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.

Fluorescence in situ Localization of Pri-miRNAs in Isolated Arabidopsis thaliana Nuclei.

Here, we present an approach combining fluorescence in situ hybridization (FISH) and immunolabeling for localization of pri-miRNAs in isolated nuclei of A. thaliana. The presented method utilizes specific DNA oligonucleotide probes, modified by addition of digoxigenin-labeled deoxynucleotides to its 3' hydroxyl terminus by terminal deoxynucleotidyl transferase (TdT). The probes are then detected by immunolabeling of digoxigenin (DIG) using specific fluorescent-labeled antibodies to visualize hybridized probes. Recently, we have applied this method to localize pri-miRNA156a, pri-miRNA163, pri-miRNA393a, and pri-miRNA414 in the nuclei isolated from leaves of 4-week-old A. thaliana. The present approach can be easily implemented to analyze nuclear distribution of diverse RNA classes, including mRNAs and pri-miRNAs in isolated fixed cells or nuclei from plant.

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.

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.

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.

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.

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.

Plastidic membrane lipids are oxidized by a lipoxygenase in Lobosphaera incisa.

Green microalgae can accumulate neutral lipids, as part of a general lipid remodeling mechanism under stress such as nitrogen starvation. Lobosphaera incisa is of special interest because of its unique TAG acyl chain composition, especially 20:4 (n-6) can reach up to 21% of dry weight after nitrogen starvation. In order to identify factors that may influence the accumulation of polyunsaturated fatty acids (PUFAs), we identified recently a linoleate 13-lipoxygenase (LiLOX). It shares highest identity with plastidic enzymes from vascular plants and is induced upon nitrogen starvation. Here, we confirmed the localization of LiLOX in the stroma of plastids via transient expression in epithelial onion cells. In order to further characterize this enzyme, we focused on the identification of the endogenous substrate of LiLOX. In this regard, an ex vivo enzymatic assay, coupled with non-targeted analysis via mass spectrometry allowed the identification of MGDG, DGDG and PC as three substrate candidates, later confirmed via in vitro assays. Further investigation revealed that LiLOX has preferences towards the lipid class MGDG, which seems in agreement with its localization in the galactolipid rich plastid. Altogether, this study shows the first characterization of plastidic LOX from green algae, showing preference for MGDGs. However, lipidomics analysis did neither reveal an endogenous LiLOX product nor the final end product of MGDG oxidation. Nevertheless, the latter is a key to understanding the role of this enzyme and since its expression is highest during the degradation of the plastidic membrane, it is tempting to assume its involvement in this process.

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.

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.

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.

Jasmonic acid biosynthesis by fungi: derivatives, first evidence on biochemical pathways and culture conditions for production.

Jasmonic acid (JA) and its derivatives called jasmonates (JAs) are lipid-derived signalling molecules that are produced by plants and certain fungi. Beside this function, JAs have a great variety of applications in flavours and fragrances production. In addition, they may have a high potential in agriculture. JAs protect plants against infections. Although there is much information on the biosynthesis and function of JA concerning plants, knowledge on these aspects is still scarce for fungi. Taking into account the practical importance of JAs, the objective of this review is to summarize knowledge on the occurrence of JAs from fungal culture media, their biosynthetic pathways and the culture conditions for optimal JA production as an alternative source for the production of these valuable metabolites.

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.

Degradation of Lipid Droplets in Plants and Algae-Right Time, Many Paths, One Goal.

In eukaryotic cells, lipids in the form of triacylglycerols (TAGs) are the major reservoir of cellular carbon and energy. These TAGs are packed into specialized organelles called lipid droplets (LDs). They can be found in most, if not all, types of cells, from bacteria to human. Recent data suggest that rather than being simple storage organelles, LDs are very dynamic structures at the center of cellular metabolism. This is also true in plants and algae, where LDs have been implicated in many processes including energy supply; membrane structure, function, trafficking; and signal transduction. Plant and algal LDs also play a vital role in human life, providing multiple sources of food and fuel. Thus, a lot of attention has been paid to metabolism and function of these organelles in recent years. This review summarizes the most recent advances on LDs degradation as a key process for TAGs release. While the initial knowledge on this process came from studies in oilseeds, the findings of the last decade revealed high complexity and specific mechanisms of LDs degradation in plants and algae. This includes identification of numerous novel proteins associated with LDs as well as a prominent role for autophagy in this process. This review outlines, systemizes, and discusses the most current data on LDs catabolism in plants and algae.

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.

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.

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.