Plant development is essentially a sequence of events, programmed by the information inherited in the DNA complement, which leads to cellular differentiation and eventually to the completion of the life cycle. The rate of development is regulated by modification of the rates of individual steps in response to stimuli from both the external and internal environments of the plant. Essential to the continued survival of the plant are negative feed-back mechanisms which regulate the responses to the environment and maintain the rates of essential developmental steps within limits which permit completion of the life cycle. One such negative feed-back mechanism has been described in some detail in elongating internodes of sugar-cane stalks. The developmental process involved is cell elongation, and the feed-back mechanism, negative feed-back regulation of the synthesis of the enzyme invertase by its product glucose. The feed-back loop maintains a steady growth rate in the presence of a variable input of photosynthetically derived carbohydrate. The work described in this thesis is concerned with further investigation of the mechanism of the response of this regulatory system in sugar cane t o external chemical stimuli, particularly plant hormones.
One of the possible mechanisms for the action of plant growth regulating hormones is through direct regulation of protein and enzyme synthesis. Many changes in protein and enzyme levels have been observed in intact plants during growth responses to applied hormones. Sugar cane invertase levels increase proportionately when the growth rate of sugar cane is increased by application of gibberellic acid. In these intact systems however, it is usually impossible to determine whether growth or enzyme synthesis is primarily affected by the hormone applied, since growth rates and enzyme levels change simultaneously.
Using the techniques developed by previous workers (Introduction 1.2) synthesis of invertase can be studied in sugarcane tissue slices under conditions in which substantial changes in enzyme levels occur without significant accompanying changes in tissue volume, dry weight or cell number. This tissue slice system was therefore used for the work described in this thesis to examine the direct effects of plant hormones on enzyme synthesis in the absence of confounding effects on growth. Simultaneous synthesis of a second enzyme, peroxidase, also occurs in these tissue slices of sugar cane (Introduction 1.3) therefore providing an opportunity to examine the specificity of hormonal effects on enzyme synthesis. Whereas a considerable amount of information was already available on the properties of invertase and invertase regulation in sugar cane, similar information on peroxidase and its regulation was lacking. Preliminary studies were therefore carried out to characterize in greater detail the regulation of this enzyme both in intact tissue and in tissue slices of sugar cane. In order to draw reasonable conclusions about the specificity of hormone action it was desirable to be able to study regulation of several enzymes simultaneously. A search was therefore also proposed to find and characterize other enzymes synthesised in these tissue slices.
The major part of this thesis is concerned with determination of the point in the sequence of events leading t o enzyme synthesis at which individual plant growth hormones act. The techniques used were partly developed by previous workers and partly developed during the course of the work reported below. Use was made of two types of specific inhibitors, those which block DNA-directed RNA synthesis and those which block the peptide-bond-forming steps of protein synthesis. The approach relied on using these specific inhibitors in combination with known pretreatments of the tissue slices to make individual partial processes of enzyme synthesis rate limiting for enzyme accumulation under known experimental conditions. The ability of hormones to regulate enzyme accumulation with various different partial processes limiting was then measured. Earlier workers had developed techniques for studying regulation of enzyme breakdown, and for separating effects of hormones on the peptide-bond-forming steps of enzyme synthesis from effects on the processes concerned with the level of messenger RNA in the tissue. Experiments were carried out both to extend the above techniques and to develop new techniques to permit separate determination of the regulation of messenger RNA synthesis and messenger RNA decay. This included estimating for the first time in a higher plant, the half - life of functional messenger RNA specific for a particular enzyme or protein.
The compounds used as specific inhibitors of RNA or protein synthesis in these studies were chosen from the wide range of compounds known to interfere with RNA and protein synthesis in other systems. The basic assumption had to be made that such compounds acted in the same way in sugar cane as in the systems on which their specificity had been directly determined. Therefore only those compounds extensively tested in other systems including plant systems were chosen for sugar-cane studies. They were critically examined for side effects in sugar-cane tissue before use.
Members from all known groups of plant regulators were tested for their effects in tissue slice experiments, regardless of their ability to modify growth or enzyme synthesis in intact sugarcane stalks. All plant hormones with the exception of ethylene affected either invertase synthesis or peroxidase synthesis in sugar-cane tissue slices in relatively short term experiments. The mechanisms by which the hormones acted on enzyme synthesis in sugar cane were found without exception t o be on steps of protein synthesis subsequent to messenger RNA (m-RNA) synthesis. Auxin and gibberellic acid were shown to stimulate invertase synthesis in sugar cane by increasing the stability of the m-RNA specifically required for its synthesis. Auxin appeared to have the opposite effect on the stability of peroxidase m-RNA. Abscisic acid and cytokinins both appeared to regulate the peptide-bond-forming or subsequent steps of enzyme format- Ion.
Two main conclusions were drawn from these studies. First, it was concluded that the mechanisms of action of these hormones in sugar cane did not involve induction or repression of DNA-directed m-RNA synthesis in the manner proposed for regulation of enzyme synthesis in bacteria. Second, the mechanisms by which these hormones cause stabilisation of m-RNA or changes in the rates of peptide-bond-forming steps may be direct, or alternatively may involve the subcellular organisation of the cell.