The 1.413 circular supercoiled mitochondrial DNA plasmid P1 from a fertile sunflower line was seq... more The 1.413 circular supercoiled mitochondrial DNA plasmid P1 from a fertile sunflower line was sequenced, and a series of 160 bp tandemly repeated sequences was observed. The P1 plasmid was detected in both fertile and cytoplasmic male-sterile (CMS) lines, but in different quantities. Two other circular plasmids, P2 and P3, each 1.8 kbp in length, were shown to share common sequences with P1. The mitochondrial plasmid P1 detected homologous sequences in the nuclear DNA of sunflower, but not in chloroplast DNA nor in main band mitochondrial DNA. RNA molecules of about 680 and 550 nucleotides were detected that were complementary to mt plasmid P1.
Plant Lipid-Transfer Proteins (LTPs) exhibit the ability to reversibly bind/transport lipids in v... more 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 analysed 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.
Nitric oxide (NO) is a major plant signaling molecule that plays key roles during plant-pathogen ... more Nitric oxide (NO) is a major plant signaling molecule that plays key roles during plant-pathogen interactions and plant development. Previous work showed the participation of NO in the development and lignin composition of sunflower roots. Thereby, we have hypothesized that NO applications could control the attack of the fungal pathogen Verticillium dahliae in sunflower. Seedlings growing hydroponically were pretreated with NO donors and further inoculated with the fungus. Evaluation of disease symptoms showed that NO pretreatments could not reduce Verticillium wilt. Strikingly, NO donors appear to promote the fungal infection. These results indicate that NO applications were unable to protect sunflower from Verticillium attack and highlight the role played by the fine tuning regulation of NO levels required to balance plant responses between development and defense.
Ha-AP10 is a basic antifungal peptide from sunflower seeds (Helianthus annuus antifungal peptide ... more Ha-AP10 is a basic antifungal peptide from sunflower seeds (Helianthus annuus antifungal peptide of 10 kDa) belonging to the family of plant lipid transfer proteins. We report here its expression in E. coli [Glutathione S-transferase (GST) system] and its phosphorylation by endogenous membrane-bound calcium-dependent protein kinases.
A mitochondrial plasmid of 1,939 bp (P2) from a cytoplasmic male-sterile line of sunflower has be... more A mitochondrial plasmid of 1,939 bp (P2) from a cytoplasmic male-sterile line of sunflower has been cloned and sequenced. It presents 437 bp of near-perfect homology to the 1.4-kb mitochondrial plasmid P1 from sunflower. Sequences homologous to P2 were found in nuclear DNA. P2 was transcribed into a major 980-nucleotide (nt) RNA molecule and two minor transcripts of 570 and
ABSTRACT Helianthinins are storage proteins present in Helianthus annuus seeds, belonging to the ... more ABSTRACT Helianthinins are storage proteins present in Helianthus annuus seeds, belonging to the 11S globulin family. Here we describe that a fraction of the helianthinins is phosphorylated. This conclusion is supported by different criteria, including identification by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry of major protein bands revealed with a specific dye for phosphoproteins, anti-phosphoserine antibody and binding to a phosphoprotein affinity matrix. Moreover, we show that the phosphorylation status of helianthinins changes following germination.
Various phosphoinositides have been implicated in plant defence signalling. Until now, such molec... more Various phosphoinositides have been implicated in plant defence signalling. Until now, such molecules have been exclusively related to intracellular signalling. Here, evidence is provided for the detection of extracellular phosphatidylinositol 4-phosphate (PI4P) in tomato cell suspensions. We have analysed and compared the intracellular and extracellular phospholipid profiles of [ 32 Pi]-prelabelled tomato cells, challenged with the fungal elicitor xylanase. These phospholipid patterns were found to be different, being phosphatidylinositol phosphate (PIP) the most abundant phospholipid in the extracellular medium. Moreover, while cells responded with a typical increase in phosphatidic acid and a decrease in intracellular PIP upon xylanase treatment, extracellular PIP level increased in a time-and dose-dependent manner. Using two experimental approaches, the extracellular PIP isoform was identified as PI4P. Addition of PI4P to tomato cell suspensions triggered the same defence responses as those induced by xylanase treatment. These include production of reactive oxygen species, accumulation of defence-related gene transcripts and induction of cell death. We demonstrate that extracellular PI4P is accumulated in xylanase-elicited cells and that exogenous application of PI4P mimics xylanase effects, suggesting its putative role as an intercellular signalling molecule.
Abbreviations: BFA, brefeldin A; DMSO, dimethylsulphoxide; FM4-64, N-(3-triethylammoniumpropyl)-4... more Abbreviations: BFA, brefeldin A; DMSO, dimethylsulphoxide; FM4-64, N-(3-triethylammoniumpropyl)-4-(6-(4diethylaminophenylhexatrienyl)pyridinium dibromide; RG-II, rhamnogalacturonan-II During seed imbibition and embryo activation, rapid change from a metabolically resting state to the activation of diverse extracellular and/or membrane bound molecules is essential and, hence, endocytosis could be activated too. In fact, we have documented endocytic internalization of the membrane impermeable endocytic tracer FM4-64 already upon 30 min of imbibition of Arabidopsis seeds. This finding suggest that endocytosis is activated early during seed imbibition in Arabidopsis. Immunolocalization of rhamnogalacturonan-II (RG-II) complexed with boron showed that whereas this pectin is localized only in the cell walls of dry seed embryos, it starts to be intracellular once the imbibition started. Brefeldin A (BFA) exposure resulted in recruitment of the intracellular RG-II pectin complexes into the endocytic BFA-induced compartments, confirming the endocytic origin of the RG-II signal detected intracellularly. Finally, germination was significantly delayed when Arabidopsis seeds were germinated in the presence of inhibitors of endocytic pathways, suggesting that trafficking of extracellular molecules might play an important role in the overcome of germination. This work constitutes the first demonstration of endocytic processes during germination and opens new perspectives about the role of the extracellular matrix and membrane components in seed germination.
Many proteins exerting antifungal activity in vitro have been isolated although in certain cases ... more Many proteins exerting antifungal activity in vitro have been isolated although in certain cases their contribution in plant resistance to fungal attack is unclear. Screening for antifungal proteins from sunflower seeds, we have detected a basic polypeptide (Ha-AP15) homologous to the previously described storage protein 2S albumin. Results reported here indicate that Ha-AP15 exerts a weak inhibition of hyphal elongation
A 16-kDa protein was isolated from Helianthus annuus flowers by its ability to inhibit the germin... more A 16-kDa protein was isolated from Helianthus annuus flowers by its ability to inhibit the germination of fungal spores. This protein, SAP16, displays an associated activity of trypsin inhibitor and was further purified to apparent homogeneity by affinity chromatography on ...
Defensins are basic antimicrobial peptides that have been implicated in plant defense against mic... more Defensins are basic antimicrobial peptides that have been implicated in plant defense against microbial attack. Looking for genes that may fulfill a protective role in Helianthus annuus L. flowers, we have isolated a full length sunflower cDNA (SD2) displaying the structural features and the consensus sequence of plant defensins. The predicted protein has a signal peptide of 31 amino acids and a mature peptide of 47 amino acids with a molecular mass of 5 300 Da. The alignment of SD2 with its orthologs revealed that 21 of 47 residues are identical and another thirteen are highly conserved, thus defining a subfamily of plant defensins. Dot and northern blot analyses showed that the highest levels of SD2 transcripts are detected in flowers. SD2 gene expression is developmentally regulated during flowering, with mRNA levels increasing during flower maturation. The putative contribution of defensins to the resistance of H. annuus flowers against a fungal pathogen is discussed.
The genetics of male fertility restoration and the RFLP of mitochondrial DNA were studied for 16 ... more The genetics of male fertility restoration and the RFLP of mitochondrial DNA were studied for 16 sunflower cytoplasms (15 male-sterile and a male-fertile).
Jasmonates are lipid-derived hormones that act as signal molecules in abiotic and biotic stresses... more 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 lM 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.
Trypomastigote cells of Trypanosoma cruzi incubated with [U-14C]glucose accumulated dolichol-P-P-... more Trypomastigote cells of Trypanosoma cruzi incubated with [U-14C]glucose accumulated dolichol-P-P-linked Man7GlcNAc2 and Man9GlcNAc2. Evidence is presented indicating that both oligosaccharides were transferred to asparagine residues in proteins. On the other hand, intracellular amastigotes behaved as epimastigotes, i.e., only Man9GlcNAc2 accumulated and was transferred to proteins under similar incubation conditions. Intracellular amastigotes differed, therefore, from amastigotes obtained from an axenic culture, which behaved as trypomastigotes. A similar processing of protein-linked Man9GlcNAc2 and Man8GlcNAc2 occurred in epimastigotes and trypomastigotes but the structure of the main Man7GlcNAc2 isomer produced by demannosylation of the above mentioned oligosaccharides differed from that of the Man7GlcNAc2 transferred in trypomastigotes and amastigotes from axenic cultures. The infective trypomastigote stage of the parasite showed, therefore, an alteration in the mechanism of protein N-glycosylation when compared to the other stages, namely epimastigote (insect vector stage) and amastigote (mammalian intracellular stage). Complex-type, asparagine-bound oligosaccharides were found to be synthesized in both epimastigotes and trypomastigotes but the amounts of those compounds were extremely low when compared to those of high mannose-type oligosaccharides.
To determine whether Ha-AP10, a member of the plant lipid transfer proteins (LTPs) family produce... more To determine whether Ha-AP10, a member of the plant lipid transfer proteins (LTPs) family produces a direct cytotoxic effect on fungal cells mediated by membrane permeabilization. LTPs can inhibit fungal growth and are considered members of the ubiquitous class of antimicrobial peptides. However, the way they exert their effects on target cells is not yet understood. Methods and Results: Viability assays demonstrate that Ha-AP10 acts as a fungicidal compound but no harmful effect is observed on plant cells. Liposome leakage assays show that the protein induces a moderate release of fluorescent probes encapsulated in model membranes, indicating its ability to interact with phospholipids. Using a fluorescent indicator of damage at the membrane level, we demonstrate that Ha-AP10 is able to induce the permeabilization of intact fungal spores in a dose-dependent manner. Conclusion: The results presented here demonstrate the permeabilization of fungal spores caused by Ha-AP10. Significance and Impact of the Study: To our knowledge, this is the first demonstration of fungal membrane damage by an LTP, giving a clue to elucidate the basis of its antimicrobial properties.
Nonspecific lipid transfer proteins (nsLTPs) belong to a large family of plant proteins whose fun... more Nonspecific lipid transfer proteins (nsLTPs) belong to a large family of plant proteins whose function in vivo remains unknown. In this research, we studied a LTP previously isolated from sunflower seeds (Ha-AP10), which displays strong antimicrobial activity against a model fungus. The protein is present during at least the first 5 days of germination, and tissue printing experiments revealed the homogeneous distribution of the protein in the cotyledons. Here we report that Ha-AP10 exerts a weak inhibitory effect on the growth of Alternaria alternata, a fungus that naturally attacks sunflower seeds. These data put into question the contribution of Ha-AP10 as an antimicrobial protein of direct effect on pathogenic fungus, and rather suggest a function related to the mobilization of lipid reserves. We also show that the levels of Ha-AP10 in germinating seeds increase upon salt stress, fungal infection and ABA treatment, indicating that it somehow participates in the adaptative responses of germinating sunflower seeds.
The 1.413 circular supercoiled mitochondrial DNA plasmid P1 from a fertile sunflower line was seq... more The 1.413 circular supercoiled mitochondrial DNA plasmid P1 from a fertile sunflower line was sequenced, and a series of 160 bp tandemly repeated sequences was observed. The P1 plasmid was detected in both fertile and cytoplasmic male-sterile (CMS) lines, but in different quantities. Two other circular plasmids, P2 and P3, each 1.8 kbp in length, were shown to share common sequences with P1. The mitochondrial plasmid P1 detected homologous sequences in the nuclear DNA of sunflower, but not in chloroplast DNA nor in main band mitochondrial DNA. RNA molecules of about 680 and 550 nucleotides were detected that were complementary to mt plasmid P1.
Plant Lipid-Transfer Proteins (LTPs) exhibit the ability to reversibly bind/transport lipids in v... more 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 analysed 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.
Nitric oxide (NO) is a major plant signaling molecule that plays key roles during plant-pathogen ... more Nitric oxide (NO) is a major plant signaling molecule that plays key roles during plant-pathogen interactions and plant development. Previous work showed the participation of NO in the development and lignin composition of sunflower roots. Thereby, we have hypothesized that NO applications could control the attack of the fungal pathogen Verticillium dahliae in sunflower. Seedlings growing hydroponically were pretreated with NO donors and further inoculated with the fungus. Evaluation of disease symptoms showed that NO pretreatments could not reduce Verticillium wilt. Strikingly, NO donors appear to promote the fungal infection. These results indicate that NO applications were unable to protect sunflower from Verticillium attack and highlight the role played by the fine tuning regulation of NO levels required to balance plant responses between development and defense.
Ha-AP10 is a basic antifungal peptide from sunflower seeds (Helianthus annuus antifungal peptide ... more Ha-AP10 is a basic antifungal peptide from sunflower seeds (Helianthus annuus antifungal peptide of 10 kDa) belonging to the family of plant lipid transfer proteins. We report here its expression in E. coli [Glutathione S-transferase (GST) system] and its phosphorylation by endogenous membrane-bound calcium-dependent protein kinases.
A mitochondrial plasmid of 1,939 bp (P2) from a cytoplasmic male-sterile line of sunflower has be... more A mitochondrial plasmid of 1,939 bp (P2) from a cytoplasmic male-sterile line of sunflower has been cloned and sequenced. It presents 437 bp of near-perfect homology to the 1.4-kb mitochondrial plasmid P1 from sunflower. Sequences homologous to P2 were found in nuclear DNA. P2 was transcribed into a major 980-nucleotide (nt) RNA molecule and two minor transcripts of 570 and
ABSTRACT Helianthinins are storage proteins present in Helianthus annuus seeds, belonging to the ... more ABSTRACT Helianthinins are storage proteins present in Helianthus annuus seeds, belonging to the 11S globulin family. Here we describe that a fraction of the helianthinins is phosphorylated. This conclusion is supported by different criteria, including identification by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry of major protein bands revealed with a specific dye for phosphoproteins, anti-phosphoserine antibody and binding to a phosphoprotein affinity matrix. Moreover, we show that the phosphorylation status of helianthinins changes following germination.
Various phosphoinositides have been implicated in plant defence signalling. Until now, such molec... more Various phosphoinositides have been implicated in plant defence signalling. Until now, such molecules have been exclusively related to intracellular signalling. Here, evidence is provided for the detection of extracellular phosphatidylinositol 4-phosphate (PI4P) in tomato cell suspensions. We have analysed and compared the intracellular and extracellular phospholipid profiles of [ 32 Pi]-prelabelled tomato cells, challenged with the fungal elicitor xylanase. These phospholipid patterns were found to be different, being phosphatidylinositol phosphate (PIP) the most abundant phospholipid in the extracellular medium. Moreover, while cells responded with a typical increase in phosphatidic acid and a decrease in intracellular PIP upon xylanase treatment, extracellular PIP level increased in a time-and dose-dependent manner. Using two experimental approaches, the extracellular PIP isoform was identified as PI4P. Addition of PI4P to tomato cell suspensions triggered the same defence responses as those induced by xylanase treatment. These include production of reactive oxygen species, accumulation of defence-related gene transcripts and induction of cell death. We demonstrate that extracellular PI4P is accumulated in xylanase-elicited cells and that exogenous application of PI4P mimics xylanase effects, suggesting its putative role as an intercellular signalling molecule.
Abbreviations: BFA, brefeldin A; DMSO, dimethylsulphoxide; FM4-64, N-(3-triethylammoniumpropyl)-4... more Abbreviations: BFA, brefeldin A; DMSO, dimethylsulphoxide; FM4-64, N-(3-triethylammoniumpropyl)-4-(6-(4diethylaminophenylhexatrienyl)pyridinium dibromide; RG-II, rhamnogalacturonan-II During seed imbibition and embryo activation, rapid change from a metabolically resting state to the activation of diverse extracellular and/or membrane bound molecules is essential and, hence, endocytosis could be activated too. In fact, we have documented endocytic internalization of the membrane impermeable endocytic tracer FM4-64 already upon 30 min of imbibition of Arabidopsis seeds. This finding suggest that endocytosis is activated early during seed imbibition in Arabidopsis. Immunolocalization of rhamnogalacturonan-II (RG-II) complexed with boron showed that whereas this pectin is localized only in the cell walls of dry seed embryos, it starts to be intracellular once the imbibition started. Brefeldin A (BFA) exposure resulted in recruitment of the intracellular RG-II pectin complexes into the endocytic BFA-induced compartments, confirming the endocytic origin of the RG-II signal detected intracellularly. Finally, germination was significantly delayed when Arabidopsis seeds were germinated in the presence of inhibitors of endocytic pathways, suggesting that trafficking of extracellular molecules might play an important role in the overcome of germination. This work constitutes the first demonstration of endocytic processes during germination and opens new perspectives about the role of the extracellular matrix and membrane components in seed germination.
Many proteins exerting antifungal activity in vitro have been isolated although in certain cases ... more Many proteins exerting antifungal activity in vitro have been isolated although in certain cases their contribution in plant resistance to fungal attack is unclear. Screening for antifungal proteins from sunflower seeds, we have detected a basic polypeptide (Ha-AP15) homologous to the previously described storage protein 2S albumin. Results reported here indicate that Ha-AP15 exerts a weak inhibition of hyphal elongation
A 16-kDa protein was isolated from Helianthus annuus flowers by its ability to inhibit the germin... more A 16-kDa protein was isolated from Helianthus annuus flowers by its ability to inhibit the germination of fungal spores. This protein, SAP16, displays an associated activity of trypsin inhibitor and was further purified to apparent homogeneity by affinity chromatography on ...
Defensins are basic antimicrobial peptides that have been implicated in plant defense against mic... more Defensins are basic antimicrobial peptides that have been implicated in plant defense against microbial attack. Looking for genes that may fulfill a protective role in Helianthus annuus L. flowers, we have isolated a full length sunflower cDNA (SD2) displaying the structural features and the consensus sequence of plant defensins. The predicted protein has a signal peptide of 31 amino acids and a mature peptide of 47 amino acids with a molecular mass of 5 300 Da. The alignment of SD2 with its orthologs revealed that 21 of 47 residues are identical and another thirteen are highly conserved, thus defining a subfamily of plant defensins. Dot and northern blot analyses showed that the highest levels of SD2 transcripts are detected in flowers. SD2 gene expression is developmentally regulated during flowering, with mRNA levels increasing during flower maturation. The putative contribution of defensins to the resistance of H. annuus flowers against a fungal pathogen is discussed.
The genetics of male fertility restoration and the RFLP of mitochondrial DNA were studied for 16 ... more The genetics of male fertility restoration and the RFLP of mitochondrial DNA were studied for 16 sunflower cytoplasms (15 male-sterile and a male-fertile).
Jasmonates are lipid-derived hormones that act as signal molecules in abiotic and biotic stresses... more 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 lM 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.
Trypomastigote cells of Trypanosoma cruzi incubated with [U-14C]glucose accumulated dolichol-P-P-... more Trypomastigote cells of Trypanosoma cruzi incubated with [U-14C]glucose accumulated dolichol-P-P-linked Man7GlcNAc2 and Man9GlcNAc2. Evidence is presented indicating that both oligosaccharides were transferred to asparagine residues in proteins. On the other hand, intracellular amastigotes behaved as epimastigotes, i.e., only Man9GlcNAc2 accumulated and was transferred to proteins under similar incubation conditions. Intracellular amastigotes differed, therefore, from amastigotes obtained from an axenic culture, which behaved as trypomastigotes. A similar processing of protein-linked Man9GlcNAc2 and Man8GlcNAc2 occurred in epimastigotes and trypomastigotes but the structure of the main Man7GlcNAc2 isomer produced by demannosylation of the above mentioned oligosaccharides differed from that of the Man7GlcNAc2 transferred in trypomastigotes and amastigotes from axenic cultures. The infective trypomastigote stage of the parasite showed, therefore, an alteration in the mechanism of protein N-glycosylation when compared to the other stages, namely epimastigote (insect vector stage) and amastigote (mammalian intracellular stage). Complex-type, asparagine-bound oligosaccharides were found to be synthesized in both epimastigotes and trypomastigotes but the amounts of those compounds were extremely low when compared to those of high mannose-type oligosaccharides.
To determine whether Ha-AP10, a member of the plant lipid transfer proteins (LTPs) family produce... more To determine whether Ha-AP10, a member of the plant lipid transfer proteins (LTPs) family produces a direct cytotoxic effect on fungal cells mediated by membrane permeabilization. LTPs can inhibit fungal growth and are considered members of the ubiquitous class of antimicrobial peptides. However, the way they exert their effects on target cells is not yet understood. Methods and Results: Viability assays demonstrate that Ha-AP10 acts as a fungicidal compound but no harmful effect is observed on plant cells. Liposome leakage assays show that the protein induces a moderate release of fluorescent probes encapsulated in model membranes, indicating its ability to interact with phospholipids. Using a fluorescent indicator of damage at the membrane level, we demonstrate that Ha-AP10 is able to induce the permeabilization of intact fungal spores in a dose-dependent manner. Conclusion: The results presented here demonstrate the permeabilization of fungal spores caused by Ha-AP10. Significance and Impact of the Study: To our knowledge, this is the first demonstration of fungal membrane damage by an LTP, giving a clue to elucidate the basis of its antimicrobial properties.
Nonspecific lipid transfer proteins (nsLTPs) belong to a large family of plant proteins whose fun... more Nonspecific lipid transfer proteins (nsLTPs) belong to a large family of plant proteins whose function in vivo remains unknown. In this research, we studied a LTP previously isolated from sunflower seeds (Ha-AP10), which displays strong antimicrobial activity against a model fungus. The protein is present during at least the first 5 days of germination, and tissue printing experiments revealed the homogeneous distribution of the protein in the cotyledons. Here we report that Ha-AP10 exerts a weak inhibitory effect on the growth of Alternaria alternata, a fungus that naturally attacks sunflower seeds. These data put into question the contribution of Ha-AP10 as an antimicrobial protein of direct effect on pathogenic fungus, and rather suggest a function related to the mobilization of lipid reserves. We also show that the levels of Ha-AP10 in germinating seeds increase upon salt stress, fungal infection and ABA treatment, indicating that it somehow participates in the adaptative responses of germinating sunflower seeds.
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Papers by Laura De La Canal