Isolation and properties of floral defensins from ornamental tobacco and petunia

Abstract

The flowers of the solanaceous plants ornamental tobacco (Nicotiana alata) and petunia (Petunia hybrida) produce high levels of defensins during the early stages of development. In contrast to the well-described seed defensins, these floral defensins are produced as precursors with C-terminal prodomains of 27 to 33 amino acids in addition to a typical secretion signal peptide and central defensin domain of 47 or 49 amino acids. Defensins isolated from N. alata and petunia flowers lack the C-terminal domain, suggesting that it is removed during or after transit through the secretory pathway. Immunogold electron microscopy has been used to demonstrate that the N. alata defensin is deposited in the vacuole. In addition to the eight canonical cysteine residues that define the plant defensin family, the two petunia defensins have an extra pair of cysteines that form a fifth disulfide bond and hence define a new subclass of this family of proteins. Expression of the N. alata defensin NaD1 is predominantly flower specific and is most active during the early stages of flower development. NaD1 transcripts accumulate in the outermost cell layers of petals, sepals, anthers, and styles, consistent with a role in protection of the reproductive organs against potential pathogens. The floral defensins inhibit the growth of Botrytis cinerea and Fusarium oxysporum in vitro, providing further support for a role in protection of floral tissues against pathogen invasion.

Figure 1

A, Alignment of the predicted amino acid sequence of NaD1 (accession no. AF509566), PhD1 (accession no. AF507975), and PhD2 (accession no. AF507976) with the predicted amino acid sequences encoded by four other flower-derived cDNA clones: FST from tobacco (accession no. Z11748), TPP3 from tomato (Lycopersicon esculentum; accession no. U20591), PPT from petunia (accession no. L27173), TGAS118 from tomato (accession no. AJ133601), and the purified seed defensins Rs-AFP2 from radish (accession no. P30230), alfAFP from alfalfa (Medicago sativa; accession no. AF31946), and γ1-P from wheat (accession no. P2015). GenBank accession numbers are given in parenthesis. The endoplasmic reticulum signal sequence has been omitted. Identical residues are boxed in black with conservative substitutions in gray. Spaces have been introduced to maximize the alignment. The arrow indicates the site of cleavage between the mature defensin and C-terminal prodomain. The disulfide bond connectivities are shown below the sequences as connecting solid lines. The additional disulfide bond in PhD1 and PhD2 is shown by a broken line. B, Comparison of the basic (Arg, Lys, and His) and acidic (Glu and Asp) amino acid composition and the net charge associated with the defensin and C-terminal domains in NaD1, PhD1, PhD2, FST, and TPP3 at neutral pH.

Figure 2

In situ location of NaD1 mRNA in flower buds. A and B, Autoradiographs of transverse sections of a stage I (10 mm in length) ornamental tobacco flower from after hybridization with a ³⁵S-labeled NaD1 antisense RNA probe. The epidermal (ep) cells of the petal (pe) and sepal (se), and the cortical cells (cc) of the style (st) and the connective tissue (ct) of the anther (a) were heavily labeled. There was no hybridization to the pollen mother cells (pmc), tapetum (ta), vascular bundle (vb), or the transmitting tissue (tt). C, Autoradiograph of a transverse section of a stage I flower bud after hybridization with a ³⁵S-labeled NaD1 sense RNA probe. The cells of the style (st), anther (a), petal (pe), and sepal (se) were not labeled.

Figure 3

Immunoblot analysis of NaD1 in ornamental tobacco flowers and floral tissues at various stages of development. A, Five stages (I–V) of flower development as described in “Materials and Methods.” Immunoblot of buffer soluble proteins (30 μg lane⁻¹) derived from petals, anthers, pistils, ovaries, and sepals at stages I through V using the α-6H.proNaD1 antibodies. B, Immunoblot of buffer soluble proteins (30 μg lane⁻¹) derived from whole flowers at stages I through V, 500 and 1,000 ng of purified NaD1 (M), and 5 and 10 ng of purified 6H.proNaD1 (P) using the α-6H.proNaD1 antibodies. Molecular mass markers are in kilodaltons. The positions of NaD1 (M) and proNaD1 (P) are marked in with arrows.

Figure 4

Purification of defensins from ornamental tobacco flower buds and petunia petals. RP-HPLC profile of gel filtration fractions from ornamental tobacco (A) and petunia (B) extracts showing percentage buffer B (%B) and retention times in minutes. Defensin peaks collected are numbered. The inset in A is an immunoblot of protein in peak 1 after incubation with α-6H.proNaD1 antibodies. C, N-Terminal sequence and electrospray mass spectrometry data for the proteins in peaks 1 (A), 2 and 3 (B), and the predicted mass of the defensin domains encoded by the NaD1, PhD1, and PhD2 cDNA clones. “x” corresponds to an unassigned amino acid that is probably a Cys as predicted from the cDNA sequence.

Figure 5

Immunogold localization of NaD1 in anthers and ovaries from stage I flowers. A, Overview of the anther showing cells of the connective tissue with electron dense deposits (arrowed) in the vacuole (v). B, Connective tissue cells of the anther and the cortical cells (C) of the ovary labeled with the α-6H.proNaD1 antibodies. The antibodies bound specifically to electron dense deposits in the vacuole and no binding was observed in the cytoplasm (cy) or cell walls (cw).

Figure 6

Effect of NaD1, PhD1, and PhD2 on the growth of F. oxysporum f. sp. dianthi Race 2 (A) and B. cinerea (B). Growth of the fungi in the test solutions is plotted relative to the growth in water for the same period (50 h). Test proteins were used at final concentrations of 2, 10, and 20 μg mL⁻¹. Growth in water is taken to be 100% growth. Ovalbumin and the 6-kD proteinase inhibitors from ornamental tobacco (NaPI) were used as negative controls. Each treatment was performed in quadruplicate. The error bars are se of the mean.