The Pollination Biology and Mating System of a Peripheral Population of Witheringia solanacea (Solanaceae)

Pollinator visitation rates over the life of a flower are determined by pollinator abundance and floral longevity. If flowers are not visited frequently enough, pollen limitation may occur, favoring the evolution of self-compatibility (SC). In plant species with varying SC levels, central populations often are self-incompatible (SI) and peripheral populations are SC. Witheringia solanacea (Solanaceae) is a species that follows this trend with the exception of one population in the Monteverde Cloud Forest Reserve, which is peripheral yet SI. I investigated this population using multiple techniques including floral bagging, pollinator observations, microsatellite analysis, and floral longevity manipulations. My results confirmed the self-incompatibility of the Monteverde population and indicated low but perhaps adequate rates of pollinator visitation per flower per hour. I found reduced genetic diversity at Monteverde and gene flow occurring unidirectionally from San Luis (a central population) to Monteverde. In the greenhouse, there was more of an effect of male than female function on floral longevity, but the largest differences were environmental. Flowers stayed open substantially longer when cool, cloudy weather was simulated and shorter when conditions were hot and sunny. The results indicate that the Monteverde population of W. solanacea is SI because 1) it is unable to maximize its fitness due to gene flow from San Luis and its relatively recent colonization of the area and 2) pollen limitation may not be severe because of supplemental pollinator availability from other Witheringia species in the area and increased floral longevities due to cool and cloudy conditions.

Professor David Firmage, Department of Biology and Environmental Studies

Professor Emeritus David Firmage, Department of Biology and Environmental Studies. Reading Looking for Alaska by Peter Jenkins

Overexpression and Purification of the Oat (Avena Fatua) Protein AFN1 and Construction of a pMAL/AFN1 Expression Plasmid

Abscisic acid (ABA) is an important phytohormone with regulatory roles in many physiological processes. ABA expression is induced by environmental stresses such as drought and it is known to be an inhibitor of seed germination. A wild oat (Avena fatua) called AFN1 has been hypothesized to initiate the early stages of germination as its mRNA accumulates in nondormant seed embryos during imbibition. The polypeptide sequence of AFN1 suggests that it is an ABA glucosyl transferase. Glucosylation by AFN1 and thereby inactivation of ABA could lead to seed germination. In order to understand the role of AFN1 in germination, an ample quantity of AFN1 polypeptide is needed to test for enzymatic ABA glucosylase activity. My work has been to overexpress recombinant AFN1containing a (His)6 tag using a pRSETC E.coli expression system followed by Purification of the AFN1 protein by means of a nickel-affinity column that bind to the (His)6 tag. Due to the insufficient yield of AFN1 fusion protein obtained with this procedure, another method using a pMAL-c2x vector is now being employed. The pMAL expression system provides a method for expressing and purifying protein by tagging proteins with maltose-binding protein (MBP). It is anticipated that MBP tag will be advantageous as it can make the fusion protein more soluble and thereby yield a larger quantity of protein. Currently, work is underway on the construction of pMAL/AFN1 plasmid.

Characterization of AFN1, a Gene Associated with Cereal Grain Germination

The AFN1 gene is transiently expressed in germinating oat grains. As AFN1 is not expressed in dormant oat grains during imbibition, we hypothesize that AFN1 may be involved in stimulating the germination process. Sequence analysis of an AFN1 cDNA clone indicates that the AFN1 polypeptide is similar to a previously identified abscisic acid (ABA) glucosyl transferase. This suggests that AFN1 may be acting to glucosylate ABA, thereby inactivating it. As the hormone ABA is known to inhibit germination, ABA glucosylation/inactivation could lead to germination in grains expressing AFN1. To test this hypothesis, we have constructed an expression plasmid that encodes an MBP::AFN1 (maltose binding protein) fusion protein. E. coli cells carrying the expression plasmid were found to produce the MBP::AFN1 fusion protein as a substantial fraction of total protein. We are currently in the process of purifying the MBP::AFN1 fusion protein by affinity chromatography, so that it can be assayed for ABA glucosyl transferase activity. We also wish to test the effect of AFN1 gene expression during grain imbibition on the germination behavior of the grains. To this end, we have constructed plasmids for the overexpression and RNAi-based suppression of AFN1 in transgenic plants. These plasmids have been introduced into oat cells by particle bombardment and we are in the process of regenerating transgenic plants for study.

Characterization of the Wheat Grain PKABA1-Interacting Protein TaWD40

Abscisic acid (ABA)-mediated gene expression is a critical component of plant responses to this important hormone, which affects plant growth, development, and responses to environmental stresses. Plant responses to ABA are mediated by a number of factors including PKABA1, an ABA induced protein kinase involved in ABA-suppressed gene expression in cereal grains, and TaWD40, which has previously been shown to physically interact with PKABA1. A full-length 1.9 kb TaWD40 cDNA, CK210682, was sequenced as part of this project. Based on the deduced protein sequence, it is thought that TaWD40 may belong to the family of E3 ubiquitin ligases, possibly targeting PKABA1 for destruction. Construction of expression plasmids for overproduction of the TaWD40 polypeptide in E. coli is currently underway. The TaWD40 cDNA has been successfully amplified from the source plasmid and inserted into an intermediate plasmid, pCR2.1. The TaWD40 cDNA is currently being cloned from the pCR2.1 intermediate plasmid into two different expression vectors, pRSET-A and pMAL-c2x, for future protein production and purification.