Gene Expression in Dwarf Mistletoe Related to Explosive Seed Dispersal with special Attention to Aquaporins

Worldwide, mistletoe plants are important forest pests, found in all continents aside from Antarctica. In North America, control of conifer parasitism by dwarf mistletoes, specifically Arceuthobium americanum, is challenging. Dwarf mistletoes are dioecious parasitic flowering plants with a unique and effective explosive seed dispersal mechanism. Due to the large accumulation of water within the fruit, which increases both the fruit size and its internal hydrostatic pressure, the transport of water into the fruit and the possible involvement of plasma membrane intrinsic proteins (PIPs), particularly aquaporins (AQPs), were the focus of the investigation. Notably, previous research also demonstrated that cuticle thickening, declining stomata number, and thermogenesis are potential “players” in forcible discharge. As such, molecular work was performed here to reveal genes involved in physiological processes potentially associated with explosive seed dispersal and to examine the expression of these genes. Therefore, the overarching goal of this project was to obtain gene sequencing and expression data for Arceuthobium spp. to gain insights into the involvement of AQPs and other proteins likely responsible for generating high hydrostatic pressure and discharge. Many methods were employed to extract the RNA from the fruit, including both traditional and commercially available methods, and following successful extraction, a cDNA library was constructed. A non-standard heterologous microarray hybridization, unconventionally used for transcription analysis of non-model species, employing both the MicroCASTer handheld system and an Affymetrix Arabidopsis Gene 1.0 ST microarray was used to determine gene sequence and expression. Five genes sequences were obtained from the handheld system: AQP2, ABC transporter, Ribulose Bisphosphate Carboxylase-Oxygenase, Alkaline/neutral invertase, and Sterol-14 demethylase. To confirm that AQP2 codes for plasma membrane intrinsic protein 2 (PIP2), gateway cloning followed by a physiological study with stopped-flow spectroscopy was undertaken. Despite sequence divergence between the two species, 24.2% of Arabidopsis Gene 1.0 ST array was hybridized to dwarf mistletoe RNA; this hybridization identified approximately 1,000 genes that are highly differentially expressed in dwarf mistletoe during fruit development. Further investigation of the Affymetrix results revealed several additional mistletoe genes, Stomatal cytokinesis defective2, Movement protein binding protein, Eceriferum1, and SHINE2, that are seasonally up and down regulated (May vs. September) and are likely involved in the explosive seed dispersal process. With these results in hand, research can advance towards molecular insights into not only explosive seed dispersal in dwarf mistletoe, but also into other processes such as development, reproduction, photosynthesis light response, and parasitism-associated gene expression within dwarf mistletoe and related plants. Ideally, this work will provide new opportunities for investigating novel avenues of plant-plant pest control