Gene expression during late embryogenesis in pea (Pisum sativum L)
A thesis submitted by Melinda Jane Mayer, B.Sc.(Bristol) in accordance with the requirements of the University of Durham for the degree of Doctor of Philosophy. Department of Biological Sciences, August 1993.Two cDNA libraries were constructed from desiccating pea cotyledons. Differential screening of the libraries with cDNA from an earlier developmental stage (physiological maturity) demonstrated that the abundant message population during dehydration shows some noticeable differences to the message populations present before desiccation. Clones hybridising to a polyubiquitin probe were isolated from a cDNA library. These clones were identified as messages for the two types of ubiquitin extension proteins (with 52 and 79 residue tails), already characterised in other species as being involved in ribosome biogenesis. The pea ubiquitin extension tail amino acid sequences showed considerable homology to tails from other plants, animals, yeast and protozoa, including a nuclear localisation site and a putative zinc-binding nucleic acid binding domain, the positions of which are conserved within the tail sequences. Sequencing of a second polyubiquitin cDNA from pea leaf demonstrated that pea contains a ubiquitin multigene family of at least four members. The expression of several genes associated with plant response to stress and two abundant seed messages (Leg A and J) was examined in developing and dehydrating cotyledons and axes. This confirmed conspicuous variations in the message levels of the genes examined as the cotyledons aged, with different members of the ubiquitin and legumin multigene families showing differential expression with age. It was also demonstrated that the expression pattern of certain messages in the cotyledons was different to that in the axes and other seed tissues. This was confirmed by an analysis of total and albumin protein fractions in cotyledons and axes. The effect on specific message and protein levels of premature desiccation treatments indicated that the temporal expression of several seed genes is related to the state of hydration of the seed, artificial desiccation leading to premature maturation. Seed storage protein message and protein levels were especially increased by premature desiccation. Legumin seed storage protein messages were also shown to be responsive to exogenous ABA applied to immature cotyledons during the seed filling stage. However, the other stress-related messages examined in pea (ubiquitin and a pea putative metallothionein) were not responsive to exogenous ABA at this developmental stage.