Structural studies of Ib-AMP1, a small antimicrobial peptide derived from the seeds of Impatiens balsamina have been performed using circular dichroism (CD) and two-dimensional proton nuclear magnetic resonance (1H NMR). This 20-residue peptide is highly basic with five arginine residues and contains four cysteines which form two intramolecular disulfide bonds. CD results reveal that the peptide may include a beta-turn but do not show evidence for either helical or beta-sheet structure over a range of temperature and pH. Structural information from NMR was obtained in the form of proton-proton internuclear distances inferred from NOEs and dihedral angle restraints from spin-spin coupling constants, which were used for distance geometry calculations. Owing to the difficulty in obtaining the correct disulfide connectivities by chemical methods, three separate calculations were performed; with no disulfides and with the two possible alternate disulfide connectivities. Results from distance geometry calculations reveal that although the peptide is small, the cysteines constrain part of it to adopt a well-defined main chain conformation. From residue 6 to 20, the backbone is well defined, whilst the N-terminal region, residues 1-5, has very few constraints and appears to be very flexible. In the defined core region, there are three beta-turns at residues 9-12, 10-13, and 12-15. The side chains show no strong interactions in the NMR spectra and are therefore thought to adopt multiple conformations. Superposition of the structures generated shows that the peptide has two hydrophilic patches which are at opposite ends of the molecule separated by a large hydrophobic patch. Little is known about the mode of action of this protein, but it is thought to interact with a membrane-bound receptor, and possible sites of interaction are discussed. The structures determined are compared with those of the alpha-conotoxins, which are also highly basic proteins with similar disulfide connectivities.

Four closely related peptides were isolated from seed of Impatiens balsamina and were shown to be inhibitory to the growth of a range of fungi and bacteria, while not being cytotoxic to cultured human cells. The peptides, designated Ib-AMP1, Ib-AMP2, Ib-AMP3, and Ib-AMP4, are 20 amino acids long and are the smallest plant-derived antimicrobial peptides isolated to date. The Ib-AMPs (I. balsamina antimicrobial peptides) are highly basic and contain four cysteine residues which form two intramolecular disulfide bonds. Searches of protein data bases have failed to identify any proteins with significant homology to the peptides described here. Characterization of isolated cDNAs reveals that all four peptides are encoded within a single transcript. The predicted Ib-AMP precursor protein consists of a prepeptide followed by 6 mature peptide domains, each flanked by propeptide domains ranging from 16 to 35 amino acids in length. Such a primary structure with repeated alternating basic mature peptide domains and acidic propeptide domains has, to date, not been reported in plants.

Radish seeds have previously been shown to contain two homologous, 5-kD cysteine-rich proteins designated Raphanus sativus-antifungal protein 1 (Rs-AFP1) and Rs-AFP2, both of which exhibit potent antifungal activity in vitro. We now demonstrate that these proteins are located in the cell wall and occur predominantly in the outer cell layers lining different seed organs. Moreover, Rs-AFPs are preferentially released during seed germination after disruption of the seed coat. The amount of released proteins is sufficient to create a microenvironment around the seed in which fungal growth is suppressed. Both the cDNAs and the intron-containing genomic regions encoding the Rs-AFP preproteins were cloned. Transcripts (0.55 kb) hybridizing with an Rs-AFP1 cDNA-derived probe were present in near-mature and mature seeds. Such transcripts as well as the corresponding proteins were barely detectable in healthy uninfected leaves but accumulated systemically at high levels after localized fungal infection. The induced leaf proteins (designated Rs-AFP3 and Rs-AFP4) were purified and shown to be homologous to seed Rs-AFPs and to exert similar antifungal activity in vitro. A chimeric Rs-AFP2 gene under the control of the constitutive cauliflower mosaic virus 35S promoter conferred enhanced resistance to the foliar pathogen Alternaria longipes in transgenic tobacco. The term "plant defensins" is proposed to denote these defense-related proteins.

From seeds of Aesculus hippocastanum, Clitoria ternatea, Dahlia merckii and Heuchera sanguinea five antifungal proteins were isolated and shown to be homologous to plant defensins previously characterised from radish seeds and γ‐thionins from Poaceae seeds. Based on the spectrum of their antimicriobial activity and the morphological distortions they induce on fungi the peptides can be divided into two classes. The peptides did not inhibit any of three different α‐amylases.

On the basis of an extensive screening of seeds from various plant species, we have isolated and characterized several different antimicrobial peptides. They were all typified by having a broad antifungal activity spectrum, a relatively low molecular weight (3-14 kDa), a high cysteine content and a high isoelectric point (pI > 10). With respect to their amino acid sequence, these peptides can be classified into six structural classes. Synergistic enhancement (up to 73-fold) of antimicrobial activity was demonstrated in some combinations of peptides belonging to different classes. cDNA clones corresponding to different antifungal peptides were isolated and used to transform tobacco plants. Extracts of these transgenic plants showed higher (up to 16-fold) antifungal activity than untransformed control plants. Such antimicrobial peptides may find applications in molecular breeding of plants with increased disease resistance.

A cDNA clone encoding an antimicrobial chitin-binding protein from amaranth (Amaranthus caudatus L.) was isolated using a cDNA library constructed from near-mature seed poly(A)+ mRNA. The deduced amino acid sequence of the cDNA clone encodes a predicted polypeptide of 86 amino acids. This polypeptide has three distinct domains: an amino-terminal putative signal peptide (25 amino acids), a domain corresponding to the mature protein (30 amino acids), and a carboxyl-terminal propeptide (31 amino acids) containing a putative N-glycosylation site. The encoded protein differs from all known members of the family of chitin-binding proteins. Transcripts of the expected size (650 bp) are present in developing seeds but not in roots, leaves or stressed leaves.

Two peptides with antimicrobial properties (Mj-AMPs) and two proteins with antifungal activity (Rs-AFPs) were isolated from seeds of Mirabilis jalapa L. and Raphanus sativus L., respectively. Both peptides and proteins are highly basic and cysteine-rich. Complete amino acid sequences were determined for the Mj-AMPs (Mj-AMP1 has 37 amino acids and Mj-AMP2 consists of 36 amino acids) and revealed homology to some invertebrata neurotoxins. N-terminal amino acid sequences of Rs-AFPs showed homology with the derived amino acid sequences of two pea genes, induced upon fungal attack, to Γ-thionins from wheat and to α-amylase inhibitors from sorghum. Both Mj-AMPs and Rs-AFPs exhibit a broad spectrum of antifungal activity. The activity of Mj-AMPs and Rs-AFPs is reduced in presence of inorganic cations, but Rs-AFPs (especially Rs-AFP2) are comparitively less sensitive to antagonism by cations. The Mj-AMPs, not the Rs-AFPs are also active against yeast and some Gram-positive bacteria. The viability of cultured human cells is not affected by Mj-AMPs nor by Rs-AFPs. In spray tests both Mj-AMP1 and Rs-AFP2 protect sugar-beet leaves against infection with Cercospora beticola (leaf spot disease).