Tobacco NtLTP1, a glandular-specific lipid transfer protein, is required for lipid secretion from glandular trichomes

Glandular trichomes are the phytochemical factories of plants, and they secrete a wide range of commercially important natural products such as lipids, terpenes and flavonoids. Herein, we report that the Nicotiana tabacum LTP1 (NtLTP1) gene, which is specifically expressed in long glandular trichomes, plays a role in lipid secretion from trichome heads. NtLTP1 mRNA is abundantly transcribed in trichomes, but NtLTP3, NtLTP4 and NtLTP5 are not. In situ hybridization revealed that NtLTP1 mRNAs accumulate specifically in long trichomes and not in short trichomes or epidermal cells. X-gluc staining of leaves from a transgenic plant expressing the NtLTP1 promoter fused to a GUS gene revealed that NtLTP1 protein accumulated preferentially on the tops of long glandular trichomes. GFP fluorescence from transgenic tobacco plants expressing an NtLTP1-GFP fusion protein was localized at the periphery of cells and in the excreted liquid droplets from the glandular trichome heads. In vitro assays using a fluorescent 2-p-toluidinonaphthalene-6-sulfonate probe indicated that recombinant NtLTP1 had lipid-binding activity. The overexpression of NtLTP1 in transgenic tobacco plants resulted in the increased secretion of trichome exudates, including epicuticular wax. In transgenic NtLTP1-RNAi lines, liquid secretion from trichomes was strongly reduced, but epicuticular wax secretion was not altered. Moreover, transgenic tobacco plants overexpressing NtLTP1 showed increased protection against aphids. Taken together, these data suggest that NtLTP1 is abundantly expressed in long glandular trichomes, and may play a role in lipid secretion from long glandular trichomes.     

Isolation and characterization of the CYP71D16 trichome-specific promoter from Nicotiana tabacum L

Trichomes are specialized epidermal cells that produce secretions that are thought to provide a first line of defence against pests and pathogens. Many trichome-secreted compounds are used commercially as flavourings, medicines, etc. Described here is the cloning and characterization of the promoter of a tobacco trichome-specific P450 gene, CYP71D16. This promoter is shown to direct the specific expression of the reporter gene, beta-glucuronidase (GUS), in glandular trichomes of Nicotiana tabacum cv. T.I. 1068 at all developmental stages. With the full promoter, GUS activity was predominantly in the gland cell, with less in the stalk cell adjacent to the gland, and in lower stalk cells. GUS staining was also observed in the most distal trichome stalk cells of non-glandular trichomes found on variety T.I. 1112. Promoter deletion analysis revealed that the region from -223 to +111 bp is sufficient to direct trichome-specific expression, but not strong gland expression. Examination of the literature suggests that this is the first characterized trichome-specific-promoter shown to function at all stages of plant development. This promoter may provide efficient bioengineering to enhance pest and pathogen resistance, and for molecular farming based on the trichome gland system.     

Serine proteinase inhibitor proteins: Exogenous and endogenous functions

Summary Proteinase inhibitor II (PIN2) proteins from the Solanaceae family have been previously used in plant transformation to acquire protection against caterpillars. Some of these PIN2 proteins have been shown to exhibit exogenous activities against trypsin and/or chymotrypsin in vitro. Despite their application in conferring insect resistance in transgenic plants, the endogenous roles of this family of proteins in various plant species have not been well defined. To investigate the exogenous and endogenous functions of PIN2 proteins, cDNAs encoding PIN2 proteins from the weed Solanum americanum (American black nightshade), designated SaPIN2a and SaPIN2b, were cloned and characterized. The localization of S. americanum SaPIN2a and SaPIN2b mRNAs and proteins in the reproductive tissues destined to undergo developmental programmed cell death subsequently led to investigations into their function during seed development. Using plant transformation of lettuce and S. americanum, it was evident that: (1) the expression of SaPIN2a in transgenic lettuce conferred resistance to cabbage looper (Trichoplusia ni) caterpillars; and (2) the expression of siRNAs from a PIN2-RNAi construct resulted in transgenic S. americanum that were impaired in seed development. These results suggest that S. americanum PIN2 proteins not only enhance resistance to caterpillars (when expressed exogenously) but they function in inhibiting endogenous proteases that are expressed during seed development. Specifically, the aborted seeds of PIN2-RNAi lines showed abnormal endothelium that subsequently affected endosperm and embryo development.     

Chloroplast-like organelles were found in enucleate sieve elements of transgenic plants overexpressing a proteinase inhibitor

SaPIN2a, a plant proteinase inhibitor from nightshade (Solanum americanum), was located to the enucleate sieve elements (SEs) of phloem. The expressed SaPIN2a in transgenic lettuce showed inhibition of plant endogenous trypsin- and chymotrypsin-like activities, suggesting that SaPIN2a can regulate proteolysis in plant cells. To further investigate the physiological role of SaPIN2a, we produced transgenic nightshade and lettuce plants overexpressing SaPIN2a from the cauliflower mosaic virus (CaMV) 35S promoter using Agrobacterium-mediated transformation. Overexpression of SaPIN2a in transgenic plants was demonstrated by northern blot and western blot analysis. SaPIN2a-overexpressing transgenic nightshade plants showed significantly lower height than wild-type plants. Transmission electron microscopy analysis showed that chloroplast-like organelles with thylakoids, which are not present in enucleate SEs of wild-type plants, were present in the enucleate SEs of SaPIN2a-overexpressing transgenic plants. This finding is discussed in terms of the possible role played by SaPIN2a in the regulation of proteolysis in SEs.     

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.