James D. Metzger

Thermoinductive Regulation of Gibberellin Metabolism in Thlaspi arvense L. (II. Cold Induction of Enzymes in Gibberellin Biosynthesis).

Vernalization of Thlaspi arvense L. results in the alteration of gibberellin (GA) metabolism such that the metabolism and turnover of the GA precursor ent-kaur-16-en-19-oic acid (kaurenoic acid) is dramatically increased. This cold-induced change in GA metabolism is restricted to the shoot tip, the site of perception of cold in this species (J.P. Hazebroek, J.D. Metzger [1990] Plant Physiol 94: 157–165). In the present report additional biochemical information about the nature of this low-temperature-regulated process is provided. The endogenous levels of kaurenoic acid in leaves and shoot tips of plants were estimated by combined gas chromatography-chemical ionization mass spectrometry at various times after 4 weeks of vernalization at 6[deg]C. The endogenous levels in shoot tips declined 10-fold by 2 d after the plants were returned to 21[deg]C; this decline continued such that there was nearly 50-fold less kaurenoic acid by 10 d after the end of vernalization. No effect of vernalization on the endogenous levels of kaurenoic acid in leaves was observed. An in vitro enzyme assay was developed to monitor changes in the ability of tissues to convert kaurenoic acid to ent-7[alpha]-hydroxykaur-16-en-19-oic acid (7-OH kaurenoic acid). The activity of this enzyme rapidly increased in microsomal extracts from shoot tips following the end of vernalization. No thermoinduced increase in activity was observed in leaves. The enzymic oxidation of ent-kaurene to ent-kaurenol was also induced in shoot tips by vernalization. However, this reaction does not appear to be rate limiting for GA biosynthesis, because substantial amounts of kaurenoic acid accumulated in noninduced shoot tips. These results corroborate our hypothesis that the conversion of kaurenoic acid to 7-OH kaurenoic acid is the primary step in GA metabolism regulated by vernalization in Thlaspi shoot tips.

Gibberellin Content of Immature Apple Seeds from Paclobutrazol-Treated Trees over Three Seasons

Seeds from heavily fruiting (“on-year”), mature untreated, and paclobutrazol-treated apple trees (Malus domestica Borkh. cv. Spartan) were sampled in mid-June 1987, mid-July 1987, and mid-July 1990. After seeds were freeze-dried, gibberellins (GAs) were extracted, purified, and fractionated via C18 reversed-phase high-performance liquid chromatography (HPLC). Nine GAs (GA1, GA3, GA4, GA7, GA8, GA9, GA19, GA20, and GA53) were quantified by the use of deuterated GA internal standards. Paclobutrazol trunk drench treatments reduced vegetative shoot elongation in the seasons that seeds were sampled by 55% or more. Between June 17, 1987 and July 15, 1987, the dry weight of seeds from both untreated and treated trees increased about 2.5 times and there were reductions, on a per seed basis, of GA4 in seeds from both untreated and treated trees, of GA7 in seeds from treated trees, and of GA9 in seeds from untreated trees. However, GA9 increased in seeds from treated trees. Changes in levels of some of the early-13-hydroxylation pathway GAs (GA15 GA3, GA8, GA19, GA20, and GA53) also occurred during the month. For mid-July harvested seeds, the pattern, with some exceptions, was that 2 years after paclobutrazol treatment (1987), levels of early-13-hydroxylation pathway GAs in seeds from treated trees were lower compared to controls but after 5 years (1990) their levels tended to increase. For the non-13-hydroxylated GAs (GA4, GA7, and GA9), 2 years after paclobutrazol treatment, GA4 levels were equal in seeds from untreated and treated trees, GA7 decreased in seeds from treated trees compared with controls, but GA9 levels increased. Levels of these three GAs were higher in seeds from treated trees 5 years after treatment (1990) but levels of GA4, GA7, and GA9 dramatically increased in seeds from treated trees 4 years after treatment (1989), as we previously reported.

Effects of dimethipin, a defoliant and desiccant, on stomatal behavior and protein synthesis

The first visible macroscopic effect of a foliar spray of dimethipin (2,3-dihydro-5,6-dimethyl-1,4-dithiin 1,1,4,4-tetraoxide) on kidney beans (Phaseolus vulgaris L. cv. Back Valentine) was a severe loss of leaf turgor followed by desiccation and, ultimately, abscission. Dimethipin-treated leaves had higher rates of transpiration than control leaves when the leaves received treatments that cause stomatal closure (e.g., darkness, water stress, or exogenous abscisic acid). The higher rates of water loss from the dimethipin-treated leaves were not due to a massive nonspecific disruption of leaf cells, since dimethipin-treated leaves maintained turgor for 24 h if the plants were placed in a chamber of 100% relative humidity. These results indicate that the dimethipin-induced loss of leaf turgor is due, at least in part, to a loss in stomatal control.The earliest detectable biochemical effect of dimethipin was an inhibition of the incorporation of14C-leucine into protein. In both kidney bean leaf discs and oat (Avena sativa L. cv. Garry) coleoptiles, greater than 50% inhibition of14C-leucine incorporation into protein was observed 1 h after the start of incubation in 1 mM dimethipin. Dimethipin had a substantially smaller effect, however, on the incorporation of3H-uridine into RNA, suggesting that dimethipin acts primarily on the processes associated with translation rather than transcription. Cycloheximide also caused a loss of stomatal control, and both dimethipin and cycloheximide retarded the degradation of chlorophyll in senescing oat leaf segments in the dark, indicating similar mechanisms of action for the two compounds. In summary, the evidence suggests that an initial inhibition of protein synthesis is responsible for the loss of stomatal control associated with high rates of transpiration and loss of leaf turgor. The possible role for dimethipin-induced loss of turgor in abscission is discussed.

Cellular basis for dimethipin-induced loss of leaf turgor and desiccation

Dimethipin-induced increase in transpiration from kidney bean leaves (Phaseolus vulgaris L. cv. Black Valentine) was not correlated with stomatal aperture. From analysis of the kinetics of water loss from excised kidney bean leaves, it was concluded that the increase in transpiration was due almost entirely to an increase in the cuticular component. Both light and scanning electron microscopic analysis of dimethipin-treated leaves indicated that the first cells to be affected by dimethipin were the epidermal cells. These results suggest that dimethipin causes a loss of leaf turgor and desiccation by disrupting epidermal cells, thereby removing a major barrier for water loss from the leaf.

Modification of dimethipin action by light

White light reduced the efficacy of dimethipin in inducing both desiccation and abscission in kidney beans (Phaseolus vulgaris L. cv. Black Valentine). Moreover, light reduced the previously reported inhibitory effect of dimethipin on protein synthesis (Metzger and Keng 1984) in a way that paralleled the reduction in dimethipin-induced morphological changes. Therefore the inhibition of protein synthesis by dimethipin was the parameter measured in experiments designed to characterize the light-induced reduction of dimethipin efficacy. The light effect was directly proportional to both the fluence rate and the duration of the light treatment. A similar effect of light was observed in cultured kidney bean cells devoid of chlorophyll, ruling out the participation of a photosynthetic related process. Moreover, light had no effect on either the metabolism of [2,3]-14C-dimethipin in kidney bean leaves or uptake of dimethipin into cultured kidney bean cells. No evidence was obtained for photochemical decomposition of dimethipin either. Thus, the light effect is possibly the result of direct modification of the biochemical processes associated with the primary mechanism(s) of dimethipin action, or perhaps promotion of the rate of repair of dimethipin-induced cellular damage.