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Plants are gaining traction as a cost-effective and scalable platform for producing recombinant proteins. However, expressing integral membrane proteins in plants is challenging due to their hydrophobic nature. In our study, we used transient and stable expression systems in Nicotiana benthamiana and Camelina sativa respectively to express SARS-CoV-2 E and M integral proteins, and target them to lipid droplets (LDs). LDs offer an ideal environment for folding hydrophobic proteins and aid in their purification through flotation. We tested various protein fusions with different linkers and tags and used three dimensional structure predictions to assess their effects. E and M mostly localized in the ER in N. benthamiana leaves but E could be targeted to LDs in oil accumulating tobacco when fused with oleosin, a LD integral protein. In Camelina sativa seeds, E and M were however found associated with purified LDs. By enhancing the accumulation of E and M within LDs through oleosin, we enriched these proteins in the purified floating fraction. This strategy provides an alternative approach for efficiently producing and purifying hydrophobic pharmaceuticals and vaccines using plant systems., (© 2023 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.)

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Exploring and deciphering the biodiversity of oil bodies (OBs) recovered from oilseeds are of growing interest in the preparation of sustainable, natural and healthy plant-based food products. This study focused on chia ( Salvia hispanica L.) and camelina ( Camelina sativa L.) seed OBs. A green refinery process including ultrasound to remove mucilage, aqueous extraction by grinding and centrifugation to recover OBs from the seeds was used. The microstructure, composition and physical stability of the OBs were examined. Confocal laser scanning microscopy images showed that chia and camelina seed OBs are spherical assemblies coated by a layer of phospholipids and proteins, which have been identified by gel electrophoresis. The mean diameters determined by laser light scattering measurements were 2.3 and 1.6 µm for chia and camelina seed OBs, respectively. The chia and camelina seed OBs were rich in lipids and other bioactive components with, respectively, 64% and 30% α-linolenic acid representing 70% and 53% of the total fatty acids in the sn -2 position of the triacylglycerols, 0.23% and 0.26% phospholipids, 3069 and 2674 mg/kg oil of β-sitosterol, and lipophilic antioxidants: 400 and 670 mg/kg oil of γ-tocopherol. Phenolic compounds were recovered from the aqueous extracts, such as rutin from camelina and caffeic acid from chia. Zeta-potential measurements showed changes from about -40 mV (pH 9) to values that were positive below the isoelectric points of pH 5.1 and 3.6 for chia and camelina seed OBs, respectively. Below pH 6.5, physical instability of the natural oil-in-water emulsions with aggregation and phase separation was found. This study will contribute to the development of innovative and sustainable food products based on natural oil-in-water emulsions containing chia and camelina seed OBs for their nutritional and health benefits.

Programmed cell death (PCD) is essential for several aspects of plant life. We previously identified the mips1 mutant of Arabidopsis thaliana, which is deficient for the enzyme catalysing myo-inositol synthesis, and that displays light-dependent formation of lesions on leaves due to Salicylic Acid (SA) over-accumulation. Rationale of this work was to identify novel regulators of plant PCD using a genetic approach. A screen for secondary mutations that abolish the mips1 PCD phenotype identified a mutation in the BIG gene, encoding a factor of unknown molecular function that was previously shown to play pleiotropic roles in plant development and defence. Physiological analyses showed that BIG is required for lesion formation in mips1 via SA-dependant signalling. big mutations partly rescued transcriptomic and metabolomics perturbations as stress-related phytohormones homeostasis. In addition, since loss of function of the ceramide synthase LOH2 was not able to abolish cell death induction in mips1, we show that PCD induction is not fully dependent of sphingolipid accumulation as previously suggested. Our results provide further insights into the role of the BIG protein in the control of MIPS1-dependent cell death and also into the impact of sphingolipid homeostasis in this pathway.

Noninvasiveness, minimal handling, and immediate response are favorable features of fluorescence readout for high-throughput phenotyping of labeled plants.Yet, remote fluorescence imaging may suffer from an autofluorescent background and artificial or natural ambient light. In this work, the latter limitations are overcome by adopting reversibly photoswitchable fluorescent proteins (RSFPs) as labels and Speed OPIOM (out-of-phase imaging after optical modulation), a fluorescence imaging protocol exploiting dynamic contrast. Speed OPIOM can efficiently distinguish the RSFP signal from autofluorescence and other spectrally interfering fluorescent reporters like GFP. It can quantitatively assess gene expressions, even when they are weak. It is as quantitative, sensitive, and robust in dark and bright light conditions. Eventually, it can be used to nondestructively record abiotic stress responses like water or iron limitations in real time at the level of individual plants and even of specific organs. Such Speed OPIOM validation could find numerous applications to identify plant lines in selection programs, design plants as environmental sensors, or ecologically monitor transgenic plants in the environment., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

Sphingolipids appear as a promising class of components susceptible to prevent the onset of the metabolic syndrome (MetS). Gut availability and effects of Camelina sativa sphingolipids were investigated in a mouse model of dietary-induced MetS. Seed meals from two Camelina sativa lines enriched, respectively, in C24- and C16-NH 2 - glycosyl-inositol-phosphoryl-ceramides (NH 2 GIPC) were used in hypercaloric diets. After 5 weeks on these two hypercaloric diets, two markers of the MetS were alleviated (adiposity and insulin resistance) as well as inflammation markers and colon barrier dysfunction. A more pronounced effect was observed with the C16-NH 2 GIPC-enriched HC diet, in particular for colon barrier function. Despite a lower digestibility, C16-NH 2 GIPC were more prevalent in the intestine wall. Sphingolipids provided as camelina meal can therefore counteract some deleterious effects of a hypercaloric diet in mice at the intestinal and systemic levels. Interestingly, these beneficial effects seem partly dependent on sphingolipid acyl chain length.

Lateral root organogenesis plays an essential role in elaborating plant root system architecture. In Arabidopsis , the AP2 family transcription factor PUCHI controls cell proliferation in lateral root primordia. To identify potential targets of PUCHI, we analyzed a time course transcriptomic dataset of lateral root formation. We report that multiple genes coding for very long chain fatty acid (VLCFA) biosynthesis enzymes are induced during lateral root development in a PUCHI-dependent manner. Significantly, several mutants perturbed in VLCFA biosynthesis show similar lateral root developmental defects as puchi-1 Moreover, puchi-1 roots display the same disorganized callus formation phenotype as VLCFA biosynthesis-deficient mutants when grown on auxin-rich callus-inducing medium. Lipidomic profiling of puchi-1 roots revealed reduced VLCFA content compared with WT. We conclude that PUCHI-regulated VLCFA biosynthesis is part of a pathway controlling cell proliferation during lateral root and callus formation., Competing Interests: The authors declare no conflict of interest.

On 5 June this year the first field trial of a CRISPR-Cas-9 gene-edited crop began at Rothamsted Research in the UK, having been approved by the UK Department for Environment, Food & Rural Affairs. However, in late July 2018, after the trial had started, the European Court of Justice ruled that techniques such as gene editing fall within the European Union's 2001 GMO directive, meaning that our gene-edited Camelina plants should be considered as genetically modified (GM). Here we describe our experience of running this trial and the legal transformation of our plants. We also consider the future of European plant research using gene-editing techniques, which now fall under the burden of GM regulation, and how this will likely impede translation of publicly funded basic research., Competing Interests: JF, JN No competing interests declared, (© 2018, Faure and Napier.)

Macroscale fluorescence imaging is increasingly used to observe biological samples. However, it may suffer from spectral interferences that originate from ambient light or autofluorescence of the sample or its support. In this manuscript, we built a simple and inexpensive fluorescence macroscope, which has been used to evaluate the performance of Speed OPIOM (Out of Phase Imaging after Optical Modulation), which is a reference-free dynamic contrast protocol, to selectively image reversibly photoswitchable fluorophores as labels against detrimental autofluorescence and ambient light. By tuning the intensity and radial frequency of the modulated illumination to the Speed OPIOM resonance and adopting a phase-sensitive detection scheme that ensures noise rejection, we enhanced the sensitivity and the signal-to-noise ratio for fluorescence detection in blot assays by factors of 50 and 10, respectively, over direct fluorescence observation under constant illumination. Then, we overcame the strong autofluorescence of growth media that are currently used in microbiology and realized multiplexed fluorescence observation of colonies of spectrally similar fluorescent bacteria with a unique configuration of excitation and emission wavelengths. Finally, we easily discriminated fluorescent labels from the autofluorescent and reflective background in labeled leaves, even under the interference of incident light at intensities that are comparable to sunlight. The proposed approach is expected to find multiple applications, from biological assays to outdoor observations, in fluorescence macroimaging., Competing Interests: The authors declare that they have no conflict of interest.

Background: Genome-wide characterization of tissue- or cell-specific gene expression is a recurrent bottleneck in biology. We have developed a sensitive approach based on ultra-low RNA sequencing coupled to laser assisted microdissection for analyzing different tissues of the small Arabidopsis embryo., Methods and Results: We first characterized the number of genes detected according to the quantity of tissue yield and total RNA extracted. Our results revealed that as low as 0.02 mm 2 of tissue and 50 pg of total RNA can be used without compromising the number of genes detected. The optimised protocol was used to compare the epidermal versus mesophyll cell transcriptomes of cotyledons at the torpedo-shaped stage of embryo development. The approach was validated by the recovery of well-known epidermal genes such AtML1 or AtPDF2 and genes involved in flavonoid and cuticular waxes pathways. Moreover, the interest and sensitivity of this approach were highlighted by the characterization of several transcription factors preferentially expressed in epidermal cells., Conclusion: This technical advance unlocks some current limitations of transcriptomic analyses and allows to investigate further and efficiently new biological questions for which only a very small amounts of cells need to be isolated. For instance, it paves the way to increasing the spatial accuracy of regulatory networks in developing small embryo of Arabidopsis or other plant tissues.

The Peer Review File associated with this Article was updated shortly after publication to redact from the authors' point-by-point response a description of unpublished work describing how Speed OPIOM may in future be used to facilitate discrimination between FRET and direct excitation.

We present speed out-of-phase imaging after optical modulation (OPIOM), which exploits reversible photoswitchable fluorophores as fluorescent labels and combines optimized periodic illumination with phase-sensitive detection to specifically retrieve the label signal. Speed OPIOM can extract the fluorescence emission from a targeted label in the presence of spectrally interfering fluorophores and autofluorescence. Up to four fluorescent proteins exhibiting a similar green fluorescence have been distinguished in cells either sequentially or in parallel. Speed OPIOM is compatible with imaging biological processes in real time in live cells. Finally speed OPIOM is not limited to microscopy but is relevant for remote imaging as well, in particular, under ambient light. Thus, speed OPIOM has proved to enable fast and quantitative live microscopic and remote-multiplexed fluorescence imaging of biological samples while filtering out noise, interfering fluorophores, as well as ambient light.Generally, fluorescence imaging needs to be done in a dark environment using molecules with spectrally separated emissions. Here, Quérard et al. develop a protocol for high-speed imaging and remote sensing of spectrally overlapping reversible photoswitchable fluorophores in ambient light.

In many plant species, gene dosage is an important cause of phenotype variation. Engineering gene dosage, particularly in polyploid genomes, would provide an efficient tool for plant breeding. The hexaploid oilseed crop Camelina sativa, which has three closely related expressed subgenomes, is an ideal species for investigation of the possibility of creating a large collection of combinatorial mutants. Selective, targeted mutagenesis of the three delta-12-desaturase (FAD2) genes was achieved by CRISPR-Cas9 gene editing, leading to reduced levels of polyunsaturated fatty acids and increased accumulation of oleic acid in the oil. Analysis of mutations over four generations demonstrated the presence of a large variety of heritable mutations in the three isologous CsFAD2 genes. The different combinations of single, double and triple mutants in the T3 generation were isolated, and the complete loss-of-function mutants revealed the importance of delta-12-desaturation for Camelina development. Combinatorial association of different alleles for the three FAD2 loci provided a large diversity of Camelina lines with various lipid profiles, ranging from 10% to 62% oleic acid accumulation in the oil. The different allelic combinations allowed an unbiased analysis of gene dosage and function in this hexaploid species, but also provided a unique source of genetic variability for plant breeding., (© 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Nannochloropsis species are oleaginous eukaryotes containing a plastid limited by four membranes, deriving from a secondary endosymbiosis. In Nannochloropsis, thylakoid lipids, including monogalactosyldiacylglycerol (MGDG), are enriched in eicosapentaenoic acid (EPA). The need for EPA in MGDG is not understood. Fatty acids are de novo synthesized in the stroma, then converted into very-long-chain polyunsaturated fatty acids (FAs) at the endoplasmic reticulum (ER). The production of MGDG relies therefore on an EPA supply from the ER to the plastid, following an unknown process. We identified seven elongases and five desaturases possibly involved in EPA production in Nannochloropsis gaditana Among the six heterokont-specific saturated FA elongases possibly acting upstream in this pathway, we characterized the highly expressed isoform Δ0-ELO1 Heterologous expression in yeast (Saccharomyces cerevisiae) showed that NgΔ0-ELO1 could elongate palmitic acid. Nannochloropsis Δ0-elo1 mutants exhibited a reduced EPA level and a specific decrease in MGDG In NgΔ0-elo1 lines, the impairment of photosynthesis is consistent with a role of EPA-rich MGDG in nonphotochemical quenching control, possibly providing an appropriate MGDG platform for the xanthophyll cycle. Concomitantly with MGDG decrease, the level of triacylglycerol (TAG) containing medium chain FAs increased. In Nannochloropsis, part of EPA used for MGDG production is therefore biosynthesized by a channeled process initiated at the elongation step of palmitic acid by Δ0-ELO1, thus acting as a committing enzyme for galactolipid production. Based on the MGDG/TAG balance controlled by Δ0-ELO1, this study also provides novel prospects for the engineering of oleaginous microalgae for biotechnological applications., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Sphingolipids and their phosphorylated derivatives are ubiquitous bio-active components of cells. They are structural elements in the lipid bilayer and contribute to the dynamic nature of the membrane. They have been implicated in many cellular processes in yeast and animal cells, including aspects of signaling, apoptosis, and senescence. Although sphingolipids have a better defined role in animal systems, they have been shown to be central to many essential processes in plants including but not limited to, pollen development, signal transduction and in the response to biotic and abiotic stress. A fuller understanding of the roles of sphingolipids within plants has been facilitated by classical biochemical studies and the identification of mutants of model species. Recently the development of powerful mass spectrometry techniques hailed the advent of the emerging field of lipidomics enabling more accurate sphingolipid detection and quantitation. This review will consider plant sphingolipid biosynthesis and function in the context of these new developments. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Very long chain fatty acids (VLCFAs) are involved in plant development and particularly in several cellular processes such as membrane trafficking, cell division and cell differentiation. However, the precise role of VLCFAs in these different cellular processes is still poorly understood in plants. In order to identify new factors associated with the biosynthesis or function of VLCFAs, a yeast multicopy suppressor screen was carried out in a yeast mutant strain defective for fatty acid elongation. Loss of function of the elongase 3 hydroxyacyl-CoA dehydratase PHS1 in yeast and PASTICCINO2 in plants prevents growth and induces cytokinesis defects. PROTEIN TYROSIN PHOSPHATASE-LIKE (PTPLA) previously characterized as an inactive dehydratase was able to restore yeast phs1 growth and VLCFAs elongation but not the plant pas2-1 defects. PTPLA interacted with elongase subunits in the Endoplasmic Reticulum (ER) and its absence induced the accumulation of 3-hydroxyacyl-CoA as expected from a dehydratase involved in fatty acid (FA) elongation. However, loss of PTPLA function increased VLCFA levels, an effect that was dependent on the presence of PAS2 indicating that PTPLA activity repressed FA elongation. The two dehydratases have specific expression profiles in the root with PAS2, mostly restricted to the endodermis, while PTPLA was confined in the vascular tissue and pericycle cells. Comparative ectopic expression of PTPLA and PAS2 in their respective domains confirmed the existence of two independent elongase complexes based on PAS2 or PTPLA dehydratase that are functionally interacting., Competing Interests: The authors have declared that no competing interests exist.

Correct gene expression requires tight RNA quality control both at transcriptional and post-transcriptional levels. Using a splicing-defective allele of PASTICCINO2 (PAS2), a gene essential for plant development, we isolated suppressor mutations modifying pas2-1 mRNA profiles and restoring wild-type growth. Three suppressor of pas2 (sop) mutations modified the degradation of mis-spliced pas2-1 mRNA species, allowing the synthesis of a functional protein. Cloning of the suppressor mutations identified the core subunit of the exosome SOP2/RRP4, the exosome nucleoplasmic cofactor SOP3/HEN2 and a novel zinc-finger protein SOP1 that colocalizes with HEN2 in nucleoplasmic foci. The three SOP proteins counteract post-transcriptional (trans)gene silencing (PTGS), which suggests that they all act in RNA quality control. In addition, sop1 mutants accumulate some, but not all of the misprocessed mRNAs and other types of RNAs that are observed in exosome mutants. Taken together, our data show that SOP1 is a new component of nuclear RNA surveillance that is required for the degradation of a specific subset of nuclear exosome targets.

Plant cytokinesis requires intense membrane trafficking and remodeling to form a specific membrane structure, the cell plate that will ultimately separate the daughter cells. The nature and the role of lipids involved in the formation of the cell plate remain unclear. Plant membranes are particularly rich in sphingolipids such as glucosyl-ceramides with long (16 carbons) or very long (24 carbons) acyl chains. We reveal here that inhibition of the synthesis of sphingolipids with very long acyl chains induces defective cell plates with persistent vesicular structures and large gaps. Golgi-derived vesicles carrying material toward the cell plate display longer vesicle-vesicle contact time and their cargos accumulate at the cell plate, suggesting membrane fusion and/or recycling defects. In vitro fusion experiments between artificial vesicles show that glycosphingolipids with very long acyl chains stimulate lipid bilayer fusion. Therefore we propose that the very long acyl chains of sphingolipids are essential structural determinants for vesicle dynamics and membrane fusion during cytokinesis., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Plant sphingolipids are a highly diverse family of structural and signal lipids. Owing to their chemical diversity and complexity, a powerful analytical method was required to identify and quantify a large number of individual molecules with a high degree of structural accuracy. By using ultra-performance liquid chromatography with a single elution system coupled to electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) in the positive multiple reaction monitoring (MRM) mode, detailed sphingolipid composition was analyzed in various tissues of two Brassicaceae species Arabidopsis thaliana and Camelina sativa. A total of 300 molecular species were identified defining nine classes of sphingolipids, including Cers, hCers, Glcs and GIPCs. High-resolution mass spectrometry identified sphingolipids including amino- and N-acylated-GIPCs. The comparative analysis of seedling, seed and oil sphingolipids showed tissue specific distribution suggesting metabolic channeling and compartmentalization., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Camelina (Camelina sativa (L.) Crantz) is a re-emergent oilseed crop that is also becoming important as a model for applied projects based on studies in Arabidopsis thaliana, since the two species are closely related members of the tribe Camelineae of the Brassicaeae. Since camelina can be transformed genetically by floral dip, genetically modified (GM) camelina is being created in many laboratories, and small-scale field trials are already being conducted in the US and Canada. Although camelina does not cross-fertilize Brassica crop species, such as oilseed rape, nothing was known about its ability to cross with other members of the tribe Camelineae, which in addition to arabidopsis includes the widespread weed, shepherd's purse (Capsella bursa-pastoris). We have tested the ability of camelina to cross with arabidopsis and C. bursa-pastoris, as well as with the more distantly related Cardamine hirsuta, tribe cardamineae. No seeds were produced in crosses with arabidopsis, and a few seeds were obtained in crosses with C. hirsuta, but the embryos aborted at an early stage of development. A few seeds were also obtained in crosses with C. bursa-pastoris, which germinated to produce plants of a phenotype intermediate to that of the parents, but the hybrids were both male and female sterile. Therefore, the likelihood of pollen-mediated gene flow from camelina to these related species is low.

Oleaginous seeds store lipids in specialized structures called oil bodies (OBs). These organelles consist of a core of neutral lipids bound by proteins embedded in a phospholipid monolayer. OB proteins are well conserved in plants and have long been grouped into only two categories: structural proteins or enzymes. Recent work, however, which identified other classes of proteins associated with OBs, clearly shows that this classification is obsolete. Proteomics-mediated OB protein identification is facilitated in plants for which the genome is sequenced and annotated. However, it is not clear whether this knowledge can be dependably transposed to less well-characterized plants, including the well-established commercial sources of seed oil as well as the many others being proposed as novel sources for biodiesel, especially in Africa and Asia. Toward an update of the current data available on OB proteins this review discusses (i) the specific difficulties for proteomic studies of organelles; (ii) a 2012 census of the proteins found in seed OBs from various crops; (iii) the oleosin composition of OBs and their role in organelle stability; (iv) PTM of OB proteins as an emerging field of investigation; and finally we describe the emerging model of the OB proteome from oilseed crops., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In land plants, very-long-chain (VLC) alkanes are major components of cuticular waxes that cover aerial organs, mainly acting as a waterproof barrier to prevent nonstomatal water loss. Although thoroughly investigated, plant alkane synthesis remains largely undiscovered. The Arabidopsis thaliana ECERIFERUM1 (CER1) protein has been recognized as an essential element of wax alkane synthesis; nevertheless, its function remains elusive. In this study, a screen for CER1 physical interaction partners was performed. The screen revealed that CER1 interacts with the wax-associated protein ECERIFERUM3 (CER3) and endoplasmic reticulum-localized cytochrome b5 isoforms (CYTB5s). The functional relevance of these interactions was assayed through an iterative approach using yeast as a heterologous expression system. In a yeast strain manipulated to produce VLC acyl-CoAs, a strict CER1 and CER3 coexpression resulted in VLC alkane synthesis. The additional presence of CYTB5s was found to enhance CER1/CER3 alkane production. Site-directed mutagenesis showed that CER1 His clusters are essential for alkane synthesis, whereas those of CER3 are not, suggesting that CYTB5s are specific CER1 cofactors. Collectively, our study reports the identification of plant alkane synthesis enzymatic components and supports a new model for alkane production in which CER1 interacts with both CER3 and CYTB5 to catalyze the redox-dependent synthesis of VLC alkanes from VLC acyl-CoAs.

Acyl chain length is thought to be crucial for biophysical properties of the membrane, in particular during cell division, when active vesicular fusion is necessary. In higher plants, the process of cytokinesis is unique, because the separation of the two daughter cells is carried out by de novo vesicular fusion to generate a laterally expanding cell plate. In Arabidopsis thaliana, very-long-chain fatty acid (VLCFA) depletion caused by a mutation in the microsomal elongase gene PASTICCINO2 (PAS2) or by application of the selective elongase inhibitor flufenacet altered cytokinesis. Cell plate expansion was delayed and the formation of the endomembrane tubular network altered. These defects were associated with specific aggregation of the cell plate markers YFP-Rab-A2a and KNOLLE during cytokinesis. Changes in levels of VLCFA also resulted in modification of endocytosis and sensitivity to brefeldin A. Finally, the cytokinesis impairment in pas2 cells was associated with reduced levels of very long fatty acyl chains in phospholipids. Together, our findings demonstrate that VLCFA-containing lipids are essential for endomembrane dynamics during cytokinesis., (@ 2011. Published by The Company of Biologists Ltd)

Sphingolipids are a class of structural membrane lipids involved in membrane trafficking and cell polarity. Functional analysis of the ceramide synthase family in Arabidopsis thaliana demonstrates the existence of two activities selective for the length of the acyl chains. Very-long-acyl-chain (C > 18 carbons) but not long-chain sphingolipids are essential for plant development. Reduction of very-long-chain fatty acid sphingolipid levels leads in particular to auxin-dependent inhibition of lateral root emergence that is associated with selective aggregation of the plasma membrane auxin carriers AUX1 and PIN1 in the cytosol. Defective targeting of polar auxin carriers is characterized by specific aggregation of Rab-A2(a)- and Rab-A1(e)-labeled early endosomes along the secretory pathway. These aggregates correlate with the accumulation of membrane structures and vesicle fragmentation in the cytosol. In conclusion, sphingolipids with very long acyl chains define a trafficking pathway with specific endomembrane compartments and polar auxin transport protein cargoes.

Sphingolipids play an essential role in the functioning of the secretory pathway in eukaryotic organisms. Their importance in the functional organization of plant cells has not been studied in any detail before. The sphingolipid synthesis inhibitor fumonisin B1 (FB1), a mycotoxin acting as a specific inhibitor of ceramide synthase, was tested for its effects on cell growth, cell polarity, cell shape, cell cycle and on the ultrastructure of BY2 cells. We used cell lines expressing different GFP-tagged markers for plant cell compartments, as well as a Golgi marker fused to the photoconvertible protein Kaede. Light and electron microscopy, combined with flow cytometry, were applied to analyse the morphodynamics and architecture of compartments of the secretory pathway. The results indicate that FB1 treatment had severe effects on cell growth and cell shape, and induced a delay in cell division processes. The cell changes were accompanied by the formation of the endoplasmic reticulum (ER)-derived tubular aggregates (FB1-induced compartments), together with an inhibition of cargo transport from the ER to the Golgi apparatus. A change in polar localization of the auxin transporter PIN1 was also observed, but endocytic processes were little affected. Electron microscopy studies confirmed that molecular FB1 targets were distinct from brefeldin A (BFA) targets. We propose that the reported effects of inhibition of ceramide biosynthesis reflect the importance of sphingolipids during cell growth and establishment of cell polarity in higher plant cells, notably through their contribution to the functional organization of the ER or its differentiation into distinct compartments., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

The role of lipids as molecular actors of protein transport and organelle morphology in plant cells has progressed over the last years through pharmacological and genetic investigations. The manuscript is reviewing the roles of various lipid families in membrane dynamics and trafficking in eukaryotic cells, and summarizes some of the related physicochemical properties of the lipids involved. The article also focuses on the specific requirements of the sphingolipid glucosylceramide (GlcCer) in Golgi morphology and protein transport through the plant secretory pathway. The use of a specific inhibitor of plant glucosylceramide synthase and selected Arabidopsis thaliana RNAi lines stably expressing several markers of the plant secretory pathway, establishes specific steps sensitive to GlcCer biosynthesis. Collectively, data of the literature demonstrate the existence of links between protein trafficking, organelle morphology, and lipid metabolism/homeostasis in eukaryotic cells including plant cells.

Cell proliferation is the main driving force for plant growth. Although genome sequence analysis revealed a high number of cell cycle genes in plants, little is known about the molecular complexes steering cell division. In a targeted proteomics approach, we mapped the core complex machinery at the heart of the Arabidopsis thaliana cell cycle control. Besides a central regulatory network of core complexes, we distinguished a peripheral network that links the core machinery to up- and downstream pathways. Over 100 new candidate cell cycle proteins were predicted and an in-depth biological interpretation demonstrated the hypothesis-generating power of the interaction data. The data set provided a comprehensive view on heterodimeric cyclin-dependent kinase (CDK)-cyclin complexes in plants. For the first time, inhibitory proteins of plant-specific B-type CDKs were discovered and the anaphase-promoting complex was characterized and extended. Important conclusions were that mitotic A- and B-type cyclins form complexes with the plant-specific B-type CDKs and not with CDKA;1, and that D-type cyclins and S-phase-specific A-type cyclins seem to be associated exclusively with CDKA;1. Furthermore, we could show that plants have evolved a combinatorial toolkit consisting of at least 92 different CDK-cyclin complex variants, which strongly underscores the functional diversification among the large family of cyclins and reflects the pivotal role of cell cycle regulation in the developmental plasticity of plants.

Very long chain fatty acids (VLCFAs) are essential components for eukaryotes. They are elongated by the elongase complex in the endoplasmic reticulum and are incorporated into four major lipid pools (triacylglycerols, waxes, phospholipids, complex sphingolipids). Functional analysis of several components of the elongase complex demonstrated the essential role of VLCFAs in plants, invertebrates and vertebrates. Although VLCFAs changes in the triacylglycerol pool has no consequence for plant development, modifications of the nature and levels of VLCFAs in waxes, phospholipids and complex sphingolipids have, collectively, profound effects on embryo, leaf, root and flower development. VLCFAs levels in epicuticular waxes are critical for the regulation of epidermal fusions during organogenesis. VLCFAs phospholipids and sphingolipids are involved in membrane structure and dynamics regulating cell size but also division and differentiation. This review summarizes the recent findings in plants but also in other organisms, highlighting the importance of very long acyl chain length during development., (Copyright (c) 2010 Académie des sciences. Published by Elsevier SAS. All rights reserved.)

Lipids have an established role as structural components of membranes or as signalling molecules, but their role as molecular actors in protein secretion is less clear. The complex sphingolipid glucosylceramide (GlcCer) is enriched in the plasma membrane and lipid microdomains of plant cells, but compared to animal and yeast cells, little is known about the role of GlcCer in plant physiology. We have investigated the influence of GlcCer biosynthesis by glucosylceramide synthase (GCS) on the efficiency of protein transport through the plant secretory pathway and on the maintenance of normal Golgi structure. We determined that GlcCer is synthesized at the beginning of the plant secretory pathway [mainly endoplasmic reticulum (ER)] and that D,L-threo-1-phenyl-2-decanoyl amino-3-morpholino-propanol (PDMP) is a potent inhibitor of plant GCS activity in vitro and in vivo. By an in vivo confocal microscopy approach in tobacco leaves infiltrated with PDMP, we showed that the decrease in GlcCer biosynthesis disturbed the transport of soluble and membrane secretory proteins to the cell surface, as these proteins were partly retained intracellularly in the ER and/or Golgi. Electron microscopic observations of Arabidopsis thaliana root cells after high-pressure freezing and freeze substitution evidenced strong morphological changes in the Golgi bodies, pointing to a link between decreased protein secretion and perturbations of Golgi structure following inhibition of GlcCer biosynthesis in plant cells.

Very-long-chain fatty acids (VLCFAs) are essential for many aspects of plant development and necessary for the synthesis of seed storage triacylglycerols, epicuticular waxes, and sphingolipids. Identification of the acetyl-CoA carboxylase PASTICCINO3 and the 3-hydroxy acyl-CoA dehydratase PASTICCINO2 revealed that VLCFAs are important for cell proliferation and tissue patterning. Here, we show that the immunophilin PASTICCINO1 (PAS1) is also required for VLCFA synthesis. Impairment of PAS1 function results in reduction of VLCFA levels that particularly affects the composition of sphingolipids, known to be important for cell polarity in animals. Moreover, PAS1 associates with several enzymes of the VLCFA elongase complex in the endoplasmic reticulum. The pas1 mutants are deficient in lateral root formation and are characterized by an abnormal patterning of the embryo apex, which leads to defective cotyledon organogenesis. Our data indicate that in both tissues, defective organogenesis is associated with the mistargeting of the auxin efflux carrier PIN FORMED1 in specific cells, resulting in local alteration of polar auxin distribution. Furthermore, we show that exogenous VLCFAs rescue lateral root organogenesis and polar auxin distribution, indicating their direct involvement in these processes. Based on these data, we propose that PAS1 acts as a molecular scaffold for the fatty acid elongase complex in the endoplasmic reticulum and that the resulting VLCFAs are required for polar auxin transport and tissue patterning during plant development.

Studies performed in animals have highlighted the major role of sphingolipids in regulating the balance between cell proliferation and cell death. Sphingolipids have also been shown to induce cell death in plants via calcium-based signalling pathways but the contribution of free cytosolic and/or nuclear calcium in the overall process has never been evaluated. Here, we show that increase in tobacco BY-2 cells of the endogenous content of Long Chain Bases (LCBs) caused by external application of d-erythro-sphinganine (DHS) is followed by immediate dose-dependent elevations of cellular free calcium concentration within the first minute in the cytosol and 10min later in the nucleus. Cells challenged with DHS enter a death process through apoptotic-like mechanisms. Lanthanum chloride, a general blocker of calcium entry, suppresses the cellular calcium variations and the PCD induced by DHS. Interestingly, dl-2-amino-5-phosphopentanoic acid (AP5) and [(+)-dizocilpine] (MK801), two inhibitors of animal and plant ionotropic glutamate receptors, suppress DHS-induced cell death symptoms by selectively inhibiting the variations of nuclear calcium concentration. The selective action of these compounds demonstrates the crucial role of nuclear calcium signature in controlling DHS-induced cell death in tobacco cells., (2009 Elsevier Ltd. All rights reserved.)

The functional genomics approach requires systematic analysis of protein subcellular distribution and interaction networks, preferably by optimizing experimental simplicity and physiological significance. Here, we present an efficient in planta transient transformation system that allows single or multiple expression of constructs containing various fluorescent protein tags in Arabidopsis cotyledons. The optimized protocol is based on vacuum infiltration of agrobacteria directly into young Arabidopsis seedlings. We demonstrate that Arabidopsis epidermal cells show a subcellular distribution of reference markers similar to that in tobacco epidermal cells, and can be used for co-localization or bi-molecular fluorescent complementation studies. We then used this new system to investigate the subcellular distribution of enzymes involved in sphingolipid metabolism. In contrast to transformation systems using tobacco epidermal cells or cultured Arabidopsis cells, our system provides the opportunity to take advantage of the extensive collections of mutant and transgenic lines available in Arabidopsis. The fact that this assay uses conventional binary vectors and a conventional Agrobacterium strain, and is compatible with a large variety of fluorescent tags, makes it a versatile tool for construct screening and characterization before stable transformation. Transient expression in Arabidopsis seedlings is thus a fast and simple method that requires minimum handling and potentially allows medium- to high-throughput analyses of fusion proteins harboring fluorescent tags in a whole-plant cellular context.

Very-long-chain fatty acids (VLCFAs) are synthesized as acyl-CoAs by the endoplasmic reticulum-localized elongase multiprotein complex. Two Arabidopsis genes are putative homologues of the recently identified yeast 3-hydroxy-acyl-CoA dehydratase (PHS1), the third enzyme of the elongase complex. We showed that Arabidopsis PASTICCINO2 (PAS2) was able to restore phs1 cytokinesis defects and sphingolipid long chain base overaccumulation. Conversely, the expression of PHS1 was able to complement the developmental defects and the accumulation of long chain bases of the pas2-1 mutant. The pas2-1 mutant was characterized by a general reduction of VLCFA pools in seed storage triacylglycerols, cuticular waxes, and complex sphingolipids. Most strikingly, the defective elongation cycle resulted in the accumulation of 3-hydroxy-acyl-CoA intermediates, indicating premature termination of fatty acid elongation and confirming the role of PAS2 in this process. We demonstrated by in vivo bimolecular fluorescence complementation that PAS2 was specifically associated in the endoplasmic reticulum with the enoyl-CoA reductase CER10, the fourth enzyme of the elongase complex. Finally, complete loss of PAS2 function is embryo lethal, and the ectopic expression of PHS1 led to enhanced levels of VLCFAs associated with severe developmental defects. Altogether these results demonstrate that the plant 3-hydroxy-acyl-CoA dehydratase PASTICCINO2 is an essential and limiting enzyme in VLCFA synthesis but also that PAS2-derived VLCFA homeostasis is required for specific developmental processes.

PASTICCINO1 (PAS1) is a high molecular weight FK506-binding protein (FKBP) involved in the control of cell proliferation and differentiation during plant development. Mutations in the C-terminal region of PAS1 result in severe developmental defects. We show here that the C-terminal domain of PAS1 controls the subcellular distribution of this protein. We also demonstrated in vitro and in vivo, by Forster resonance energy transfer, that this C-terminal region is required for interaction with FAN (FKBP-associated NAC), a new member of the plant-specific family of NAC transcription factors. PAS1 and FAN are translocated into the nucleus upon auxin treatment in plant seedlings. The nuclear translocation of PAS1 is dependent on the presence of the C terminus of the protein. Finally, we showed that FAN is involved in PAS1-regulated processes because FAN overproduction partly complemented the pas1 phenotype. We suggest that PAS1 regulates the function of this NAC-like transcription factor by controlling its targeting to the nucleus upon plant cell division.

PASTICCINO2 (PAS2), a member of the protein Tyr phosphatase-like family, is conserved among all eukaryotes and is characterized by a mutated catalytic site. The cellular functions of the Tyr phosphatase-like proteins are still unknown, even if they are essential in yeast and mammals. Here, we demonstrate that PAS2 interacts with a cyclin-dependent kinase (CDK) that is phosphorylated on Tyr and not with its unphosphorylated isoform. Phosphorylation of the conserved regulatory Tyr-15 is involved in the binding of CDK to PAS2. Loss of the PAS2 function dephosphorylated Arabidopsis thaliana CDKA;1 and upregulated its kinase activity. In accordance with its role as a negative regulator of the cell cycle, overexpression of PAS2 slowed down cell division in suspension cell cultures at the G2-to-M transition and early mitosis and inhibited Arabidopsis seedling growth. The latter was accompanied by altered leaf development and accelerated cotyledon senescence. PAS2 was localized in the cytoplasm of dividing cells but moved into the nucleus upon cell differentiation, suggesting that the balance between cell division and differentiation is regulated through the interaction between CDKA;1 and the antiphosphatase PAS2.

The dual-specificity CDC25 phosphatases are critical positive regulators of cyclin-dependent kinases (CDKs). Even though an antagonistic Arabidopsis thaliana WEE1 kinase has been cloned and tyrosine phosphorylation of its CDKs has been demonstrated, no valid candidate for a CDC25 protein has been reported in higher plants. We identify a CDC25-related protein (Arath;CDC25) of A. thaliana, constituted by a sole catalytic domain. The protein has a tyrosine-phosphatase activity and stimulates the kinase activity of Arabidopsis CDKs. Its tertiary structure was obtained by NMR spectroscopy and confirms that Arath;CDC25 belongs structurally to the classical CDC25 superfamily with a central five-stranded beta-sheet surrounded by helices. A particular feature of the protein, however, is the presence of an additional zinc-binding loop in the C-terminal part. NMR mapping studies revealed the interaction with phosphorylated peptidic models derived from the conserved CDK loop containing the phosphothreonine-14 and phosphotyrosine-15. We conclude that despite sequence divergence, Arath;CDC25 is structurally and functionally an isoform of the CDC25 superfamily, which is conserved in yeast and in plants, including Arabidopsis and rice.

Normal embryo development is required for correct seedling formation. The Arabidopsis gurke and pasticcino3 mutants were isolated from different developmental screens and the corresponding embryos exhibit severe defects in their apical region, affecting bilateral symmetry. We have recently identified lethal acc1 mutants affected in acetyl-CoA carboxylase 1 (ACCase 1) that display a similar embryo phenotype. A series of crosses showed that gk and pas3 are allelic to acc1 mutants, and direct sequencing of the ACC1 gene revealed point mutations in these new alleles. The isolation of leaky acc1 alleles demonstrated that ACCase 1 is essential for correct plant development and that mutations in ACCase affect cellular division in plants, as is the case in yeast. Interestingly, significant metabolic complementation of the mutant phenotype was obtained by exogenous supply of malonate, suggesting that the lack of cytosolic malonyl-CoA is likely to be the initial factor leading to abnormal development in the acc1 mutants.

The FKBP12 (FK506-binding protein 12 kD) immunophilin interacts with several protein partners in mammals and is a physiological regulator of the cell cycle. In Arabidopsis, only one specific partner of AtFKBP12, namely AtFIP37 (FKBP12 interacting protein 37 kD), has been identified but its function in plant development is not known. We present here the functional analysis of AtFIP37 in Arabidopsis. Knockout mutants of AtFIP37 show an embryo-lethal phenotype that is caused by a strong delay in endosperm development and embryo arrest. AtFIP37 promoter::beta-glucuronidase reporter gene constructs show that the gene is expressed during embryogenesis and throughout plant development, in undifferentiating cells such as meristem or embryonic cells as well as highly differentiating cells such as trichomes. A translational fusion with the enhanced yellow fluorescent protein indicates that AtFIP37 is a nuclear protein localized in multiple subnuclear foci that show a speckled distribution pattern. Overexpression of AtFIP37 in transgenic lines induces the formation of large trichome cells with up to six branches. These large trichomes have a DNA content up to 256C, implying that these cells have undergone extra rounds of endoreduplication. Altogether, these data show that AtFIP37 is critical for life in Arabidopsis and implies a role for AtFIP37 in the regulation of the cell cycle as shown for FKBP12 and TOR (target of rapamycin) in mammals.

PASTICCINO (PAS) genes are required for coordinated cell division and differentiation during plant development. In loss-of-function pas mutants, plant aerial tissues showed ectopic cell division that was specifically enhanced by cytokinins, leading to disorganized tumor-like tissue. To determine the role of the PAS genes in controlling cell proliferation, we first analyzed the expression profiles of several genes involved in cell division and meristem function. Differentiated and meristematic cells of the pas mutants were more competent for cell division as illustrated by the ectopic and enlarged expression profiles of CYCLIN-DEPENDENT KINASE A and CYCLIN B1. The expression of meristematic homeobox genes KNOTTED-LIKE IN ARABIDOPSIS (KNAT2, KNAT6), and SHOOT MERISTEMLESS was also increased in pas mutants. Moreover, the loss of meristem function caused by shoot meristemless mutation can be suppressed by pas2. The KNAT2 expression pattern defines an enlarged meristematic zone in pas mutants that can be mimicked in wild type by cytokinin treatment. Cytokinin induction of the primary cytokinin response markers, ARABIDOPSIS RESPONSE REGULATOR (ARR5 and ARR6), was enhanced and lasted longer in pas mutants, suggesting that PAS genes in wild type repress cytokinin responses. The expression of the cytokinin-regulated cyclin D, cyclin D3.1, was nonetheless not modified in pas mutants. However, primary auxin response genes were down-regulated in pas mutants, as shown by a lower auxin induction of IAA4 and IAA1 genes, demonstrating that the auxin response was also modified. Altogether, our results suggest that PAS genes are involved in the hormonal control of cell division and differentiation.

The pasticcino2 (pas2) mutant shows impaired embryo and seedling development associated with cell de-differentiation and proliferation. This process is specifically enhanced in presence of cytokinins leading to callus-like structure of the apical part of the seedling. Cell proliferation concerns localized and stochastic nodules of dividing cells. In absence of cytokinins, cell proliferation leads to small calli on stems but, most often, cell proliferation is associated with post-genital organ fusion. The PAS2 gene was identified by positional cloning. PAS2 expression was found in every plant organ and was not regulated by PAS1 and PAS3 genes. PAS2 encodes the Arabidopsis member of the protein tyrosine phosphatase-like (Ptpl) family, a new PTP family originally described in mice and humans and characterized by a mutated PTP active site. This family of proteins has a yeast homolog that is essential for cell viability. The absence of yeast PAS2 homolog can be functionally replaced by the Arabidopsis PAS2 protein, demonstrating that PAS2 function is conserved between higher and lower eukaryotes.

FK506-binding proteins (FKBPs) belong to the large family of peptidyl-prolyl cis-trans isomerases, which are known to be involved in many cellular processes, such as cell signalling, protein trafficking and transcription. FKBPs associate into protein complexes, although the involvement and precise role of their foldase activity remain to be elucidated. FKBPs represent a large gene family in plants that is involved in growth and development. Disruption of genes encoding FKBPs in plants and animals has underlined the importance of this family of proteins in the regulation of cell division and differentiation.

AtFKBP12 is an Arabidopsis cDNA that encodes a protein similar to the mammalian immunophilin, FKBP12. AtFKBP12 was used as 'bait' in a yeast 2-hybrid system to screen for cDNAs in Arabidopsis encoding proteins that bind to FKBP12. Two partial cDNAs were recovered encoding the C-terminus of a protein we have called Arabidopsis thaliana FKBP12 interacting protein 37 (AtFIP37). AtFIP37 is similar to a mammalian protein, FAP48, that also binds to FKBP12. The interaction between AtFKBP12 and AtFIP37 in the 2-hybrid system, as assessed by histidine auxotrophy and beta-galactosidase activity, was disrupted by FK506, but not by cyclosporin A, a drug that binds to cyclophilin A. AtFIP37 was also shown to bind in vitro to AtFKBP12 in GST-fusion protein binding assays. The binding was abolished by prior incubation of AtFKBP12 with FK506. These findings indicate that an Arabidopsis FKBP12 ortholog encodes a protein that binds FK506 and that the interaction between AtFKBP12 and AtFIP37 may involve the FK506 binding site of AtFKBP12. The interaction provides interesting new opportunities for controlling protein:protein interactions in vivo in plants.

The pasticcino (pas) mutants of Arabidopsis thaliana are a new class of plant developmental mutants; members of this class show ectopic cell proliferation in cotyledons, extra layers of cells in the hypocotyl, and an abnormal apical meristem. This phenotype is correlated with both cell division and cell elongation defects. There are three complementation groups of pas mutants (pas1, pas2, and pas3, with, respectively 2, 1, and 4 alleles). Here we describe in more detail the pas1-1 allele, which was obtained by insertional mutagenesis. The PAS1 gene has been cloned and characterized; it encodes an immunophilin-like protein similar to the p59 FK506-binding protein (FKBP52). PAS1 is characterized by an FKBP-like domain and three tetratricopeptide repeat units. Although the presence of immunophilins in plants has already been demonstrated, the pas1-1 mutant represents the first inactivation of an FKBP-like gene in plants. PAS1 expression is altered in pas1 mutants and in the pas2 and pas3 mutants. The expression of the PAS1 gene is increased in the presence of cytokinins, a class of phytohormones originally discovered because of their ability to stimulate cell division. These results are of particular relevance as they show for the first time that an FKBP-like protein plays an important role in the control of plant development.

The control of cell division by growth regulators is critical to proper plant development. The isolation of single-gene mutants altered in the response to plant hormones should permit the identification of essential genes controlling the growth and development of plants. We have isolated mutants pasticcino belonging to 3 complementation groups (pas1, pas2, pas3) in the progeny of independent ethyl methane sulfonate and T-DNA mutagenized Arabidopsis thaliana plants. The screen was performed in the presence or absence of cytokinin. The mutants isolated were those that showed a significant hypertrophy of their apical parts when grown on cytokinin-containing medium. The pas mutants have altered embryo, leaf and root development. They display uncoordinated cell divisions which are enhanced by cytokinin. Physiological and biochemical analyses show that cytokinins are probably involved in pas phenotypes. The PAS genes have been mapped respectively to chromosomes 3, 5 and 1 and represent new plant genes involved in the control of cell division and plant development.

In Nicotiana plumbaginifolia cytokinins affect seedling development by inhibiting root growth and hypocotyl elongation and by stimulating cotyledon expansion. The zea3.1 mutant was selected for its inability to grow in conditions of low nitrogen and for its ability to grow independently on inhibitory concentrations of zeatin (J.D. Faure, M. Jullien, M. Caboche [1994] Plant J 5: 481-491). The zea3.1 growth response to cytokinins is reflected by an increase in cotyledon expansion due to cell division and by a swelling of the hypocotyl due to cell enlargement. An analysis of the seedling's root length and fresh weight over a wide range of benzyladenine concentrations showed that zea3.1 plants exhibit a higher sensitivity and an amplified response to cytokinins. A similar response of zea3.1 to benzyladenine was also seen in the expression of msr1, a cytokinin-regulated gene. Regulation of msr1 expression by protein phosphorylation was unaffected by the zea3.1 mutation. No significant differences in cytokinin and auxin levels were found between zea3.1 and wild-type seedlings, suggesting that the mutant phenotype is not caused by an alteration of these hormone levels. The data presented suggest that ZEA3 negatively modulates cytokinin responses and may function as a broad regulator of seedling development.

When photomorphogenesis takes place during early plant development, the cotyledons undergo a metabolic transition from heterotrophic sink metabolism to autotrophic source metabolism. A mutant screen was devised for seedlings affected in the regulation of nitrate assimilation during this early sink-source transition in Nicotiana plumbaginifolia. A mutant (EMS 203.6) was isolated for its inability to grow on low nitrate concentration. In contrast to wild-type (WT) plants, the mutant cotyledons remained tightly attached to each other throughout seedling development. It was found that a low carbon/nitrogen ratio (C/N ratio) in the medium was required for mutant growth. The higher the ratio was, the more the growth was inhibited. Mutant EMS 203.6 accumulated all amino acids in permissive conditions (low C/N ratio), and all amino acids and sugars also in selective (high C/N ratio) conditions. In addition, sucrose in the medium repressed light-regulated genes involved in nitrate assimilation and in photosynthesis in the mutant but not in the WT plants. The mutation was mapped to the Zea3 complementation group which confers resistance to zeatin. This zeatin resistance was associated with a hypertrophy of mutant cotyledons in response to cytokinin. Both cytokinin resistance and sensitivity to a high C/N ratio were not observed in etiolated mutant seedlings and were restricted to the jointed-cotyledon developmental stage. Previous physiological studies showed evidence for a role of cytokinins in the expression of nitrate reductase. Here, the first genetic evidence for a link between carbohydrate/nitrogen metabolism and cytokinin action during early development is provided.

Reporter gene techniques and mutant analysis were used to identify the molecular basis of the regulation of the expression of nitrate reductase (NR) by nitrate and nitrate-, or ammonium-derived metabolites (N-metabolites), in the true diploïd species Nicotiana plumbaginifolia and in the amphidiploïd species Nicotiana tabacum. The N. plumbaginifolia mutant E23 results from the insertion of a Tnt1-like retrotransposon (Tnp2) in the first exon of the single-copy nia gene, which encodes nitrate reductase. One of the resulting transcripts ends in the 5' LTR (long terminal repeat) sequence of this retrotransposon, and another one in the 3' LTR. Nitrate and N-metabolites modulate the expression of these truncated transcripts, indicating that intron splicing and termination processes are not essential to these regulatory events. A GUS reporter sequence was transcriptionally linked to the promoter of the nia-1 gene of N. tabacum. This fusion was functional in transient expression assays done with protoplasts derived from mesophyll cells of N. tabacum. However none of the regulatory mechanisms known to affect steady-state levels of the nia-1 transcript were operative under these experimental conditions. Transgenic plants carrying either this fusion or translational fusions of GUS linked to the promoter of either the nia-1 or nia-2 gene of N. tabacum were obtained by Agrobacterium-mediated transfer. A low proportion of the transgenic plants (22 out of 105 independent transformants) expressed GUS activity although at a low level. Only 4 plants exhibited a detectable level of GUS mRNA. The concentration of this mRNA increased significantly in an NR-deficient background, indicating regulation by N-metabolites. Only 2 plants, however, showed regulation (induction) by nitrate. Attempts to use aux2 or nptII reporter sequences linked to either the nia-1 or nia-2 promoter as marker genes for the selection of regulatory mutants of the nitrate assimilation pathway were unsuccessful because of our inability to isolate transgenic plants in which these reporter genes were properly regulated by nitrate. The implications of these results are discussed.

Six independent mutant lines of Nicotiana plumbaginifolia resistant to ethanol, designated E3, E8, E101, E112, E144 and E251, were isolated as germinating seedlings on selective medium. In all cases, resistance to ethanol was conferred by a single recessive nuclear mutation at the same locus. Mutant seeds and pollen lacked detectable ADH activity, with the exception of E251 where a residual activity was detected. An antiserum directed against Arabidopsis thaliana ADH detected an ADH-related polypeptide of 44 kDa present in wild-type seeds and, to a lesser extent, in the seeds of the leaky mutant E251. No ADH-related polypeptide could be detected in seeds of the other mutants. However, all of them had a nearly normal level of ADH mRNA except one which did not synthesize any mRNA. These results suggest that these ethanol-resistant mutants are impaired in one of the structural genes coding for alcohol dehydrogenase. The corresponding locus has been designated Adh1.