Marcela Pinedo

Vesicular fractions of sunflower apoplastic fluids are associated with potential exosome marker proteins.

Based on the presence of phospholipids in the extracellular fluids (EFs) of sunflower seeds, we have hypothesized on the existence of vesicles in the apoplastic compartment of plants. Ultracentrifugation of sunflower EF allowed the isolation of particles of 50‐200 nm with apparent membrane organization. A small GTPase Rab was putatively identified in this vesicular fraction. Since Rab proteins are involved in vesicular traffic and their presence in exosomes from animal fluids has been demonstrated, evidence presented here supports the existence of exosome‐like vesicles in apoplastic fluids of sunflower. Their putative contribution to intercellular communication in plants is discussed.

Apoplastic exosome-like vesicles: a new way of protein secretion in plants?

The presence of apoplastic proteins without predicted signal peptide in the gene sequence suggests the existence of protein secretion independent of the ER/Golgi classical route. In animals, one of the pathways proposed for alternative protein secretion involves the release of exosomes to the extracellular space. Although this pathway has not been dissected in plants some indirect evidence is emerging. We have reported that apoplastic fractions of sunflower seeds contain exosome-like vesicles. Besides, these vesicles are enriched in the lectin Helja, which is immunolocalized in the extracellular space even if it the protein has no predicted signal peptide. Here we show that Helja is not glycosylated and its secretion is insensitive to brefeldin A, two of the major characteristics to discard ER/Golgi-mediated protein transport. Moreover, the levels of Helja in sunflower extracellular vesicles are not affected by brefeldin A treatment. Our results suggest that Helja could be exported through an exosome-mediated pathway and point out that this mechanism may be responsible for the secretion of at least part of the leaderless proteins detected in the extracellular compartment of plants.

Extracellular Sunflower Proteins: Evidence on Non-classical Secretion of a Jacalin-Related Lectin

Extracellular proteins from sunflower seedlings were analyzed by electrophoresis followed by peptide mass fingerprinting. Tentative identification revealed novel proteins for this crop. A significant number of those proteins were not expected to be extracellular because they lacked the typical signal peptide responsible for secretion. In silico analysis showed that some members of this group presented the characteristic disordered structures of certain non-classical and leaderless mammalian secretory proteins. Among these proteins, a putative jacalin-related lectin (Helja) with a mannose binding domain was further isolated from extracellular fluids by mannose-affinity chromatography, thus validating its identification. Besides, immunolocalization assays confirmed its extracellular location. These results showed that a lectin, not predicted to be secreted in strict requirement of the N-terminal signal peptide, occurs in a sunflower extracellular compartment. The implications of this finding are discussed.

Nitric oxide is required for determining root architecture and lignin composition in sunflower. Supporting evidence from microarray analyses

Nitric oxide (NO) is a signal molecule involved in several physiological processes in plants, including root development. Despite the importance of NO as a root growth regulator, the knowledge about the genes and metabolic pathways modulated by NO in this process is still limited. A constraint to unravel these pathways has been the use of exogenous applications of NO donors that may produce toxic effects. We have analyzed the role of NO in root architecture through the depletion of endogenous NO using the scavenger cPTIO. Sunflower seedlings growing in liquid medium supplemented with cPTIO showed unaltered primary root length while the number of lateral roots was deeply reduced; indicating that endogenous NO participates in determining root branching in sunflower. The transcriptional changes induced by NO depletion have been analyzed using a large-scale approach. A microarray analysis showed 330 genes regulated in the roots (p≤0.001) upon endogenous NO depletion. A general cPTIO-induced up-regulation of genes involved in the lignin biosynthetic pathway was observed. Even if no detectable changes in total lignin content could be detected, cell walls analyses revealed that the ratio G/S lignin increased in roots treated with cPTIO. This means that endogenous NO may control lignin composition in planta. Our results suggest that a fine tuning regulation of NO levels could be used by plants to regulate root architecture and lignin composition. The functional implications of these findings are discussed.

Sunflower root growth regulation: the role of jasmonic acid and its relation with auxins

Jasmonates are lipid-derived hormones that act as signal molecules in abiotic and biotic stresses and influence several aspects of plant growth and development. In this work we have investigated the effect of jasmonic acid (JA) on the root architecture of Helianthus annuus seedlings and if JA and auxins interact to modulate the growth of the primary root (PR) and lateral roots (LR). The addition of μM concentrations of JA to the growing medium of sunflower seedlings decreased the growth of the PR and LR, and also reduced the number of LR. Moreover, treatment with ibuprofen, an inhibitor of JA synthesis, increased PR and LR root length causing a deep effect on root architecture. Hence, not only exogenous but also the endogenous JA regulates sunflower root growth. Microscopic analysis showed that the application of JA reduces the cortex cell length and the estimated cell production rate in root meristem while ibuprofen only affects the cell elongation. A possible interaction between JA and auxins to regulate root growth was further analyzed. We show that JA produced its phenotype even in the presence of reduced levels of auxin generated by treatment with an auxin transport inhibitor. Besides, the auxin produced its phenotype even when ibuprofen was applied. In conclusion, JA may induce primary and lateral root growth inhibition in sunflower by an auxin-independent pathway.

Plant extracellular vesicles are incorporated by a fungal pathogen and inhibit its growth

Extracellular vesicles (EV) are membrane particles released by cells into their environment and are considered to be key players in intercellular communication. EV are produced by all domains of life but limited knowledge about EV in plants is available, although their implication in plant defense has been suggested. We have characterized sunflower EV and tested whether they could interact with fungal cells. EV were isolated from extracellular fluids of seedlings and characterized by transmission electron microscopy and proteomic analysis. These nanovesicles appeared to be enriched in cell wall remodeling enzymes and defense proteins. Membrane-labeled EV were prepared and their uptake by the phytopathogenic fungus Sclerotinia sclerotiorum was verified. Functional tests further evaluated the ability of EV to affect fungal growth. Spores treated with plant EV showed growth inhibition, morphological changes, and cell death. Conclusive evidence on the existence of plant EV is presented and we demonstrate their ability to interact with and kill fungal cells. Our results introduce the concept of cell-to-cell communication through EV in plants.

Chlorogenic acid is a fungicide active against phytopathogenic fungi

Plants synthesize diverse types of secondary metabolites and some of them participate in plant protection against pathogen attack. These compounds are biodegradable and renewable alternatives, which may be envisaged for the control of plant pests and diseases. Chlorogenic acid (CGA) is a phenolic secondary metabolite which accumulates in diverse plant tissues and can be found in several agro-industrial by-products and waste. The aim of this work was to determine whether CGA could control the growth of various plant pathogenic fungi, gaining insight into its mechanism of action. Microscopic analysis showed the complete inhibition of spore germination or reduction of mycelial growth for Sclerotinia sclerotiorum, Fusarium solani, Verticillium dahliae, Botrytis cinerea and Cercospora sojina. CGA concentrations that did not completely abolish spore germination were able to produce a partial inhibition of mycelial growth. Viability tests and vital dye staining demonstrate that CGA induces fungal cell lysis. Its fungicidal activity involves an early membrane permeabilization of the spores. These results show the antifungal activity of CGA against phytopathogenic fungi relevant in horticulture and agriculture highlighting the potential of CGA-enriched wastes and by-products to be used as biofungicides.

Molecular characterization of Helja, an extracellular jacalin-related protein from Helianthus annuus: Insights into the relationship of this protein with unconventionally secreted lectins

Jacalin-related lectins (JRLs) encompass cytosolic, nuclear and vacuolar members displaying the jacalin domain in one or more copies or in combination with unrelated domains. Helianthus annuus jacalin (Helja) is a mannose-specific JRL previously identified in the apoplast of Helianthus annuus seedlings, and this protein has been proposed to follow unconventional secretion. Here, we describe the full-length Helja cDNA sequence, which presents a unique jacalin domain (merolectin) and the absence of a signal peptide, confirming that the protein cannot follow the classical ER-dependent secretory pathway. Helja mRNA is present in seeds, cotyledons, roots and hypocotyls, but no transcripts were detected in the leaves. Searches for sequence similarity showed that Helja is barely similar to other JRLs present in H. annuus databases and less than 45% identical to other monocot or dicot JRLs. Strikingly, most of the merolectins recovered through data mining using Helja as a query were predicted as apoplastic, although most of these proteins lack the signal peptide required for classical secretion. Thus, Helja is the first bait identified to recover putative unconventionally secreted lectins. Because the recovered JRLs are widely distributed among the plant kingdom, an as yet unknown role for jacalin lectins in the apoplast is emerging.

Antimicrobial activity and mechanism of action of a thionin-like peptide from Capsicum annuum fruits and combinatorial treatment with fluconazole against Fusarium solani

Many Fusarium species are able to cause severe infections in plants as well as in animals and humans. Therefore, the discovery of new antifungal agents is of paramount importance. CaThi belongs to the thionins, which are cationic peptides with low molecular weights (∼5 kDa) that have toxic effects against various microorganisms. Herein, we study the mechanism of action of CaThi and its combinatory effect with fluconazole (FLC) against Fusarium solani. The mechanism of action of CaThi was studied by growth inhibition, viability, plasma membrane permeabilization, ROS induction, caspase activation, localization, and DNA binding capability, as assessed with Sytox green, DAB, FITC‐VAD‐FMK, CaThi‐FITC, and gel shift assays. The combinatory effect of CaThi and FLC was assessed using a growth inhibition assay. Our results demonstrated that CaThi present a dose dependent activity and at the higher used concentration (50 µg mL−1) inhibits 83% of F. solani growth, prevents the formation of hyphae, permeabilizes membranes, induces endogenous H2O2, activates caspases, and localizes intracellularly. CaThi combined with FLC, at concentrations that alone do not inhibit F. solani, result in 100% death of F. solani when combined. The data presented in this study demonstrate that CaThi causes death of F. solani via apoptosis; an intracellular target may also be involved. Combined treatment using CaThi and FLC is a strong candidate for studies aimed at improved targeting of F. solani. This strategy is of particular interest because it minimizes selection of resistant microorganisms.

On the role of a Lipid-Transfer Protein. Arabidopsis ltp3 mutant is compromised in germination and seedling growth.

Plant Lipid-Transfer Proteins (LTPs) exhibit the ability to reversibly bind/transport lipids in vitro. LTPs have been involved in diverse physiological processes but conclusive evidence on their role has only been presented for a few members, none of them related to seed physiology. Arabidopsis seeds rely on storage oil breakdown to supply carbon skeletons and energy for seedling growth. Here, Arabidopsis ltp3 mutant was analyzed for its ability to germinate and for seedling establishment. Ltp3 showed delayed germination and reduced germination frequency. Seedling growth appeared reduced in the mutant but this growth restriction was rescued by the addition of an exogenous carbon supply, suggesting a defective oil mobilization. Lipid breakdown analysis during seedling growth revealed a differential profile in the mutant compared to the wild type. The involvement of LTP3 in germination and seedling growth and its relationship with the lipid transfer ability of this protein is discussed.