chilling sensitivity in plants and cyanobacteria: the

Nishida & MurataCHILLING SENSITIVITY AND MEMBRANESAnnu. Rev. Plant Physiol. Plant Mol. Biol. 1996. 47:541–68Copyright © 1996 by Annual Reviews Inc. All rights reserved

CHILLIN G SENSITIV ITY INPLANTS ANDCYANOBACTERIA: The CrucialContribution of Membrane Lipids

I. Nishida and N. MurataNational Institute for Basic Biology, Okazaki, 444 Japan

KEY WORDS: acyl-lip id desaturase, genetic manipulation, glycerol-3-phosphateacyltransferase, lipid molecular species, photoinhibition

ABSTRACT

The contribution of membrane lipids, particularly the level of unsaturation offatty acids, to chilling sensitivity of plants has been intensively discussed formany years. We have demonstrated that the chilling sensitivity can be manipu-lated by modulating levels of unsaturation of fatty acids of membrane lipids bythe action of acyl-lipid desaturases and glycerol-3-phosphate acyltransferase.This review covers recent studies on genetic manipulation of these enzymes intransgenic tobacco and cyanobacteria with special emphasis on the crucial im-portance of the unsaturation of membrane lipids in protecting the photosyntheticmachinery from photoinhibition under cold conditions. Furthermore, we reviewthe molecular mechanism of temperature-induced desaturation of fatty acids andintroduce our hypothesis that changes in the membrane fluidity is the initial eventof the expression of desaturase genes.

CONTENTS

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 542

UNSATURATION OF FATTY ACIDS AND CHILLING SENSITIVITY . . . . . . . . . . . 543

ENZYMES INVOLVED IN THE REGULATION OF FATTY ACIDUNSATURATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 544

Biosynthesis of Lipids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 544Acyltransferases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 545Desaturases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 546

GENETIC MANIPULATION OF THE UNSATURATION OF MEMBRANE LIPIDSAND CHILLING SENSITIVITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 548

Overexpression of cDNA for Glycerol-3-Phosphate Acyltransferases in TobaccoPlants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 548

Overexpression of the plsB Gene and Mutation of Fatty Acid Elongase inArabidopsis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 550

Disruption of Genes for Desaturases in Synechocystis sp. PCC6803 . . . . . . . . . . . . . 551Expression of the Gene for a Desaturase in Synechococcus sp. PCC7942 . . . . . . . . . 554

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MOLECULAR MECHANISM OF LOW-TEMPERATURE PHOTOINHIBITIONREGULATED BY THE UNSATURATION OF MEMBRANE LIPIDS. . . . 555

Photoinhibition in Transgenic Tobacco Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 555Photoinhibition in Synechocystis sp. PCC6803. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 556Turnover of the D1 Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557

CHILLING SENSITIVITY OF ARABIDOPSIS MUTANTS AND OTHERTRANSGENIC PLANTS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557

TEMPERATURE ACCLIMATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 558Desaturation of Membrane Lipids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 558The Signal Transduction Pathway to the Expression of Cold-Inducible Genes. . . . . . 559

CONCLUSIONS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 560

INTRODUCTION

Chillin g sensitiv ity is manifested by certain plants whose germination, growth,development of reproductive organs, and postharvest longevity are restrictedwithin a range of chillin g temperatures from 0°C (nonfreezing) to about 15°C(68, 148). Certain stages in the lif e cycle of a plant are more sensitive to chillin gthan are other stages of development. For example, seedlings appear to bemore susceptible to chillin g than plants at advanced stages of development(68), and the maturation of pollen is the process that is the most sensitive tochillin g temperatures during the entire lif e cycle of chillin g-sensitive plants(97, 112).

In the past several decades, extensive attention has been paid to the molecu-lar mechanisms of the chillin g sensitiv ity of plants (68, 148) because of theagricultural demands for improvements in the chillin g tolerance of horticulturalcrops. In particular, the role of the unsaturation of membrane lipids in toleranceof plants to chillin g has been discussed for many years. In 1973, Lyons (68)and Raison (100) first proposed the hypothesis that the thermotropic phasetransition of membrane lipids might play an initiative role in the chillin gsensitiv ity of plants. With further exposure to chillin g, the phase-separatedbiomembranes become incapable of maintaining ionic gradients and cellularmetabolism becomes disrupted, with the death of plant cells being the finalresult (77). The occurrence of phase separation as the initial event in chillin ginjury has been demonstrated in the unicellular cyanobacterium Anacystisnidulans (80). However, because plant cells contain high levels of polyunsatu-rated fatty acids in their membranes, it has been argued that the chillin g-in-duced phase transition would not occur in lipids in intact membranes. In ourprevious work (76, 82), we found a positive correlation between the chillin gsensitiv ity of herbaceous plants and the level of saturated and trans-monoun-saturated molecular species of phosphatidylglycerol (PG), which are alsotermed “high-melting point molecular species” in thylakoid membranes. Asimilar correlation was observed in other herbaceous plants (6, 64, 105), alpineplants (18), and woody plants (137). However, such studies do not indicate

542 NISHIDA & MURATA

more than a correlation, and there remains the question of whether thesehigh-melting point molecular species are directly related to the chillin g sensi-tivity of plants.

In recent years, there have been extensive advances in our understanding ofthe regulation of the unsaturation of the membrane lipids of plants and cyano-bacteria. The successful genetic manipulation of enzymes responsible for thebiosynthesis (126) and desaturation (83) of fatty acids has been achieved, aswell as the isolation of mutants with defective enzymes (8, 129). In this context,the roles of the unsaturation of membrane lipids in tolerance and response tolow temperature have been reexamined using transgenic and mutant strains.In this review, we briefly summarize recent progress in the genetic manipula-tion of acyltransferases and acyl-lipid desaturases in transgenic plants andcyanobacteria and effects of lipid changes caused by transformation on thechillin g sensitiv ity. It appears that the unsaturation of lipids in thylakoidmembranes protects the photosynthetic machinery from photoinhibition at lowtemperature. We also review the molecular mechanisms of desaturation of fattyacids in response to low temperature and introduce our hypothesis that changesin membrane fluidity are the initial events that lead to the expression ofdesaturases.

UNSATURATION OF FATTY ACIDS AND CHILLI NGSENSITIVITY

It has been well-established in a wide range of organisms, such as microor-ganisms, plants, and poikilotherms, that the level of unsaturated fatty acids inthe glycerolipids of membranes changes with changes in growth temperature(14). A downward shif t in growth temperature generally increases the degreeof unsaturation of membrane lipids. By measuring a number of physical pa-rameters that define the thermal motility of lipid molecules, such an increasein the degree of unsaturation of membrane lipids can be demonstrated tocompensate for the decrease in the fluidity of membrane lipids that is broughtabout by the downward shif t in temperature (15, 101). The increase in thedegree of unsaturation of membrane lipids is also correlated with the sustainedactivity of membrane-bound enzymes at lower temperature (100, 101). Thus,the unsaturation of membrane lipids is considered to be one of the most criticalparameters for the functioning of biological membranes and, therefore, for thesurvival of organisms at lower temperatures (15).

The above argument might, however, be invalid because low temperatureinduces a number of other changes in cellular metabolism, e.g. in proteinsynthesis (13, 34, 72) and in the levels of expression of a number of novelgenes, the so-called cold-inducible genes or cold-responsive genes (10, 11, 27,28, 44, 45, 58, 60, 61, 72a,b, 86, 92, 93, 96, 108, 113, 114, 150, 151, 153,

CHILLING SENSITIVIT Y AND MEMBRANES 543

154). Therefore, to evaluate the role of unsaturated fatty acids in cold tolerance,it is essential to manipulate the level of unsaturated fatty acids independentlyof low temperature, as has recently been achieved both by manipulating thegenes in question (57, 79, 83, 155) and also by creating mutant strains that arepartially defective in cellular lipid metabolism (8).

ENZYMES INVOLVED IN THE REGULATION OF FATTYACID UNSATURATION

Biosynthesis of LipidsBefore attempts can be made to manipulate the unsaturation of fatty acids, itis necessary to understand the biosynthetic pathway and the enzymes involvedin the biosynthesis and desaturation of fatty acids. In plant cells, saturated fattyacids are synthesized within plastids (133, 135). The major reactions arecatalyzed by members of the so-called type II fatty acid synthase system (36),which consists of multiple enzymes that are required for fatty acid biosynthesisand which requires a protein cofactor, acyl-carrier protein (ACP). In contrastwith the bacterial type II fatty acid synthase system in Escherichia coli, thefatty acid synthase system of plastids cannot produce unsaturated fatty acidsas products of reactions that lead to fatty acid synthesis de novo (69). Palmi-toyl-ACP (16:0-ACP) and stearoyl-ACP (18:0-ACP) are synthesized by thissystem as two major and key substrates for further metabolism.

The biosynthesis of unsaturated fatty acids in plant cells has been reviewedelsewhere (8, 36, 50). In the first committed step toward the biosynthesis ofC18-unsaturated fatty acids, plant cells convert 18:0-ACP to oleoyl-ACP(18:1-ACP) in a reaction catalyzed by stearoyl-ACP desaturase in plastids (50,133). By contrast, 16:0-ACP is not a good substrate for the stearoyl-ACPdesaturase. Further desaturation of 18:1(9) into polyunsaturated fatty acids iscatalyzed by acyl-lipid desaturases once the fatty acids have been esterif iedon glycerolipids (83, 117). Therefore, in plant cells, the level of polyunsatu-rated fatty acids in the membrane lipids depends to a considerable extent onthe activities of acyl-lipid desaturases.

Fatty acids are incorporated into glycerolipids via the stepwise esterif icationof glycerol 3-phosphate (106). The reaction is catalyzed by glycerol-3-phos-phate acyltransferase (GPAT) and 1-acylglycerol-3-phosphate acyltransferase(106). The phosphatidic acid produced is a common precursor to cellularglycerolipids. Plant cells contain separate sets of acyltransferases in the en-doplasmic reticulum (ER), chloroplasts (or plastids), and mitochondria. Oncefatty acids have been esterif ied to the sn-1 and sn-2 positions in phosphatidicacid, they are hardly ever reorganized by the further metabolism of glycero-lipids in plant cells (106). Furthermore, in plant cells, saturated fatty acids


esterif ied on the glycerol backbone of most lipids cannot be desaturated (81).Thus, the specificities of the acyltransferases to fatty acids and to the sn-po-sitions of the glycerol backbone become key factors in the regulation of thelevels of unsaturated molecular species of glycerolipids. In plastids, the sn-2position of glycerol 3-phosphate is exclusively esterif ied with 16:0 (106), andlevels of high-melting-point molecular species of PG are regulated by thespecificity to fatty acids of the GPAT in the plastids (23, 24).

In cyanobacterial cells, fatty acids are synthesized via a mechanism similarto that in the cells of higher plants (80). 16:0-ACP and 18:0-ACP are synthe-sized as end-products by the fatty acid synthase system (62, 63, 130, 131).Neither acyl-ACP desaturase nor acyl-ACP hydrolase has been found in cy-anobacterial cells. The acyl groups of acyl-ACPs are directly incorporated intoglycerolipids by acyltransferases and then converted to unsaturated fatty acidsby acyl-lipid desaturases (83). Thus, the levels of unsaturated fatty acids inthe membrane lipids of cyanobacterial cells are determined exclusively by theacyl-lipid desaturases.

Acyltransferases

GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE IN THE PLASTIDS The GPAT inplastids is a nucleus-encoded stromal protein that utilizes acyl-ACP as asubstrate in vivo. This enzyme in chillin g-resistant plants, such as pea andspinach, is specific to 18:1-ACP (23, 24). By contrast, the enzyme in chillin g-sensitive plants, such as squash, utilizes both 18:1-ACP and 16:0-ACP as itssubstrate with a preference for unsaturated acyl-ACP. The enzyme was firstpurif ied to homogeneity from squash cotyledons (88), and the cDNA for theprecursor to this GPAT was isolated immunochemically from a cDNA expres-sion library of squash cotyledons using antibodies raised against the purif iedprotein (48). The gene and the cDNA of Arabidopsis thaliana for the precursorto GPAT were then isolated by heterologous hybridization (89). To date,cDNAs for GPAT have also been isolated from cucumber (51) and Frenchbean (24a), two chillin g-sensitive plants, and from pea (149) and spinach (49),two chillin g-resistant plants.

GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE FROM ESCHERICHIA COLI Thisenzyme, encoded by the plsB gene (65), is bound to the cytoplasmic membraneand utilizes both acyl-ACP and acyl-CoA as substrates (33). It is selective forsaturated acyl-ACP as compared to unsaturated acyl-ACP. As we discussbelow, the level of unsaturated molecular species of PG can be successfullymanipulated when the product of the plsB gene is targeted to the chloroplastsof Arabidopsis (155).


DesaturasesFatty acid desaturases play a central role in regulating the level of unsaturationof fatty acids in membrane lipids. The desaturases can be classif ied into threegroups according to their specificities to the biochemical form of their fattyacid substrates, namely, acyl-lipid desaturases, acyl-ACP desaturases, andacyl-CoA desaturases (83). Acyl-lipid desaturases, which are specific to fattyacids esterif ied to glycerolipids (50, 117, 147), are most common in plants andcyanobacteria. They are transmembrane proteins in the ER and chloroplastmembranes of plant cells (50). The desaturase in the ER uses cytochrome b5

as the electron donor (54, 128), whereas the desaturases in chloroplasts andcyanobacteria use ferredoxin as the electron donor (119, 120, 147). We dem-onstrated recently by immunogold labeling and electron microscopy that acyl-lipid desaturases are located in both the plasma and thylakoid membranes ofthe cells of Synechocystis sp. PCC6803 (L Mustardy, D Los, Z Gombos & NMurata, unpublished information). Acyl-ACP desaturases are present in thestroma of plastids (70) but have not been found in cyanobacteria (62, 131).Neither plants nor cyanobacteria contains acyl-CoA desaturases (42).

Each desaturase introduces a double bond into fatty acids specifically at thespecific ∆ (counting from the carboxyl group) or ω (counting from the methylend) position of the fatty-acyl chain and at specific sn positions of glycerides.The positional specificity of the desaturation of fatty acids in Synechocystissp. PCC6803, as determined by feeding of cells with heptanoic acid (39),revealed that this cyanobacterium contains four desaturases, which are desig-nated ∆6, ∆9, ∆12, and ω3 acyl-lipid desaturases (39).

ACYL-LIPID DESATURASES FROM CYANOBACTERIA The desA gene, which en-codes a ∆12 acyl-lipid desaturase, was first isolated from a genomic library ofSynechocystis sp. PCC6803 (142) by complementation (20) of a mutant, des-ignated Fad12 (145), that was unable to desaturate the ∆12 position of C18fatty acids esterif ied at the sn-1 position of all classes of glycerolipids. ThedesA gene has also been isolated from other cyanobacterial strains, namely,Synechococcus sp. PCC7002 (111), Synechocystis sp. PCC6714 (111), An-abaena variabilis (111), and Spirulina platensis (78).

The desC gene, which encodes a ∆9 acyl-lipid desaturase, was found in the5′ upstream region of the desA gene on the chromosome of A. variabilis (110).The desC gene of Synechocystis sp. PCC6803 was then cloned by heterologoushybridization (110). The deduced amino acid sequences of the products ofthose desC genes are about 25% identical to those of ∆9 acyl-CoA desaturasesfrom rat (138), mouse (52, 94), and yeast (132).

The desB gene, which encodes an ω3 acyl-lipid desaturase, has been clonedfrom a desA-depleted strain of Synechocystis sp. PCC6803 by heterologous


hybridization with a probe derived from the desA gene (109). The desD gene,which encodes a ∆6 desaturase from Synechocystis sp. PCC6803, has beencloned (104) by a gain-of-function method using Anabaena sp. PCC7120,which does not contain a ∆6 desaturase. Table 1 summarizes the desaturasesthat have been cloned from cyanobacteria.

ACYL-LIPID DESATURASES FROM HIGHER PLANTS Acyl-lipid de-saturases have also been cloned from several plant sources by dif ferent tech-niques, such as a cloning method based on the polymerase chain reaction (PCR)(118) with a primer designed by reference to the amino acid sequence of thepurif ied protein (121), chromosome walking (3, 47), dif ferential screening(158, 159), and T-DNA tagging (21, 95, 157) (Table 2). In contrast with thesesuccessful results, the molecular cloning of enzymes responsible for either the∆3-trans-unsaturation of 16:0 esterif ied at the sn-2 position of PG or the ∆7(ω9) unsaturation of 16:0 esterif ied at the sn-2 position of monogalacto-syldiacylglycerol has not been successful.

ACYL-ACP DESATURASES FROM HIGHER PLANTS A ∆9 acyl-ACPdesaturase (or stearoyl-ACP desaturase) was partially purif ied from maturingsafflower seeds (70) and was purif ied to homogeneity from avocado mesocarp(123) and developing embryos of safflower seeds (140). cDNAs encodingstearoyl-ACP desaturases have been isolated from castor bean (55, 123), saf-

Table 1 Genes for acyl-lip id desaturases that have been cloned from cyanobacteria

Strain

Amino acidsencoded bythe openreading frame Reference

∆12 Desaturase (desA)Synechocystis sp. PCC6803 351 Wada et al (142)Synechocystis sp. PCC6714 349 Sakamoto et al (111)Synechococcus sp. PCC7002 347 Sakamoto et al (111)Anabaena variabilis 350 Sakamoto et al (111)Spirulina platensis 351 Murata et al (78)

ω3 Desaturase (desB)Synechocystis sp. PCC6803 359 Sakamoto et al (109)

∆9 Desaturase (desC)Synechocystis sp. PCC6803 318 Sakamoto et al (110)Anabaena variabilis 272 Sakamoto et al (110)

∆6 Desaturase (desD)Synechocystis sp. PCC6803 359 Reddy et al (104)Spirulina platensis 368 Murata et al (78)


flower seeds (140), cucumber (122), spinach (87), Brassica rapa (56), andBrassica napus (127). The stearoyl-ACP desaturase of castor bean is detectedas an active homodimer that contains four iron atoms when its cDNA isoverexpressed in E. coli (22).

GENETIC MANIPULATION OF THE UNSATURATIONOF MEMBRANE LIPIDS AND CHILLI NG SENSITIVITY

Overexpression of cDNA for Glycerol-3-PhosphateAcyltransferases in Tobacco Plants

CHANGES IN LEVEL OF UNSATURATED PG MOLECULAR SPECIESThe isolation of cDNA for the acyltransferase allows the selective manipulationof the unsaturation of fatty acids of PG. Tobacco is one of the most usefulplants for the establishment of transgenic plants (38). In addition, both thechillin g sensitiv ity and the extent of fatty acid saturation of PG in tobacco

Table 2 cDNAs and genes for acyl-lip id desaturases that have been cloned fromhigher plants

Strain

cDNAorgene

Amino acidsencoded bythe openreading frame Reference

∆12 Desaturase (ER-located, FAD2)Arabidopsis thaliana cDNA 383 Okuley et al (95)Glycine max cDNA-1 387 Heppard et al (37)

cDNA-2 387 Heppard et al (37)∆12 Desaturase (chloroplast-located, FAD6)

Arabidopsis thaliana cDNA 418 Falcone et al (20a)Spinacia oleracea cDNA 447 Schmidt et al (118)Glycine max cDNA 424 Hitz et al (41)Brassica napus cDNA 443 Hitz et al (41)

∆15 (ω3) Desaturase (ER-located, FAD3)Vigna radiata (ARG1) cDNA 380 Yamamoto (158)Arabidopsis thaliana gene 386 Yadav et al (157)

cDNA 386 Yadav et al (157)Brassica napus cDNA-1 383 Arondel et al (3)

cDNA-2 377 Yadav et al (157)Glycine soja cDNA 380 Yadav et al (157)

∆15 (ω3) Desaturase (chloroplast-located, FAD7 or 8)Arabidopsis thaliana gene FAD7 446 Iba et al (47)

cDNA FAD7 446 Yadav et al (157)cDNA FAD8 435 Gibson et al (26)

Brassica napus cDNA >404 Yadav et al (157)Glycine soja cDNA 453 Yadav et al (157)


plants are intermediate between those of squash and Arabidopsis. Therefore,we transformed tobacco plants with respect to these traits by inducing theoverexpression of cDNAs for the plastid-located GPATs from squash andArabidopsis (79). The transformation of tobacco plants with cDNAs for GPATsfrom squash and Arabidopsis signif icantly altered the molecular species com-position of PG (Figure 1A), but it did not signif icantly affect the relativecomposition of lipid classes or the fatty acid composition of the otherglycerolipids. The level of cis-unsaturated molecular species of PG in tobacco

Figure 1 Changes in relative levels of cis-unsaturated molecular species of PG. (A) Transgenictobacco plants transformed with pBI121 (control), with pSQ (cDNA for squash GPAT), and withpAR (cDNA for Arabidopsis GPAT). Values were calculated from the data in Reference 79. (B)Arabidopsis plants: wild type, a transgenic plant transformed with the plsB gene, and the fab1mutant. Values were calculated from the data in References 155 and 156. Open areas correspond tothe sum of all the cis-unsaturated molecular species, and the shaded areas correspond to the sum ofthe saturated and trans-monounsaturated molecular species.


leaves was 64% of the total molecular species of PG, whereas in leaves oftransgenic tobacco plants transformed with pBI121 (the control plasmid), thecDNA for squash GPAT (pSQ), and the cDNA for Arabidopsis GPAT (pARA),it was 64%, 24%, and 72%, respectively. These results support our hypothesisthat the GPAT in the plastids plays a major role in determining the level ofcis-unsaturated molecular species of PG (76).

CHILLING SENSITIVITY In transgenic tobacco plants that had been trans-formed with pBI121, pSQ, and pARA, the photosynthesis of leaf discs wasinhibited by 25 ±11%, 88 ±12% and 7 ±3%, respectively, during incubationfor 4 h at 1°C under strong illumination. Under similar conditions, photosyn-thesis of spinach and squash leaf discs was inhibited by 7% and 82%, respec-tively. These findings demonstrate that the chillin g tolerance of tobacco withrespect to leaf disc photosynthesis was enhanced by transformation with pARAwhile it was diminished by transformation with pSQ. The chillin g sensitiv ityof intact plants has also been estimated directly by exposing plants in a plantbox to a temperature of 1°C for 10 days under continuous illumination andthen incubating them at 25°C for another two days. Chlorosis was observedin leaves from control (pBI121) and pSQ-transformed tobacco plants. Theseresults demonstrate that the chillin g sensitiv ity of tobacco leaves can be ma-nipulated by use of GPAT, which alters the level of unsaturated molecularspecies of PG in chloroplasts.

SENSITIVITY TO HIGH TEMPERATURE The effect of suppressed levelsof cis-unsaturated molecular species of PG on the stability of the photosystemII (PSII) complex was investigated with tobacco plants that had been trans-formed with pBI121 and pSQ (75). No signif icant dif ferences were found inthe sensitiv ity to high temperature of the oxygen-evolving activity betweenthe transformed plants. This result stands in marked contrast to the inferencesmade from results of some previous studies (98, 102), in which the heat stabilityof the photosynthetic machinery increased in parallel with the saturation offatty acids in the thylakoid membrane lipids. However, because such changesin the composition of membrane lipids in these studies were achieved byaltering the growth temperature, which might also affect the levels of othercellular metabolites and enzymes, the proposal of a direct link between thechanges in lipids and the thermal stability of photosynthetic activities seemsunjustif ied.

Overexpression of the plsB Gene and Mutation of Fatty AcidElongase in Arabidopsis

Wolter et al (155) succeeded in reducing the level of cis-unsaturated molecular


species of PG by targeting the product of the plsB gene of E. coli to thechloroplasts of Arabidopsis. The level of cis-unsaturated molecular species inPG fell from 90% in the wild-type plants to <50% in the transgenic plants(Figure 1B). In this experiment, changes were also observed in the fatty acidcomposition—to a minor extent—in all the other glycerolipids. Arabidopsisplants transformed with the plsB gene exhibited a chillin g-sensitive phenotypewhen they were incubated in darkness for seven days at 4°C and then returnedto 20°C. Their older leaves wilted and became brown and necrotic after twodays at 20°C. The chillin g-sensitive phenotype was enhanced by light, and theplants under illumination started to wilt during incubation for three to fourdays at 4°C and eventually died. These observations provide further evidencefor the contribution of the cis-unsaturated molecular species of PG in chloro-plasts to the chillin g tolerance of higher plants.

In contrast with the result of Wolter et al (155), Wu & Browse (156) reportedthat, in a fab1 mutant of Arabidopsis (defective in palmitoyl-ACP elongaseand having, therefore, an elevated level of 16:0 in its cellular lipids), the levelof cis-unsaturated molecular species of PG was greatly reduced from 91% inthe wild-type plant to 57% in the mutant plant (Figure 1B). The wild-typeplant and the fab1 mutant showed no dif ferences in growth and in chillin ginjury when they were incubated at 2°C for seven days under continuous,moderately intense illumination (156). Because chillin g-sensitive plants, suchas cucumber or mung bean, died under the same conditions, they implied thatthe level of unsaturated molecular species of PG would not be a major factorfor defining the chillin g resistance of plants, at least in Arabidopsis. However,their experiments also showed that the fab1 mutant plant displayed retardationof growth after incubation at 2°C under illumination for more than two weeks,and that the mutant plant eventually died after incubation for more than fourweeks. Under these conditions the wild-type plant survived normally. As isdiscussed in a later section (Molecular Mechanism of Low-Temperature Pho-toinhibition Regulated by the Unsaturation of Membrane Lipids), we recentlyfound that increased levels of cis-unsaturated molecular species of PG intransgenic tobacco relieve the photoinhibition of the photosystem II comlexat low temperature by accelerating a recovery process from the low-tempera-ture-induced photoinactivated state. In such experiments, the intensity of lightduring incubation at low temperature plays a key role. When the intensity oflight is relatively low as in experiments by Wu & Browse (156), photoinhibi-tion in vivo is not apparent.

Disruption of Genes for Desaturases in Synechocystis sp.PCC6803

DECREASE IN LEVELS OF POLYUNSATURATED FATTY ACIDS Thecyanobacterium, Synechocystis sp. PCC6803, contains four desaturases that


control the levels of polyunsaturated fatty acids in membrane lipids (84). Agreat advantage to using this organism is that a specific function of a gene canbe defined by use of the method known as insertional mutation (152). Usingthis method, we created cyanobacterial strains with dif ferent levels of unsatu-rated fatty acids in their membrane lipids (109, 142, 144). These strains allowus to examine the roles of the unsaturation of membrane lipids in variousphenomena, such as growth, chillin g tolerance, and temperature acclimation(29–32, 143). Figure 2A shows a typical example of changes in levels ofunsaturated molecular species of membrane lipids. In this experiment, the desAand desD genes of the wild-type strain of Synechocystis sp. PCC6803 weredisrupted by inserting antibiotic-resistance gene cassettes (e.g. Kmr, a kanamy-cin-resistance gene cassette) into the genome. The results demonstrated thatindividual unsaturated bonds of membrane lipids in this Synechocystis straincan be specifically deleted by the stepwise disruption of the genes for de-saturases.

GROWTH RATE The wild-type, Fad6 (defective in the expression of thedesD gene) and Fad6/desA::Kmr cells of Synechocystis sp. PCC6803 grow atsimilar rates during the exponential phase at 34°C. At 22°C, the wild-type andFad6 cells grow at about the same rate, but the Fad6/desA::Kmr cells grow ata markedly lower rate. These observations suggest that disruption of the desAgene or the elimination of the ∆12 double bond from the membrane lipids hasa deleterious effect on the growth of this cyanobacterium at the low temperature(144).

CHILLING SENSITIVITY When wild-type Fad6 and Fad6/desA::Kmr cellsthat had been grown at 34°C were exposed to a low temperature, the extentof photoinhibition of photosynthesis, which was assayed at 34°C (31, 32, 144),depended on the extent of unsaturation of membrane lipids (Figure 3). At 10°C,the Fad6/desA::Kmr cells, which contained only monounsaturated and satu-rated lipid molecular species, suffered the most severe photoinhibition amongthe three types of cell, whereas the wild-type and Fad6 cells were indistin-guishable, and levels of photoinhibition were insignif icant. At room tempera-ture, the photoinhibition was less signif icant than at low temperatures in allthe strains, but the Fad6/desA::Kmr cells showed the highest degree of pho-toinhibition among the three types of cell (Figure 3) (31, 32, 144). Photosyn-thetic electron-transport activities were, by contrast, unaffected by the changesin the extent of unsaturation of membrane lipids (31). These results suggestthat introduction of the second double bond into the membrane lipids protectsthe cells against photoinhibition, in particular at low temperature.

HIGH-TEMPERATURE SENSITIVITY In contrast with the severe damage


to the photosynthetic machinery at low temperature with decreases in theunsaturation of membrane lipids, the sensitiv ity to high temperature of thephotosynthetic machinery was barely affected or was only slightly reduced bya decrease in the unsaturation of membrane lipids (29, 143). Again, our resultsclearly refute the possibility that in cyanobacteria unsaturated membrane lipidsmight be unfavorable for high-temperature tolerance (98, 102). We have found

Figure 2 Changes in molecular species composition after manipulation of genes for desaturasesin cyanobacteria. Reproduced from Murata & Wada (83). (A) Mutation of the ∆6 and ∆12desaturases in Synechocystis sp. PCC6803 (144). (B) Introduction of the ∆12 desaturase bytransformation with the desA gene from Synechocystis sp. PCC6803 (143). T-desA, a straintransformed with the desA gene.


recently that the high-temperature tolerance of photosynthesis that is acquiredby a change in the growth temperature is caused by protein factors (90, 91).

Expression of the Gene for a Desaturase in Synechococcus sp.PCC7942

INCREASES IN LEVELS OF POLYUNSATURATED FATTY ACIDSSynechococcus sp. PCC7942 (Anacystis nidulans strain R2) is a Group 1 strainof cyanobacteria (84), and it is unable to introduce a second double bond intomonounsaturated fatty acids (84). We transformed wild-type cells of this strainwith the desA gene of Synechocystis sp. PCC6803 in an attempt to increasethe extent of unsaturation of membrane lipids (109, 142). Figure 2B shows thechanges in the composition of lipid molecular species in cells of the wild-typestrain and in cells that had been transformed with the desA gene. The desAgene endowed the Synechococcus strain with the novel ability to synthesize16:2(9, 12) and 18:2(9, 12) at the sn-1 position.

CHILLING SENSITIVITY When wild-type cells and cells that had been trans-formed with a vector plasmid were exposed to temperatures below 10°C, thephotosynthetic activity decreased irreversibly; more than 50% of the activitywas lost during incubation at 5°C for 60 min (142, 143). The cells transformedwith the desA gene, by contrast, did not show any signif icant changes inphotosynthetic activity under the same conditions (142). These observationsdemonstrate that an elevated level of unsaturation of membrane lipids enhancesthe low-temperature tolerance of Synechococcus sp. PCC7942. This result is

Figure 3 Photoinhibition of photosynthesis in wild-type and Fad6/desA::Kmr cells of Synecho-cystis sp. PCC6803 grown at 34°C. •−• , Wild type; °−°, Fad6/desA::Kmr. Reproduced fromMurata & Wada (83) and based on results of Gombos et al (32).


consistent with the observations in Synechocystis sp. PCC6803, in which theextent of unsaturation of membrane lipids was decreased, with a loss oflow-temperature tolerance, by the disruption of genes for the desaturases.

HIGH-TEMPERATURE SENSITIVITY We have also compared wild-type and trans-formed cells of Synechococcus with respect to the high-temperature toleranceof the photosynthetic machinery and photosynthetic electron transport (143).No detectable changes accompanied the increases in the extent of unsaturationof membrane lipids.

MOLECULAR MECHANISM OF LOW-TEMPERATUREPHOTOINHIBITION REGULATED BY THEUNSATURATION OF MEMBRANE LIPIDS

For oxygenic photosynthetic organisms, light is an absolute prerequisite forphotosynthesis, but, at the same time, it is harmful to the photosyntheticapparatus (2). There is a general consensus that the light-induced damage tothe photosynthetic machinery is particularly serious in photosystem II (PSII)(2, 99). The photoinhibition of photosynthesis is such a major stress that therate of light-induced inactivation of the reaction center exceeds the rate of therecovery process from the light-induced inactivated state (2). Furthermore,photoinhibition is aggravated if strong light is combined with other stresses,such as low temperature (2). Then the question arises as to how unsaturatedmembrane lipids might alleviate the low-temperature-induced photoinhibition.Do they suppress the light-induced inactivation process, or do they acceleratethe recovery process?

Photoinhibition in Transgenic Tobacco Plants

The level of cis-unsaturated molecular species of PG does not affect the processof light-induced inactivation of photoinhibition in transgenic tobacco. Thisconclusion is based on the observation that the photoinhibition at high and lowtemperatures of leaf discs from both pSQ- and pBI121-transformed plantsoccurs at about the same rate if the recovery from the photoinhibited state isblocked by administration of lincomycin, an inhibitor of protein synthesis (75).In contrast, the recovery from the photoinhibited state does occur under weakillumination in leaf discs that have been photoinhibited to a certain level (about80%). During the recovery period, wild-type and pBI121-transformed tobaccoplants resume photosynthesis at a rate equal to 50–60% of the original rate,whereas the recovery of the pSQ-transformed plants is much slower than thatof the pBI121-transformed plants (Figure 4). These results indicate that cis-unsaturated molecular species of PG accelerate the recovery of the photosyn-


thetic machinery from chillin g-induced photoinhibition. Recently, it was re-ported (9) that there are no signif icant dif ferences in the fluorescence parame-ters of photosynthesis between Arabidopsis plants transformed with the plsBgene and wild-type plants. However, in the cited study, the chillin g-inducedphotoinhibition was not examined by dissecting the photoinactivation processand the recovery process.

Photoinhibition in Synechocystis sp. PCC6803

Results similar to those described above have been obtained with Synechocystis

Figure 4 The recovery of the photosynthetic machinery from photoinhibition in leaves of pBI121-transformed (°−°) and pSQ-transformed plants (•−•). Reproduced from Moon et al (75).


sp. PCC6803 strains with dif ferent levels of unsaturation of membrane lipids(32). Under photoinactivation conditions with recovery blocked by lincomycin,there is no signif icant dif ference among the rates of photoinhibition amongdif ferent strains with dif ferent levels of unsaturation of membrane lipids (32).By contrast, under recovery conditions when the photoinactivation process isnegligible, an elevated level of unsaturation of membrane lipids considerablyenhances the rate of recovery from photoinhibition. Therefore, it seems verylikely that the apparent enhancement of the photoinhibition of photosynthesisin intact cells at low temperature might be caused by disruption of the recoveryprocess (32). This conclusion is consistent with that drawn from transgenictobacco plants, and it suggests that the unsaturation of membrane lipids isdefinitely important for the survival of oxygenic photosynthetic organisms atlow temperature.

Turnover of the D1 Protein

According to a current hypothetical molecular mechanism of photoinhibitionin vivo (1, 2), the photoinactivation process is caused by the light-induceddamage to the D1 protein of the PSII reaction center (85), and the recoveryprocess involves several steps, as follows: (a) removal of the light-inactivatedD1 protein from the PSII complex by proteolysis, (b) synthesis de novo of theprecursor to the D1 protein (pre-D1 protein), (c) sorting of the pre-D1 proteininto the PSII complex, (d) maturation of the pre-D1 protein by processing, and(e) reconstitution of the oxygen-evolving complex and recovery of the fullyactive PSII complex. Therefore, we can assume that one of the above recoverysteps is accelerated by the unsaturation of membrane lipids.

The proteolytic digestion of the damaged D1 protein was investigated inwild-type, Fad6, and Fad6/desA::Kmr cells of Synechocystis sp. PCC6803 byimmunoblotting (53). It was clear that the unsaturation of membrane lipids didnot limit the removal of the damaged D1 protein. Transcription of the psbAgene (the structural gene for the pre-D1 protein) was not affected by theunsaturation of membrane lipids. It is likely that either reassembly of the pre-D1

protein with the PSII complex or the processing of the pre-D1 protein thatyields the mature D1 protein is accelerated by the unsaturation of membranelipids.

CHILLI NG SENSITIVITY OF ARABIDOPSIS MUTANTSAND OTHER TRANSGENIC PLANTS

Several Arabidopsis mutants that are defective in lipid-synthesizing activitieshave been shown to exhibit an altered growth phenotype at low temperature.The fad5 (previously designated fadB; defective in chloroplast sn-2-palmitoyl-


MGDG desaturase) and fad6 (previously designated fadC; defective in chlo-roplast ∆12 desaturase) mutant plants are characterized, at low temperatures,by leaf chlorosis, a reduced growth rate, and changes in chloroplast morphol-ogy, such as a decrease in size and appressed regions of thylakoid membranes(46). The chillin g-induced phenotypes of these mutants are similar to those ofcyanobacterial strains with decreased levels of the ∆12 double bond. The fad2mutant (defective in microsomal ∆12 desaturase) had a greatly reduced rateof stem elongation at 12°C and died at 6°C (71).

Kodama et al (57) reported that chillin g-induced damage to very youngtobacco seedlings could be alleviated by a slight increase in levels of 18:3 asa result of overexpression of the Fad7 gene of Arabidopsis that encodes aplastid-located ω3 desaturase. Because the changes in the extent of the unsatu-ration are very minor, it is very unlikely that the change in chillin g sensitiv ityin this case is caused by a membrane-related mechanism.

TEMPERATURE ACCLIMATION

Desaturation of Membrane LipidsIt is important for liv ing organisms to maintain the fluidity of their cell mem-branes at a certain level, and this requirement is achieved by changes in thelevels of unsaturated fatty acids in membrane lipids that are catalyzed by fattyacid desaturases (15, 107). Several mechanisms for the regulation of low-tem-perature-induced desaturation of fatty acids have been reviewed, as follows(15, 25, 139). 1. The concentration of oxygen, a substrate of desaturases, limitsthe rate of desaturation of fatty acids in some plant systems, e.g. nonphoto-synthetic plant tissues (35) and cultured sycamore cells (103). 2. The impor-tance of the synthesis de novo of desaturases in response to low temperaturewas first demonstrated by Fulco and coworkers (25), who observed the low-temperature-induced synthesis of an enzyme responsible for the ∆5 desatura-tion of 16:0 in Bacillus megaterium. A similar response in terms of the syn-thesis of fatty acid desaturases has been found in Tetrahymena (139). 3.Although the second mechanism is considered the most likely in the regulationof the unsaturation of membrane lipids in Tetrahymena, there remains thepossibility that at an early stage of the low-temperature response, the desaturaseitself is activated by the decrease in the fluidity of the membrane lipids thatis caused by low temperature (43, 125, 139). 4. It has also been proposed thatthe relative rates of biosynthesis of saturated vs unsaturated fatty acids controlthe levels of unsaturated fatty acids in safflower seeds (7).

Cyanobacterial cells respond to low temperature by an increase in the levelof unsaturated fatty acids in membrane lipids (77, 80, 115, 146). Therefore,the regulation of the activities of acyl-lipid desaturases has been examined in


Synechocystis sp. PCC6803, from which all four acyl-lipid desaturases havebeen cloned. The crucial step in the regulation of the unsaturation of membranelipids in cyanobacterial cells is the synthesis de novo of acyl-lipid desaturases.The chillin g-induced desaturation of fatty acids can be inhibited by rifampicinand chloramphenicol but not by cerulenin, an inhibitor of the synthesis of fattyacids (67, 116, 146). Northern blot analyses (67) have indicated that the levelof the mRNA for the ∆12 desaturase (desA) increases tenfold upon a decreasein ambient temperature from 36°C to 22°C (67), which suggests that thechillin g-induced desaturation of membrane lipids might be caused by upregu-lation of the expression of the gene for the desaturase. Further studies suggestedthat the increase in the level of desA mRNA at low temperature is caused bythe stabilization of the mRNA and by the enhanced transcription of the desAgene (D Los, M Ray & N Murata, unpublished information).

The effect of low temperature on the upregulation of expression of the desAgene can be mimicked by the catalytic hydrogenation of only a small portionof the plasma membrane lipids in cells of Synechocystis sp. PCC6803 (141).After this intervention, the level of desA mRNA increased tenfold within 1 h(141). This time course was similar to that observed after a shif t from 36°Cto 22°C. Therefore, it appears that changes in membrane fluidity could beinvolved in the initial event that leads to the expression of genes for desaturasesin cyanobacterial cells.

The effect of temperature on the activity of desaturases has been investigatedin extracts of E. coli cells that overexpress the product of a desA gene (SPampoon, D Los & N Murata, unpublished information). The desaturase ex-hibited lower activity at lower temperatures, as is observed with most otherenzymes.

The Signal Transduction Pathway to the Expression ofCold-Inducible Genes

Low temperature induces a number of changes in cellular metabolism. Thesechanges include increases in the extent of unsaturation of fatty acids andphospholipid content, changes in protein composition, and induction of cold-inducible genes. Cold-inducible genes have been cloned from numerous plantspecies (10, 11, 27, 28, 44, 45, 58, 60, 61, 72a,b, 86, 92, 93, 96, 108, 150,151, 153, 154) and from cyanobacteria (113, 114). The genes can be classif iedas dhn-, lea-, and rab-relabled genes [dehydrin- (5, 12), late embryogenesis-abundant- (4), and responsive to abscisic acid- (124) related genes]. Othercold-inducible genes encode polypeptides that are homologous to the productof a human tumor gene, bbc1 (108), to a lipid transfer protein (45), and to thetranslation elongation factor 1α (19). In addition, RNA-binding proteins andthe ribosomal protein S21 have been identif ied as products of cold-inducible


genes in cyanobacteria (113, 114). The expression of these genes at lowtemperature is controlled by various mechanisms, and some of the genes arealso induced by environmental stresses other than low temperature. However,all attempts to improve chillin g tolerance by inducing overexpression of anyone of these genes have been unsuccessful, which perhaps indicates thatcooperation of products of cold-inducible genes is necessary for expression ofa chillin g-tolerant phenotype.

Much current attention is being focused on the way in which a low tem-perature is perceived by the cell and the way in which the signal is transducedto the nucleus to induce the expression of cold-inducible genes. To understandhow the temperature signal might regulate the levels of transcripts of genesfor the desaturases, we have to take into consideration the following results.Monroy & Dhindsa (73) demonstrated that an influx of Ca2+ ions from theextracellular apoplasmic space into the cytoplasm is involved in the expressionof the cas (cold-acclimation specific) genes in alfalfa cells in suspensionculture. They also examined the possible involvement of protein phosphory-lation in cold acclimation (74) and have cloned several Ca2+-dependent proteinkinases from alfalfa by a consensus sequence-based amplif ication method (73).Shinozaki and his coworkers (40) succeeded in amplif ying the gene for phos-phatidylinositol-specific phospholipase C from A. thaliana by PCR. This geneis induced by cold, drought, salt, and abscisic acid.

CONCLUSIONS

1. The weight of evidence indicates that the unsaturation of membrane lipidsis correlated with the chillin g sensitiv ity of plants and cyanobacteria. Theunsaturation apparently protects the photosystem II complex from low-tem-perature photoinhibition by accelerating the recovery of the complex fromthe photoinhibited state. The processing of the pre-D1 protein to the matureD1 protein and the sorting of the pre-D1 protein to the thylakoid membranesare possible sites of the acceleration of the turnover cycle of the D1 protein.It is noteworthy in this regard that acidic lipids, such as PG, are involvedin the sorting of proteins to membranes in microbial systems (16, 17, 59,66).

2. Membrane lipids are not the sole factors that regulate the chillin g sensitiv ityof plants. At low temperatures, most plants accumulate polyols and aminoacids (or amino acid derivatives) as compatible solutes, which might con-tribute to the chillin g sensitiv ity of these plants. It is also possible that someother factors, such as specific proteins, are responsible for chillin g toler-ance.

3. The degree of chillin g sensitiv ity of a plant depends on the stage of its lif ecycle. Maturation of pollen is the most chillin g-sensitive of all stages. The


imbibition of seeds is also a very chillin g-sensitive process. It is unclearas yet whether the unsaturation of membrane lipids is related to the chillin gsensitiv ity of plants at these stages.

4. We have suggested that the temperature sensor of cyanobacterial cell islocated in the plasma membrane. However, the sensor, the signal trans-ducers, the details of the regulation of expression of genes for desaturases,and a role for Ca2+ ions in the signaling all remain mysteries that remainto be resolved.

5. Most crops of tropical origin are sensitive to chillin g temperatures. Thissensitiv ity limits the worldwide production of agricultural materials. Abetter understanding of the molecular mechanisms of chillin g sensitiv ity isimportant if we are to improve the chillin g tolerance of crops by geneticengineering.

ACKNOWLEDGMENTS

This work was supported, in part, by Grants-in-Aid for Scientif ic Research onPriority Areas (Nos. 04273102 and 04273103) to N. Murata and by a grantfor General Scientif ic Research (No. 06640854) to I. Nishida from the Ministryof Education, Science, Sports and Culture, Japan.

Any Annual Review chapter , as well as anyarticle cited in an Annual Review chapter ,

may be purchased from the Annual Reviews Pre-pr ints and Repr ints service.

1-800-347-8007; 415-259-5017; email:[email protected]

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