In contrast to the extracts of leaves of untreated tobacco plants, extracts of leaves of tobacco plants that were treated with jasmonic acid methyl ester (JAME) for 3 days clearly show agglutinating activity, due to expression of the Nicotiana tabacum agglutinin (also called Nictaba). This lectin is one of the recently discovered inducible plant lectins that possibly play a role in cell signalling in plant cells. The localisation and expression of Nictaba will be examined during this thesis. The coding sequence (CDS) of wild-type (WT) Nictaba and the coding sequences of mutant Nictaba constructs were cloned in vectors through the use of the GatewayTM technology. During this cloning, the constructs were fused with the CDS of EGFP that was already present in the vector. This makes it possible for the constructs to be expressed in transgenic plant cells as EGFP-fusion proteins. The obtained expression vectors were transformed in Bright-Yellow 2 (BY-2) cells or onion epidermal cells using particle bombardment. Confocal fluorescence laser scanning microscopy was performed to study the expression and localisation of the EGFP-fusion-proteins.
A first construct was built by cloning the WT Nictaba CDS into a vector. The obtained expression vector was then transformed in BY-2 cells. Approximately 24 h after the transformation fluorescence was detected in the cytoplasm, around the nucleus and in the nucleus of transformed BY-2 cells. After 40 h no more fluorescence was detected in the nucleus though fluorescence was still clearly visible around the nucleus and in the cytoplasm what indicated a possible migration of the EGFP-fusion-product. A second construct was made by introducing a mutation in the putative nuclear localisation signal of Nictaba. This construct was then cloned into a vector whereupon the obtained expression vector was transformed in BY-2 cells. Fluorescence was detected in the cytoplasm and around the nucleus of transformed cells. The difference in localisation between these two constructs confirmed the functionality of the NLS in the CDS of WT Nictaba. Two more constructs were made by fusing the WT Nictaba CDS to: 1) the sequence of the sorting signal of sporamin for vacuolar targeting or 2) the sequence of the sorting signal of rubisco for chloroplast targeting. Expression of Nictaba in the vacuole or the chloroplast will clearly interfere with the role of Nictaba in the cytoplasm. Only the construct with the vacuolar targeting signal showed expression in transgenic BY-2 cells. Fluorescence was detected in prevacuolar compartments and in the ER of transgenic BY-2 cells. Finally one construct was made by fusing the CDS of the Nictaba NLS mutant tot the CDS of this VTS to study the possible interactions of the NLS and the VTS. However, no expression was detected after transformation of this construct in BY-2 cells or in onion epidermal cells.