CALEOSIN 1 interaction with AUTOPHAGY-RELATED PROTEIN 8 facilitates lipid droplet microautophagy in seedlings.

Lipid droplets (LDs) of seed tissues are storage organelles for triacylglycerols (TAGs) that provide the energy and carbon for seedling establishment. In the major route of LD degradation (lipolysis), TAGs are mobilized by lipases. However, LDs may also be degraded via lipophagy, a type of selective autophagy, which mediates LD delivery to vacuoles or lysosomes. The exact mechanisms of LD degradation and the mobilization of their content in plants remain unresolved. Here, we provide evidence that LDs are degraded via a process morphologically resembling microlipophagy in Arabidopsis (Arabidopsis thaliana) seedlings. We observed the entry and presence of LDs in the central vacuole as well as their breakdown. Moreover, we show co-localization of AUTOPHAGY-RELATED PROTEIN 8b (ATG8b) and LDs during seed germination and localization of lipidated ATG8 (ATG8-PE) to the LD fraction. We further demonstrate that structural LD proteins from the caleosin family, CALEOSIN 1 (CLO1), CALEOSIN 2 (CLO2), and CALEOSIN 3 (CLO3), interact with ATG8 proteins and possess putative ATG8-interacting motifs (AIMs). Deletion of the AIM localized directly before the proline knot disrupts the interaction of CLO1 with ATG8b, suggesting a possible role of this region in the interaction between these proteins. Collectively, we provide insights into LD degradation by microlipophagy in germinating seeds with a particular focus on the role of structural LD proteins in this process.

Multi-omics analysis of xylem sap uncovers dynamic modulation of poplar defenses by ammonium and nitrate.

Xylem sap is the major transport route for nutrients from roots to shoots. In the present study, we investigated how variations in nitrogen (N) nutrition affected the metabolome and proteome of xylem sap and the growth of the xylem endophyte Brennaria salicis, and we also report transcriptional re-wiring of leaf defenses in poplar (Populus × canescens). We supplied poplars with high, intermediate or low concentrations of ammonium or nitrate. We identified 288 unique proteins in xylem sap. Approximately 85% of the xylem sap proteins were shared among ammonium- and nitrate-supplied plants. The number of proteins increased with increasing N supply but the major functional categories (catabolic processes, cell wall-related enzymes, defense) were unaffected. Ammonium nutrition caused higher abundances of amino acids and carbohydrates, whereas nitrate caused higher malate levels in xylem sap. Pipecolic acid and N-hydroxy-pipecolic acid increased, whereas salicylic acid and jasmonoyl-isoleucine decreased, with increasing N nutrition. Untargeted metabolome analyses revealed 2179 features in xylem sap, of which 863 were differentially affected by N treatments. We identified 124 metabolites, mainly from specialized metabolism of the groups of salicinoids, phenylpropanoids, phenolics, flavonoids, and benzoates. Their abundances increased with decreasing N, except coumarins. Brennaria salicis growth was reduced in nutrient-supplemented xylem sap of low- and high- NO3- -fed plants compared to that of NH4+ -fed plants. The drastic changes in xylem sap composition caused massive changes in the transcriptional landscape of leaves and recruited defenses related to systemic acquired and induced systemic resistance. Our study uncovers unexpected complexity and variability of xylem composition with consequences for plant defenses.

Wounding Triggers Wax Biosynthesis in Arabidopsis Leaves in an Abscisic Acid and Jasmonoyl-Isoleucine Dependent Manner.

Wounding caused by insects or abiotic factors such as wind and hail can cause severe stress for plants. Intrigued by the observation that wounding induces expression of genes involved in surface wax synthesis in a jasmonoyl-isoleucine (JA-Ile)-independent manner, the role of wax biosynthesis and respective genes upon wounding was investigated. Wax, a lipid-based barrier, protects plants both from environmental threats as well as from an uncontrolled loss of water. Its biosynthesis is described to be regulated by abscisic acid (ABA), whereas the main wound-signal is the hormone JA-Ile. We show in this study, that genes coding for enzymes of surface wax synthesis are induced upon wounding in Arabidopsis thaliana leaves in a JA-Ile-independent but ABA-dependent manner. Furthermore, the ABA-dependent transcription factor MYB96 is a key regulator of wax biosynthesis upon wounding. On the metabolite level, wound-induced wax accumulation is strongly reduced in JA-Ile-deficient plants, but this induction is only slightly decreased in ABA-reduced plants. To further analyze the ABA-dependent wound response, we conducted wounding experiments in high humidity. They show that high humidity prevents the wound-induced wax accumulation in A. thaliana leaves. Together the data presented in this study show that wound-induced wax accumulation is JA-Ile-dependent on the metabolite level, but the expression of genes coding for enzymes of wax synthesis is regulated by ABA.

Cell wall-localized BETA-XYLOSIDASE4 contributes to immunity of Arabidopsis against Botrytis cinerea.

Plant cell walls constitute physical barriers that restrict access of microbial pathogens to the contents of plant cells. The primary cell wall of multicellular plants predominantly consists of cellulose, hemicellulose, and pectin, and its composition can change upon stress. BETA-XYLOSIDASE4 (BXL4) belongs to a seven-member gene family in Arabidopsis (Arabidopsis thaliana), one of which encodes a protein (BXL1) involved in cell wall remodeling. We assayed the influence of BXL4 on plant immunity and investigated the subcellular localization and enzymatic activity of BXL4, making use of mutant and overexpression lines. BXL4 localized to the apoplast and was induced upon infection with the necrotrophic fungal pathogen Botrytis cinerea in a jasmonoyl isoleucine-dependent manner. The bxl4 mutants showed a reduced resistance to B. cinerea, while resistance was increased in conditional overexpression lines. Ectopic expression of BXL4 in Arabidopsis seed coat epidermal cells rescued a bxl1 mutant phenotype, suggesting that, like BXL1, BXL4 has both xylosidase and arabinosidase activity. We conclude that BXL4 is a xylosidase/arabinosidase that is secreted to the apoplast and its expression is upregulated under pathogen attack, contributing to immunity against B. cinerea, possibly by removal of arabinose and xylose side-chains of polysaccharides in the primary cell wall.

Chlamydomonas CHT7 Is Required for an Effective Quiescent State by Regulating Nutrient-Responsive Cell Cycle Gene Expression.

COMPROMISED HYDROLYSIS OF TRIACYLGLYCEROLS7 (CHT7) in Chlamydomonas (Chlamydomonas reinhardtii) was previously shown to affect the transcription of a subset of genes during nitrogen (N)-replete growth and following N refeeding. Here, we show that an extensive derepression of genes involved in DNA metabolism and cell cycle-related processes, as well as downregulation of genes encoding oxidoreductases and nutrient transporters, occurs in the cht7 mutant during N deprivation. Cellular mutant phenotypes are consistent with the observed transcriptome misregulation, as cht7 cells fail to properly arrest growth, nuclear replication, and cell division following N deprivation. Reduction in cht7 colony formation following N refeeding is explained by its compromised viability during N deprivation and by the occurrence of abortive divisions during N refeeding. Surprisingly, the largely unstructured C-terminal half of CHT7 with predicted protein binding domains, but not the canonical CXC DNA binding domain, is essential for the ability of CHT7 to form stable complexes and reverse the cellular phenotypes and transcription levels in the cht7 mutant. Hence, although lacking the presumed DNA binding domain, CHT7 modulates the expression of cell cycle genes in response to N availability, which is essential for establishing an effective quiescent state and the coordinated resumption of growth following N refeeding.

Chemokine-like MDL proteins modulate flowering time and innate immunity in plants.

Human macrophage migration inhibitory factor (MIF) is an atypical chemokine implicated in intercellular signaling and innate immunity. MIF orthologs (MIF/D-DT-like proteins, MDLs) are present throughout the plant kingdom, but remain experimentally unexplored in these organisms. Here, we provide an in planta characterization and functional analysis of the three-member gene/protein MDL family in Arabidopsis thaliana. Subcellular localization experiments indicated a nucleo-cytoplasmic distribution of MDL1 and MDL2, while MDL3 is localized to peroxisomes. Protein-protein interaction assays revealed the in vivo formation of MDL1, MDL2, and MDL3 homo-oligomers, as well as the formation of MDL1-MDL2 hetero-oligomers. Functionally, Arabidopsismdl mutants exhibited a delayed transition from vegetative to reproductive growth (flowering) under long-day conditions, but not in a short-day environment. In addition, mdl mutants were more resistant to colonization by the bacterial pathogen Pseudomonas syringae pv. maculicola. The latter phenotype was compromised by the additional mutation of SALICYLIC ACID INDUCTION DEFICIENT 2 (SID2), a gene implicated in the defense-induced biosynthesis of the key signaling molecule salicylic acid. However, the enhanced antibacterial immunity was not associated with any constitutive or pathogen-induced alterations in the levels of characteristic phytohormones or defense-associated metabolites. Interestingly, bacterial infection triggered relocalization and accumulation of MDL1 and MDL2 at the peripheral lobes of leaf epidermal cells. Collectively, our data indicate redundant functionality and a complex interplay between the three chemokine-like Arabidopsis MDL proteins in the regulation of both developmental and immune-related processes. These insights expand the comparative cross-kingdom analysis of MIF/MDL signaling in human and plant systems.

Sphingolipid-Induced Programmed Cell Death is a Salicylic Acid and EDS1-Dependent Phenotype in Arabidopsis Fatty Acid Hydroxylase (Fah1, Fah2) and Ceramide Synthase (Loh2) Triple Mutants.

Ceramides (Cers) and long-chain bases (LCBs) are plant sphingolipids involved in the induction of plant programmed cell death (PCD). The fatty acid hydroxylase mutant fah1 fah2 exhibits high Cer levels and moderately elevated LCB levels. Salicylic acid glucoside level is increased in this mutant, but no cell death can be detected by trypan blue staining. To determine the effect of Cers with different chain lengths, fah1 fah2 was crossed with ceramide synthase mutants longevity assurance gene one homologue1-3 (loh1, loh2 and loh3). Surprisingly, only triple mutants with loh2 show cell death detected by trypan blue staining under the selected conditions. Sphingolipid profiling revealed that the greatest differences between the triple mutant plants are in the LCB and LCB-phosphate (LCB-P) fraction. fah1 fah2 loh2 plants accumulate LCB d18:0, LCB t18:0 and LCB-P d18:0. Crossing fah1 fah2 loh2 with the salicylic acid (SA) synthesis mutant sid2-2 and with the SA signaling mutants enhanced disease susceptibility 1-2 (eds1-2) and phytoalexin deficient 4-1 (pad4-1) revealed that lesions are SA- and EDS1-dependent. These quadruple mutants also confirm that there may be a feedback loop between SA and sphingolipid metabolism as they accumulated less Cers and LCBs. In conclusion, PCD in fah1 fah2 loh2 is a SA- and EDS1-dependent phenotype, which is likely due to accumulation of LCBs.

Here, there and everywhere - the importance of neutral lipids in plant growth and development.

In plants, lipids serve as one of the major and vital cellular constituents. Neutral lipids reserves play an essential role in the plant life cycle by providing carbon and energy equivalents for periods of active metabolism. The most common form of lipid storage are triacylglycerols (TAGs) packed into specialized organelles called lipid droplets (LDs). They have been observed in diverse plant organs and tissues, like oil seeds or pollen grains. LDs consist of a core, composed mostly of TAGs, enclosed by a single layer of phospholipids that is decorated by a unique set of structural proteins. Moreover, the recent advances in exploration of LDs proteome revealed a plethora of diverse proteins interacting with LDs. This is likely the result of a highly dynamic nature of these organelles and their involvement in many diverse aspect of cellular metabolism, tightly synchronized with plant developmental programs and directly related to plant-environment interactions. In this review we summarize and discuss the current progress in understanding the role of LDs and their cargo during plants life cycle, with a special emphasis on developmental aspects.

Signal peptide peptidase activity connects the unfolded protein response to plant defense suppression by Ustilago maydis.

The corn smut fungus Ustilago maydis requires the unfolded protein response (UPR) to maintain homeostasis of the endoplasmic reticulum (ER) during the biotrophic interaction with its host plant Zea mays (maize). Crosstalk between the UPR and pathways controlling pathogenic development is mediated by protein-protein interactions between the UPR regulator Cib1 and the developmental regulator Clp1. Cib1/Clp1 complex formation results in mutual modification of the connected regulatory networks thereby aligning fungal proliferation in planta, efficient effector secretion with increased ER stress tolerance and long-term UPR activation in planta. Here we address UPR-dependent gene expression and its modulation by Clp1 using combinatorial RNAseq/ChIPseq analyses. We show that increased ER stress resistance is connected to Clp1-dependent alterations of Cib1 phosphorylation, protein stability and UPR gene expression. Importantly, we identify by deletion screening of UPR core genes the signal peptide peptidase Spp1 as a novel key factor that is required for establishing a compatible biotrophic interaction between U. maydis and its host plant maize. Spp1 is dispensable for ER stress resistance and vegetative growth but requires catalytic activity to interfere with the plant defense, revealing a novel virulence specific function for signal peptide peptidases in a biotrophic fungal/plant interaction.

Ustilago maydis effector Jsi1 interacts with Topless corepressor, hijacking plant jasmonate/ethylene signaling.

Ustilago maydis is the causal agent of maize smut disease. During the colonization process, the fungus secretes effector proteins that suppress immune responses and redirect the host metabolism in favor of the pathogen. As effectors play a critical role during plant colonization, their identification and functional characterization are essential to understanding biotrophy and disease. Using biochemical, molecular, and transcriptomic techniques, we performed a functional characterization of the U. maydis effector Jasmonate/Ethylene signaling inducer 1 (Jsi1). Jsi1 interacts with several members of the plant corepressor family Topless/Topless related (TPL/TPR). Jsi1 expression in Zea mays and Arabidopsis thaliana leads to transcriptional induction of the ethylene response factor (ERF) branch of the jasmonate/ethylene (JA/ET) signaling pathway. In A. thaliana, activation of the ERF branch leads to biotrophic susceptibility. Jsi1 likely activates the ERF branch via an EAR (ET-responsive element binding-factor-associated amphiphilic repression) motif, which resembles EAR motifs from plant ERF transcription factors, that interacts with TPL/TPR proteins. EAR-motif-containing effector candidates were identified from different fungal species, including Magnaporthe oryzae, Sporisorium scitamineum, and Sporisorium reilianum. Interaction between plant TPL proteins and these effector candidates from biotrophic and hemibiotrophic fungi indicates the convergent evolution of effectors modulating the TPL/TPR corepressor hub.

Pheophorbide a May Regulate Jasmonate Signaling during Dark-Induced Senescence.

Chlorophyll degradation is one of the most visible signs of leaf senescence. During senescence, chlorophyll is degraded in the multistep pheophorbide a oxygenase (PAO)/phyllobilin pathway. This pathway is tightly regulated at the transcriptional level, allowing coordinated and efficient remobilization of nitrogen toward sink organs. Using a combination of transcriptome and metabolite analyses during dark-induced senescence of Arabidopsis (Arabidopsis thaliana) mutants deficient in key steps of the PAO/phyllobilin pathway, we show an unanticipated role for one of the pathway intermediates, i.e. pheophorbide a Both jasmonic acid-related gene expression and jasmonic acid precursors specifically accumulated in pao1, a mutant deficient in PAO. We propose that pheophorbide a, the last intact porphyrin intermediate of chlorophyll degradation and a unique pathway "bottleneck," has been recruited as a signaling molecule of chloroplast metabolic status. Our work challenges the assumption that chlorophyll breakdown is merely a result of senescence, and proposes that the flux of pheophorbide a through the pathway acts in a feed-forward loop that remodels the nuclear transcriptome and controls the pace of chlorophyll degradation in senescing leaves.

The Microalga Nannochloropsis during Transition from Quiescence to Autotrophy in Response to Nitrogen Availability.

The marine microalgae Nannochloropsis oceanica (CCMP1779) is a prolific producer of oil and is considered a viable and sustainable resource for biofuel feedstocks. Nitrogen (N) availability has a strong impact on the physiological status and metabolism of microalgal cells, but the exact nature of this response is poorly understood. To fill this gap we performed transcriptomic profiling combined with cellular and molecular analyses of N. oceanica CCMP1779 during the transition from quiescence to autotrophy. N deprivation-induced quiescence was accompanied by a strong reorganization of the photosynthetic apparatus and changes in the lipid homeostasis, leading to accumulation of triacylglycerol. Cell cycle activation and re-establishment of photosynthetic activity observed in response to resupply of the growth medium with N were accompanied by a rapid degradation of triacylglycerol stored in lipid droplets (LDs). Besides observing LD translocation into vacuoles, we also provide evidence for direct interaction between the LD surface protein (NoLDSP) and AUTOPHAGY-RELATED8 (NoATG8) protein and show a role of microlipophagy in LD turnover in N. oceanica CCMP1779. This knowledge is crucial not only for understanding the fundamental mechanisms controlling the cellular energy homeostasis in microalgal cells but also for development of efficient strategies to achieve higher algal biomass and better microalgal lipid productivity.

Sphingolipid Δ4-desaturation is an important metabolic step for glycosylceramide formation in Physcomitrium patens.

Glycosylceramides are abundant membrane components in vascular plants and are associated with cell differentiation, organogenesis, and protein secretion. Long-chain base (LCB) Δ4-desaturation is an important structural feature for metabolic channeling of sphingolipids into glycosylceramide formation in plants and fungi. In Arabidopsis thaliana, LCB Δ4-unsaturated glycosylceramides are restricted to pollen and floral tissue, indicating that LCB Δ4-desaturation has a less important overall physiological role in A. thaliana. In the bryophyte Physcomitrium patens, LCB Δ4-desaturation is a feature of the most abundant glycosylceramides of the gametophyte generation. Metabolic changes in the P. patens null mutants for the sphingolipid Δ4-desaturase (PpSD4D) and the glycosylceramide synthase (PpGCS), sd4d-1 and gcs-1, were determined by ultra-performance liquid chromatography coupled with nanoelectrospray ionization and triple quadrupole tandem mass spectrometry analysis. sd4d-1 plants lacked unsaturated LCBs and the most abundant glycosylceramides. gcs-1 plants lacked all glycosylceramides and accumulated hydroxyceramides. While sd4d-1 plants mostly resembled wild-type plants, gcs-1 mutants were impaired in growth and development. These results indicate that LCB Δ4-desaturation is a prerequisite for the formation of the most abundant glycosylceramides in P. patens. However, loss of unsaturated LCBs does not affect plant viability, while blockage of glycosylceramide synthesis in gcs-1 plants causes severe plant growth and development defects.

Galactoglycerolipid Lipase PGD1 Is Involved in Thylakoid Membrane Remodeling in Response to Adverse Environmental Conditions in Chlamydomonas.

Photosynthesis occurs in the thylakoid membrane, where the predominant lipid is monogalactosyldiacylglycerol (MGDG). As environmental conditions change, photosynthetic membranes have to adjust. In this study, we used a loss-of-function Chlamydomonas reinhardtii mutant deficient in the MGDG-specific lipase PGD1 (PLASTID GALACTOGLYCEROLIPID DEGRADATION1) to investigate the link between MGDG turnover, chloroplast ultrastructure, and the production of reactive oxygen species (ROS) in response to different adverse environmental conditions. The pgd1 mutant showed altered MGDG abundance and acyl composition and altered abundance of photosynthesis complexes, with an increased PSII/PSI ratio. Transmission electron microscopy showed hyperstacking of the thylakoid grana in the pgd1 mutant. The mutant also exhibited increased ROS production during N deprivation and high light exposure. Supplementation with bicarbonate or treatment with the photosynthetic electron transport blocker DCMU protected the cells against oxidative stress in the light and reverted chlorosis of pgd1 cells during N deprivation. Furthermore, exposure to stress conditions such as cold and high osmolarity induced the expression of PGD1, and loss of PGD1 in the mutant led to increased ROS production and inhibited cell growth. These findings suggest that PGD1 plays essential roles in maintaining appropriate thylakoid membrane composition and structure, thereby affecting growth and stress tolerance when cells are challenged under adverse conditions.

Wood Formation under Severe Drought Invokes Adjustment of the Hormonal and Transcriptional Landscape in Poplar.

Drought is a severe environmental stress that exerts negative effects on plant growth. In trees, drought leads to reduced secondary growth and altered wood anatomy. The mechanisms underlying wood stress adaptation are not well understood. Here, we investigated the physiological, anatomical, hormonal, and transcriptional responses of poplar to strong drought. Drought-stressed xylem was characterized by higher vessel frequencies, smaller vessel lumina, and thicker secondary fiber cell walls. These changes were accompanied by strong increases in abscisic acid (ABA) and antagonistic changes in salicylic acid in wood. Transcriptional evidence supported ABA biosynthesis and signaling in wood. Since ABA signaling activates the fiber-thickening factor NST1, we expected upregulation of the secondary cell wall (SCW) cascade under stress. By contrast, transcription factors and biosynthesis genes for SCW formation were down-regulated, whereas a small set of cellulose synthase-like genes and a huge array of genes involved in cell wall modification were up-regulated in drought-stressed wood. Therefore, we suggest that ABA signaling monitors normal SCW biosynthesis and that drought causes a switch from normal to "stress wood" formation recruiting a dedicated set of genes for cell wall biosynthesis and remodeling. This proposition implies that drought-induced changes in cell wall properties underlie regulatory mechanisms distinct from those of normal wood.

The glycosyltransferase UGT76E1 significantly contributes to 12-O-glucopyranosyl-jasmonic acid formation in wounded Arabidopsis thaliana leaves.

Jasmonoyl-isoleucine (JA-Ile) is a phytohormone that orchestrates plant defenses in response to wounding, feeding insects, or necrotrophic pathogens. JA-Ile metabolism has been studied intensively, but its catabolism as a potentially important mechanism for the regulation of JA-Ile-mediated signaling is not well-understood. Especially the enzyme(s) responsible for specifically glycosylating 12-hydroxy-jasmonic acid (12-OH-JA) and thereby producing 12-O-glucopyranosyl-jasmonic acid (12-O-Glc-JA) is still elusive. Here, we used co-expression analyses of available Arabidopsis thaliana transcriptomic data, identifying four UDP-dependent glycosyltransferase (UGT) genes as wound-induced and 12-OH-JA-related, namely, UGT76E1, UGT76E2, UGT76E11, and UGT76E12 We heterologously expressed and purified the corresponding proteins to determine their individual substrate specificities and kinetic parameters. We then used an ex vivo metabolite-fingerprinting approach to investigate these proteins in conditions as close as possible to their natural environment, with an emphasis on greatly extending the range of potential substrates. As expected, we found that UGT76E1 and UGT76E2 are 12-OH-JA-UGTs, with UGT76E1 contributing a major in vivo UGT activity, as deduced from Arabidopsis mutants with abolished or increased UGT gene expression. In contrast, recombinant UGT76E11 acted on an unidentified compound and also glycosylated two other oxylipins, 11-hydroxy-7,9,13-hexadecatrienoic acid (11-HHT) and 13-hydroxy-9,11,15-octadecatrienoic acid (13-HOT), which were also accepted by recombinant UGT76E1, UGT76E2, and UGT76E12 enzymes. UGT76E12 glycosylated 12-OH-JA only to a low extent, but also accepted an artificial hydroxylated fatty acid and low amounts of kaempferol. In conclusion, our findings have elucidated the missing step in the wound-induced synthesis of 12-O-glucopyranosyl-jasmonic acid in A. thaliana.

Ectomycorrhizal fungi induce systemic resistance against insects on a nonmycorrhizal plant in a CERK1-dependent manner.

Below-ground microbes can induce systemic resistance against foliar pests and pathogens on diverse plant hosts. The prevalence of induced systemic resistance (ISR) among plant-microbe-pest systems raises the question of host specificity in microbial induction of ISR. To test whether ISR is limited by plant host range, we tested the ISR-inducing ectomycorrhizal fungus Laccaria bicolor on the nonmycorrhizal plant Arabidopsis thaliana. We used the cabbage looper Trichoplusia ni and bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pto) as readouts for ISR on Arabidopsis. We found that root inoculation with L. bicolor triggered ISR against T. ni and induced systemic susceptibility (ISS) against the bacterial pathogen Pto. We found that L. bicolor-triggered ISR against T. ni was dependent on jasmonic acid signaling and salicylic acid biosynthesis and signaling. Heat-killed L. bicolor and chitin were sufficient to trigger ISR against T. ni and ISS against Pto. The chitin receptor CERK1 was necessary for L. bicolor-mediated effects on systemic immunity. Collectively our findings suggest that some ISR responses might not require intimate symbiotic association, but rather might be the result of root perception of conserved microbial signals.

Identification of client iron-sulfur proteins of the chloroplastic NFU2 transfer protein in Arabidopsis thaliana.

Iron-sulfur (Fe-S) proteins have critical functions in plastids, notably participating in photosynthetic electron transfer, sulfur and nitrogen assimilation, chlorophyll metabolism, and vitamin or amino acid biosynthesis. Their maturation relies on the so-called SUF (sulfur mobilization) assembly machinery. Fe-S clusters are synthesized de novo on a scaffold protein complex and then delivered to client proteins via several transfer proteins. However, the maturation pathways of most client proteins and their specificities for transfer proteins are mostly unknown. In order to decipher the proteins interacting with the Fe-S cluster transfer protein NFU2, one of the three plastidial representatives found in Arabidopsis thaliana, we performed a quantitative proteomic analysis of shoots, roots, and seedlings of nfu2 plants, combined with NFU2 co-immunoprecipitation and binary yeast two-hybrid experiments. We identified 14 new targets, among which nine were validated in planta using a binary bimolecular fluorescence complementation assay. These analyses also revealed a possible role for NFU2 in the plant response to desiccation. Altogether, this study better delineates the maturation pathways of many chloroplast Fe-S proteins, considerably extending the number of NFU2 clients. It also helps to clarify the respective roles of the three NFU paralogs NFU1, NFU2, and NFU3.

Arabidopsis mlo3 mutant plants exhibit spontaneous callose deposition and signs of early leaf senescence.

KEY MESSAGE: Arabidopsis thaliana mlo3 mutant plants are not affected in pathogen infection phenotypes but-reminiscent of mlo2 mutant plants-exhibit spontaneous callose deposition and signs of early leaf senescence. The family of Mildew resistance Locus O (MLO) proteins is best known for its profound effect on the outcome of powdery mildew infections: when the appropriate MLO protein is absent, the plant is fully resistant to otherwise virulent powdery mildew fungi. However, most members of the MLO protein family remain functionally unexplored. Here, we investigate Arabidopsis thaliana MLO3, the closest relative of AtMLO2, AtMLO6 and AtMLO12, which are the Arabidopsis MLO genes implicated in the powdery mildew interaction. The co-expression network of AtMLO3 suggests association of the gene with plant defense-related processes such as salicylic acid homeostasis. Our extensive analysis shows that mlo3 mutants are unaffected regarding their infection phenotype upon challenge with the powdery mildew fungi Golovinomyces orontii and Erysiphe pisi, the oomycete Hyaloperonospora arabidopsidis, and the bacterial pathogen Pseudomonas syringae (the latter both in terms of basal and systemic acquired resistance), indicating that the protein does not play a major role in the response to any of these pathogens. However, mlo3 genotypes display spontaneous callose deposition as well as signs of early senescence in 6- or 7-week-old rosette leaves in the absence of any pathogen challenge, a phenotype that is reminiscent of mlo2 mutant plants. We hypothesize that de-regulated callose deposition in mlo3 genotypes might be the result of a subtle transient aberration of salicylic acid-jasmonic acid homeostasis during development.

The type 2 acyl-CoA:diacylglycerol acyltransferase family of the oleaginous microalga Lobosphaera incisa.

BACKGROUND: Oleaginous microalgae are promising sources of energy-rich triacylglycerols (TAGs) for direct use for food, feed and industrial applications. Lobosphaera incisa is a fresh water unicellular alga, which in response to nutrient stress accumulates a high amount of TAGs with a high proportion of arachidonic acid (ARA). The final committed step of de novo TAG biosynthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferases (DGATs), which add a fatty acid (FA) to the final sn-3 position of diacylglycerol (DAG). RESULTS: Genome analysis revealed the presence of five putative DGAT isoforms in L. incisa, including one DGAT of type 1, three DGATs of type 2 and a single isoform of a type 3 DGAT. For LiDGAT1, LiDGAT2.1, LiDGAT2.2 and LiDGAT2.3 enzyme activity was confirmed by expressing them in the TAG-deficient yeast strain H1246. Feeding experiments of yeast transformants with fatty acids suggest a broad substrate specificity spectrum for LiDGAT1. A significant TAG production in response to exogenous ARA was found for LiDGAT2.2. Cellular localization of the four type 1 and type 2 DGATs expressed in yeast revealed that they all localize to distinct ER domains. A prominent association of LiDGAT1 with ER domains in close proximity to forming lipid droplets (LDs) was also observed. CONCLUSIONS: The data revealed a distinct molecular, functional and cellular nature of type 1 and type 2 DGATs from L. incisa, with LiDGAT1 being a major contributor to the TAG pool. LiDGATs of type 2 might be in turn involved in the incorporation of unusual fatty acids into TAG and thus regulate the composition of TAG. This report provides a valuable resource for the further research of microalgae DGATs oriented towards production of fresh-water strains with higher oil content of valuable composition, not only for oil industry but also for human and animal nutrition.