in accordance with environmental conditions. In particular, increasing and decreasing in TA levels have been observed in a variety of plant species exposed to low and high temperatures, respectively (2Smoleńska G. Kuiper P.J.C. Physiol. Plant. 1977; 41: 29-35Crossref Scopus (65) Google Scholar, 3Willemot C. Hope H.J. Williams R.J. Michaud R. Cryobiology. 1977; 14: 87-93Crossref PubMed Scopus (40) Google Scholar, 4Horiguchi G. Kodama H. Nishimura M. Iba K. Planta. 1996; 199: 439-442Crossref Google Scholar). TAs are formed from DAs in the endoplasmic reticulum (ER) and in plastids by ω-3 fatty acid desaturases (5Ohlrogge J. Browse J. Plant Cell. 1995; 7: 957-970Crossref PubMed Scopus (1258) Google Scholar). In Arabidopsis, FAD3 encodes an ER-localized ω-3 desaturase (6Arondel V. Lemieux B. Hwang I. Gibson S. Goodman H.M. Somerville C.R. Science. 1992; 258: 1353-1355Crossref PubMed Scopus (393) Google Scholar), whereas FAD7 and FAD8 encode plastidial isozymes of this enzyme (7Iba K. Gibson S. Nishiuchi T. Fuse T. Nishimura M. Arondel V. Hugly S. Somerville C. J. Biol. Chem. 1993; 268: 24099-24105Abstract Full Text PDF PubMed Google Scholar, 8Gibson S. Arondel V. Iba K. Somerville C. Plant Physiol. 1994; 106: 1615-1621Crossref PubMed Scopus (221) Google Scholar). The physiological relevance of the responses to temperature has been demonstrated in transgenic plants with modified leaf TA contents. For example, FAD7-deficient tobacco plants showed reduced leaf TA levels but performed better regarding growth and photosynthesis at high temperature (9Murakami Y. Tsuyama M. Kobayashi Y. Kodama H. Iba K. Science. 2000; 287: 476-479Crossref PubMed Scopus (338) Google Scholar). Thus, regulation of plastidial ω-3 desaturase activity is likely associated with the adaptation of plants to elevated temperatures. The production of TAs in root tissues depends predominantly on the activity of ER-localized FAD3 desaturase (10Lemieux B. Miquel M. Somerville C. Browse J. Theor. Appl. Genet. 1990; 80: 234-240Crossref PubMed Scopus (229) Google Scholar). According to previous studies in Arabidopsis and wheat, it is likely that temperature-regulated post-transcriptional mechanisms are involved in FAD3 expression (11Somerville C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6215-6218Crossref PubMed Scopus (107) Google Scholar, 12Horiguchi G. Fuse T. Kawakami N. Kodama H. Iba K. Plant J. 2000; 24: 805-813Crossref PubMed Google Scholar). In wheat root tips, the enhanced accumulation of TAs upon exposure to lower temperatures was accompanied by an increase in the amount of FAD3 protein, which was not preceded by a concurrent increase in its transcript level (12Horiguchi G. Fuse T. Kawakami N. Kodama H. Iba K. Plant J. 2000; 24: 805-813Crossref PubMed Google Scholar). In contrast to the metabolism of TAs in the non-photosynthetic root tissues, the plastidial FAD7 and FAD8 desaturases are the major contributors for TA production in leaf tissues (13McConn M. Hugly S. Browse J. Somerville C. Plant Physiol. 1994; 106: 1609-1614Crossref PubMed Scopus (121) Google Scholar). FAD8 was originally identified as a temperature-sensitive plastidial isozyme by phenotypic analysis of the fad7 mutant (14Browse J. McCourt P. Somerville C. Plant Physiol. 1986; 81: 859-864Crossref PubMed Google Scholar), in which FAD8 is the only functional ω-3 desaturase in plastids. Reduction in leaf TA level caused by the fad7 mutation is less pronounced at lower temperatures, implying that FAD8 activity is induced by low temperature. Consistent with this observation, the steady-state level of FAD8 transcripts increases in response to temperature drops within the physiological range, whereas FAD7 transcript accumulation is almost unresponsive to changing temperature (8Gibson S. Arondel V. Iba K. Somerville C. Plant Physiol. 1994; 106: 1615-1621Crossref PubMed Scopus (221) Google Scholar). It has therefore been postulated that temperature-dependent changes in leaf TA level are mostly due to transcriptional regulation of FAD8 expression. Despite this convincing explanation, there has been no direct evidence showing that the transcriptional regulation of FAD8 expression is actually the decisive event controlling the temperature sensitivity of FAD8 activity. 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 ω-3 desaturase. To overcome these difficulties, a series of FAD7-FAD8 chimeric genes, each encoding a functional plastidial ω-3 desaturase, either with or without a c-Myc epitope tag, was introduced into the Arabidopsis fad7fad8 double mutant. The functionality of each chimeric gene in response to temperature was analyzed from the transcriptional to post-translational levels. Here we describe a novel post-translational mechanism in which the C-terminal region of FAD8 desaturase acts in an autoregulatory fashion to destabilize the protein at high temperature, leading to a reduction of the concentration of this enzyme in the absence of a concurrent decrease in its transcript level. Plant Materials and Growth Conditions—All lines of Arabidopsis thaliana described here were derived from the Columbia wild type. The mutant lines fad7fad8 (Ref. 13McConn M. Hugly S. Browse J. Somerville C. Plant Physiol. 1994; 106: 1609-1614Crossref PubMed Scopus (121) Google Scholar, stock number CS8036) and fad7 (Ref. 14Browse J. McCourt P. Somerville C. Plant Physiol. 1986; 81: 859-864Crossref PubMed Google Scholar, stock number CS3108) are available from the Arabidopsis Biological Resource Center at Ohio State University. The mutant fad8 line was isolated from the progeny of a cross between the fad7fad8 and wild-type Columbia plants using restriction enzyme polymorphisms specific to the fad7 (15Kusumi J. Iba K. J. Plant Res. 1998; 111: 87-91Crossref Google Scholar) and fad8 (8Gibson S. Arondel V. Iba K. Somerville C. Plant Physiol. 1994; 106: 1615-1621Crossref PubMed Scopus (221) Google Scholar) alleles. All transgenic lines were confirmed to be homozygous for the transgenes after two rounds of backcrossing. Unless otherwise described, plants were routinely grown at 22 °C under continuous white light on solid Murashige-Skoog (MS) medium (16Murashige T. Skoog F. Physiol. Plant. 1962; 15: 473-497Crossref Scopus (53969) Google Scholar) containing 1% (w/v) sucrose and 0.5% (w/v) gellan gum (Kanto Chemical, Tokyo, Japan) and were harvested on day 18 for subsequent of used in this were as described in J. T. A The to of the number and the FAD8 was by from of wild-type Columbia The to of the number and the FAD7 was by from the All chimeric in were on these two to described in the The FAD8-derived region encoding the C-terminal amino acids of was in with a are To the chimeric for c-Myc-tagged a encoding the epitope of c-Myc protein G. Cell. Biol. PubMed Scopus Google Scholar) is was at the of the at of the FAD7 protein and of the FAD8 protein between the and and of the at was by the as described in R. A to and Academic Press, All products were to the absence of of chimeric used in this are available on of Arabidopsis chimeric gene in was to the of the with of Arabidopsis plants was performed by the N. J. G. C. R. Acad. Sci. 1993; Scholar). transformants were for as described C. Iba K. Planta. PubMed Scopus Google Scholar). fatty acid of total leaf lipids was by as described H. T. G. Nishimura M. Iba K. Plant Physiol. 1994; PubMed Scopus Google Scholar). For transgenic and mutant plants were to °C on day and in that after the temperature were harvested on day 18 for fatty acid plants were at 22 °C analysis on day were performed for each line to the and and of leaf TA and levels at each temperature The TA level was as the of and whereas the level was as the of and levels. of these are available as in leaf TA and upon temperature from 22 to °C and R.J. J. Biol. Chem. Full Text PDF PubMed Google were as and was with the of of Reduction and Analysis for the New Scholar). 22 22 changes in leaf TA and levels to FAD7 or expression relative to the fad7fad8 double mutant and were as of and and are for in transgenic and mutant lines are in of FAD7-FAD8 chimeric used in analysis of the fad7fad8 double mutant. and in and derived from of FAD7 and FAD8, containing coding and lines lines containing and transcriptional and the of with The with in a modified FAD8 encoding the C-terminal this were introduced to the of FAD7 lines encoding the C-terminal of the FAD7 at by the H. G. Sci. PubMed Scopus Google Scholar), or the of the FAD8 protein at of the c-Myc within and encoding c-Myc-tagged desaturases lines used as for Northern analysis that containing c-Myc and are by the FAD7 whereas are by the FAD8 The is to of fatty acid changes in leaf TA and levels to FAD8 changes in leaf TA and levels to FAD8 changes in leaf TA and levels to FAD7 changes in leaf TA and levels to FAD7 changes in leaf TA and levels to FAD8 changes in leaf TA and levels to FAD7 expression. in a Northern was from leaf tissues as described in C. 1990; PubMed Google of was on a 1% (w/v) and were performed on the of 1986; PubMed Scopus Google Scholar) as described (15Kusumi J. Iba K. J. Plant Res. 1998; 111: 87-91Crossref Google Scholar). The of FAD7 and FAD8, to of and of were used as were to °C on day and harvested on day 18 for Northern plants were at 22 °C on day Western leaf were by the as described in Plant J. PubMed Google concentration was by the of R.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar) as modified in A. PubMed Scopus Google of were on a (w/v) and to a membrane used in this and with were by the to the total were by the with were a solid medium on day and to at 22 °C The the from into the solid day plants were to medium containing in The concentration of has been to of N. J. Plant Cell. PubMed Scopus Google Scholar), under plants after the at either 22 or °C for the in total leaf were and used for Western analysis as described for FAD8 on of the fad7fad8 an for temperature-sensitive FAD8 of the fad7fad8 double mutant was using functional of FAD7 and FAD8. It was that expression of FAD8 in this in of an fad7 which is by its fatty acid composition. For this of the two the region associated with the and containing and were from the wild-type and were into the fad7fad8 double mutant. fad7fad8 lines harboring of FAD7 and FAD8, are as and To the temperature-sensitive FAD8 plants of mutant and the transgenic lines were grown at two temperatures, 22 and to of fatty acid this temperature range, the leaf TA level changes in the fad7 but only in wild-type plants (14Browse J. McCourt P. Somerville C. Plant Physiol. 1986; 81: 859-864Crossref PubMed Google Scholar). The fad8 mutant has been to be almost from the wild on the of fatty acid (13McConn M. Hugly S. Browse J. Somerville C. Plant Physiol. 1994; 106: 1609-1614Crossref PubMed Scopus (121) Google Scholar). a temperature from 22 to °C caused a decrease in leaf TA level in the fad7 mutant but only in wild-type In the fad8 the decrease in leaf TA level from the was an amount lower the in the fad7 mutant In of the and the levels of TAs in leaf tissues were in the fad8 and fad7 respectively Despite changes in leaf TA level in response to temperature were in of the transgenic lines harboring the for the low levels of FAD7 and FAD8 we the analyses of a transgenic line of each In the and the in temperature in decreases of leaf TA levels by and respectively from each whereas are to the in the fad8 and fad7 This suggests the of regulatory for temperature-sensitive expression within the that are not in the C-terminal of FAD8 for FAD8 the regulatory for the temperature-sensitive FAD8 a series of FAD7-FAD8 chimeric genes, each encoding a functional plastidial ω-3 desaturase, were on structural between the two described for the and each was introduced into the fad7fad8 double and the level of in response to temperature was assayed by examining the fatty acid composition. All transformants showed a temperature response to that observed in either the or and of the transformants an and To the of temperature response that was by expression of a chimeric changes in leaf TA level to expression were and were by the of FAD3 activity to the fad7fad8 the of TAs and DAs from the plastidial to the membrane is low J. M. Miquel M. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar), it is that this desaturase activity in the is by the activity of plastidial ω-3 desaturase derived from expression. upon the in temperature, and expression and to the changes in leaf TA levels. the of FAD7 and FAD8 expression in the fad8 and fad7 be to be and and Thus, the was in the temperature sensitivity of and expression. of expression of FAD8 expression from the level of expression at the lower temperature The two chimeric genes, and were on and by the with each a FAD7 under the of the FAD8 whereas FAD8 by the FAD7 The changes in leaf TA levels to and expression were and to in the and lines and It that the temperature-sensitive FAD8 expression was regulated the region the the of the transcriptional In with this it was that the expression of be to the a structural within the The were from by the FAD8-derived region that the which encodes the and of the desaturase these chimeric genes, and leading to changes in leaf TA levels to expression by and respectively and the FAD8-derived C-terminal coding region within the was likely a for expression. this region was in of the region of to an chimeric gene this by the FAD7 an of a in leaf TA level upon the in temperature and these that the C-terminal coding region of FAD8 is to an temperature-sensitive expression. In activity of ω-3 desaturase to production of TAs in with of This was between and the after two rounds of is from between and of expression to decreases of leaf TA level was accompanied by a to increases of leaf level This of the is of the and suggests that TA as as levels a for the degree of temperature sensitivity of expression of the chimeric C-terminal of FAD8 for the temperature-sensitive FAD8 regulated the C-terminal coding is associated with transcriptional regulation of FAD8 steady-state transcript levels of FAD7-FAD8 chimeric were In the fad7fad8 double transcripts from the FAD7 and FAD8 are decay (15Kusumi J. Iba K. J. Plant Res. 1998; 111: 87-91Crossref Google Scholar). only the transcripts derived from expression are by FAD7 or FAD8 in the transgenic fad7fad8 by Northern analysis in steady-state levels of and transcripts in response to temperature were to of FAD8 and FAD7 in wild-type with the expression of and as confirmed by the fatty acid analysis and transcript accumulation of these chimeric was almost unresponsive to temperature was in the expression of not that the transcriptional regulation of FAD8 expression is not necessarily the decisive event controlling the temperature sensitivity of FAD8 activity. To the that the C-terminal coding region of FAD8 acts in to a of was containing a within the FAD8-derived C-terminal coding region this region of the FAD8 were introduced to the of FAD7 Despite an this expression to an to the The in leaf TA level to expression upon the in temperature was to be and This is not but when in with the of Northern analysis it suggests that the C-terminal region of FAD8 protein, its encoding is for the desaturase activity in response to temperature, at the post-translational level. of c-Myc-tagged ω-3 in the of FAD8 activity was accompanied by a in the amount of FAD8 protein or in the specific activity of this transgenic fad7fad8 lines of plastidial ω-3 desaturases were In it was that epitope at the of the desaturase in a of activity not To an epitope tag without the and functional of the desaturase, a that was with was to the of the desaturase protein on an analysis using the H. G. Sci. PubMed Scopus Google Scholar), FAD7 and FAD8 were to for The of the were and amino acids for FAD7 and FAD8, respectively To the of of the epitope tag after the the c-Myc was the of FAD7 and the of FAD8 and encoding c-Myc-tagged desaturases were for of the transgenes in and modified were as and Western of in of transgenic of were only in the transgenic that these derived from expression. In the and as a with a to This was in with the of at the by the In in the and lines were as of and the the major of in the to have been from the of phenotypic of the fad7fad8 double mutant by functional expression of The leaf TA levels in of the transgenic lines were the level in the fad7fad8 double at 22 each of transgenic fad7fad8 lines either the or the no was in of leaf TA levels. were for temperatures °C not that specific activities to of that the expression of as by Western and fatty acid provides regarding expression of FAD7 and FAD8 C-terminal of FAD8 for at regulatory of the C-terminal region on the accumulation of FAD8 protein, of temperature on steady-state levels of derived from the transgenes were tested. growth at 22 plants the were to by Western analysis in the of of in the line were by the temperature In the of in the transgenic in the at This decrease was to levels after at 22 were with the wild-type not are with the in that the of FAD8 activity is due to a in the amount of FAD8 protein, a in its specific activity. To the mechanisms the accumulation of FAD8 protein at high temperature, the of the decay of after with an of protein was at temperatures. in the amount of not the of at 22 and the the products of and were to at 22 The decay of these was at after the of the were of at 22 that the C-terminal region of FAD8 desaturase acts in an autoregulatory fashion to destabilize the protein at high temperature, a reduction in the amount of enzyme in the absence of a concurrent decrease in its transcript level In this we an the of post-transcriptional mechanisms that the activities of FAD7 and FAD8 desaturases in response to temperature. on structural relatedness, a series of FAD7-FAD8 chimeric were that a functional plastidial ω-3 desaturase, either with or without a c-Myc epitope tag of these chimeric into the Arabidopsis fad7fad8 double mutant in accumulation of transcripts and and by enhanced production of TAs in leaf tissues which to with the ω-3 desaturase activity of the gene each chimeric gene either temperature-sensitive or an was between changes in the of transcripts and A and by Northern it that transcript levels of chimeric the of the containing the transcriptional In as by Western and fatty acid changes in the of products and TAs and were regulated the C-terminal coding was at in due to post-translational of the ω-3 desaturase protein at high temperature, which was only the desaturase the FAD8-derived C-terminal region the temperature-sensitive expression of FAD8 desaturase is regulated at the transcriptional and post-translational the response of its activity to temperature changes within the physiological depends on post-translational it is to the C-terminal amino acids of FAD8 desaturase as the temperature-sensitive regulatory the mechanism of its on the stability of ω-3 desaturase protein has to be In the activities of desaturases were by examining the of leaf fatty acid in the fad7fad8 double mutant. to the low of this it was to this region to structural or amino acid for temperature the that the of FAD7 and FAD8 is for the C-terminal region (8Gibson S. Arondel V. Iba K. Somerville C. Plant Physiol. 1994; 106: 1615-1621Crossref PubMed Scopus (221) Google Scholar), it is convincing that this region in the physiological of the two of fatty acid desaturase expression is regulated post-translational stability has been for A desaturases in and in J. Full Text Full Text PDF PubMed Scopus Google Scholar). amino acids of the exposed to the of the were to as a for its H. G. J. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). A protein, associated with the has been in the of this desaturase G. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). it the with the it is of mechanisms in plastidial FAD7 and FAD8 desaturases are M. S. S. S. M. J. J. N. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Res. PubMed Scopus Google Scholar). the protein desaturase in the P.K. Stumpf P.K. Conn E.E. The Biochemistry of Plants: A Comprehensive Treatise. 4. 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PubMed Scopus Google Scholar). it is to this to plastidial for the of C-terminal epitope into the desaturase in a of activity. we in the fad7fad8 double mutant functional expression of c-Myc-tagged FAD7 and FAD8 desaturases a of transgenic plants the of the ω-3 desaturase isozymes it be to regulation from the transcriptional to post-translational levels. is from functional of the two plastidial isozymes to be To these structural are to in to temperature and physiological we are to the and and of the using A of the mechanisms the regulation of ω-3 desaturase activity to the of plants in elevated temperatures. are to K. for on with