The nightshade proteinase inhibitor IIb gene is constitutively expressed in glandular trichomes.

The best known property of plant proteinase inhibitor II (PIN2) genes is their wound-inducible expression in leaves and constitutive expression in flowers. Here we show by promoter analysis in transgenic plants and in situ reverse transcription-PCR (RT-PCR) analysis that SaPIN2b, a member of the PIN2 gene family of nightshade (Solanum americanum), is also constitutively expressed in glandular trichomes. SaPIN2b promoter and its deletions were cloned and fused upstream of beta-glucuronidase (GUS) to transform the nightshade and tobacco (Nicotiana tabacum) plants. Histochemical staining assays indicated that SaPIN2b:GUS was expressed constitutively in glandular trichomes, predominantly in the gland cells, of both transgenic nightshade and tobacco plants. Constitutive expression of SaPIN2b in glandular trichomes was further confirmed by liquid phase in situ RT-PCR analysis of nightshade leaves. Deletion analysis from the 5' end of the SaPIN2b promoter revealed that separate regulatory elements control SaPIN2b expression in gland cells and stalk cells of glandular trichomes. Fluorometric GUS assays showed that SaPIN2b:GUS expression was significantly increased in transgenic plant leaves after mechanical wounding or methyl jasmonate treatment. The SaPIN2b promoter sequence contains six MYB-binding motifs and an L1 box that are involved in trichome differentiation and development. Overexpression of SaPIN2b in tobacco resulted in a significant increase in glandular trichome density and promotion of trichome branching. These results suggest that, as well as being an induced defensive protein of the well-known PIN2 family, SaPIN2b could also play roles in trichome-based defense by functioning as a constitutive component of trichome chemical defense and/or by regulating the development of glandular trichomes.