Graduate Thesis Or Dissertation


Cellular events conditioned by the Np gene of Pisum sativum L. in response to reduced UV light, weevil oviposition, and bruchins Public Deposited

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  • In Pisum, the Np gene conditions two mitotic responses - to bruchid weevil oviposition on the pod and to reduced UV light. Oviposition by the weevil results in tumorous or neoplastic growth under the egg. Biochemically active compounds, called bruchins, were isolated from two bruchid insects (Doss et al, 2000). Femtomolar concentrations of bruchin result in programmed cell death (PCD) and neoplasm formation on the pod at the application site. PCD is evident at the application site within four hours and a large (several mg) neoplasm in five to seven days. Low UV light results in growth of neoplasm, termed spontaneous neoplasm (SN), over the entire surface of the pod and, as with bruchin application, appears to begins with PCD followed by mitosis. Genotypes containing np/np do not develop SN and respond only weakly to bruchin. Both responses conditioned by Np initiate at the stomatal complex and are first detected by increased auto-fluorescence. In both cases, this is accompanied by increased peroxide/peroxidase activity first at the stomata, then radiating outward across the epidermal surface and into the mesophyll cells. Nuclei in the epidermis stained strongly for peroxide/peroxidase within 3 h of bruchin application. Nuclei in the mesophyll stained for peroxide/peroxidase by 24 h. Rose Bengal, which generates singlet oxygen, stimulated site-specific neoplasm formation on greenhouse grown pods. Lanthanum, an inhibitor of Ca2+ influx, inhibited both ROS production and bruchin action. ZnCl2 inhibited ROS production and bruchin response less effectively than LaCl3. Epidermal cell death demonstrated hallmarks of apoptosis. TUNEL demonstrated the presence of oligonucleosomal fragmentation, which is associated with PCD. Pods treated with bruchin demonstrated progressive TUNEL staining. The first areas to test positive for endogenous nuclease activity were the subsidiary cells of the stomatal complex. Transmission electron microscopy (TEM) found nuclear blebbing, chromatin condensation, mitochondrial swelling, changes in cytosol density and increased vacuolization. This work demonstrates that the bruchin and SN responses conditioned by the Np gene originate from similar cellular events: increased auto-fluorescence, increased peroxide/peroxidase, and PCD.
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