Genetic study and further biochemical characterization of a tobacco mutant that overproduces sterols

Summary A genetic and biochemical characterization is presented of a tobacco mutant that was previously shown to have an increased sterol content with an accumulation of biosynthetic intermediates. We first show that a precise regulation of the membrane sterol composition occurs in this mutant, via a selective esterification process. Indeed, sterols representing the usual end-products of the biosynthetic pathway are preferably integrated into the membranes as free sterols, whereas most of the intermediates pool is esterified and stored in cytoplasmic lipid droplets. It is further demonstrated that overproduction of sterols by the LAB1-4 mutant is due to a single nuclear and semi-dominant mutation. Finally, increase of biosynthesis and esterification of unusual sterols are shown to be responsible for the resistance of LAB1-4 calli to LAB170 250F, the triazole pesticide used to select this mutant. However, differentiated LAB1-4 tissues do not express the resistance trait, suggesting that sterol biosynthesis might not be the only site of action for the triazole at the plant level.     

Increased sterol biosynthesis in tobacco calli resistant to a triazole herbicide which inhibits demethylation of 14α-methyl sterols

The γ-keto triazole derivative 4,4-dimethyl-1-(2-methoxyphenyl)-1-(1,2,4-triazol-1-yl)-1-penten-3-one is toxic to Nicotiana tabacum L. cv. Xanthi plants or cell cultures. Analysis of the sterol composition of treated wild-type plant material demonstrates that this herbicide is an inhibitor of the C-14α-methyl demethylation process in sterol biosynthesis. Selection experiments, consisting of screening large populations of microcalli derived from UV-mutagenized tobacco protoplasts for resistance to a lethal dose (1 mg · 1^?1) of the γ-keto triazole, have resulted in the recovery of two groups of resistant calli. In the first group, selected calli show a sterol composition in the absence or presence of the inhibitor very similar to that of wild-type sensitive calli, whereas in the second group the main feature of the selected calli is a new sterol profile. These calli present an overproduction of sterols with a concomitant esterification of overproduced metaolites, just as it was demonstrated for calli previously selected in our laboratory for resistance to LAB 170250F, a triazole fungicide (Maillot-Vernier et al., 1991, Mol. Gen. Genet. 231, 33–40).     

Expression of the Hevea brasiliensis (H.B.K.) Mull. Arg. 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase 1 in Tobacco Results in Sterol Overproduction

A genomic fragment encoding one (HMGR1) of the three 3-hydroxy-3-methylglutaryl coenzyme A reductases (HMGRs) from Hevea brasiliensis (H.B.K.) Mull. Arg. (M.-L. Chye, C.-T. Tan, N.-H. Chua [1992] Plant Mol Biol 19: 473-484) was introduced into Nicotiana tabacum L. cv xanthi via Agrobacterium transformation to study the influence of the hmg1 gene product on plant isoprenoid biosynthesis. Transgenic plants were morphologically indistinguishable from control wild-type plants and displayed the same developmental pattern. Transgenic lines showed an increase in the level of total sterols up to 6-fold, probably because of an increased expression level of hmg1 mRNA and a corresponding increased enzymatic activity for HMGR, when compared with the level of total sterols from control lines not expressing the hmg1 transgene. In addition to the pathway end products, campesterol, sitosterol, and stigmasterol, some biosynthetic intermediates such as cycloartenol also accumulated in transgenic tissues. Most of the overproduced sterols were detected as steryl-esters and were likely to be stored in cytoplasmic lipid bodies. These data strongly support the conclusion that plant HMGR is a key limiting enzyme in phytosterol biosynthesis.     

The effects of altered membrane sterol composition on oxidative phosphorylation in a haem mutant of Saccharomyces cerevisiae

1. The sterol, unsaturated fatty acid and cytochrome contents of cells of a delta-aminolaevulinate synthase mutant of Saccharomyces cerevisiae are manipulated by growing the organism in media containing defined supplements of delta-aminolaevulinate and other porphyrin intermediates. 2. If unsaturated fatty acids are added to the growth medium as Tween 80, sterol content and respiratory cytochromes alone are manipulated. 3. In the presence of delta-aminolaevulinate (10-50mg/1) cells exhibit moderate to high respiratory activity, but growth yields are low, indicating a loss of oxidative phosphorylation. This is associated with the depletion of membrane lipids, either unsaturated fatty acids and sterols together or sterols alone. 4. Sterol depletion leads to the loss of coupled mitochondrial oxidative phosphorylation in vitro. 5. The lesion in oxidative phosphorylation is associated with an increase in the passive permeability of sterol-depleted mitochondria to protons. 6. Arrhenius plots of mitochondrial permeability to protons indicate that the activation energy for proton entry increases as the sterol content of the membranes decreases. 7. Studies on a cytoplasmic petite mutant isolated from strain ole-3, which lacks a functional membrane-bound protein-translocating adenosine triphosphatase, indicate that proton permeability of the petite mitochondria varies as a function of sterol composition in the same way as that of ole-3 grande mitochondria. This indicates that sterols alone are probably directly responsible for the increased proton entry, owing to a reorganization of the lipid in the membrane. 8. Supplemented ole-3 cells with a normal lipid composition and normal or higher than normal respiratory activities have a growth efficiency only 65% of that of the wild-type, indicating that a further lesion in energy metabolism may be present.     

Cellular sterol ester synthesis in plants is performed by an enzyme (phospholipid:sterol acyltransferase) different from the yeast and mammalian acyl-CoA:sterol acyltransferases

A gene encoding a sterol ester-synthesizing enzyme was identified in Arabidopsis. The cDNA of the Arabidopsis gene At1g04010 (AtPSAT) was overexpressed in Arabidopsis behind the cauliflower mosaic virus 35S promoter. Microsomal membranes from the leaves of overexpresser lines catalyzed the transacylation of acyl groups from phosphatidylethanolamine to sterols. This activity correlated with the expression level of the AtPSAT gene, thus demonstrating that this gene encodes a phospholipid:sterol acyltransferase (PSAT). Properties of the AtPSAT were examined in microsomal fractions from the tissues of an overexpresser. The enzyme did not utilize neutral lipids, had the highest activity with phosphatidylethanolamine, had a 5-fold preference for the sn-2 position, and utilized both saturated and unsaturated fatty acids. Various sterols and sterol intermediates, including triterpenic precursors, were acylated by the PSAT, whereas other triterpenes were not. Sterol selectivity studies showed that the enzyme is activated by end product sterols and that sterol intermediates are preferentially acylated by the activated enzyme. This indicates that PSAT both regulates the pool of free sterols as well as limits the amount of free sterol intermediates in the membranes. Two T-DNA insertion mutants in the AtPSAT gene, with strongly reduced (but still measurable) levels of sterol esters in their tissues, had no detectable PSAT activity in the microsomal fractions, suggesting that Arabidopsis possess other enzyme(s) capable of acylating sterols. The AtPSAT is the only intracellular enzyme found so far that catalyzes an acyl-CoA-independent sterol ester formation. Thus, PSAT has a similar physiological function in plant cells as the unrelated acyl-CoA:sterol acyltransferase has in animal cells.     

In Vitro Selection of Calli Resistant to a Triazole Cytochrome-P-450-Obtusifoliol-14-Demethylase Inhibitor from Protoplasts of Nicotiana tabacum L. cv Xanthi

The selection of biochemical mutants has been undertaken in order to elucidate regulatory and functional aspects of sterol biosynthesis in plants. 2-(4-Chlorophenyl)-3-phenyl-1-(1H-1,2,4- triazol-1-yl)-2,3-oxidopropane (LAB170250F), an experimental fungicide of the triazole family, was used as a selective agent. Indeed, this compound is a strong inhibitor of the cytochrome-P-450-obtusifoliol-14-demethylase in sterol biosynthesis. The selection strategy consisted of screening large populations of microcalli derived from ultraviolet-mutagenized protoplasts of Nicotiana tabacum L. cv Xanthi for resistance to a lethal concentration of LAB170250F. The best selective conditions were first determined, i.e. strength of the selection pressure as well as the time and duration of its application in the developmental process from protoplast to whole plant. Selection experiments resulted in the recovery of 40 resistant calli. These calli were divided into three classes according to the modification of their sterol content in response to LAB170250F. Some of these calli might be impaired in sterol biosynthesis, but most have a sterol profile identical to that of the control calli. This suggests that the toxic properties of LAB170250F are due to the parallel inhibition of sterol biosynthesis and of at least one additional unidentified target in the plant cell.     

A chilling sensitive mutant of Arabidopsis with altered steryl-ester metabolism

A chilling-sensitive mutant of Arabidopsis thaliana was isolated and subjected to genetic, physiological, and biochemical analysis. The chilling-sensitive nature of the mutant line is due to a single recessive nuclear mutation at a locus designated chs1. In contrast to wild-type plants, which are not adversely affected by low temperatures, the chs1 mutant is killed by several days of exposure to temperatures below 18°C. Following exposure to chilling temperatures, the mutant displays two common symptoms of chilling injury—leaf chlorosis and electrolyte leakage. In these respects, the physiological response of the mutant to low temperatures mimics the response observed in some naturally occurring chilling sensitive species. The biochemical basis of chilling sensitivity was explored by examining the pattern of incorporation of ¹⁴CO₂ into soluble metabolites and lipids in wild-type and mutant plants. The only difference observed between the mutant and wild type was that following low temperature treatment, the mutant accumulated 10-fold more radioactivity in a specific class of neutral lipids which were identified by a variety of criteria to be steryl-esters. The accumulation of radioactivity in the steryl-ester fraction occurs 24 hours before there is any visible evidence of chilling injury. These results suggest one of two possible explanations: either the mutation directly affects sterol metabolism, which in turn leads to chilling sensitivity, or the mutation affects another unidentified function and the accumulation of radioactivity in steryl-esters is a secondary consequence of chilling injury.     

Fragility of plasma membranes in Saccharomyces cerevisiae enriched with different sterols.

Saccharomyces cerevisiae NCYC 366, grown under strictly anaerobic conditions to induce requirements for an unsaturated fatty acid (supplied by Tween 80) and a sterol, contained free sterol fractions enriched to the extent of 67 to 93% with the exogenously supplied sterol (campesterol, cholesterol, 7-dehydrocholesterol, 22, 23-dihydrobrassicasterol, beta-sitosterol, or stigmasterol). Cells enriched in any one of the sterols did not differ in volume, growth rate, contents of free sterol, esters and phospholipids, or phospholipid composition. Cholesterol-enriched cells contained about 2% more lipid than cells enriched in any of the other sterols, which was largely accounted for by increased contents of triacylglycerols and, to a lesser extent, esterified sterols. Phospholipids were enriched to the extent of about 52 to 63% with C18:1 residues. Cells enriched in ergosterol or stigmasterol were slightly less susceptible to the action of a wall-digesting basidiomycete glucanase than cells enriched with any one of the other sterols. The capacity of the plasma membrane to resist stretching, as indicated by the stability and volume of spheroplasts suspended in hypotonic solutions of buffered sorbitol (particularly in the range 0.9 to 0.7 M), was greater with spheroplasts enriched in sterols with an unsaturated side chain at C17 (ergosterol or stigmasterol) than with any of the other sterols. Plasma membranes were obtained from spheroplasts enriched in cholesterol or stigmasterol and had free sterol fractions containing 70 and 71%, respectively, of the sterol supplied exogenously to the cells. The sterol-phospholipid molar ratios in these membranes were, respectively, 1:7 and 1:8.     

Lipids of plasma membranes prepared from oat root cells : effects of induced water-deficit tolerance

Plasma membranes were isolated from oat (Avena sativa) roots by the phase-partitioning method. The membranes were exposed to repeated periods of moderate water-deficit stress, and a water-deficit tolerance was induced (acclimated plants). The plasma membranes of the controls (nonacclimated plants) were characterized by a high phospholipid content, 79% of total lipids, cerebrosides (9%) containing hydroxy fatty acids (>90% 24:1-OH) and free sterols, acylated sterylglucosides, sterylglucosides, and steryl esters, together amounting to 12%. Major phospholipids were phosphatidylcholine and phosphatidylethanolamine with lesser amounts of phosphatidylglycerol, phosphatidylinositol, and phosphatidic acid. After the membranes were acclimated to dehydration, the lipid to protein ratio decreased from 1.3 to 0.7 micromoles per milligram. Furthermore, the cerebrosides decreased to 5% and free sterols increased from 9% (nonacclimated plants) to 14%. Because the total phospholipids did not change significantly, the free sterol to phospholipid ratio increased from 0.12 to 0.19. There was no change in the relative distribution of sterols after acclimation. The ratio of phosphatidylcholine to phosphatidylethanolamine changed from 1.1 in the nonacclimated plants to 0.69 in the acclimated plants. The results show that acclimation to dehydration implies substantial alterations in the lipid composition of the plasma membrane.     

Reduction of cholesterol and glycoalkaloid levels in transgenic potato plants by overexpression of a type 1 sterol methyltransferase cDNA

Transgenic potato (Solanum tuberosum cv Désirée) plants overexpressing a soybean (Glycine max) type 1 sterol methyltransferase (GmSMT1) cDNA were generated and used to study sterol biosynthesis in relation to the production of toxic glycoalkaloids. Transgenic plants displayed an increased total sterol level in both leaves and tubers, mainly due to increased levels of the 24-ethyl sterols isofucosterol and sitosterol. The higher total sterol level was due to increases in both free and esterified sterols. However, the level of free cholesterol, a nonalkylated sterol, was decreased. Associated with this was a decreased glycoalkaloid level in leaves and tubers, down to 41% and 63% of wild-type levels, respectively. The results show that glycoalkaloid biosynthesis can be down-regulated in transgenic potato plants by reducing the content of free nonalkylated sterols, and they support the view of cholesterol as a precursor in glycoalkaloid biosynthesis.     

Overexpression of Arabidopsis thaliana farnesyl diphosphate synthase (FPS1S) in transgenic Arabidopsis induces a cell death/senescence-like response and reduced cytokinin levels

To investigate the contribution of farnesyl diphosphate synthase (FPS) to the overall control of the mevalonic acid pathway in plants, we have generated transgenic Arabidopsis thaliana overexpressing the Arabidopsis FPS1S isoform. Despite high levels of FPS activity in transgenic plants (8- to 12-fold as compared to wild-type plants), the content of sterols and the levels of 3-hydroxy-3-methylglutaryl-CoA reductase activity in leaves were similar to those in control plants. Plants overexpressing FPS1S showed a cell death/senescence-like phenotype and grew less vigorously than wild-type plants. The onset and the severity of these phenotypes directly correlated with the levels of FPS activity. In leaves of plants with increased FPS activity, the expression of the senescence activated gene SAG12 was prematurely induced. Transgenic plants grown in the presence of either mevalonic acid (MVA) or the cytokinin 2-isopentenyladenine (2-iP) recovered the wild-type phenotype. Quantification of endogenous cytokinins demonstrated that FPS1S overexpression specifically reduces the levels of endogenous zeatin-type cytokinins in leaves. Altogether these results support the notion that increasing FPS activity without a concomitant increase of MVA production leads to a reduction of IPP and DMAPP available for cytokinin biosynthesis. The reduced cytokinin levels would be, at least in part, responsible for the phenotypic alterations observed in the transgenic plants. The finding that wild-type and transgenic plants accumulated similar increased amounts of sterols when grown in the presence of exogenous MVA suggests that FPS1S is not limiting for sterol biosynthesis.     

Lipid composition of plasma membranes isolated from sunflower seedlings grown under water-stress

Plasma membranes were isolated by aqueous two-phase-partitioning from sunflower ( Helianthus annuus cv. Isabel) seedlings grown both under field irrigation and dryland conditions. Water-stressed plants showed a decrease in the leaf water potential and in the osmotic potential at full turgor, with the turgor pressure remaining at positive values. Dryland conditions also induced a reduction in the bulk modulus of elasticity. Plasma membranes of irrigated plants were characterized by high contents of phospholipids (68% of total lipids), free sterols (15. 7%) and glycolipids (9. 1%), mainly glycosphingolipids and steryl glycosides. Diacylglycerols, triacylglycerols and free fatty acids were also present. The major phospholipids were phosphatidylcholine and phosphatidylethanolamine with smaller amounts of phosphatidylinositol and phosphatidylglycerol. Following water stress, the plasma membranes showed a reduction of about 24 and 31% in total lipids and phospholipids, respectively. Also the amounts of glycolipids and diacylglycerols decreased significantly upon water stress. There was no change in free fatty acids, however, and triacylglycerols and free sterols increased. As a consequence, the free sterol to phospholipid molar ratio increased from 0. 4 to 0. 7 under water deficit conditions. The ratio of phosphatidylcholine to phosphatidylethanolamine increased from 1. 1 (control plants) to 1. 6 (water-stressed plants), while phosphatidic acid rose to 4% of total phospholipids. Dehydration did not result in any substantial change in the unsaturation level of the individual lipid classes, however. The results show that dryland conditions resulted in a marked alteration in the lipid composition of the sunflower leaf plasma membrane     

Functional expression of Saccharomyces cerevisiae CYP51A1 encoding lanosterol-14-demethylase in tobacco results in bypass of endogenous sterol biosynthetic pathway and resistance to an obtusifoliol-14-demethylase herbicide inhibitor

Nicotiana tabacum protoplasts have been transformed by Agrobacterium tumefaciens containing a T-DNA in which the gene CYP51A1 encoding lanosterol-14-demethylase (LAN14DM) from Saccharomyces cerevisiae is under the control of a cauliflower mosaic virus (CaMV) 35S promoter. Two transformants strongly expressed the LAN14DM as shown by Northern and Western experiments. These transgenic calli were killed by LAB 170250F (LAB) (a phytotoxic fungicide inhibiting both plant obtusifoliol-14-demethylase (OBT14DM) and LAN14DM) but were resistant to γ-ketotriazole (γ-kt), a herbicide which has been shown to inhibit OBT14DM but not LAN14DM at a concentration that was lethal to control calli. However, these transgenic calli were killed by mixtures of γ-kt plus fungicide inhibitors of LAN14DM such as ketoconazole, itraconazole or flusilazole which alone were not effective. Further analysis of the transgenic calli grown in the presence of γ-kt showed that their Δ⁵-sterol content was close to that of untreated control calli obtained from protoplasts transformed with control plasmid; this is in agreement with evidence that the LAN14DM expressed from the transgene could bypass the blocked OBT14DM by using the plant substrate obtusifoliol. In contrast, control calli when treated with γ-kt, displayed a sterol content strongly enriched in 14α-methyl sterols and depressed in physiological Δ⁵-sterols. When the transgenic calli were cultured in mixtures of γ-kt and LAN14DM inhibitors sterol compositions enriched in 14α-methyl sterols were obtained, reflecting a strong inhibition of both ‘endogenous’ OBT14DM and ‘exogenous’ LAN14DM. Taken together these results show that in tobacco calli transformed with CYP51A1, resistance to a triazole herbicide arises from expression of a functional LAN14DM enzyme; its activity in transgenic tissues creates a bypass of the sterol biosynthetic pathway at the 14-demethylase level when this latter is blocked by an OBT14DM herbicide inhibitor.     

Targeted mutation of the MLN64 START domain causes only modest alterations in cellular sterol metabolism

The StAR-related lipid transfer (START) domain, first identified in the steroidogenic acute regulatory protein (StAR), is involved in the intracellular trafficking of lipids. Sixteen mammalian START domain-containing proteins have been identified to date. StAR, a protein targeted to mitochondria, stimulates the movement of cholesterol from the outer to the inner mitochondrial membranes, where it is metabolized into pregnenolone in steroidogenic cells. MLN64, the START domain protein most closely related to StAR, is localized to late endosomes along with other proteins involved in sterol trafficking, including NPC1 and NPC2, where it has been postulated to participate in sterol distribution to intracellular membranes. To investigate the role of MLN64 in sterol metabolism, we created mice with a targeted mutation in the Mln64 START domain, expecting to find a phenotype similar to that in humans and mice lacking NPC1 or NPC2 (progressive neurodegenerative symptoms, free cholesterol accumulation in lysosomes). Unexpectedly, mice homozygous for the Mln64 mutant allele were viable, neurologically intact, and fertile. No significant alterations in plasma lipid levels, liver lipid content and distribution, and expression of genes involved in sterol metabolism were observed, except for an increase in sterol ester storage in mutant mice fed a high fat diet. Embryonic fibroblast cells transfected with the cholesterol side-chain cleavage system and primary cultures of granulosa cells from Mln64 mutant mice showed defects in sterol trafficking as reflected in reduced conversion of endogenous cholesterol to steroid hormones. These observations suggest that the Mln64 START domain is largely dispensable for sterol metabolism in mice.     

Role of sterol structure in the thermotropic behavior of plasma membranes of Saccharomyces cerevisiae

Plasma membranes from Saccharomyces cerevisiae were prepared by a new procedure involving lyticase treatment of the yeast cells. The plasma membranes were right-side-out, closed vesicles of uniform appearance with a sterol to phospholipid molar ratio of 0.365. The thermotropic behavior of these plasma membranes from wild-type yeast and from sterol mutants was examined by differential scanning calorimetry, fluorescence anisotropy and Arrhenius kinetics of plasma membrane enzymes. While differential scanning calorimetry failed to demonstrate any lipid transition, fluorescence anisotropy data indicated that lipid transitions were occurring in the plasma membranes of the yeast sterol mutants but not the sterol wild-type. The temperature dependence of the plasma membrane enzymes, chitin synthase and Mg²⁺-ATPase, was also investigated. The Arrhenius kinetics of chitin synthase did not reveal any transitions in either the sterol mutant or wild-type plasma membranes, yet the Arrhenius kinetics of the Mg²⁺-ATPase suggested that lipid transitions were occurring in both cases.     

Effects of triadimefon and osmotic stress on plasma membrane composition and ATPase activity in white spruce (Picea glauca) needles

White spruce [ Picea glauca (Moench) Voss.] seedlings were grown in solution culture and treated with 20 mg I^-1 triadimefon [1-(chlorophenoxy)-3,3-dimethyl-1-(1,2,4-triazol-1-yl)-2-butanol] for 4 weeks and then subjected to osmotic stress with polyethylene glycol 3350. Water potentials and electrolyte leakage were measured in control and triadimefon-treated seedlings before and after the plants were subjected to osmotic stress. The plasma membranes were isolated from needles to study their lipid composition and the activity of plasma-membrane bound ATPase. Triadimefon treatment reduced water potentials and increased leakage of electrolytes from seedlings. However, when the seedlings were exposed to osmotic stress, triadimefon-treated plants maintained higher water potentials and leaked less electrolytes compared with the control plants. Both triadimefon and osmotic stress treatments inhibited the activity of plasma membrane-bound ATPase and altered the composition of free sterols in the plasma membranes. Triadimefon-treated plants contained traces of campesterol, which was not present in control. Osmotic stress drastically reduced phospholipid:protein and sterol:protein ratios and increased sterol:phospholipid ratios in the plasma membranes     

Mechanisms of sterol uptake and transport in yeast

Sterols are essential lipid components of eukaryotic membranes. Here we summarize recent advances in understanding how sterols are transported between different membranes. Baker's yeast is a particularly attractive organism to dissect this lipid transport pathway, because cells can synthesize their own major sterol, ergosterol, in the membrane of the endoplasmic reticulum from where it is then transported to the plasma membrane. However, Saccharomyces cerevisiae is also a facultative anaerobic organism, which becomes sterol auxotroph in the absence of oxygen. Under these conditions, cells take up sterol from the environment and transport the lipid back into the membrane of the endoplasmic reticulum, where the free sterol becomes esterified and is then stored in lipid droplets. Steryl ester formation is thus a reliable readout to assess the back-transport of exogenously provided sterols from the plasma membrane to the endoplasmic reticulum. Structure/function analysis has revealed that the bulk membrane function of the fungal ergosterol can be provided by structurally related sterols, including the mammalian cholesterol. Foreign sterols, however, are subject to a lipid quality control cycle in which the sterol is reversibly acetylated. Because acetylated sterols are efficiently excreted from cells, the substrate specificity of the deacetylating enzymes determines which sterols are retained. Membrane-bound acetylated sterols are excreted by the secretory pathway, more soluble acetylated sterol derivatives such as the steroid precursor pregnenolone, on the other hand, are excreted by a pathway that is independent of vesicle formation and fusion. Further analysis of this lipid quality control cycle is likely to reveal novel insight into the mechanisms that ensure sterol homeostasis in eukaryotic cells. Article from a special issue on Steroids and Microorganisms.     

Plant sterols: Diversity,biosynthesis, and physiological functions

Sterols, which are isoprenoid derivatives, are structural components of biological membranes. Special attention is now being given not only to their structure and function, but also to their regulatory roles in plants. Plant sterols have diverse composition; they exist as free sterols, sterol esters with higher fatty acids, sterol glycosides, and acylsterol glycosides, which are absent in animal cells. This diversity of types of phytosterols determines a wide spectrum of functions they play in plant life. Sterols are precursors of a group of plant hormones, the brassinosteroids, which regulate plant growth and development. Furthermore, sterols participate in transmembrane signal transduction by forming lipid microdomains. The predominant sterols in plants are β-sitosterol, campesterol, and stigmasterol. These sterols differ in the presence of a methyl or an ethyl group in the side chain at the 24th carbon atom and are named methylsterols or ethylsterols, respectively. The balance between 24-methylsterols and 24-ethylsterols is specific for individual plant species. The present review focuses on the key stages of plant sterol biosynthesis that determine the ratios between the different types of sterols, and the crosstalk between the sterol and sphingolipid pathways. The main enzymes involved in plant sterol biosynthesis are 3-hydroxy-3methylglutaryl-CoA reductase, C24-sterol methyltransferase, and C22-sterol desaturase. These enzymes are responsible for maintaining the optimal balance between sterols. Regulation of the ratios between the different types of sterols and sterols/sphingolipids can be of crucial importance in the responses of plants to stresses.     

Biochemical and immunohistochemical analysis of pectic polysaccharides in the cell walls of Arabidopsis mutant QUASIMODO 1 suspension-cultured cells: implications for cell adhesion

Mutation in the Arabidopsis thaliana QUASIMODO 1 gene (QUA1), which encodes a putative glycosyltransferase, reduces cell wall pectin content and cell adhesion. Suspension-cultured calli were generated from roots of wild-type (wt) and qua1-1 A. thaliana plants. The altered cell adhesion phenotype of the qua1-1 plant was also found with its suspension-cultured calli. Cell walls of both wt and qua1-1 calli were analysed by chemical, enzymatic and immunohistochemical techniques in order to assess the role of pectic polysaccharides in the mutant phenotype. Compared with the wt, qua1-1 calli cell walls contained more arabinose (23.6 versus 21.6 mol%), rhamnose (3.1 versus 2.7 mol%), and fucose (1.4 versus 1.2 mol%) and less uronic acid (24.2 versus 27.6 mol%), and they were less methyl-esterified (DM: 22.9% versus 30.3%). When sequential pectin extraction of calli cell walls was performed, qua1-1 water-soluble and chelator-soluble extracts contained more arabinose and less uronic acid than wt. Water-soluble pectins were less methyl-esterified in qua1-1 than in wt. Chelator-soluble pectins were more acetyl-esterified in qua1-1. Differences in the cell wall chemistry of wt and mutant calli were supported by a reduction in JIM7 labelling (methyl-esterified homogalacturonan) of the whole wall in small cells and particularly by a reduced labelling with 2F4 (calcium-associated homogalacturonan) in the middle lamella at tricellular junctions of large qua1-1 cells. Differences in the oligosaccharide profile obtained after endopolygalacturonase degradation of alkali extracts from qua1-1 and wt calli indicated variations in the structure of covalently bonded homogalacturonan. About 29% more extracellular polymers rich in pectins were recovered from the calli culture medium of qua1-1 compared with wt. These results show that perturbation of QUASIMODO 1-1 gene expression in calli resulted in alterations of homogalacturonan content and cell wall location. The consequences of these structural variations are discussed with regard to plant cell adhesion.     

Sterol-induced upregulation of phosphatidylcholine synthesis in cultured fibroblasts is affected by the double-bond position in the sterol tetracyclic ring structure.

We have examined how a specific enrichment of cultured fibroblasts with various sterols (cholesterol, lathosterol, 7-dehydrocholesterol, allocholesterol and dihydrocholesterol) regulate synthesis de novo of phosphatidylcholine, cholesterol and cholesteryl (or steryl) esters in human skin fibroblasts. When human skin fibroblasts were incubated for 1 h with 130 microM cholesterol/CyD complexes, the mass of cellular free cholesterol increased by 100 nmol.mg-1 protein (from 90 nmol.mg-1 to 190 nmol.mg-1 protein). A similar exposure of cells to different sterol/CyD complexes increased the cell sterol content between 38 and 181 nmol sterol per mg cell protein. In cholesterol-enriched cells, the rate of phosphatidylcholine synthesis was doubled compared to control cells, irrespective of the type of precursor used ([3H]choline, [3H]palmitic acid, or [14C]glycerol). Enrichment of fibroblasts with 7-dehydrocholesterol, allocholesterol, or dihydrocholesterol also upregulated phosphatidylcholine synthesis, whereas cells enriched with lathosterol failed to upregulate their phosphatidylcholine synthesis. The activity of membrane-bound CTP:phosphocholine cytidylyltransferase, the rate-limiting enzyme, was increased by 47 +/- 4% in cholesterol-enriched cells whereas its activity was unchanged in lathosterol-enriched cells. Sterol enrichment with all tested sterols (including lathosterol) down-regulated acetate-incorporation into cholesterol, and upregulated sterol esterification in the sterol-enriched fibroblasts. Using 31P-NMR to measure the lamellar-to-hexagonal (Lalpha-HII) phase transition in multilamellar lipid dispersions, lathosterol-containing membranes underwent their transition at significantly higher temperatures compared to membranes containing any of the other sterols. In a system with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and either cholesterol or lathosterol (70:30 mol/mol), differential scanning calorimetry also revealed that the Lalpha-HII-transition occurred at a higher temperature with lathosterol compared to either cholesterol, allocholesterol, or dihydrocholesterol. These findings together suggest that there may exist a correlation between the propensity of a sterol to stabilize the Lalpha-HII-transition and its capacity to upregulate the activity of CTP:phosphocholine cytidylyltransferase in cells.