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Gene discovery in pursuit of carbohydrate metabolic genes in the parasitic plant Cuscuta pentagona: Cloning of a sucrose phosphate synthase-like gene. Research supported by the STEM-Talent Expansion Program, Summer 2007 Biology Department, University of Nebraska at Omaha, Omaha, Nebraska 68182-0040, USA. Introduction: The plant as a parasite Just as some microbes and animals make their living parasitizing other organisms, many species of plants make their living by parasitizing other plants. The parasitic habit has arisen several times among flowering plant lineages. One feature that all parasitic plants share is the use of penetrating organs, called haustoria, to connect to the host in order to draw upon its water and nutrient supply. Beyond the occurrence of haustoria, however, parasitic plants can vary widely in their degree of modification, and in the degree to which they are dependent upon their hosts. Some parasitic plants can complete their life cycles without engaging in parasitism, while others are wholly dependent upon their hosts. Species of Cuscuta, commonly called the dodders, lack roots, have only vestiges of leaves, and perform very little or no photosynthesis in most tissues. As such, they are wholly dependent upon their hosts for water and mineral nutrients, as well as for a majority of their energy needs. In order to determine the physiological consequences of adopting the parasitic habit, we are investigating the core metabolic processes common to all photosynthesizing plants. These processes include photosynthetic carbon assimilation, sugar and starch metabolism, nitrogen assimilation, and amino acid biosynthesis. As a starting point in these investigations, we are identifying the genes that encode the enzymes that mediate these processes. In this study, we attempted to identify the sucrose phosphate synthase gene, involved in carbohydrate metabolism. To provide a point of comparison, we are also pursuing this gene in Cuscuta’s nonparasitic relative, ivy-leaf morning glory (Ipomoea hederacea). The 2007 Biology-STEP molecular research team Left to right: Mark Schoenbeck (instructor), James Lucas, Kimberly Meints, Kristina Wiley, Craig Harrison, Sarah Faltin, Lacy Jacobsen, Aaron Zach, Andria Bethelmie, Sobia Rasool, Kirk Larson, Sydney Brommer (technical and research assistant), and Julie Kowal (lab assistant). plastid triose phosphate starch granule hexose phosphates starch synthase cytoplasm triose phosphate hexose phosphates sucrose for export sucrose phosphate synthase Figure 2. Plant cells may be either sources of, or sinks for, fixed carbon in the form of carbohydrates. Cells may store carbohydrates in the form of starch (left) employing enzymatic activities such as starch synthase. Alternatively, cells may generate sucrose, a primary form of translocated carbohydrate, for mobilization to other plant organs, through the activity of sucrose phosphate synthase. Because dodder functions as a strong sink in order to draw carbohydrate resources from its host, it would be of interest to understand the functioning of starch synthase and sucrose phosphate synthase genes in the parasite, relative to their functions in non-parasitic plants. A dodder starch synthase gene was identified by the 2005 Biology-STEP molecular research team. The sucrose phosphate synthase gene was chosen as an objective for this work. Conclusions and future work Sequence analysis of recovered clones shows a high degree of similarity between C. pentagona sucrose phosphate synthase (SPS) and sequences reported from Convolvulaceae. Recovery and analysis of the complete SPS gene, and proximal genomic sequences from dodder and non-parasitic relatives may reveal changes in putative promoter regions. Figure 1. The parasitic angiosperm Cuscuta pentagona on its host (left) and its non-parasitic relative Ipomoea hederacea. Recent molecular investigations place the genus Cuscuta in the family Convolvulaceae with Ipomoea. Molecular and physiological investigations may provide clues as to the changes that accompanied the adoption of the parasitic habit, which included the loss of functional leaves and roots, and, likely, the modification of core metabolic processes. Figure 3. A flow chart describing the strategy for identifying genes involved in core metabolic processes in dodder and ivy-leaf morning glory. The gene discovery strategy employed a range of skills, comprising the use of bioinformatic tools as well as standard molecular research methods. Both the SPS and previously identified starch synthase gene fragments will be employed to monitor levels of cognate gene transcipts, providing information on how carbohydrate metabolic genes may be regulated in parasitic and non-parasitic plants. Acknowledgements Funds for this research were provided by an NSF STEP grant, NSF-033642. Laboratory equipment was provided by NIH Grant Number P20 RR16469 from the INBRE Program of the National Center for Research Resources.