Wheat Etioplasts Research Articles

Association of the NADPH:protochlorophyllide oxidoreductase (POR) with isolated etioplast inner membranes from wheat.

Membrane association of NADPH:protochlorophyllide oxidoreductase (POR, EC: 1.6.99.1) with isolated prolamellar bodies (PLBs) and prothylakoids (PTs) from wheat etioplasts was investigated. In vitro-expressed radiolabelled POR, with or without transit peptide, was used to characterize membrane association conditions. Proper association of POR with PLBs and PTs did not require the presequence, whereas NADPH and hydrolysable ATP were vital for the process. After treating the membranes with thermolysin, sodium hydroxide or carbonate, a firm attachment of the POR protein to the membrane was found. Although the PLBs and PTs differ significantly in their relative amount of POR in vivo, no major differences in POR association capacity could be observed between the two membrane systems when exogenous NADPH was added. Experiments run with only an endogenous NADPH source almost abolished association of POR with both PLBs and PTs. In addition, POR protein carrying a mutation in the putative nucleotide-binding site (ALA06) was unable to bind to the inner membranes in the presence of NADPH, which further demonstrates that the co-factor is essential for proper membrane association. POR protein carrying a mutation in the substrate-binding site (ALA24) showed less binding to the membranes as compared to the wild type. The results presented here introduce studies of a novel area of protein-membrane interaction, namely the association of proteins with a paracrystalline membrane structure, the PLB.

Comparative Absorption, Translocation, and Metabolism of Foliar-Applied Oxyfluorfen in Wheat and Barley

Wheat is known to be relatively tolerant to diphenyl ether herbicides. The absorption, translocation, and metabolism of foliar-applied oxyfluorfen in wheat were examined in comparison with those of oxyfluorfen-susceptible barley. Epicuticular wax contents in the first fully expanded leaves were similar in wheat and barley, but the wheat leaves had a 1.74-fold higher cuticle content than the barely leaves. Absorption of [ 14 C]oxyfluorfen, as estimated by the amount of [ 14 C]oxyfluorfen penetrating the cuticle, appeared to be similar in the leaves of both species, although most of the radioactivity remained in the epicuticular wax. Little translocation of the herbicide out of the treated leaf was observed in either species, but slightly more translocation of [ 14 C]oxyfluorfen to shoots other than the treated leaf or to roots was found in barley than in wheat leaves. Autoradiographs of the [ 14 C]oxyfluorfen-treated leaves of both species also showed that the radioactivity was distributed mainly in the treated site. Thin-layer chromatographic analysis of leaf extracts revealed that oxyfluorfen metabolism did not occur in intact leaves of either species. The binding constants for the herbicide were estimated as 44.2 and 191 nM, respectively, for wheat and barley etioplasts, showing that the herbicide had higher affinity to barley than to wheat etioplasts. These results indicate that the differential susceptibilities of wheat and barley to oxyfluorfen are not due to the differential absorption, translocation, and metabolism of the herbicide, but to the differential affinity of the herbicide.

Analysis of Fluorescence Lifetime of Protochlorophyllide and Chlorophyllide in Isolated Etioplast Membranes Measured from Multifrequency Cross‐correlation Phase Fluorometry

AbstractThe fluorescence decays of protochlorophyllide (Pchlide) and of chlorophyllide (Chlide) in wheat etioplast membranes were analyzed using a multiexponential fluorescence decay model. Using different excitation wavelengths from 430 to 470 nm, we found that a triple‐exponential model at 14°C and a double‐exponential model at — 170°C were adequate to describe the Pchlide fluorescence decay. We discuss the origin of the three fluorescence lifetime components at 14°C on the basis of the dependence of their fractional intensities on the excitation wavelength and by correlating the fractional intensities with integrated fluorescence intensities of different Pchlide forms in steady‐state fluorescence spectra. The fluorescence decay of the main Pchlide form, photoactive Pchlide‐F657, is shown to have a complex character with a fast component of 0.25 ns and a slower component of about 2 ns. Two lifetime components of 2 ns and 5.5–6.0 ns are ascribed to the second photoactive form, Pchlide‐F645, and to nonphotoactive Pchlide forms, respectively. In etioplast membranes preilluminated by a short saturating light pulse, we found a single 5.0 ns component for Chlide‐F688 (the Chlide‐NADPH: protochlorophyllide oxidoreductase [PORJ‐NADP+complex) and an additional 1.6 ns component when the formation of Chlide‐F696 (the Chlide‐POR‐NADPH complex) was promoted by exogenous NADPH. From the fluorescence lifetime results we evaluated the quantum yield of the primary photoreaction by Chlide‐F696 as being 70%.

Differential Susceptibilities of Wheat and Barley to Diphenyl Ether Herbicide Oxyfluorfen

Wheat is known to be relatively tolerant to diphenyl ether herbicides. Growth and physiological responses of wheat to diphenyl ether herbicide oxyfluorfen were examined in comparison with those of oxyfluorfensusceptible barley. Compared to barley, wheat was significantly less susceptible to the herbicide with preemergence and postemergence treatments. The differential susceptibilities of wheat and barley to the herbicide were more apparent with postemergence than with preemergence treatment. The effects of the herbicide on causing cellular leakage, chlorophyll loss, and lipid peroxidation were much lower in wheat than in barley leaves. Protoporphyrin IX accumulated in both wheat and barley leaves treated with oxyfluorfen, in a concentration-dependent manner. However, the magnitude of the protoporphyrin IX accumulation was much lower in wheat than in barley leaves treated with oxyfluorfen at a concentration of higher than 0.33 μM. Protoporphyrinogen oxidase from wheat leaves was found to be less susceptible to oxyfluorfen than that from barley leaves. The I50 concentrations of oxyfluorfen on protoporphyrinogen oxidase activity were approximately 0.2 and 0.06 μM for wheat and barley etioplasts, respectively. Whereas the inhibition of protoporphyrinogen oxidase in barley etioplasts increased with increasing concentration of oxyfluorfen, protoporphyrinogen oxidase in wheat etioplasts was not further inhibited beyond 0.33 μM oxyfluorfen concentrations. In addition, both plant species similarly responded to superoxide anion-generating paraquat and to singlet oxygen-generating rose bengal, implying that the relative tolerance of wheat to oxyfluorfen is not due to protective mechanism against active oxygen species. Our results suggest that the differential susceptibilities of wheat and barley to oxyfluorfen are at least partly due to the differential inhibition of protoporphyrinogen oxidase by the herbicide, and thereby wheat is relatively tolerant to the herbicide.

Use of fluorescence probes 1-aniline-8-naphthalene sulfonate and pyrene for studying the localisation of proteins in inner membranes from wheat etioplasts

The fluorescence probes 1-aniline-8-naphthalene sulfonate (ANS) and pyrene were applied for characterisation of the light-induced changes in etioplast inner membranes (EPIMs) from 7 d-old dark-grown wheat seedlings (Triticum aestivum L. cv. Pobeda). The major aim was to obtain information about the localisation of membrane proteins in the EPIMs, using probes situated in different regions of the membranes. The quenching of tryptophan fluorescence showed tha the main parts of proteins were accessible to the pyrene buried in the lipid bilayer which suggests that most of the proteins also enter the lipid bilayer. The substantial quenching of the tryptophan fluorescence by the surface-situated ANS demonstrated that a part of the tryptophan residues was probably localised close to the membrane surface. The registered changes after irradiation could be explained by the presence of large aggregates of NADPH-protochlorophyllide oxidoreductase (POR), protochlorophyllide (PChlide) and NADPH in membranes that start to disconnect and redistribute along the prothylakoids.

Carotenoid‐deficient young wheat etioplasts are able to bind precursor proteins on the plastid surface but are impaired in their translocation ability

Young carotenoid‐deficient etioplasts, isolated from Norflurazon (NF)‐treated wheat seedlings, were used to study the role of coloured carotenoids in the binding and import reactions of different nuclear‐encoded plastid proteins. Plastids from control seedlings exhibited significantly higher import efficiencies than did plastids from NF‐treated plants. Etioplasts containing normal levels of carotenoids imported approximately 2000 and 800 molecules per plastid of the precursors of the small Rubisco subunit (pSS) and the Rieske FeS protein (pFeS), respectively. Plastids from NF‐treated plants imported approximately 100 and 70 pSS and pFeS molecules per plastid, respectively. In addition, a maximum binding capacity of NF‐treated plastids of 1200 protein molecules per plastid was observed for both pSS and pFeS when assayed at 25°C: and a maximum binding capacity of approximately 1300 molecules per plastid was noted at 4°C. For control plastids, a similar amount of binding, or approximately 1400 protein molecules per plastid, could only be observed if import was inhibited by low ATP concentrations at 4°C. When these plastids were washed and transferred to conditions promoting import at 25°C and 10 mM Mg‐ATP, close to 60% of the envelope‐associated precursor protein molecules were imported. These results indicate that control and NF‐treated young etioplasts contain similar amounts of binding sites for precursor proteins. However, only in the case of control plastids the binding was productive and lead to import and processing in the stroma upon transfer to conditions promoting import. Plastids isolated from wheat seedlings grown in weak red light and containing different amounts of carotenoids, were assayed for their ability to bind and import a protein with unusual import characteristics, the Chlamydomonas reinhardtii PsaF precursor of PSI (pPsaF) and transit peptide deletion constructs. The PsaF protein was imported in a transit peptide‐dependent manner into control etioplasts, whereas import of pPsaF into young wheat etioplasts isolated from NF‐treated plants was inhibited at low levels of plastid carotenoids.

Carotenoid-deficient young wheat etioplasts are able to bind precursor proteins on the plastid surface but are impaired in their translocation ability

Carotenoid-deficient young wheat etioplasts are able to bind precursor proteins on the plastid surface but are impaired in their translocation ability

The light intensity dependence of protochlorophyllide photoconversion and its significance to the catalytic mechanism of protochlorophyllide reductase

The light-dependent step in chlorophyll synthesis by higher plants involves hydrogen transfer from NADPH + to the porphyrin protochlorophyllide catalysed by the photoenzyme protochlorophyllide reductase. The light intensity dependence of the process has been studied in vitro using wheat etioplast membranes. The results suggest that a single photochemical event is involved in the photoconversion. In support of this conclusion we also demonstrate that illumination of these membranes with a train of ultrashort (150 fs) flashes resulted in chlorophyll accumulation. The significance of the findings in terms of possible mechanisms for the reductase are discussed.

Photoreduction of zinc protopheophorbide b with NADPH-protochlorophyllide oxidoreductase from etiolated wheat (Triticum aestivum L.).

A preparation of prolamellar bodies from wheat etioplasts was used as a source for NADPH-protochlorophyllide oxidoreductase (pchlide reductase). The enzyme was solubilized with Triton X-100 after reduction of the endogenous photoconvertible protochlorophyllide a to chlorophyllide a by saturating illumination. Protochlorophylls a and b, protochlorophyllide a and zinc protopheophorbide b were added to the soluble enzyme preparation to determine if they were reduced in the dark or in the light. None of the compounds were reduced (with NADPH) in the dark; however, light-dependent reduction was demonstrated with protochlorophyllide a and zinc protopheophorbide b. The yield was approximately 50% for both substrates. Photoreduction did not occur with the esterified protochlorophylls a and b. Photoreduction of zinc protopheophorbide b, the zinc analogue of protochlorophyllide b, is the first demonstration of the reduction of a chlorophyll-b-related compound by pchlide reductase.

Optical Multichannel Analysis of Protochlorophyllide Phototransformation in Detergent-Solubilized Etioplast Membranes of Wheat

Extracts of wheat etioplast membranes obtained after treatment with 7 mᴍ n-octyl-β-ᴅ-glucopyranoside (OG), n-dodecyl-β-ᴅ-maltoside (DM) or Triton X-100 contained the three spectral forms of Pchlide (the photoactive Pchlide638 and Pchlide650 and the inactive Pchlide630) in various relative amounts . The OG extract had a Pchlide composition close to that of the intact membranes whereas the DM extract was enriched in Pchlide638 and the Triton extract was enriched in Pchlide630. Measurements of the kinetics of phototransformation and of timeresolved absorbance spectra during phototransformation in continuous light shows that the inactive Pchlide630 is in fact slowly transformed to Chlide, especially in the Triton extract where this form is more abundant. Addition of NADPH favours the phototransformation of Pchlide630 and the slow regeneration of Pchlide638 and Pchlide650 from Pchlide630 in darkness after illumination. No such regeneration was however observed in the Triton extract. NADPH had only slight effects on the Chlide shift towards shorter wavelengths after phototransformation in solubilized membranes.

Differential expression of the psbB and psbH genes encoding the 47 kDa chlorophyll a-protein and the 10 kDa phosphoprotein of photosystem II during chloroplast development in wheat

The nucleotide sequence of a region of wheat chloroplast DNA containing the psbB gene for the 47 kDa chlorophyll a-binding protein of photosystem II has been determined. The gene encodes a polypeptide of 508 amino acid residues which is predicted to contain six hydrophobic membrane-spanning regions. The psbB gene is located 562 bp upstream of the psbH gene for the 10 kDa phosphoprotein of photosystem II. A small open reading frame of 38 codons is located between psbB and psbH, and on the opposite strand the psbN gene, encoding a photosystem II polypeptide of 43 amino acid residues, is located between orf38 and psbH. S1 nuclease mapping indicated that the 5' ends of transcripts were located 371 and 183 bp upstream of the psbB translation initiation codon. Predominant transcripts of 2.1 kb and 1.8 kb for psbB and 0.4 kb for psbH were present in RNA isolated from etiolated and greening wheat seedlings. Immunodecoration of Western blots indicated that the 47 kDa polypeptide was absent, or present in very low amounts, in dark-grown tissue and accumulated on greening, whereas the 10 kDa polypeptide was present in similar amounts in both dark-grown and greening seedlings. The 10 kDa polypeptide was phosphorylated in vitro by incubating wheat etioplast membranes with [gamma 32P] ATP.

On the aggregational states of protochlorophyllide and its protein complexes in wheat etioplasts

The inner membranes from wheat (Triticum aestivum L. cv. Walde) etioplasts were separated into membrane fractions representative of prolamellar bodies and prothylakoids by differential and gradient centrifugations. The isolated fractions were characterized by absorption‐, low‐temperature fluorescence‐, and circular dichroism (CD) spectroscopy, by high performancy liquid chromatography and by sodium dodecyl sulphate polyacrylamide gel electrophoresis.The prolamellar body fraction was enriched in NADPH‐protochlorophyllide oxidoreductase (E.C. 1.6.99.1), and in protochlorophyllide showing an absorption maximum at 650 nm and a fluorescence emission maximum at 657 nm. Esterified protochlorophyllide was mainly found in the prothylakoid fraction. The carotenoid content was qualitatively the same in the two fractions. On a protein basis the carotenoid content was about three times higher in the prolamellar body fraction than in the prothylakoid fraction. The CD spectra of the membrane fractions showed a CD couplet with a positive band at 655 nm, a zero crossing at 643–644 nm and a negative band at 623–636 nm. These results differ from earlier CD measurements on protochlorophyllide holochrome preparations. The results support the interpretation that protochlorophyllide is present as large aggregates in combination with NADPH and NADPH‐protochlorophyllide oxidoreductase in the prolamellar bodies.

Prolamellar bodies of oat, wheat, and rye: Structure, lipid composition, and adsorption of saponins

Membranolytic active saponins (26-desglucoavenacosides) derived from isolated oat-avenacosides adsorb to oat prolamellar bodies (PLBs), but not to wheat or rye etioplast inner membranes. Thus, the saponin adsorption is a peculiarity of oat PLBs but not a general tool for PLB-analysis. The qualitative and quantitative analysis of free sterols and galactolipids of isolated PLBs did not explain the exclusive adsorption of saponins to oat PLBs. In addition, isolated PLBs and prothylakoids (PTs) of all three plants did not differ with respect to the molecular species composition of mono- (MGDG) or di-galactosyldiacylglyceride (DGDG). Recrystallization leading to a structural reorganization of wheat PLBs (wide type to narrow type) also had no influence on the molecular species composition of the isolated etioplast inner membranes. These observations as well as earlier investigations raise the question, whether the composition of PLBs is responsible for the tubular, semicrystalline construction. Experiments with the chelating agent EDTA indicate an external influence on the PLB construction and underline the idea that the PLB construction is not influenced by the membrane components themselves. Low concentrations of EDTA in the isolation media decompose the paracrystalline structure of the PLBs into membranes, resembling those of greening plastids. Based on these experiments we assume the ionic environment in plastids to be responsible for the construction and decomposition of PLBs and that changes of the membrane components are of minor importance.

The use of continuous assays to characterize the oxidative cyclase that synthesizes the chlorophyll isocyclic ring

A continuous spectroscopic assay has been developed for magnesium protoporphyrin monomethyl ester oxidative cyclase, which records either the dark formation of both free and protein-bound magnesium phaeoporphyrin or, following flash illumination, its corresponding chlorin. The properties of the enzyme were studied in wheat etioplasts. When plastids were pre-illuminated in the presence of NADPH all endogenous protochlorophyllide was converted into chlorophyllide and the product of dark incubation with magnesium protoporphyrin monomethyl ester was protein-bound magnesium 2-vinyl phaeoporphyrin a5 monomethyl ester with either a vinyl or an ethyl group at position 4 of the macrocycle alone. Rates of chlorin production from magnesium protoporphyrin monomethyl ester (up to 1240 pmol/h per mg of protein) were adequate to support known rates of plant chlorophyll synthesis. The enzyme required NADPH and O2 and had an approximate Km of 0.5 microM for magnesium protoporphyrin IX monomethyl ester. Lipid-soluble metal-complexing agents inhibited enzyme activity: hydrophilic agents were ineffective. The strong inhibition of mycobactin suggested the involvement of iron ions. Zinc protoporphyrin monomethyl ester, but not copper or nickel or metal-free protoporphyrin monomethyl esters, was a substrate; magnesium protoporphyrin dimethyl ester was inhibitory. The activity of the enzyme was unchanged by prior greening of the plants. The activity in isolated etioplasts was very dependent upon intactness of the plastid structure.

The NADPH-protochlorophyllide oxidoreductase is the major protein constituent of prolamellar bodies in wheat (Triticum aestivum L.)

A fraction of highly purified prolamellar bodies was isolated from etioplasts of wheat (Triticum aestivum L. cv. Starke II, Weibull), as previously described by Ryberg and Sundqvist (1982, Physiol. Plant., 56, 125-132). Studies on the protein composition revealed that only one major polypeptide of an apparent molecular weight of 36000 is present in the fraction of prolamellar bodies. This polypeptide was identified as the NADPH-protochlorophyllide oxidoreductase. The highest specific activity of the enzyme in etiolated leaf tissue was confirmed to be in the fraction of prolamellar bodies.

A Comparison between Prolamellar Bodies and Prothylakoid Membranes of Etioplasts of Dark-Grown Wheat Concerning Lipid and Polypeptide Composition

The aim of the present investigation was to find factors critical for the co-existence of prolamellar bodies and prothylakoids in etioplasts of wheat (Triticum aestivum L. cv Starke II). The lipid composition of the prolamellar body and prothylakoid fractions was qualitatively similar. However, the molar ratio of monogalactosyl diacylglycerol to digalactosyl diacylglycerol was higher in the prolamellar body fraction (1.6 +/- 0.1), as was the lipid content on a protein basis. Protochlorophyllide was present in both fractions. The dominating protein of the prolamellar body fraction was protochlorophyllide oxidoreductase. This protein was present also in prothylakoid fractions. The other major protein of the prothylakoid fraction was the coupling factor 1, subunit of the chloroplast ATPase. From the lipid and protein data, we conclude that prolamellar bodies are formed when monogalactosyl diacylglycerol is present in larger amounts than can be stabilized into planar bilayer prothylakoid membranes by lamellar lipids or proteins.

Lipid composition of prolamellar bodies and prothylakoids of wheat etioplasts

Prolamellar bodies and prothylakoids were fractionated from etioplasts of wheat (Triticum aestivum L., cv. Starke II, Weibull) and characterized with emphasis on lipid composition. The two fractions contained the same lipid classes. Glycolipids (monogalactosyl diacylglycerol, digalactosyl diacylglycerol, and sulphoquinovosyl diacylglycerol) were the dominating complex lipids. Phospholipids (mainly phosphatidyl choline and phosphatidyl glycerol) constituted between 10 and 15 mol% of the total amounts of polar lipids. Free sterols and sterol esters were present in low amounts (ca 6 mol%). Saponins could not be detected. The contents of glycolipids and protochlorophyllide were higher in the prolamellar body fraction than in the prothylakoid fraction on a protein basis, as was the protochlorophyllide content on a glycolipid basis. The molar ratio of monogalactosyl diacylglycerol to digalactosyl diacylglycerol was higher in the prolamellar body fraction (1.8) than in the prothylakoid fraction (1.2).Since the same chemical constituents were found in the two membrane fractions we propose that the difference in ultrastructure between prolamellar bodies and prothylakoids is due to different relative amounts of lipids (glycolipids), protochlorophyllide, and proteins in the two membrane systems.

Characterization of prolamellar bodies and prothylakoids fractionated from wheat etioplasts

Prolamellar bodies and prothylakoids from etioplasts of wheat (Triticum aestivum L. cv. Starke II, Weibull) were separated by sucrose density gradient centrifugation. Top‐loaded and bottom‐loaded sucrose gradients were compared. As a consequence of avoiding long time exposure of the membranes to low sucrose concentrations, separation in bottom‐loaded gradients, as compared to separation in top‐loaded gradients, resulted in a sharper and more narrow band of prothylakoids, and in better preservation of phototransformable protochlorophyllide, especially in the prothylakoids. In bottom‐loaded gradients, the prothylakoids were found concentrated in a band at a density of 1.20 g'ml−1. The prolamellar bodies were found at a density of 1.17 g'ml−1. In top‐loaded gradients the prothylakoids were found at a lower density than the prolamellar bodies. The prothylakoid fraction contained about 60% of the recovered protochlorophyllide and about 85% of the recovered protein. Absorption and fluorescence emission spectra revealed a higher amount of phototransformable protochlorophyllide, in relation to non‐phototransformable, in the prolamellar body fraction than in the prothylakoid fraction. Polyacrylamide gel electrophoresis indicated a high proportion of protochlorophyllide reductase in the prolamellar bodies. Chloroplast ATPase (CF1) was found predominantly in the prothylakoid fraction. Thus, our results strongly indicate the presence of phototransformable protochlorophyllide in the prolamellar bodies proper, while the main bulk of proteins are located in the prothylakoids.

Spectral forms of protochlorophyllide in prolamellar bodies and prothylakoids fractionated from wheat etioplasts

The relation between the different protochlorophyllide (PChlide) forms in isolated etioplast inner membranes was dependent on the concentration of sucrose and NADPH in the isolation media. Etioplasts were prepared from wheat (Triticum aestivum L. cv. Starke II, Weibull) by differential centrifugation. The etioplasts were freed of envelope and stroma and the etioplast inner membranes were exposed to a concentration series of sucrose. Fluorescence emission spectra revealed a positive correlation between the emission ratio 657/633 nm and the sucrose concentration in which the membranes were suspended. Addition of NADPH prevented the degradation of 657 nm emission caused by low sucrose concentrations. PChlide already altered to PChide628–632 could not re‐form PChlide650–657 after the addition of NADPH in darkness. Prolamellar bodies and prothylakoids were separated in a bottom‐loaded sucrose density gradient in the presence of NADPH. The dominating PChlide‐protein complex in the prolamellar bodies was PClide650–657. Only minor amounts of PChlide628–632 were found in these membranes. The prothylakoids had a higher content of PChlide628–632, relative to PChlide650–657, than the prolamellar bodies, as judged from absorption and fluorescence spectra. After phototransformation the fluorescence emission at 633 nm increased relative to the emission from phototransformed PChlide indicating an efficient energy transfer between PChlide628–632 and PChlide650–657 before irradiation.

Ultrastructure and lipid composition of etioplasts in developing dark-grown wheat leaves

Changes in the ultrastructure and lipid composition of etioplasts have been evaluated in three regions from the base to the tip of 8-day-old darkgrown wheat leaves and in the upper-2/3 region of etiolated leaves of different ages. In developing darkgrown tissues, the main morphological changes that etioplasts undergo consist of an increase in the amount of thylakoïds which, in the most mature etioplasts, align in parallel arrays. Concomitantly, galactolipids and sulfolipid form an increasing proportion of the total lipids. Trans-3-hexadecenoic acid was not detectable in phosphatidylglycerol (PG) of etioplasts showing appressed thylakoïds isolated from 5-day-old leaves, but was present in significant amounts in etioplasts in the basal part of 8-day-ols leaves in which membrane appression was barely visible. The proportions of this acid increase as etioplasts develop, reaching 25% of the PG fatty acids (1.2% of the total fatty acids) in the most differentiated etioplasts. In wheat etioplasts, it appears that trans-3-hexadecenoic acid may accumulate in considerable amounts in darkgrown tissues and that its accumulation is not directly involved in membrane appression.