Search

Background: Sterols are structural and functional components of eukaryotic cell membranes. Plants produce a complex mixture of sterols, among which β-sitosterol, stigmasterol, campesterol, and cholesterol in some Solanaceae, are the most abundant species. Many reports have shown that the stigmasterol to β-sitosterol ratio changes during plant development and in response to stresses, suggesting that it may play a role in the regulation of these processes. In tomato (Solanum lycopersicum), changes in the stigmasterol to β-sitosterol ratio correlate with the induction of the only gene encoding sterol C22-desaturase (C22DES), the enzyme specifically involved in the conversion of β-sitosterol to stigmasterol. However, despite the biological interest of this enzyme, there is still a lack of knowledge about several relevant aspects related to its structure and function., Results: In this study we report the subcellular localization of tomato C22DES in the endoplasmic reticulum (ER) based on confocal fluorescence microscopy and cell fractionation analyses. Modeling studies have also revealed that C22DES consists of two well-differentiated domains: a single N-terminal transmembrane-helix domain (TMH) anchored in the ER-membrane and a globular (or catalytic) domain that is oriented towards the cytosol. Although TMH is sufficient for the targeting and retention of the enzyme in the ER, the globular domain may also interact and be retained in the ER in the absence of the N-terminal transmembrane domain. The observation that a truncated version of C22DES lacking the TMH is enzymatically inactive revealed that the N-terminal membrane domain is essential for enzyme activity. The in silico analysis of the TMH region of plant C22DES revealed several structural features that could be involved in substrate recognition and binding., Conclusions: Overall, this study contributes to expand the current knowledge on the structure and function of plant C22DES and to unveil novel aspects related to plant sterol metabolism.

Free and glycosylated sterols are both structural components of the plasma membrane that regulate their biophysical properties and consequently different plasma membrane-associated processes such as plant adaptation to stress or signaling. Several reports relate changes in glycosylated sterols levels with the plant response to abiotic stress, but the information about the role of these compounds in the response to biotic stress is scarce. In this work, we have studied the response to the necrotrophic fungus Botrytis cinerea in an Arabidopsis mutant that is severely impaired in steryl glycosides biosynthesis due to the inactivation of the two sterol glucosyltransferases (UGT80A2 and UGT80B1) reported in this plant. This mutant exhibits enhanced resistance against B. cinerea when compared to wild-type plants, which correlates with increased levels of jasmonic acid (JA) and up-regulation of two marker genes ( PDF1.2 and PR4 ) of the ERF branch of the JA signaling pathway. Upon B. cinerea infection, the ugt80A2;B1 double mutant also accumulates higher levels of camalexin, the major Arabidopsis phytoalexin, than wild-type plants. Camalexin accumulation correlates with enhanced transcript levels of several cytochrome P450 camalexin biosynthetic genes, as well as of their transcriptional regulators WRKY33 , ANAC042 , and MYB51 , suggesting that the Botrytis -induced accumulation of camalexin is coordinately regulated at the transcriptional level. After fungus infection, the expression of genes involved in the indole glucosinolate biosynthesis is also up-regulated at a higher degree in the ugt80A2;B1 mutant than in wild-type plants. Altogether, the results of this study show that glycosylated sterols play an important role in the regulation of Arabidopsis response to B. cinerea infection and suggest that this occurs through signaling pathways involving the canonical stress-hormone JA and the tryptophan-derived secondary metabolites camalexin and possibly also indole glucosinolates., (Copyright © 2019 Castillo, Pastor, Chávez, Arró, Boronat, Flors, Ferrer and Altabella.)

Steryl esters (SEs) serve as a storage pool of sterols that helps to maintain proper levels of free sterols (FSs) in cell membranes throughout plant growth and development, and participates in the recycling of FSs and fatty acids released from cell membranes in aging tissues. SEs are synthesized by sterol acyltransferases, a family of enzymes that catalyze the transfer of fatty acil groups to the hydroxyl group at C-3 position of the sterol backbone. Sterol acyltransferases are categorized into acyl-CoA:sterol acyltransferases (ASAT) and phospholipid:sterol acyltransferases (PSAT) depending on whether the fatty acyl donor substrate is a long-chain acyl-CoA or a phospolipid. Until now, only Arabidopsis ASAT and PSAT enzymes (AtASAT1 and AtPSAT1) have been cloned and characterized in plants. Here we report the identification, cloning, and functional characterization of the tomato ( Solanum lycopersicum cv. Micro-Tom) orthologs. SlPSAT1 and SlASAT1 were able to restore SE to wild type levels in the Arabidopsis psat1-2 and asat1-1 knock-out mutants, respectively. Expression of SlPSAT1 in the psat1-2 background also prevented the toxicity caused by an external supply of mevalonate and the early senescence phenotype observed in detached leaves of this mutant, whereas expression of SlASAT1 in the asat1-1 mutant revealed a clear substrate preference of the tomato enzyme for the sterol precursors cycloartenol and 24-methylene cycloartanol. Subcellular localization studies using fluorescently tagged SlPSAT1 and SlASAT1 proteins revealed that SlPSAT1 localize in cytoplasmic lipid droplets (LDs) while, in contrast to the endoplasmic reticulum (ER) localization of AtASAT1, SlASAT1 resides in the plasma membrane (PM). The possibility that PM-localized SlASAT1 may act catalytically in trans on their sterol substrates, which are presumably embedded in the ER membrane, is discussed. The widespread expression of SlPSAT1 and SlASAT1 genes in different tomato organs together with their moderate transcriptional response to several stresses suggests a dual role of SlPSAT1 and SlASAT1 in tomato plant and fruit development and the adaptive responses to stress. Overall, this study contributes to enlarge the current knowledge on plant sterol acyltransferases and set the basis for further studies aimed at understanding the role of SE metabolism in tomato plant growth and development.

Isoprenoids comprise the largest class of natural compounds and are found in all kinds of organisms. In plants, they participate in both primary and specialized metabolism, playing essential roles in nearly all aspects of growth and development. The enormous diversity of this family of compounds is extensively exploited for biotechnological and biomedical applications as biomaterials, biofuels or drugs. Despite their variety of structures, all isoprenoids derive from the common C 5 precursor isopentenyl diphosphate (IPP). Plants synthesize IPP through two different metabolic pathways, the mevalonic acid (MVA) and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways that operate in the cytosol-RE and plastids, respectively. MEP-derived isoprenoids include important compounds for chloroplast function and as such, knock-out mutant plants affected in different steps of this pathway display important alterations in plastid structure. These alterations often lead to albino phenotypes and lethality at seedling stage. MVA knock-out mutant plants show, on the contrary, lethal phenotypes already exhibited at the gametophyte or embryo developmental stage. However, the recent characterization of conditional knock-down mutant plants of farnesyl diphosphate synthase (FPS), a central enzyme in cytosolic and mitochondrial isoprenoid biosynthesis, revealed an unexpected role of this pathway in chloroplast development and plastidial isoprenoid metabolism in post-embryonic stages. Upon FPS silencing, chloroplast structure is severely altered, together with a strong reduction in the levels of MEP pathway-derived major end products. This phenotype is associated to misregulation of genes involved in stress responses predominantly belonging to JA and Fe homeostasis pathways. Transcriptomic experiments and analysis of recent literature indicate that sterols are the cause of the observed alterations through an as yet undiscovered mechanism.

In plants, sterols are found in free form (free sterols, FSs) and conjugated as steryl esters (SEs), steryl glycosides (SGs) and acyl steryl glycosides (ASGs). Conjugated sterols are ubiquitously found in plants but their relative contents highly differ among species and their profile may change in response to developmental and environmental cues. SEs play a central role in membrane sterol homeostasis and also represent a storage pool of sterols in particular plant tissues. SGs and ASGs are main components of the plant plasma membrane (PM) that specifically accumulate in lipid rafts, PM microdomains known to mediate many relevant cellular processes. There are increasing evidences supporting the involvement of conjugated sterols in plant stress responses. In spite of this, very little is known about their metabolism. At present, only a limited number of genes encoding enzymes participating in conjugated sterol metabolism have been cloned and characterized in plants. The aim of this review is to update the current knowledge about the tissue and cellular distribution of conjugated sterols in plants and the enzymes involved in their biosynthesis. We also discuss novel aspects on the role of conjugated sterols in plant development and stress responses recently unveiled using forward- and reverse-genetic approaches., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Sterol glycosyltransferases (SGTs) catalyze the glycosylation of the free hydroxyl group at C-3 position of sterols to produce sterol glycosides. Glycosylated sterols and free sterols are primarily located in cell membranes where in combination with other membrane-bound lipids play a key role in modulating their properties and functioning. In contrast to most plant species, those of the genus Solanum contain very high levels of glycosylated sterols, which in the case of tomato may account for more than 85% of the total sterol content. In this study, we report the identification and functional characterization of the four members of the tomato ( Solanum lycopersicum cv. Micro-Tom) SGT gene family. Expression of recombinant SlSGT proteins in E. coli cells and N. benthamiana leaves demonstrated the ability of the four enzymes to glycosylate different sterol species including cholesterol, brassicasterol, campesterol, stigmasterol, and β-sitosterol, which is consistent with the occurrence in their primary structure of the putative steroid-binding domain found in steroid UDP-glucuronosyltransferases and the UDP-sugar binding domain characteristic for a superfamily of nucleoside diphosphosugar glycosyltransferases. Subcellular localization studies based on fluorescence recovery after photobleaching and cell fractionation analyses revealed that the four tomato SGTs, like the Arabidopsis SGTs UGT80A2 and UGT80B1, localize into the cytosol and the PM, although there are clear differences in their relative distribution between these two cell fractions. The SlSGT genes have specialized but still largely overlapping expression patterns in different organs of tomato plants and throughout the different stages of fruit development and ripening. Moreover, they are differentially regulated in response to biotic and abiotic stress conditions. SlSGT4 expression increases markedly in response to osmotic, salt, and cold stress, as well as upon treatment with abscisic acid and methyl jasmonate. Stress-induced SlSGT2 expression largely parallels that of SlSGT4 . On the contrary, SlSGT1 and SlSGT3 expression remains almost unaltered under the tested stress conditions. Overall, this study contributes to broaden the current knowledge on plant SGTs and provides support to the view that tomato SGTs play overlapping but not completely redundant biological functions involved in mediating developmental and stress responses.

Farnesyl diphosphate synthase (FPS) catalyzes the synthesis of farnesyl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. Arabidopsis (Arabidopsis thaliana) contains two genes (FPS1 and FPS2) encoding FPS. Single fps1 and fps2 knockout mutants are phenotypically indistinguishable from wild-type plants, while fps1/fps2 double mutants are embryo lethal. To assess the effect of FPS down-regulation at postembryonic developmental stages, we generated Arabidopsis conditional knockdown mutants expressing artificial microRNAs devised to simultaneously silence both FPS genes. Induction of silencing from germination rapidly caused chlorosis and a strong developmental phenotype that led to seedling lethality. However, silencing of FPS after seed germination resulted in a slight developmental delay only, although leaves and cotyledons continued to show chlorosis and altered chloroplasts. Metabolomic analyses also revealed drastic changes in the profile of sterols, ubiquinones, and plastidial isoprenoids. RNA sequencing and reverse transcription-quantitative polymerase chain reaction transcriptomic analysis showed that a reduction in FPS activity levels triggers the misregulation of genes involved in biotic and abiotic stress responses, the most prominent one being the rapid induction of a set of genes related to the jasmonic acid pathway. Down-regulation of FPS also triggered an iron-deficiency transcriptional response that is consistent with the iron-deficient phenotype observed in FPS-silenced plants. The specific inhibition of the sterol biosynthesis pathway by chemical and genetic blockage mimicked these transcriptional responses, indicating that sterol depletion is the primary cause of the observed alterations. Our results highlight the importance of sterol homeostasis for normal chloroplast development and function and reveal important clues about how isoprenoid and sterol metabolism is integrated within plant physiology and development., (© 2016 American Society of Plant Biologists. All rights reserved.)

The first transgenes were introduced in a plant genome more than 30 years ago. Since then, the capabilities of the plant scientific community to engineer the genome of plants have progressed at an unparalleled speed. Plant genetic engineering has become a central technology that has dramatically incremented our basic knowledge of plant biology and has enabled the translation of this knowledge into a number of increasingly complex and sophisticated biotechnological applications, which in most cases rely on the simultaneous co-expression of multiple recombinant proteins from different origins. To meet the new challenges of modern plant biotechnology, the plant scientific community has developed a vast arsenal of innovative molecular tools and genome engineering strategies. In this chapter we review a variety of tools, technologies, and strategies developed to transfer and simultaneously co-express multiple transgenes and proteins in a plant host. Their potential advantages, disadvantages, and future prospects are also discussed.

The existence of multigenic families in the mevalonate pathway suggests divergent functional roles for pathway components involved in the biosynthesis of plant sterols. Squalene epoxidases (SQEs) are key components of this pathway, and Squalene Epoxidase 1 (SQE1) has been identified as a fundamental enzyme in this biosynthetic step. In the present work, we extended the characterization of the remaining SQE family members, phylogenetically resolving between true SQEs and a subfamily of SQE-like proteins that is exclusive to Brassicaceae. Functional characterization of true SQE family members, Squalene Epoxidase 2 (SQE2) and Squalene Epoxidase 3 (SQE3), indicates that SQE3, but not SQE2, contributes to the bulk SQE activity in Arabidopsis, with sqe3-1 mutants accumulating squalene and displaying sensitivity to terbinafine. We genetically demonstrated that SQE3 seems to play a particularly significant role in embryo development. Also, SQE1 and SQE3 both localize in the endoplasmic reticulum, and SQE3 can functionally complement SQE1. Thus, SQE1 and SQE3 seem to be two functionally unequal redundant genes in the promotion of plant SQE activity in Arabidopsis., (Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.)

The enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyzes the NADPH-mediated reductive deacylation of HMG-CoA to mevalonic acid, which is the first committed step of the mevalonate pathway for isoprenoid biosynthesis. In agreement with its key regulatory role in the pathway, plant HMG-CoA reductase is modulated by many diverse external stimuli and endogenous factors and can be detected to variable levels in every plant tissue. A fine determination of HMG-CoA reductase activity levels is required to understand its contribution to plant development and adaptation to changing environmental conditions. Here, we report a procedure to reliably determine HMG-CoA reductase activity in plants. The method includes the sample collection and homogenization strategies as well as the specific activity determination based on a classical radiochemical assay.

Farnesyl diphosphate synthase (FPS) catalyzes the sequential head-to-tail condensation of isopentenyl diphosphate (IPP, C5) with dimethylallyl diphosphate (DMAPP, C5) and geranyl diphosphate (GPP, C10) to produce farnesyl diphosphate (FPP, C15). This short-chain prenyl diphosphate constitutes a key branch-point of the isoprenoid biosynthetic pathway from which a variety of bioactive isoprenoids that are vital for normal plant growth and survival are produced. Here we describe a protocol to obtain highly purified preparations of recombinant FPS and a radiochemical assay method for measuring FPS activity in purified enzyme preparations and plant tissue extracts.

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme catalyzes the major rate-limiting step of the mevalonic acid (MVA) pathway from which sterols and other isoprenoids are synthesized. In contrast with our extensive knowledge of the regulation of HMGR in yeast and animals, little is known about this process in plants. To identify regulatory components of the MVA pathway in plants, we performed a genetic screen for second-site suppressor mutations of the Arabidopsis thaliana highly drought-sensitive drought hypersensitive2 (dry2) mutant that shows decreased squalene epoxidase activity. We show that mutations in SUPPRESSOR OF DRY2 DEFECTS1 (SUD1) gene recover most developmental defects in dry2 through changes in HMGR activity. SUD1 encodes a putative E3 ubiquitin ligase that shows sequence and structural similarity to yeast Degradation of α factor (Doα10) and human TEB4, components of the endoplasmic reticulum-associated degradation C (ERAD-C) pathway. While in yeast and animals, the alternative ERAD-L/ERAD-M pathway regulates HMGR activity by controlling protein stability, SUD1 regulates HMGR activity without apparent changes in protein content. These results highlight similarities, as well as important mechanistic differences, among the components involved in HMGR regulation in plants, yeast, and animals.

Arabidopsis thaliana contains two genes encoding farnesyl diphosphate (FPP) synthase (FPS), the prenyl diphoshate synthase that catalyzes the synthesis of FPP from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In this study, we provide evidence that the two Arabidopsis short FPS isozymes FPS1S and FPS2 localize to the cytosol. Both enzymes were expressed in E. coli, purified and biochemically characterized. Despite FPS1S and FPS2 share more than 90% amino acid sequence identity, FPS2 was found to be more efficient as a catalyst, more sensitive to the inhibitory effect of NaCl, and more resistant to thermal inactivation than FPS1S. Homology modelling for FPS1S and FPS2 and analysis of the amino acid differences between the two enzymes revealed an increase in surface polarity and a greater capacity to form surface salt bridges of FPS2 compared to FPS1S. These factors most likely account for the enhanced thermostability of FPS2. Expression analysis of FPS::GUS genes in seeds showed that FPS1 and FPS2 display complementary patterns of expression particularly at late stages of seed development, which suggests that Arabidopsis seeds have two spatially segregated sources of FPP. Functional complementation studies of the Arabidopsis fps2 knockout mutant seed phenotypes demonstrated that under normal conditions FPS1S and FPS2 are functionally interchangeable. A putative role for FPS2 in maintaining seed germination capacity under adverse environmental conditions is discussed.

The enzyme HMG-CoA reductase (HMGR) has a key regulatory role in the mevalonate pathway for isoprenoid biosynthesis, critical not only for normal plant development, but also for the adaptation to demanding environmental conditions. Consistent with this notion, plant HMGR is modulated by many diverse endogenous signals and external stimuli. Protein phosphatase 2A (PP2A) is involved in auxin, abscisic acid, ethylene and brassinosteroid signaling and now emerges as a positive and negative multilevel regulator of plant HMGR, both during normal growth and in response to a variety of stress conditions. The interaction with HMGR is mediated by B" regulatory subunits of PP2A, which are also calcium binding proteins. The new discoveries uncover the potential of PP2A to integrate developmental and calcium-mediated environmental signals in the control of plant HMGR.

Plants synthesize a myriad of isoprenoid products that are required both for essential constitutive processes and for adaptive responses to the environment. The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes a key regulatory step of the mevalonate pathway for isoprenoid biosynthesis and is modulated by many endogenous and external stimuli. In spite of that, no protein factor interacting with and regulating plant HMGR in vivo has been described so far. Here, we report the identification of two B'' regulatory subunits of protein phosphatase 2A (PP2A), designated B''α and B''β, that interact with HMGR1S and HMGR1L, the major isoforms of Arabidopsis thaliana HMGR. B''α and B''β are Ca²⁺ binding proteins of the EF-hand type. We show that HMGR transcript, protein, and activity levels are modulated by PP2A in Arabidopsis. When seedlings are transferred to salt-containing medium, B''α and PP2A mediate the decrease and subsequent increase of HMGR activity, which results from a steady rise of HMGR1-encoding transcript levels and an initial sharper reduction of HMGR protein level. In unchallenged plants, PP2A is a posttranslational negative regulator of HMGR activity with the participation of B''β. Our data indicate that PP2A exerts multilevel control on HMGR through the five-member B'' protein family during normal development and in response to a variety of stress conditions.

Farnesyl diphosphate (FPP) synthase (FPS) catalyses the synthesis of FPP, the major substrate used by cytosolic and mitochondrial branches of the isoprenoid pathway. Arabidopsis contains two farnesyl diphosphate synthase genes, FPS1 and FPS2, that encode isozymes FPS1L (mitochondrial), FPS1S and FPS2 (both cytosolic). Here we show that simultaneous knockout of both FPS genes is lethal for Arabidopsis, and embryo development is arrested at the pre-globular stage, demonstrating that FPP-derived isoprenoid metabolism is essential. In addition, lack of FPS enzyme activity severely impairs male genetic transmission. In contrast, no major developmental and metabolic defects were observed in fps1 and fps2 single knockout mutants, demonstrating the redundancy of the genes. The levels of sterols and ubiquinone, the major mitochondrial isoprenoid, are only slightly reduced in the single mutants. Although one functional FPS gene is sufficient to support isoprenoid biosynthesis for normal growth and development, the functions of FPS1 and FPS2 during development are not completely redundant. FPS1 activity has a predominant role during most of the plant life cycle, and FPS2 appears to have a major role in seeds and during the early stages of seedling development. Lack of FPS2 activity in seeds, but not of FPS1 activity, is associated with a marked reduction in sitosterol content and positive feedback regulation of 3-hydroxy-3-methylglutaryl CoA reductase activity that renders seeds hypersensitive to the 3-hydroxy-3-methylglutaryl CoA reductase inhibitor mevastatin., (© 2010 The Authors. Journal compilation © 2010 Blackwell Publishing Ltd.)

3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR, EC 1.1.1.34) catalyzes the major rate-limiting step in the mevalonate (MVA) pathway for isoprenoid biosynthesis. Its activity is regulated at different levels, from transcriptional to post-translational. Treatment of Arabidopsis thaliana plants with myriocin, a specific inhibitor of serine palmitoyltransferase (SPT), the first enzyme of sphingolipid biosynthesis, resulted in a concomitant reduction of both HMGR activity and the sterol content, which reveals regulatory cross-talk between these two lipid biosynthesis pathways. Myriocin-induced down-regulation of HMGR activity is exerted at the post-translational level, like the regulatory response of HMGR to enhancement or depletion of the flux through the sterol pathway.

Squalene synthase (SQS) catalyzes the condensation of two molecules of farnesyl diphosphate (FPP) to produce squalene (SQ), the first committed precursor for sterol, brassinosteroid, and triterpene biosynthesis. Arabidopsis thaliana contains two SQS-annotated genomic sequences, At4g34640 (SQS1) and At4g34650 (SQS2), organized in a tandem array. Here we report that the SQS1 gene is widely expressed in all tissues throughout plant development, whereas SQS2 is primarily expressed in the vascular tissue of leaf and cotyledon petioles, and the hypocotyl of seedlings. Neither the complete A. thaliana SQS2 protein nor the chimeric SQS resulting from the replacement of the 69 C-terminal residues of SQS2 by the 111 C-terminal residues of the Schizosaccharomyces pombe SQS were able to confer ergosterol prototrophy to a Saccharomyces cerevisiae erg9 mutant strain lacking SQS activity. A soluble form of SQS2 expressed in Escherichia coli and purified was unable to synthesize SQ from FPP in the presence of NADPH and either Mg2+ or Mn2+. These results demonstrated that SQS2 has no SQS activity, so that SQS1 is the only functional SQS in A. thaliana. Mutational studies revealed that the lack of SQS activity of SQS2 cannot be exclusively attributed to the presence of an unusual Ser replacing the highly conserved Phe at position 287. Expression of green fluorescent protein (GFP)-tagged versions of SQS1 in onion epidermal cells demonstrated that SQS1 is targeted to the endoplasmic reticulum (ER) membrane and that this location is exclusively dependent on the presence of the SQS1 C-terminal hydrophobic trans-membrane domain.

Arv1p is involved in the regulation of cellular lipid homeostasis in the yeast Saccharomyces cerevisiae. Here, we report the characterization of the two Arabidopsis thaliana ARV genes and the encoded proteins, AtArv1p and AtArv2p. The functional identity of AtArv1p and AtArv2p was demonstrated by complementation of the thermosensitive phenotype of the arv1Delta yeast mutant strain YJN1756. Both A. thaliana proteins contain the bipartite Arv1 homology domain (AHD), which consists of an NH(2)-terminal cysteine-rich subdomain with a putative zinc-binding motif followed by a C-terminal subdomain of 33 amino acids. Removal of the cysteine-rich subdomain has no effect on Arvp activity, whereas the presence of the C-terminal subdomain of the AHD is critical for Arvp function. Localization experiments of AtArv1p and AtArv2p tagged with green fluorescent protein (GFP) and expressed in onion epidermal cells demonstrated that both proteins are exclusively targeted to the endoplasmic reticulum. Analysis of beta-glucuronidase (GUS) activity in transgenic A. thaliana plants carrying chimeric ARV1::GUS and ARV2::GUS genes showed that ARV gene promoters direct largely overlapping patterns of expression that are restricted to tissues in which cells are actively dividing or expanding. The results of this study support the notion that plants, yeast and mammals share common molecular mechanisms regulating intracellular lipid homeostasis.

To investigate the role of mitochondrial farnesyl diphosphate synthase (FPS) in plant isoprenoid biosynthesis we characterized transgenic Arabidopsis thaliana plants overexpressing FPS1L isoform. This overexpressed protein was properly targeted to mitochondria yielding a mature and active form of the enzyme of 40 kDa. Leaves from transgenic plants grown under continuous light exhibited symptoms of chlorosis and cell death correlating to H(2)O(2) accumulation, and leaves detached from the same plants displayed accelerated senescence. Overexpression of FPS in mitochondria also led to altered leaf cytokinin profile, with a reduction in the contents of physiologically active trans-zeatin- and isopentenyladenine-type cytokinins and their corresponding riboside monophosphates as well as enhanced levels of cis-zeatin 7-glucoside and storage cytokinin O-glucosides. Overexpression of 3-hydroxy-3-methylglutaryl coenzyme A reductase did not prevent chlorosis in plants overexpressing FPS1L, but did rescue accelerated senescence of detached leaves and restored wild-type levels of cytokinins. We propose that the overexpression of FPS1L leads to an enhanced uptake and metabolism of mevalonic acid-derived isopentenyl diphosphate and/or dimethylallyl diphosphate by mitochondria, thereby altering cytokinin homeostasis and causing a mitochondrial dysfunction that renders plants more sensitive to the oxidative stress induced by continuous light.

Plants produce diverse isoprenoids, which are synthesized in plastids, mitochondria, endoplasmic reticulum (ER), and the nonorganellar cytoplasm. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) catalyzes the synthesis of mevalonate, a rate-limiting step in the cytoplasmic pathway. Several branches of the pathway lead to the synthesis of structurally and functionally varied, yet essential, isoprenoids. Several HMGR isoforms have been identified in all plants examined. Studies based on gene expression and on fractionation of enzyme activity suggested that subcellular compartmentalization of HMGR is an important intracellular channeling mechanism for the production of the specific classes of isoprenoids. Plant HMGR has been shown previously to insert in vitro into the membrane of microsomal vesicles, but the final in vivo subcellular localization(s) remains controversial. To address the latter in Arabidopsis (Arabidopsis thaliana) cells, we conducted a multipronged microscopy and cell fractionation approach that included imaging of chimeric HMGR green fluorescent protein localizations in transiently transformed cell leaves, immunofluorescence confocal microscopy in wild-type and stably transformed seedlings, immunogold electron microscopy examinations of endogenous HMGR in seedling cotyledons, and sucrose density gradient analyses of HMGR-containing organelles. Taken together, the results reveal that endogenous Arabidopsis HMGR is localized at steady state within ER as expected, but surprisingly also predominantly within spherical, vesicular structures that range from 0.2- to 0.6-microm diameter, located in the cytoplasm and within the central vacuole in differentiated cotyledon cells. The N-terminal region, including the transmembrane domain of HMGR, was found to be necessary and sufficient for directing HMGR to ER and the spherical structures. It is believed, although not directly demonstrated, that these vesicle-like structures are derived from segments of HMGR-ER. Nevertheless, they represent a previously undescribed subcellular compartment likely capable of synthesizing mevalonate, which provides new evidence for multiorganelle compartmentalization of the isoprenoid biosynthetic pathways in plants.

Overexpression of Arabidopsis thaliana farnesyl diphosphate synthase isoform 1S (FPS1S) in transgenic A. thaliana (L.) Heynh. leads to necrotic lesion formation in leaves in planta and to premature senescence in detached leaves [A. Masferrer et al. (2002) Plant J 30:123-132]. Here we report that leaves of plants overexpressing FPS1S with symptoms of necrosis show increased H2O2 formation and induction of both the pathogenesis-related 1 (PR-1) and the alternative oxidase 1a (AOX1a) genes. These findings indicate that plants overexpressing FPS1S should be considered as lesion-mimic mutants and lead us to propose that H2O2 is the main inducing agent of necrosis in these plants. The onset of necrosis appears in a developmentally regulated manner that correlates with the developmental decline of endogenous 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) activity. Accordingly, constitutive overexpression of HMGR in plants overexpressing FPS1S prevents both necrosis and premature senescence. These observations demonstrate that both phenotypes are due to an insufficient supply of mevalonic acid and support the notion that the metabolic imbalance associated with FPS1S overexpression is, in fact, triggered by the developmental decline of HMGR activity. We also show that overexpression of FPS1S alleviates growth inhibition caused by overexpression of the catalytic domain of isoform HMGR1S. Overall, our results reinforce the view that the levels of specific intermediates of the mevalonic acid pathway must be strictly controlled, particularly those located at branch-point positions, in order to avoid deleterious effects on plant growth and development.

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.

The enzyme dehydrodolichyl diphosphate (dedol-PP) synthase is a cis-prenyltransferase that catalyzes the synthesis of dedol-PP, the long-chain polyprenyl diphosphate used as a precursor for the synthesis of dolichyl phosphate. Here we report the cloning and characterization of a cDNA from Arabidopsis thaliana encoding dedol-PP synthase. The identity of the cloned enzyme was confirmed by functional complementation of a yeast mutant strain defective in dedol-PP synthase activity together with the detection of high levels of dedol-PP synthase activity in the transformed yeast mutant. The A. thaliana dedol-PP synthase mRNA was detected at high levels in roots but was hardly detected in flowers, leaves, stems and in A. thaliana suspension-cultured cells.

Mevalonate kinase (MVK), the enzyme that catalyzes the phosphorylation of mevalonate to produce mevalonate 5-phosphate, is considered as a potential regulatory enzyme of the isoprenoid biosynthetic pathway. The Arabidopsis thaliana MVK gene corresponding to the MVK cDNA previously isolated has been cloned and characterized. RNAse protection analysis indicated that the expression of the MVK gene generates three mRNA populations with 5' ends mapping 203, 254 and 355 nt upstream of the MVK ATG start codon. Northern blot analysis showed that the MVK mRNA accumulates preferentially in roots and influorescences. Histochemical analysis, with transgenic A. thaliana plants containing a translational fusion of a 1.8 kb fragment of the 5' region of the MVK gene to the beta-glucuronidase (GUS) reporter gene, indicated that the MVK 5'-flanking region directs widespread expression of the GUS gene throughout development, although the highest levels of GUS activity are detected in roots (meristematic region) and flowers (sepals, petals, anthers, style and stigmatic papillae). The expression pattern of the MVK gene suggests that the role of the encoded MVK is the production of a general pool of mevalonate-5-phosphate for the synthesis of different classes of isoprenoids involved in both basic and specialized plant cell functions. Functional promoter deletion analysis in transfected A. thaliana protoplasts indicated that regulatory elements between positions -295 and -194 of the MVK 5'-flanking region are crucial for high-level MVK gene expression.

Squalene synthase (SQS) catalyzes the first committed step of the sterol biosynthetic pathway. A full-length Arabidopsis thaliana SQS cDNA has been isolated by combining library screening and PCR-based approaches. Arabidopsis SQS is encoded by a small gene family of two genes (SQS1 and SQS2) which are organized in a tandem array. SQS1 and SQS2 have an identical organization with regard to intron positions and exon sizes and encode SQS isoforms showing a high level of sequence conservation (79% identity and 88% similarity). The isolated cDNA has been assigned to the SQS1 gene product, SQS1. RNA blot analysis has shown that the 1.6-kb SQS1 mRNA is detected in all plant tissues analyzed (inflorescenses, leaves, stems and roots) although the transcript is especially abundant in roots. Arabidopsis SQS1 isoform is unable to complement the SQS-defective Saccharomyces cerevisiae strain 5302, although SQS activity was detected in the microsomal fraction of the transformed yeast strain. However, a chimeric SQS resulting from the replacement of the 66 C-terminal residues of the Arabidopsis enzyme by the 111 C-terminal residues of the Schizosaccharomyces pombe enzyme was able to confer ergosterol prototrophy to strain 5302. Labeling studies using [3H]farnesyl-P2 and microsomal fractions obtained from yeast strains expressing either Arabidopsis SQS1 or chimeric Arabidopsis/S. pombe SQS derivatives indicated that the C-terminal region of the enzyme is involved in the channeling of squalene through the yeast sterol pathway.

The catalytic domain of 3-hydroxy-3-methylglutaryl-CoA reductase isoform 1 (HMGR1cd) from Arabidopsis thaliana has been expressed in Escherichia coli in a catalytically active form and purified. The high efficiency of the bacterial expression system together with the simplicity of the purification procedure used in this study resulted in the attainment of large quantities of pure enzyme (about 5 mg/l culture) with a final specific activity of up to 17 U/mg. This specific activity is higher than that reported to date for any 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) purified from a plant source. HMGR1cd activity was completely blocked by the HMGR inhibitor mevinolin (IC50 = 12.5 nM). No significant differences were observed between the Km values of HMGR1cd for NADPH (71 +/- 7 microM) and (S)-3-hydroxy-3-methylglutaryl-CoA (8.3 +/- 1.5 microM) and those of pure HMGR preparations obtained from different plant sources. The purified HMGR1cd was reversibly inactivated by phosphorylation at a single site by Brassica oleracea HMGR kinase A, which is functionally related to the mammalian AMP-activated protein kinase. The site of phosphorylation is Ser577 in the complete sequence of A. thaliana HMGR1. The results in this paper represent the first evidence that a higher plant HMGR is regulated by direct phosphorylation, at least in a cell-free system. Our results also reinforce the view that the AMP-activated protein kinase/SNF1 family is an ancient and highly conserved protein kinase system.

Tobacco proteins that share homology with thaumatin, a sweet protein of Thaumatococcus daniellii Benth., are produced in various physiological situations such as pathogenesis-related stress or water deficit stress. Using purified polyclonal anti-thaumatin antibodies, we have detected other thaumatin-like proteins in tobacco (Nicotiana tabacum var Samsun) that have been related with floral differentiation. Thaumatin-like proteins with apparent molecular masses of 42.6, 31.6, and 26.3 kilodaltons were found in immature and mature flower organs in vivo, and others of 46.7, 41.7, and 27.5 kilodaltons were exclusively detected in thin cell layer explants forming flowers. In situ immunolocalization revealed their synthesis in newly differentiated floral meristems, in tracheids, and in parenchyma cells.

32P-labeled glycogen synthase specifically immunoprecipitated from 32P-phosphate incubated rat hepatocytes contains, in addition to [32P] phosphoserine, significant levels of [32P] phosphothreonine (7% of the total [32P] phosphoaminoacids). When the 32P-immunoprecipitate was cleaved with CNBr, the [32P] phosphothreonine was recovered in the large CNBr fragment (CB-2, Mapp 28 Kd). Homogeneous rat liver glycogen synthase was phosphorylated by all the protein kinases able to phosphorylate CB-2 "in vitro" (casein kinases I and II, cAMP-dependent protein kinase and glycogen synthase kinase-3). After analysis of the immunoprecipitated enzyme for phosphoaminoacids, it was observed that only casein kinase II was able to phosphorylate on threonine and 32P-phosphate was only found in CB-2. These results demonstrate that rat liver glycogen synthase is phosphorylated at threonine site(s) contained in CB-2 and strongly indicate that casein kinase II may play a role in the "in vivo" phosphorylation of liver glycogen synthase. This is the first protein kinase reported to phosphorylate threonine residues in liver glycogen synthase.