A proteinase inhibitor II of Solanum americanum is expressed in phloem

Although proteinase inhibitor proteins are known to confer insect resistance in transgenic plants, their endogenous roles remain undefined. Here, we describe the expression of a proteinase inhibitor II (PIN2) protein from Solanum americanum in phloem of stems, roots and leaves suggesting a novel endogenous role for PIN2 in phloem. The phloem consists of parenchyma cells, sieve elements (SE), and companion cells (CC) which are in close association with SE. We isolated two cDNAs encoding PIN2, SaPIN2a and SaPIN2b, from a S. americanum cDNA library using a tomato PIN2 cDNA as hybridization probe. SaPIN2a shows 73.6% identity to SaPIN2b. Southern blot analysis confirmed that two genes occur in S. americanum. Northern blot analysis showed that both are wound-inducible and are expressed in flowers. Unlike SaPIN2b and other previously characterized plant PIN2 proteins, SaPIN2a is abundantly expressed in stems. In situ hybridization studies on stem sections showed that SaPIN2a mRNA is expressed in CC and some SE, likely the immature developing SE, of external and internal phloem. Western blot analysis using SaPIN2a-specific antibodies showed SaPIN2a accumulation in stems, leaf midribs and fruits. Immunohistochemical localization, using these antibodies, revealed SaPIN2a expression in external and internal phloem of stem. Immunoelectron microscopy of stem, root and leaf sections further localized SaPIN2a to the CC and predominantly to the SE, particularly the parietal cytoplasm adjacent to the cell wall, the lumen and the sieve-area pores. These results suggest that, other than a possible role in plant defense, SaPIN2a could be involved in regulating proteolysis in the SE.