A cytosolic bifunctional geranyl/farnesyl diphosphate synthase provides MVA-derived GPP for geraniol biosynthesis in rose flowers.

Geraniol derived from essential oils of various plant species is widely used in the cosmetic and perfume industries. It is also an essential trait of the pleasant smell of rose flowers. In contrast to other monoterpenes which are produced in plastids via the methyl erythritol phosphate pathway, geraniol biosynthesis in roses relies on cytosolic NUDX1 hydrolase which dephosphorylates geranyl diphosphate (GPP). However, the metabolic origin of cytosolic GPP remains unknown. By feeding Rosa chinensis "Old Blush" flowers with pathway-specific precursors and inhibitors, combined with metabolic profiling and functional characterization of enzymes in vitro and in planta, we show that geraniol is synthesized through the cytosolic mevalonate (MVA) pathway by a bifunctional geranyl/farnesyl diphosphate synthase, RcG/FPPS1, producing both GPP and farnesyl diphosphate (FPP). The downregulation and overexpression of RcG/FPPS1 in rose petals affected not only geraniol and germacrene D emissions but also dihydro-ß-ionol, the latter due to metabolic cross talk of RcG/FPPS1-dependent isoprenoid intermediates trafficking from the cytosol to plastids. Phylogenetic analysis together with functional characterization of G/FPPS orthologs revealed that the G/FPPS activity is conserved among Rosaceae species. Site-directed mutagenesis and molecular dynamic simulations enabled to identify two conserved amino acids that evolved from ancestral FPPSs and contribute to GPP/FPP product specificity. Overall, this study elucidates the origin of the cytosolic GPP for NUDX1-dependent geraniol production, provides insights into the emergence of the RcG/FPPS1 GPPS activity from the ancestral FPPSs, and shows that RcG/FPPS1 plays a key role in the biosynthesis of volatile terpenoid compounds in rose flowers.

RNA-dependent synthesis of ergosteryl-3ß-O-glycine in Ascomycota expands the diversity of steryl-amino acids.

A wide range of bacteria possess virulence factors such as aminoacyl-tRNA transferases (ATTs) that are capable of rerouting aminoacyl-transfer RNAs away from protein synthesis to conjugate amino acids onto glycerolipids. We recently showed that, although these pathways were thought to be restricted to bacteria, higher fungi also possess ergosteryl-3ß-O-L-aspartate synthases (ErdSs), which transfer the L-Asp moiety of aspartyl-tRNAAsp onto the 3ß-OH group of ergosterol (Erg), yielding ergosteryl-3ß-O-L-aspartate (Erg-Asp). Here, we report the discovery, in fungi, of a second type of fungal sterol-specific ATTs, namely, ergosteryl-3ß-O-glycine (Erg-Gly) synthase (ErgS). ErgS consists of a freestanding DUF2156 domain encoded by a gene distinct from and paralogous to that of ErdS. We show that the enzyme only uses Gly-tRNAGly produced by an independent glycyl-tRNA synthetase (GlyRS) to transfer glycine onto the 3ß-OH of Erg, producing Erg-Gly. Phylogenomics analysis also show that the Erg-Gly synthesis pathway exists only in Ascomycota, including species of biotechnological interest, and more importantly, in human pathogens, such as Aspergillus fumigatus. The discovery of a second type of Erg-aa not only expands the repertoire of this particular class of fungal lipids but suggests that Erg-aa synthases might constitute a genuine subfamily of lipid-modifying ATTs.

Daidzein Hydroxylation by CYP81E63 Is Involved in the Biosynthesis of Miroestrol in Pueraria mirifica.

White Kwao Krua (Pueraria candollei var. mirifica), a Thai medicinal plant, is a rich source of phytoestrogens, especially isoflavonoids and chromenes. These phytoestrogens are well known; however, their biosynthetic genes remain largely uncharacterized. Cytochrome P450 (P450) is a large protein family that plays a crucial role in the biosynthesis of various compounds in plants, including phytoestrogens. Thus, we focused on P450s involved in the isoflavone hydroxylation that potentially participates in the biosynthesis of miroestrol. Three candidate P450s were isolated from the transcriptome libraries by considering the phylogenetic and expression data of each tissue of P. mirifica. The candidate P450s were functionally characterized both in vitro and in planta. Accordingly, the yeast microsome harboring PmCYP81E63 regiospecifically exhibited either 2' or 3' daidzein hydroxylation and genistein hydroxylation. Based on in silico calculation, PmCYP81E63 had higher binding energy with daidzein than with genistein, which supported the in vitro result of the isoflavone specificity. To confirm in planta function, the candidate P450s were then transiently co-expressed with isoflavone-related genes in Nicotiana benthamiana. Despite no daidzein in the infiltrated N. benthamiana leaves, genistein and hydroxygenistein biosynthesis were detectable by liquid Chromatography with tandem mass spectrometry (LC-MS/MS). Additionally, we demonstrated that PmCYP81E63 interacted with several enzymes related to isoflavone biosynthesis using bimolecular fluorescence complementation studies and a yeast two-hybrid analysis, suggesting a scheme of metabolon formation in the pathway. Our findings provide compelling evidence regarding the involvement of PmCYP81E63 in the early step of the proposed miroestrol biosynthesis in P. mirifica.

Elucidating the Mesocarp Drupe Transcriptome of Açai (Euterpe oleracea Mart.): An Amazonian Tree Palm Producer of Bioactive Compounds.

Euterpe oleracea palm, endemic to the Amazon region, is well known for açai, a fruit violet beverage with nutritional and medicinal properties. During E. oleracea fruit ripening, anthocyanin accumulation is not related to sugar production, contrarily to grape and blueberry. Ripened fruits have a high content of anthocyanins, isoprenoids, fibers, and proteins, and are poor in sugars. E. oleracea is proposed as a new genetic model for metabolism partitioning in the fruit. Approximately 255 million single-end-oriented reads were generated on an Ion Proton NGS platform combining fruit cDNA libraries at four ripening stages. The de novo transcriptome assembly was tested using six assemblers and 46 different combinations of parameters, a pre-processing and a post-processing step. The multiple k-mer approach with TransABySS as an assembler and Evidential Gene as a post-processer have shown the best results, with an N50 of 959 bp, a read coverage mean of 70x, a BUSCO complete sequence recovery of 36% and an RBMT of 61%. The fruit transcriptome dataset included 22,486 transcripts representing 18 Mbp, of which a proportion of 87% had significant homology with other plant sequences. Approximately 904 new EST-SSRs were described, and were common and transferable to Phoenix dactylifera and Elaeis guineensis, two other palm trees. The global GO classification of transcripts showed similar categories to that in P. dactylifera and E. guineensis fruit transcriptomes. For an accurate annotation and functional description of metabolism genes, a bioinformatic pipeline was developed to precisely identify orthologs, such as one-to-one orthologs between species, and to infer multigenic family evolution. The phylogenetic inference confirmed an occurrence of duplication events in the Arecaceae lineage and the presence of orphan genes in E. oleracea. Anthocyanin and tocopherol pathways were annotated entirely. Interestingly, the anthocyanin pathway showed a high number of paralogs, similar to in grape, whereas the tocopherol pathway exhibited a low and conserved gene number and the prediction of several splicing forms. The release of this exhaustively annotated molecular dataset of E. oleracea constitutes a valuable tool for further studies in metabolism partitioning and opens new great perspectives to study fruit physiology with açai as a model.

The Arabidopsis thaliana-Streptomyces Interaction Is Controlled by the Metabolic Status of the Holobiont.

How specific interactions between plant and pathogenic, commensal, or mutualistic microorganisms are mediated and how bacteria are selected by a plant are important questions to address. Here, an Arabidopsis thaliana mutant called chs5 partially deficient in the biogenesis of isoprenoid precursors was shown to extend its metabolic remodeling to phenylpropanoids and lipids in addition to carotenoids, chlorophylls, and terpenoids. Such a metabolic profile was concomitant to increased colonization of the phyllosphere by the pathogenic strain Pseudomonas syringae pv. tomato DC3000. A thorough microbiome analysis by 16S sequencing revealed that Streptomyces had a reduced colonization potential in chs5. This study revealed that the bacteria-Arabidopsis interaction implies molecular processes impaired in the chs5 mutant. Interestingly, our results revealed that the metabolic status of A. thaliana was crucial for the specific recruitment of Streptomyces into the microbiota. More generally, this study highlights specific as well as complex molecular interactions that shape the plant microbiota.

Cholesterol asymmetry at the tip of filopodia during cell adhesion.

During adhesion, cells develop filopodia to facilitate the attachment to the extracellular matrix. The small guanosine triphosphate (GTP)-binding protein, Cdc42, plays a central role in the formation of filopodia. It has been reported that Cdc42 activity is regulated by cholesterol (Chol). We examined Chol distribution in filopodia using Chol-binding domain 4 (D4) fragment of bacterial toxin, perfringolysin O that senses high membrane concentration of Chol. Our results indicate that fluorescent D4 was enriched at the tip of the outer leaflet of filopodia in the initiation phase of cell adhesion. This enrichment was accompanied by a defect of D4 labeling in the inner leaflet. Steady phase adhered cell experiment indicated that both Cdc42 and ATP-binding cassette transporter, ABCA1, were involved in the binding of D4 to the cell surface. Depletion of Chol activated Cdc42. Our results suggest that asymmetric distribution of Chol at the tip of filopodia induces activation of Cdc42, and thus, facilitates filopodia formation.

The specific molecular architecture of plant 3-hydroxy-3-methylglutaryl-CoA lyase.

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase (HMGL) is involved in branched-chain amino acid catabolism leading to acetyl-CoA production. Here, using bioinformatics analyses and protein sequence alignments, we found that in Arabidopsis thaliana a single gene encodes two HMGL isoforms differing in size (51 kDa, HMGL51 and 46 kDa, HMGL46). Similar to animal HMGLs, both isoforms comprised a C-terminal type 1 peroxisomal retention motif, and HMGL51 contained a mitochondrial leader peptide. We observed that only a shortened HMGL (35 kDa, HMGL35) is conserved across all kingdoms of life. Most notably, all plant HMGLs also contained a specific N-terminal extension (P100) that is located between the N-terminal mitochondrial targeting sequence TP35 and HMGL35 and is absent in bacteria and other eukaryotes. Interestingly, using HMGL enzyme assays, we found that rather than HMGL46, homodimeric recombinant HMGL35 is the active enzyme catalyzing acetyl-CoA and acetoacetate synthesis when incubated with (S)-HMG-CoA. This suggested that the plant-specific P100 peptide may inactivate HMGL according to specific physiological requirements. Therefore, we investigated whether the P100 peptide in HMGL46 alters its activity, possibly by modifying the HMGL46 structure. We found that induced expression of a cytosolic HMGL35 version in A. thaliana delays germination and leads to rapid wilting and chlorosis in mature plants. Our results suggest that in plants, P100-mediated HMGL inactivation outside of peroxisomes or mitochondria is crucial, protecting against potentially cytotoxic effects of HMGL activity while it transits to these organelles.

Inhibition of Phytosterol Biosynthesis by Azasterols.

Inhibitors of enzymes in essential cellular pathways are potent probes to decipher intricate physiological functions of biomolecules. The analysis of Arabidopsis thaliana sterol profiles upon treatment with a series of azasterols reveals a specific in vivo inhibition of SMT2, a plant sterol-C-methyltransferase acting as a branch point between the campesterol and sitosterol biosynthetic segments in the pathway. Side chain azasteroids that modify sitosterol homeostasis help to refine its particular function in plant development.

Transcriptome analysis of Pueraria candollei var. mirifica for gene discovery in the biosyntheses of isoflavones and miroestrol.

BACKGROUND: Pueraria candollei var. mirifica, a Thai medicinal plant used traditionally as a rejuvenating herb, is known as a rich source of phytoestrogens, including isoflavonoids and the highly estrogenic miroestrol and deoxymiroestrol. Although these active constituents in P. candollei var. mirifica have been known for some time, actual knowledge regarding their biosynthetic genes remains unknown. RESULTS: Miroestrol biosynthesis was reconsidered and the most plausible mechanism starting from the isoflavonoid daidzein was proposed. A de novo transcriptome analysis was conducted using combined P. candollei var. mirifica tissues of young leaves, mature leaves, tuberous cortices, and cortex-excised tubers. A total of 166,923 contigs was assembled for functional annotation using protein databases and as a library for identification of genes that are potentially involved in the biosynthesis of isoflavonoids and miroestrol. Twenty-one differentially expressed genes from four separate libraries were identified as candidates involved in these biosynthetic pathways, and their respective expressions were validated by quantitative real-time reverse transcription polymerase chain reaction. Notably, isoflavonoid and miroestrol profiling generated by LC-MS/MS was positively correlated with expression levels of isoflavonoid biosynthetic genes across the four types of tissues. Moreover, we identified R2R3 MYB transcription factors that may be involved in the regulation of isoflavonoid biosynthesis in P. candollei var. mirifica. To confirm the function of a key-isoflavone biosynthetic gene, P. candollei var. mirifica isoflavone synthase identified in our library was transiently co-expressed with an Arabidopsis MYB12 transcription factor (AtMYB12) in Nicotiana benthamiana leaves. Remarkably, the combined expression of these proteins led to the production of the isoflavone genistein. CONCLUSIONS: Our results provide compelling evidence regarding the integration of transcriptome and metabolome as a powerful tool for identifying biosynthetic genes and transcription factors possibly involved in the isoflavonoid and miroestrol biosyntheses in P. candollei var. mirifica.

Triterpenoids in Echinoderms: Fundamental Differences in Diversity and Biosynthetic Pathways.

Echinoderms form a remarkable phylum of marine invertebrates that present specific chemical signatures unique in the animal kingdom. It is particularly the case for essential triterpenoids that evolved separately in each of the five echinoderm classes. Indeed, while most animals have Δ5-sterols, sea cucumbers (Holothuroidea) and sea stars (Asteroidea) also possess Δ7 and Δ9(11)-sterols, a characteristic not shared with brittle stars (Ophiuroidea), sea urchins (Echinoidea), and crinoids (Crinoidea). These particular Δ7 and Δ9(11) sterols emerged as a self-protection against membranolytic saponins that only sea cucumbers and sea stars produce as a defense mechanism. The diversity of saponins is large; several hundred molecules have been described in the two classes of these saponins (i.e., triterpenoid or steroid saponins). This review aims to highlight the diversity of triterpenoids in echinoderms by focusing on sterols and triterpenoid glycosides, but more importantly to provide an updated view of the biosynthesis of these molecules in echinoderms.

Metabolism and Biological Activities of 4-Methyl-Sterols.

4,4-Dimethylsterols and 4-methylsterols are sterol biosynthetic intermediates (C4-SBIs) acting as precursors of cholesterol, ergosterol, and phytosterols. Their accumulation caused by genetic lesions or biochemical inhibition causes severe cellular and developmental phenotypes in all organisms. Functional evidence supports their role as meiosis activators or as signaling molecules in mammals or plants. Oxygenated C4-SBIs like 4-carboxysterols act in major biological processes like auxin signaling in plants and immune system development in mammals. It is the purpose of this article to point out important milestones and significant advances in the understanding of the biogenesis and biological activities of C4-SBIs.

Analysis of CFB, a cytokinin-responsive gene of Arabidopsis thaliana encoding a novel F-box protein regulating sterol biosynthesis.

Protein degradation by the ubiquitin-26S proteasome pathway is important for the regulation of cellular processes, but the function of most F-box proteins relevant to substrate recognition is unknown. We describe the analysis of the gene Cytokinin-induced F-box encoding (CFB, AT3G44326), identified in a meta-analysis of cytokinin-related transcriptome studies as one of the most robust cytokinin response genes. F-box domain-dependent interaction with the E3 ubiquitin ligase complex component ASK1 classifies CFB as a functional F-box protein. Apart from F-box and transmembrane domains, CFB contains no known functional domains. CFB is expressed in all plant tissues, predominantly in root tissue. A ProCFB:GFP-GUS fusion gene showed strongest expression in the lateral root cap and during lateral root formation. CFB-GFP fusion proteins were mainly localized in the nucleus and the cytosol but also at the plasma membrane. cfb mutants had no discernible phenotype, but CFB overexpressing plants showed several defects, such as a white upper inflorescence stem, similar to the hypomorphic cycloartenol synthase mutant cas1-1. Both CFB overexpressing plants and cas1-1 mutants accumulated the CAS1 substrate 2,3-oxidosqualene in the white stem tissue, the latter even more after cytokinin treatment, indicating impairment of CAS1 function. This suggests that CFB may link cytokinin and the sterol biosynthesis pathway.

Diversification of sterol methyltransferase enzymes in plants and a role for ß-sitosterol in oriented cell plate formation and polarized growth.

Phytosterols are classified into C24-ethylsterols and C24-methylsterols according to the different C24-alkylation levels conferred by two types of sterol methyltransferases (SMTs). The first type of SMT (SMT1) is widely conserved, whereas the second type (SMT2) has diverged in charophytes and land plants. The Arabidopsis smt2 smt3 mutant is defective in the SMT2 step, leading to deficiency in C24-ethylsterols while the C24-methylsterol pathway is unchanged. smt2 smt3 plants exhibit severe dwarfism and abnormal development throughout their life cycle, with irregular cell division followed by collapsed cell files. Preprophase bands are occasionally formed in perpendicular directions in adjacent cells, and abnormal phragmoplasts with mislocalized KNOLLE syntaxin and tubulin are observed. Defects in auxin-dependent processes are exemplified by mislocalizations of the PIN2 auxin efflux carrier due to disrupted cell division and failure to distribute PIN2 asymmetrically after cytokinesis. Although endocytosis of PIN2-GFP from the plasma membrane (PM) is apparently unaffected in smt2 smt3, strong inhibition of the endocytic recycling is associated with a remarkable reduction in the level of PIN2-GFP on the PM. Aberrant localization of the cytoplasmic linker associated protein (CLASP) and microtubules is implicated in the disrupted endocytic recycling in smt2 smt3. Exogenous C24-ethylsterols partially recover lateral root development and auxin distribution in smt2 smt3 roots. These results indicate that C24-ethylsterols play a crucial role in division plane determination, directional auxin transport, and polar growth. It is proposed that the divergence of SMT2 genes together with the ability to produce C24-ethylsterols were critical events to achieve polarized growth in the plant lineage.

ATP citrate lyase activity is post-translationally regulated by sink strength and impacts the wax, cutin and rubber biosynthetic pathways.

Cytosolic acetyl-CoA is involved in the synthesis of a variety of compounds, including waxes, sterols and rubber, and is generated by the ATP citrate lyase (ACL). Plants over-expressing ACL were generated in an effort to understand the contribution of ACL activity to the carbon flux of acetyl-CoA to metabolic pathways occurring in the cytosol. Transgenic Arabidopsis plants synthesizing the polyester polyhydroxybutyrate (PHB) from cytosolic acetyl-CoA have reduced growth and wax content, consistent with a reduction in the availability of cytosolic acetyl-CoA to endogenous pathways. Increasing the ACL activity via the over-expression of the ACLA and ACLB subunits reversed the phenotypes associated with PHB synthesis while maintaining polymer synthesis. PHB production by itself was associated with an increase in ACL activity that occurred in the absence of changes in steady-state mRNA or protein level, indicating a post-translational regulation of ACL activity in response to sink strength. Over-expression of ACL in Arabidopsis was associated with a 30% increase in wax on stems, while over-expression of a chimeric homomeric ACL in the laticifer of roots of dandelion led to a four- and two-fold increase in rubber and triterpene content, respectively. Synthesis of PHB and over-expression of ACL also changed the amount of the cutin monomer octadecadien-1,18-dioic acid, revealing an unsuspected link between cytosolic acetyl-CoA and cutin biosynthesis. Together, these results reveal the complexity of ACL regulation and its central role in influencing the carbon flux to metabolic pathways using cytosolic acetyl-CoA, including wax and polyisoprenoids.

S-carvone suppresses cellulase-induced capsidiol production in Nicotiana tabacum by interfering with protein isoprenylation.

S-Carvone has been described as a negative regulator of mevalonic acid (MVA) production by interfering with 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGR) activity, a key player in isoprenoid biosynthesis. The impact of this monoterpene on the production of capsidiol in Nicotiana tabacum, an assumed MVA-derived sesquiterpenoid phytoalexin produced in response to elicitation by cellulase, was investigated. As expected, capsidiol production, as well as early stages of elicitation such as hydrogen peroxide production or stimulation of 5-epi-aristolochene synthase activity, were repressed. Despite the lack of capsidiol synthesis, apparent HMGR activity was boosted. Feeding experiments using (1-13C)Glc followed by analysis of labeling patterns by 13C-NMR, confirmed an MVA-dependent biosynthesis; however, treatments with fosmidomycin, an inhibitor of the MVA-independent 2-C-methyl-D-erythritol 4-phosphate (MEP) isoprenoid pathway, unexpectedly down-regulated the biosynthesis of this sesquiterpene as well. We postulated that S-carvone does not directly inhibit the production of MVA by inactivating HMGR, but possibly targets an MEP-derived isoprenoid involved in the early steps of the elicitation process. A new model is proposed in which the monoterpene blocks an MEP pathway-dependent protein geranylgeranylation necessary for the signaling cascade. The production of capsidiol was inhibited when plants were treated with some inhibitors of protein prenylation or by further monoterpenes. Moreover, S-carvone hindered isoprenylation of a prenylable GFP indicator protein expressed in N. tabacum cell lines, which can be chemically complemented with geranylgeraniol. The model was further validated using N. tabacum cell extracts or recombinant N. tabacum protein prenyltransferases expressed in Escherichia coli. Our study endorsed a reevaluation of the effect of S-carvone on plant isoprenoid metabolism.

Isoprenoid biosynthesis is required for miRNA function and affects membrane association of ARGONAUTE 1 in Arabidopsis.

Plant and metazoan microRNAs (miRNAs) guide ARGONAUTE (AGO) protein complexes to regulate expression of complementary RNAs via base pairing. In the plant Arabidopsis thaliana, the main miRNA effector is AGO1, but few other factors required for miRNA activity are known. Here, we isolate the genes defined by the previously described miRNA action deficient (mad) mutants, mad3 and mad4. Both genes encode enzymes involved in isoprenoid biosynthesis. MAD3 encodes 3-hydroxy-3-methylglutaryl CoA reductase (HMG1), which functions in the initial C(5) building block biogenesis that precedes isoprenoid metabolism. HMG1 is a key regulatory enzyme that controls the amounts of isoprenoid end products. MAD4 encodes sterol C-8 isomerase (HYDRA1) that acts downstream in dedicated sterol biosynthesis. Using yeast complementation assays and in planta application of lovastatin, a competitive inhibitor of HMG1, we show that defects in HMG1 catalytic activity are sufficient to inhibit miRNA activity. Many isoprenoid derivatives are indispensable structural and signaling components, and especially sterols are essential membrane constituents. Accordingly, we provide evidence that AGO1 is a peripheral membrane protein. Moreover, specific hypomorphic mutant alleles of AGO1 display compromised membrane association and AGO1-membrane interaction is reduced upon knockdown of HMG1/MAD3. These results suggest a possible basis for the requirement of isoprenoid biosynthesis for the activity of plant miRNAs, and unravel mechanistic features shared with their metazoan counterparts.

Sterols are required for cell-fate commitment and maintenance of the stomatal lineage in Arabidopsis.

Asymmetric cell division is important for regulating cell proliferation and fate determination during stomatal development in plants. Although genes that control asymmetric division and cell differentiation in stomatal development have been reported, regulators controlling the process from asymmetric division to cell differentiation remain poorly understood. Here, we report a weak allele (fk-J3158) of the Arabidopsis sterol C-14 reductase gene FACKEL (FK) that shows clusters of small cells and stomata in leaf epidermis, a common phenomenon that is often seen in mutants defective in stomatal asymmetric division. Interestingly, the physical asymmetry of these divisions appeared to be intact in fk mutants, but the cell-fate asymmetry was greatly disturbed, suggesting that the FK pathway links these two crucial events in the process of asymmetric division. Sterol profile analysis revealed that the fk-J3158 mutation blocked downstream sterol production. Further investigation indicated that cyclopropylsterol isomerase1 (cpi1), sterol 14α-demethylase (cyp51A2) and hydra1 (hyd1) mutants, corresponding to enzymes in the same branch of the sterol biosynthetic pathway, displayed defective stomatal development phenotypes, similar to those observed for fk. Fenpropimorph, an inhibitor of the FK sterol C-14 reductase in Arabidopsis, also caused these abnormal small-cell and stomata phenotypes in wild-type leaves. Genetic experiments demonstrated that sterol biosynthesis is required for correct stomatal patterning, probably through an additional signaling pathway that has yet to be defined. Detailed analyses of time-lapse cell division patterns, stomatal precursor cell division markers and DNA ploidy suggest that sterols are required to properly restrict cell proliferation, asymmetric fate specification, cell-fate commitment and maintenance in the stomatal lineage cells. These events occur after physical asymmetric division of stomatal precursor cells.

ABCG9, ABCG11 and ABCG14 ABC transporters are required for vascular development in Arabidopsis.

In order to obtain insights into the regulatory pathways controlling phloem development, we characterized three genes encoding membrane proteins from the G sub-family of ABC transporters (ABCG9, ABCG11 and ABCG14), whose expression in the phloem has been confirmed. Mutations in the genes encoding these dimerizing 'half transporters' are semi-dominant and result in vascular patterning defects in cotyledons and the floral stem. Co-immunoprecipitation and bimolecular fluorescence complementation experiments demonstrated that these proteins dimerize, either by flexible pairing (ABCG11 and ABCG9) or by forming strict heterodimers (ABCG14). In addition, metabolome analyses and measurement of sterol ester contents in the mutants suggested that ABCG9, ABCG11 and ABCG14 are involved in lipid/sterol homeostasis regulation. Our results show that these three ABCG genes are required for proper vascular development in Arabidopsis thaliana.

Methyl-Jasmonate Functions as a Molecular Switch Promoting Cross-Talk between Pathways for the Biosynthesis of Isoprenoid Backbones Used to Modify Proteins in Plants.

In plants, the plastidial mevalonate (MVA)-independent pathway is required for the modification with geranylgeranyl groups of CaaL-motif proteins, which are substrates of protein geranylgeranyltransferase type-I (PGGT-I). As a consequence, fosmidomycin, a specific inhibitor of 1-deoxy-d-xylulose (DX)-5 phosphate reductoisomerase/DXR, the second enzyme in this so-called methylerythritol phosphate (MEP) pathway, also acts as an effective inhibitor of protein prenylation. This can be visualized in plant cells by confocal microscopy by expressing GFP-CaM-CVIL, a prenylation sensor protein. After treatment with fosmidomycin, the plasma membrane localization of this GFP-based sensor is altered, and a nuclear distribution of fluorescence is observed instead. In tobacco cells, a visual screen of conditions allowing membrane localization in the presence of fosmidomycin identified jasmonic acid methyl esther (MeJA) as a chemical capable of gradually overcoming inhibition. Using Arabidopsis protein prenyltransferase loss-of-function mutant lines expressing GFP-CaM-CVIL proteins, we demonstrated that in the presence of MeJA, protein farnesyltransferase (PFT) can modify the GFP-CaM-CVIL sensor, a substrate the enzyme does not recognize under standard conditions. Similar to MeJA, farnesol and MVA also alter the protein substrate specificity of PFT, whereas DX and geranylgeraniol have limited or no effect. Our data suggest that MeJA adjusts the protein substrate specificity of PFT by promoting a metabolic cross-talk directing the origin of the prenyl group used to modify the protein. MVA, or an MVA-derived metabolite, appears to be a key metabolic intermediate for this change in substrate specificity.