Determination of the Cellular Mechanisms Regulating Thermo-induced Stem Growth in Thiaspi

Field pennycress (Thiaspi arvense L.) is a species with a cold requirement for the initiation of reproductive development (thermoinduction). Work in this laboratory has been focused on elucidating the biochemical and molecular mechanisms underlying the bolting or rapid stem elongation response that is an intricate part of reproductive development in this species. In the present paper the cellular basis for thermo-induced stem growth was determined. Evidence is presented indicating that bolting results from the production of new cells that elongate to their original length before thermoinduction. This increase in cell division occurs in the pith and cortex approximately 0.5 to 5.0 millimeters below the stem apex. For at least the early stages of thermoinduced stem growth, enhanced cell elongation does not appear to be a factor because average lengths of pith cells from stems of thermo-induced plants were similar or less than noninduced controls. In addition, both the amount of increase in the production of new pith cells and stem growth were positively correlated with the length of the cold treatment. Two other lines of evidence are presented corroborating previous assertions (JD Metzger [1985] Plant Physiol 78: 8-13) that gibberellins mediate thermo-induced stem growth in field pennycress. First, treatment of noninduced plants with gibberellin A3 completely mimicked the effects of a 4week cold treatment on mitotic activity in the pith and cortex. Second, very little increase in the production of new cells was observed in the pith and cortex of thermo-induced plants of a gibberellin-deficient dwarf mutant of field pennycress. It is also shown that the influence of photoperiod on stem growth is mediated by an effect on the final length that cells ultimately attain. It is well known that environmental factors play an important role in the regulation of of stem growth. Some of the most dramatic responses to environmental stimuli occur in rosette plants in which internode expansion is minimal until the plants are subjected to changes in specific environmental conditions such as daylength or temperature. The inductive stimuli trigger a sequence of events that result in rapid stem growth (bolting). At least initially, the increase in stem growth is the result of expansion of internodes that existed before the inductive treatment. The bolting response is an integral part of reproductive development and is closely associated with floral initiation (15, 26). In many instances, GAs' are an obligate part of the signal transduction pathway leading to bolting, and although the ' Abbreviation: GA, gibberellin. molecular details of GA involvement are not yet clear, it seems likely that alterations in GA biosynthesis and metabolism induced by the stimulus have an important role (15, 26). In spinach, for example, rapid stem growth resulting from a lengthening of the photoperiod is associated with dramatic changes in endogenous GA levels and metabolism (3, 20). Moreover, certain enzymes in the GA biosynthetic pathway of this species have been shown to be light regulated (4, 5). Our work in this area has centered on elucidating the biochemical and molecular events that underlie thermoor cold-induced stem elongation in field pennycress (Thlaspi arvense L.). This species has a requirement for a period of low temperature (0-1 5°C) before stem elongation is initiated (14). Previous results demonstrated that thermo-induced stem growth in field pennycress is mediated by GAs (14). More recently, we (7) showed that metabolism of the GA precursor, ent-kaurenoic acid is under thermoinductive regulation in the shoot tips, the site of perception of low temperatures (17), but not in the leaves. We further hypothesized that increased conversion of this compound to GAs following thermoinduction forms the biochemical basis for thermo-induced stem growth in field pennycress (7, 18). However, the correlation between specific changes in GA metabolism and macroscopically measurable increases in stem growth is not a straightforward indication of a cause and effect relationship because stem growth is the product of cell elongation and the production of new cells. Thus, the assignment of a causal role for a specific alteration in GA metabolism could be erroneous if it occurred between the onset of measurable stem growth and the cellular events that form the basis of this process. The purpose of the research described in this paper was to determine the cellular basis for thermoinduced stem growth in field pennycress and to compare the timing of cellular changes with other thermo-induced processes. MATERIALS AND METHODS