0 1 . 00 / 0 Gibberellins and Light Regulated Petiole Growth in Thlaspi arvense

Petiole growth in Thlaspi arvense L. was stimulated when a basic 8 hour photoperiod (4.20 milliwatts per square centimeter) was extended with low intensity light (0.16 milliwatt per square centimeter) from incandescent lamps. The day length extension was effective only when the light contained high proportions of far red light. Exogenous gibberellin A3 (GA3) could partially substitute for the promotive effect of the extended photoperiod. Moreover, the GA biosynthesis inhibitor 2-chlorocholine chloride inhibited the increase in petiole growth induced by the extended photoperiod. However, evidence was obtained indicating that gibberellins do not mediate the effect of the extended photoperiod. First, petiole growth was greater in plants receiving both exogenous GA3 and a day length extension than the sum of the effects of the two treatments alone. Second, petioles were sensitive to exogenous GA3 only during the early stages of leaf development, whereas mature (but not senescent) leaves continued to respond to an extension of the photoperiod. Third, the cellular basis for growth induced by extending the photoperiod was different from that observed with GA3. It was concluded that light and gibberellins are both important in the overall regulation of petiole growth, but act through independent mechanisms. Field pennycress (Thlaspi arvense L.) is a winter annual weed that has a cold requirement for stem elongation and flowering. Work in this laboratory has been focused on the role of GAs' in the environmental control of stem elongation in this species (8, 9). During the course of these studies we observed that photoperiod exerts a strong influence on leaf growth, particularly petiole length. Petioles from plants that had been transferred to LD were longer than from plants maintained under SD. Furthermore, application of GA3 to plants in SD caused petioles to elongate, thereby mimicking the effect ofLD. These observations suggest that GAs may mediate the observed LD-induced increase in petiole growth. Indeed, evidence for a role of GAs in this phenomenon in spinach has been published (21). Thus, in the present paper we report the results of experiments with two objectives in mind: first, to characterize more completely the role of light in the regulation of petiole growth in field pennycress; and second, to test the hypothesis that GAs mediate LD-induced petiole growth. MATERIALS AND METHODS Plant Material. Field pennycress seeds were germinated in Petri dishes and 7-d-old seedlings transferred to 10 cm plastic ' Abbreviations: GA(s), gibberellin(s); RL, red light; FRL, far red light; CCC, 2-chlorocholine chloride. pots filled with vermiculite. The pots were continuously subirrigated with one-quarter strength Hoagland solution (1). The plants were grown under SD conditions at 21°C until they were ready for use, approximately 6 weeks after germination. Light Treatments. SD treatments consisted of 8 h of light from fluorescent and incandescent lamps (4.20 mW cm-2) followed by 16 h of darkness. LD treatments were composed of the same basic 8 h photoperiod as SD followed by 16 h of low intensity illumination from incandescent lamps (0.16 mW cm-2). The spectral dependency of the LD response was investigated by subjecting plants to 16 h of low intensity light enriched with either RL or FRL following the basic 8 h high intensity photoperiod. The RL treatments were achieved by filtering light from eight 20 W fluorescent lamps (Gro-lux,2 GTE Products, Inc., St. Marys, PA) through a CBS-Red 650 absorbtion filter (Carolina Biological Supply, Burlington, NC) and 10 cm ofdeionized H20. FRL was similarly obtained by filtering light from four 40 W incandescent appliance lamps through a CBS-Far red 750 filter and 10 cm of deionized H20. Since a spectroradiometer was not available, light levels were determined daily with a YSI model 65 radiometer (YSI-Kettering, Yellow Springs, OH). Similar energy levels (approximately 0.16 mW cm-2) impinging on the tops of all plants were obtained by adjusting plant height appropriately. Application of Chemicals. Leaves were treated with GA3 by applying 10 Iul of a solution containing 10 Mg of GA3, 10% (v/v) acetone, and 0.05% (v/v) Tween 20 to blades of leaves on alternate days. GA biosynthesis was inhibited by supplying CCC to roots in subirrigation solution at a concentration of 1 mm (8). Treatment with CCC was initiated 3 d prior to the start of an experiment. Growth Measurements. At the start of an experiment, leaves with petioles 5 to 10 mm long were marked with a small spot of indelible ink. Petiole length was measured daily with a ruler at the end of the high intensity portion of the photoperiod. Anatomical Observations. The lengths of the pith cells in longitudinal sections of petioles were determined using a microscope equipped with an eye piece micrometer. Following a light treatment, petioles were excised, and fixed in a mixture of formalin:ethanol:acetic acid:H20 (10:50:5:35). The fixed tissues were dehydrated in a graded series of t-butanol in H20 and then embedded in paraffin (6). Longitudinal sections, 8 Mrm thick, were cut, stained with safranin and fast green (6). The average cell length in each section was determined from 75 randomly chosen pith cells. Three petioles from each treatment were analyzed with similar results. 2 Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by the United States Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable. 237 www.plantphysiol.org on July 22, 2017 Published by Downloaded from Copyright © 1988 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 86, 1988 The number of cells per mm of petiole was determined by the method described by Van Volkenburgh and Cleland (18). Briefly, each petiole was placed in a test tube containing 10 ml of 5% (v/v) chromic acid for 2 d at room temperature. The petioles were broken into smaller pieces by vigorous agitation with a Vortex Genie (Fischer Scientific, Pittsburgh, PA). Individual cells were than liberated from the pieces by triterating with a Pasteur pipet. One ml of the cell suspension was diluted with 2 ml H20 and the number of cells determined with a haemocytometer. The number of cells permm was then calculated by dividing the total number of cells in each petiole by its length. Petioles from all treatments had similar cross-sectional areas so valid comparisons could be made. Each treatment had five replicates.