Phytochrome Regulation of Greening in Barley-effects on Chlorophyll Accumulation

Red light treatment of dark-grown 6-day-old barley seedlings (Hordeum vulgare L.) strongly reduces the lag in chlorophyll accumulation in subsequent white light over that found in dark control seedlings placed under white light. Fluence-response studies show that the effect has both very low fluence and low fluence components. Kinetic studies indicate that the reduction in lag begins immediately following either a low fluence or a very low fluence red irradiation, with the initial rate of change significantly lower after the very low fluence treatment and showing sharp far red-absorbing form of phytochrome dependence. In both cases, the effect is maximal after roughly 4 hours, either remaining fairly constant (very low fluence) or declining somewhat (low fluence) thereafter. Saturating far red light alone yields a response equivalent to very low fluence red, and will reverse only the low fluence component of the red response. Escape from far red reversibility occurs gradually over about a 3 hour period. Since the kinetics described here differ from those in the literature related to phytochrome effects on transcription of the mRNA for the light-harvesting chlorophyll a/b-binding protein, we conclude that the phytochrome-regulated component of chlorophyll accumulation is not limited by transcription of the mRNA for its major apoprotein. Leaf segments vacuum-infiltrated with water retain the capacity to green in white light. If they are infiltrated with mannitol solutions of various concentrations, their capacity to green declines sharply at concentrations above 0.2 molar. These results bear on interpretation of run-on transcription experiments with isolated nuclei: preparation of the nuclei involves enzymic digestion of the tissue in the presence of 0.7 molar mannitol for 2.5 hours, to obtain protoplasts prior to breaking the cells. The results here make it unlikely that normal transcriptional regulation is occurring during this procedure.