PDF1.5 Enhances Adaptation to Low Nitrogen Levels and Cadmium Stress.

Environmental acclimation ability plays a key role in plant growth, although the mechanism remains unclear. Here, we determined the involvement of Arabidopsis thaliana PLANT DEFENSIN 1 gene AtPDF1.5 in the adaptation to low nitrogen (LN) levels and cadmium (Cd) stress. Histochemical analysis revealed that AtPDF1.5 was mainly expressed in the nodes and carpopodium and was significantly induced in plants exposed to LN conditions and Cd stress. Subcellular localization analysis revealed that AtPDF1.5 was cell wall- and cytoplasm-localized. AtPDF1.5 overexpression significantly enhanced adaptation to LN and Cd stress and enhanced the distribution of metallic elements. The functional disruption of AtPDF1.5 reduced adaptations to LN and Cd stress and impaired metal distribution. Under LN conditions, the nitrate transporter AtNRT1.5 expression was upregulated. Nitrate transporter AtNRT1.8 expression was downregulated when AtPDF1.5 was overexpressed, resulting in enhanced transport of NO3- to shoots. In response to Cd treatment, AtPDF1.5 regulated the expression of metal transporter genes AtHMP07, AtNRAMP4, AtNRAMP1, and AtHIPP3, resulting in higher Cd accumulation in the shoots. We conclude that AtPDF1.5 is involved in the processing or transmission of signal substances and plays an important role in the remediation of Cd pollution and LN adaptation.

Fungicidal Activity of Recombinant Javanicin against Cryptococcus neoformans Is Associated with Intracellular Target(s) Involved in Carbohydrate and Energy Metabolic Processes.

The occurrence of Cryptococcus neoformans, the human fungal pathogen that primarily infects immunocompromised individuals, has been progressing at an alarming rate. The increased incidence of infection of C. neoformans with antifungal drugs resistance has become a global concern. Potential antifungal agents with extremely low toxicity are urgently needed. Herein, the biological activities of recombinant javanicin (r-javanicin) against C. neoformans were evaluated. A time-killing assay was performed and both concentration- and time-dependent antifungal activity of r-javanicin were indicated. The inhibitory effect of the peptide was initially observed at 4 h post-treatment and ultimately eradicated within 36 to 48 h. Fungal outer surface alteration was characterized by the scanning electron microscope (SEM) whereas a negligible change with slight shrinkage of external morphology was observed in r-javanicin treated cells. Confocal laser scanning microscopic analysis implied that the target(s) of r-javanicin is conceivably resided in the cell thereby allowing the peptide to penetrate across the membrane and accumulate throughout the fungal body. Finally, cryptococcal cells coped with r-javanicin were preliminarily investigated using label-free mass spectrometry-based proteomics. Combined with microscopic and proteomics analysis, it was clearly elucidated the peptide localized in the intracellular compartment where carbohydrate metabolism and energy production associated with glycolysis pathway and mitochondrial respiration, respectively, were principally interfered. Overall, r-javanicin would be an alternative candidate for further development of antifungal agents.

Plant defensin antibacterial mode of action against Pseudomonas species.

BACKGROUND: Though many plant defensins exhibit antibacterial activity, little is known about their antibacterial mode of action (MOA). Antimicrobial peptides with a characterized MOA induce the expression of multiple bacterial outer membrane modifications, which are required for resistance to these membrane-targeting peptides. Mini-Tn5-lux mutant strains of Pseudomonas aeruginosa with Tn insertions disrupting outer membrane protective modifications were assessed for sensitivity against plant defensin peptides. These transcriptional lux reporter strains were also evaluated for lux gene expression in response to sublethal plant defensin exposure. Also, a plant pathogen, Pseudomonas syringae pv. syringae was modified through transposon mutagenesis to create mutants that are resistant to in vitro MtDef4 treatments. RESULTS: Plant defensins displayed specific and potent antibacterial activity against strains of P. aeruginosa. A defensin from Medicago truncatula, MtDef4, induced dose-dependent gene expression of the aminoarabinose modification of LPS and surface polycation spermidine production operons. The ability for MtDef4 to damage bacterial outer membranes was also verified visually through fluorescent microscopy. Another defensin from M. truncatula, MtDef5, failed to induce lux gene expression and limited outer membrane damage was detected with fluorescent microscopy. The transposon insertion site on MtDef4 resistant P. syringae pv. syringae mutants was sequenced, and modifications of ribosomal genes were identified to contribute to enhanced resistance to plant defensin treatments. CONCLUSIONS: MtDef4 damages the outer membrane similar to polymyxin B, which stimulates antimicrobial peptide resistance mechanisms to plant defensins. MtDef5, appears to have a different antibacterial MOA. Additionally, the MtDef4 antibacterial mode of action may also involve inhibition of translation.

Plant Defensins from a Structural Perspective.

Plant defensins form a family of proteins with a broad spectrum of protective activities against fungi, bacteria, and insects. Furthermore, some plant defensins have revealed anticancer activity. In general, plant defensins are non-toxic to plant and mammalian cells, and interest in using them for biotechnological and medicinal purposes is growing. Recent studies provided significant insights into the mechanisms of action of plant defensins. In this review, we focus on structural and dynamics aspects and discuss structure-dynamics-function relations of plant defensins.

A non-secreted plant defensin AtPDF2.6 conferred cadmium tolerance via its chelation in Arabidopsis.

KEY MESSAGE: Plant defensin AtPDF2.6 is not secreted to the apoplast and localized in cytoplasm. AtPDF2.6 is mainly expressed in root vascular bundles of xylem parenchyma cell, and significantly induced by Cd stress. AtPDF2.6 detoxicate cytoplasmic Cd via chelation, thus enhanced Cd tolerance in Arabidopsis. In order to detoxify the heavy metal cadmium (Cd), plants have evolved several mechanisms, among which chelation represents the major Cd-detoxification mechanism. In this study, we aimed to identify a new defensin protein involved in cytoplasmic Cd detoxification by using plant molecular genetics and physiological methods. The results of bioinformatic analysis showed that the Arabidopsis thaliana defensin gene AtPDF2.6 has a signal peptide that may mediate its secretion to the cell wall. Subcellular localization analysis revealed that AtPDF2.6 is localized to the cytoplasm and is not secreted to the apoplast, whereas histochemical analysis indicated that AtPDF2.6 is mainly expressed in the root xylem parenchyma cells and that its expression is significantly induced by Cd. An in vitro Cd-binding assay revealed that AtPDF2.6 has Cd-chelating activity. Heterologous overexpression of AtPDF2.6 increased Cd tolerance in Escherichia coli and yeast, and AtPDF2.6 overexpression significantly enhanced Cd tolerance in Arabidopsis, whereas functional disruption of AtPDF2.6 decreased Cd tolerance. These data suggest that AtPDF2.6 detoxifies cytoplasmic Cd via chelation and thereby enhances Cd tolerance in Arabidopsis. Our findings accordingly challenge the commonly accepted view of defensins as secreted proteins.

Proteomic changes in the xylem sap of Brassica napus under cadmium stress and functional validation.

BACKGROUND: The xylem sap of vascular plants primarily transports water and mineral nutrients from the roots to the shoots and also transports heavy metals such as cadmium (Cd). Proteomic changes in xylem sap is an important mechanism for detoxifying Cd by plants. However, it is unclear how proteins in xylem sap respond to Cd. Here, we investigated the effects of Cd stress on the xylem sap proteome of Brassica napus using a label-free shotgun proteomic approach to elucidate plant response mechanisms to Cd toxicity. RESULTS: We identified and quantified 672 proteins; 67% were predicted to be secretory, and 11% (73 proteins) were unique to Cd-treated samples. Cd stress caused statistically significant and biologically relevant abundance changes in 28 xylem sap proteins. Among these proteins, the metabolic pathways that were most affected were related to cell wall modifications, stress/oxidoreductases, and lipid and protein metabolism. We functionally validated a plant defensin-like protein, BnPDFL, which belongs to the stress/oxidoreductase category, that was unique to the Cd-treated samples and played a positive role in Cd tolerance. Subcellular localization analysis revealed that BnPDFL is cell wall-localized. In vitro Cd-binding assays revealed that BnPDFL has Cd-chelating activity. BnPDFL heterologous overexpression significantly enhanced Cd tolerance in E. coli and Arabidopsis. Functional disruption of Arabidopsis plant defensin genes AtPDF2.3 and AtPDF2.2, which are mainly expressed in root vascular bundles, significantly decreased Cd tolerance. CONCLUSIONS: Several xylem sap proteins in Brassica napus are differentially induced in response to Cd treatment, and plant defensin plays a positive role in Cd tolerance.

The plant defensin NaD1 introduces membrane disorder through a specific interaction with the lipid, phosphatidylinositol 4,5 bisphosphate.

Plant defensins interact with phospholipids in bilayers as part of their cytotoxic activity. Solanaceous class II defensins with the loop 5 sequence pattern "S-[KR]-[ILVQ]-[ILVQ]-[KR]-[KR]" interact with PI(4,5)P2. Here, the prototypical defensin of this class, NaD1, is used to characterise the biophysical interactions between these defensins and phospholipid bilayers. Binding of NaD1 to bilayers containing PI(4,5)P2 occurs rapidly and the interaction is very strong. Dual polarisation interferometry revealed that NaD1 does not dissociate from bilayers containing PI(4,5)P2. Binding of NaD1 to bilayers with or without PI(4,5)P2 induced disorder in the bilayer. However, permeabilisation assays revealed that NaD1 only permeabilised liposomes with PI(4,5)P2 in the bilayer, suggesting a role for this protein-lipid interaction in the plasma membrane permeabilising activity of this defensin. No defensins in the available databases have the PI(4,5)P2 binding sequence outside the solanaceous class II defensins, leading to the hypothesis that PI(4,5)P2 binding co-evolved with the C-terminal propeptide to protect the host cell against the effects of the tight binding of these defensins to their cognate lipid as they travel along the secretory pathway. This data has allowed us to develop a new model to explain how this class of defensins permeabilises plasma membranes to kill target cells.

NMR structure, conformational dynamics, and biological activity of PsDef1 defensin from Pinus sylvestris.

Plants have developed a complex defense response system against pests and pathogens. Defensins, produced by plants as part of their innate immune response, form the family of small, basic, cysteine-rich proteins with activity primarily directed against fungal pathogens. In addition, plant defensins can show antibacterial activity and protease and insect amylase inhibitory activities. However, in gymnosperms, only antifungal activity of defensins has been described thus far. Here, we report antibacterial and insect α-amylase inhibition activities for defensin PsDef1 from P. sylvestris, the first defensin from gymnosperms with a broad range of biological activities described. We also report the solution NMR structure of PsDef1 and its dynamics properties assessed by a combination of experimental NMR and computational techniques. Collectively, our data provide an insight into structure, dynamics, and functional properties of PsDef1 that could be common between defensins from this taxonomic group.

Expression of apoplast-targeted plant defensin MtDef4.2 confers resistance to leaf rust pathogen Puccinia triticina but does not affect mycorrhizal symbiosis in transgenic wheat.

Rust fungi of the order Pucciniales are destructive pathogens of wheat worldwide. Leaf rust caused by the obligate, biotrophic basidiomycete fungus Puccinia triticina (Pt) is an economically important disease capable of causing up to 50 % yield losses. Historically, resistant wheat cultivars have been used to control leaf rust, but genetic resistance is ephemeral and breaks down with the emergence of new virulent Pt races. There is a need to develop alternative measures for control of leaf rust in wheat. Development of transgenic wheat expressing an antifungal defensin offers a promising approach to complement the endogenous resistance genes within the wheat germplasm for durable resistance to Pt. To that end, two different wheat genotypes, Bobwhite and Xin Chun 9 were transformed with a chimeric gene encoding an apoplast-targeted antifungal plant defensin MtDEF4.2 from Medicago truncatula. Transgenic lines from four independent events were further characterized. Homozygous transgenic wheat lines expressing MtDEF4.2 displayed resistance to Pt race MCPSS relative to the non-transgenic controls in growth chamber bioassays. Histopathological analysis suggested the presence of both pre- and posthaustorial resistance to leaf rust in these transgenic lines. MtDEF4.2 did not, however, affect the root colonization of a beneficial arbuscular mycorrhizal fungus Rhizophagus irregularis. This study demonstrates that the expression of apoplast-targeted plant defensin MtDEF4.2 can provide substantial resistance to an economically important leaf rust disease in transgenic wheat without negatively impacting its symbiotic relationship with the beneficial mycorrhizal fungus.

Peanut defensins: Novel allergens isolated from lipophilic peanut extract.

BACKGROUND: Peanut is one of the most hazardous sources of food allergens. Unknown allergens are still hidden in the complex lipophilic matrix. These allergens need to be discovered to allow estimation of the allergenic risk for patients with peanut allergy and to further improve diagnostic measures. OBJECTIVE: We performed detection, isolation, and characterization of novel peanut allergens from lipophilic peanut extract. METHODS: Extraction of roasted peanuts were performed under defined extraction conditions and examined by means of 2-dimensional PAGE. Subsequently, chromatographic methods were adapted to isolate low-molecular-weight components. Proteins were studied by using SDS-PAGE and immunoblotting with sera from patients with peanut allergy. For allergen identification protein sequencing, homology search and mass spectrometry were applied. Functional characterization for allergenicity was performed by using the basophil activation assay and for antimicrobial activity by using inhibition assays of different bacteria and fungi. RESULTS: IgE-reactive proteins of 12, 11, and 10 kDa were first detected after chloroform/methanol extraction in the flow through of hydrophobic interaction chromatography. The proteins were able to activate basophils of patients with peanut allergy. N-terminal sequencing and homology search in the expressed sequence tag database identified the allergens as peanut defensins, which was confirmed by using mass spectrometry. On microbial cell cultures, the peanut defensins showed inhibitory effects on the mold strains of the genera Cladosporium and Alternaria but none on bacteria. CONCLUSIONS: We identified defensins as novel peanut allergens (Ara h 12 and Ara h 13) that react in particular with IgE of patients with severe peanut allergy. Their antimicrobial activity is solely antifungal.

Characterization and functional analysis of gerbera plant defensin (PDF) genes reveal the role of GhPDF2.4 in defense against the root rot pathogen Phytophthora cryptogea.

Gerbera (Gerbera hybrida), a major fresh cut flower crop, is very susceptible to root rot disease. Although plant defensins (PDFs), a major group of plant antimicrobial peptides, display broad-spectrum antifungal and antibacterial activities, PDF genes in gerbera have not been systematically characterized. Here, we identified and cloned nine PDF genes from gerbera and divided them into two classes based on phylogenetic analysis. Most Class I GhPDF genes were highly expressed in petioles, whereas all Class II GhPDF genes were highly expressed in roots. Phytophthora cryptogea inoculation strongly upregulated all Class II GhPDF genes in roots and upregulated all Class I GhPDF genes in petioles. Transient overexpression of GhPDF1.5 and GhPDF2.4 inhibited P. cryptogea infection in tobacco (Nicotiana benthamiana) leaves. Transient overexpression of GhPDF2.4, but not GhPDF1.5, significantly upregulated ACO and LOX gene expression in tobacco leaves, indicating that overexpressing GhPDF2.4 activated the jasmonic acid/ethylene defense pathway and that the two types of GhPDFs have different modes of action. Prokaryotically expressed recombinant GhPDF2.4 inhibited mycelial growth and delayed the hyphal swelling of P. cryptogea, in vitro, indicating that GhPDF2.4 is a morphogenetic defensin. Moreover, the addition of GhPDF2.4 to plant culture medium alleviated the root rot symptoms of in vitro-grown gerbera seedlings and greatly reduced pathogen titer in P. cryptogea-inoculated gerbera roots in the early stages of treatment. Our study provides a basis for the use of GhPDFs, especially GhPDF2.4, for controlling root rot disease in gerbera. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-024-00146-8.

Hyperpolarisation of Mitochondrial Membranes Is a Critical Component of the Antifungal Mechanism of the Plant Defensin, Ppdef1.

Plant defensins are a large family of small cationic proteins with diverse functions and mechanisms of action, most of which assert antifungal activity against a broad spectrum of fungi. The partial mechanism of action has been resolved for a small number of members of plant defensins, and studies have revealed that many act by more than one mechanism. The plant defensin Ppdef1 has a unique sequence and long loop 5 with fungicidal activity against a range of human fungal pathogens, but little is known about its mechanism of action. We screened the S. cerevisiae non-essential gene deletion library and identified the involvement of the mitochondria in the mechanism of action of Ppdef1. Further analysis revealed that the hyperpolarisation of the mitochondrial membrane potential (MMP) activates ROS production, vacuolar fusion and cell death and is an important step in the mechanism of action of Ppdef1, and it is likely that a similar mechanism acts in Trichophyton rubrum.

Plant Defensin type 1 (PDF1): protein promiscuity and expression variation within the Arabidopsis genus shed light on zinc tolerance acquisition in Arabidopsis halleri.

Plant defensins are recognized for their antifungal properties. However, a few type 1 defensins (PDF1s) were identified for their cellular zinc (Zn) tolerance properties after a study of the metal extremophile Arabidopsis halleri. In order to investigate whether different paralogues would display specialized functions, the A. halleri PDF1 family was characterized at the functional and genomic levels. Eleven PDF1s were isolated from A. halleri. Their ability to provide Zn tolerance in yeast cells, their activity against Fusarium oxysporum f. sp. melonii, and their level of expression in planta were compared with those of the seven A. thaliana PDF1s. The genomic organization of the PDF1 family was comparatively analysed within the Arabidopsis genus. AhPDF1s and AtPDF1s were able to confer Zn tolerance and AhPDF1s also displayed antifungal activity. PDF1 transcripts were constitutively more abundant in A. halleri than in A. thaliana. Within the Arabidopsis genus, the PDF1 family is evolutionarily dynamic, in terms of gain and loss of gene copy. Arabidopsis halleri PDF1s display no superior abilities to provide Zn tolerance. A constitutive increase in AhPDF1 transcript accumulation is proposed to be an evolutionary innovation co-opting the promiscuous PDF1 protein for its contribution to Zn tolerance in A. halleri.

The Plant Defensin Ppdef1 Is a Novel Topical Treatment for Onychomycosis.

Onychomycosis, or fungal nail infection, causes not only pain and discomfort but can also have psychological and social consequences for the patient. Treatment of onychomycosis is complicated by the location of the infection under the nail plate, meaning that antifungal molecules must either penetrate the nail or be applied systemically. Currently, available treatments are limited by their poor nail penetration for topical products or their potential toxicity for systemic products. Plant defensins with potent antifungal activity have the potential to be safe and effective treatments for fungal infections in humans. The cystine-stabilized structure of plant defensins makes them stable to the extremes of pH and temperature as well as digestion by proteases. Here, we describe a novel plant defensin, Ppdef1, as a peptide for the treatment of fungal nail infections. Ppdef1 has potent, fungicidal activity against a range of human fungal pathogens, including Candida spp., Cryptococcus spp., dermatophytes, and non-dermatophytic moulds. In particular, Ppdef1 has excellent activity against dermatophytes that infect skin and nails, including the major etiological agent of onychomycosis Trichophyton rubrum. Ppdef1 also penetrates human nails rapidly and efficiently, making it an excellent candidate for a novel topical treatment of onychomycosis.

PaDef (Persea americana var. drymifolia), a Plant Antimicrobial Peptide, Triggers Apoptosis, and Induces Global Epigenetic Modifications on Histone 3 in an Acute Lymphoid Leukemia Cell Line.

In recent years, it has been recognized that epigenetic alterations play an important role in the development and maintenance of cancer, including leukemias. Furthermore, it is known that these alterations are involved in the emergence of resistance to conventional chemotherapeutics. Consequently, molecules with an anticancer activity whose activity is ruled by epigenetic modifications are attractive to search for new therapies against cancer. The plant antimicrobial peptides have been widely evaluated as molecules with anticancer activity; however, the analysis of the epigenetic regulation induced by these molecules associated with this activity is scarce and still is an unexplored field. In this work, we show that the PaDef defensin, a plant antimicrobial peptide from Mexican avocado fruit (Persea americana var. drymifolia) is cytotoxic for Jurkat cell line from acute lymphoid leukemia cells, through an apoptotic process. PaDef inhibited cell viability in a concentration-dependent manner, with an IC50 = 47.3 µM. Treatment of Jurkat cells with PaDef (IC50) induced cell death by apoptosis dependent on caspases 8 and 9; besides, it was related to an increase in the production of reactive oxygen species and the loss of mitochondrial membrane potential. Interestingly, the inhibition of caspase activation by inhibitors of caspases 8 and 9 does not revert the reduction in viability, suggesting that other mechanisms, in addition to caspase activity, could be participating in the PaDef cytotoxic effect. Also, the modifications in the histone 3 tails induced by PaDef in Jurkat cells were evaluated, specifically acetylation and methylation. PaDef increased global histone 3 acetylation and lysine 9 specific marks (2-fold and up to 4-fold, respectively). These effects correlated with the reduction of the Histone Deacetylase activity (HDAC, ∼50%). Based on methylation marks, PaDef treatment increased lysine 9 di- and tri-methylation tags (2-fold in both cases). The epigenetic modulation induced by PaDef on Jurkat cells could be related to the chromatin compaction-decompaction promoting gene expression or repression; however, further studies are necessary to correlate these marks with the transcription of specific genes. Therefore, the study of new molecules that may have anticancer activity through epigenetic modulation is interesting.

Structure, dynamics, and function of PsDef2 defensin from Pinus sylvestris.

Plant defensins demonstrate high structural stability at extreme temperatures and pH values and, in general, are non-toxic to mammalian cells. These properties make them attractive candidates for use in biotechnology and biomedicine. Knowing the structure-function relationship is desirable to guide the design of plant defensin-based applications. Thus far, the broad range of biological activities was described only for one defensin from gymnosperms, the defensin PsDef1 from Scots pine. Here, we report that closely related defensin from the same taxonomy group, PsDef2, differing from PsDef1 by six amino acids, also possesses antimicrobial, antibacterial, and insect α-amylase inhibitory activities. We also report the solution structure and dynamics properties of PsDef2 assessed using a combination of experimental nuclear magnetic resonance (NMR) techniques. Lastly, we perform a comparative analysis of PsDef2 and PsDef1 gaining a molecular-level insight into their structure-dynamics-function relationship.

Antifungal Activity, Structural Stability, and Immunomodulatory Effects on Human Immune Cells of Defensin from the Lentil Lens culinaris.

An increase in the frequency of mycoses and spreading of multidrug-resistant fungal pathogens necessitates the search for new antifungal agents. Earlier, we isolated the novel defensin from lentil Lensculinaris seeds, designated as Lc-def, which inhibited the growth of phytopathogenic fungi. Here, we studied an antifungal activity of Lc-def against human pathogenic Candida species, structural stability of the defensin, and its immunomodulatory effects that may help to prevent fungal infection. We showed that Lc-def caused 50% growth inhibition of clinical isolates of Candida albicans, C. krusei, and C. glabrata at concentrations of 25-50 µM, but was not toxic to different human cells. The lentil defensin was resistant to proteolysis by C. albicans and was not cleaved during simulated gastroduodenal digestion. By using the multiplex xMAP assay, we showed for the first time for plant defensins that Lc-def increased the production of such essential for immunity to candidiasis pro-inflammatory cytokines as IL-12 and IL-17 at the concentration of 2 µM. Thus, we hypothesized that the lentil Lc-def and plant defensins in general may be effective in suppressing of mucocutaneous candidiasis due to their antifungal activity, high structural stability, and ability to activate a protective immune response.

Genome-scale identification of plant defensin (PDF) family genes and molecular characterization of their responses to diverse nutrient stresses in allotetraploid rapeseed.

Plant defensins (PDFs), short peptides with strong antibacterial activity, play important roles in plant growth, development, and stress resistance. However, there are few systematic analyses on PDFs in Brassica napus. Here, bioinformatics methods were used to identify genome-wide PDFs in Brassica napus, and systematically analyze physicochemical properties, expansion pattern, phylogeny, and expression profiling of BnaPDFs under diverse nutrient stresses. A total of 37 full-length PDF homologs, divided into two subgroups (PDF1s and PDF2s), were identified in the rapeseed genome. A total of two distinct clades were identified in the BnaPDF phylogeny. Clade specific conserved motifs were identified within each clade respectively. Most BnaPDFs were proved to undergo powerful purified selection. The PDF members had enriched cis-elements related to growth and development, hormone response, environmental stress response in their promoter regions. GO annotations indicate that the functional pathways of BnaPDFs are mainly involved in cells killing and plant defense responses. In addition, bna-miRNA164 and bna-miRNA172 respectively regulate the expression of their targets BnaA2.PDF2.5 and BnaC7.PDF2.6. The expression patterns of BnaPDFs were analyzed in different tissues. BnaPDF1.2bs was mainly expressed in the roots, whereas BnaPDF2.2s and BnaPDF2.3s were both expressed in stamen, pericarp, silique, and stem. However, the other BnaPDF members showed low expression levels in various tissues. Differential expression of BnaPDFs under nitrate limitation, ammonium excess, phosphorus starvation, potassium deficiency, cadmium toxicity, and salt stress indicated that they might participate in different nutrient stress resistance. The genome-wide identification and characterization of BnaPDFs will enrich understanding of their molecular characteristics and provide elite gene resources for genetic improvement of rapeseed resistance to nutrient stresses.

Overexpression of a Defensin-Like Gene CAL2 Enhances Cadmium Accumulation in Plants.

Accumulation and detoxification of cadmium in rice shoots are of great importance for adaptation to grow in cadmium contaminated soils and for limiting the transport of Cd to grains. However, the molecular mechanisms behind the processes involved in this regulation remain largely unknown. Defensin proteins play important roles in heavy metal tolerance and accumulation in plants. In rice, the cell wall-localized defensin protein (CAL1) is involved in Cd efflux and partitioning to the shoots. In the present study, we functionally characterized the CAL2 defensin protein and determined its contribution to Cd accumulation. CAL2 shared 66% similarity with CAL1, and its mRNA accumulation is mainly observed in roots and is unaffected by Cd stress, but its transcription level was lower than that of CAL1 based on the relative expression of CAL2/Actin1 observed in this study and that reported previously. A promoter-GUS assay revealed that CAL2 is expressed in root tips. Stable expression of the CAL2-mRFP fusion protein indicated that CAL2 is also localized in the cell walls. An in vitro Cd binding experiment revealed that CAL2 has Cd chelation activity. Overexpression of CAL2 increased Cd accumulation in Arabidopsis and rice shoots, but it had no effect on the accumulation of other essential elements. Heterologous expression of CAL2 enhanced Cd sensitivity in Arabidopsis, whereas overexpression of CAL2 had no effect on Cd tolerance in rice. These findings indicate that CAL2 positively regulates Cd accumulation in ectopic overexpression lines of Arabidopsis and rice. We have identified a new gene regulating Cd accumulation in rice grain, which would provide a new genetic resource for molecular breeding.

Histidine-Rich Defensins from the Solanaceae and Brasicaceae Are Antifungal and Metal Binding Proteins.

Plant defensins are best known for their antifungal activity and contribution to the plant immune system. The defining feature of plant defensins is their three-dimensional structure known as the cysteine stabilized alpha-beta motif. This protein fold is remarkably tolerant to sequence variation with only the eight cysteines that contribute to the stabilizing disulfide bonds absolutely conserved across the family. Mature defensins are typically 46-50 amino acids in length and are enriched in lysine and/or arginine residues. Examination of a database of approximately 1200 defensin sequences revealed a subset of defensin sequences that were extended in length and were enriched in histidine residues leading to their classification as histidine-rich defensins (HRDs). Using these initial HRD sequences as a query, a search of the available sequence databases identified over 750 HRDs in solanaceous plants and 20 in brassicas. Histidine residues are known to contribute to metal binding functions in proteins leading to the hypothesis that HRDs would have metal binding properties. A selection of the HRD sequences were recombinantly expressed and purified and their antifungal and metal binding activity was characterized. Of the four HRDs that were successfully expressed all displayed some level of metal binding and two of four had antifungal activity. Structural characterization of the other HRDs identified a novel pattern of disulfide linkages in one of the HRDs that is predicted to also occur in HRDs with similar cysteine spacing. Metal binding by HRDs represents a specialization of the plant defensin fold outside of antifungal activity.