Localization of the production of 1-aminocyclopropane-1-carboxylic acid and its conversion to ethylene during the rhythmic production of the gas in Sorghum bicolor seedlings

Abstract

Studies were conducted to determine where in the plant 1-aminocyclopropane-1-carboxylic acid (ACC) is made and converted to ethylene in Sorghum bicolor seedlings producing the gas in circadian rhythms. For the first time, a natural enzyme was used to convert ACC to ethylene for assay by gas chromatography. Parameters like Km and Vmax of ACC oxidase in the standard assay were well complemented in more rigorous Eddie Hofstee and Lineweaver-Burk plots. A conversion formula was derived allowing use of constants and variable product to calculate the unknown ACC concentration. The new method proved far simpler and efficient than the NaOCl and Hg⁺² method by eliminating slow purification steps and interference. Measuring rates of ethylene production, free and conjugated ACC contents from detached as well as intact plant organs tested the role of roots in rhythmic ethylene synthesis. Variation in ethylene synthesis in the phytochrome B mutant and its wild-type cultivar under different light regimes suggested that phytochrome regulates ethylene synthesis. Occurrence of rhythmic phenomena in detached shoots made it clear that regulation of ethylene synthesis takes place in shoots. Pulses of ethylene may be signaling the plant as the "hands" of the biological clock and may have some role in the phenotype of the mutant cultivar. Detachment did not create a significant difference in ACC levels between intact and detached shoots of either cultivar. Especially, ACC levels did not decrease in detached shoots as would be expected if their ACC originated to a major degree in roots. Similar results were found in roots, suggesting that adequate amounts of ACC to convert into ethylene are present in detached shoots. Ethylene rhythms may be controlled by a self-inhibitory mechanism taking place at the time of ACC synthesis. Conjugation of ACC (MACC) may be indirectly regulating ethylene synthesis by preventing excessive accumulation of ACC in roots and shoots. Probing of mRNA isolated during a time course with be353050 transcript gave two putative ACC synthase genes, tentatively named sbACS2 and sbACS3. Flooding and injury treatments did not induce statement of either of the transcripts. sbACS3 is expressed constitutively whereas sbACS2 is diurnally expressed. The combined effect of both the transcripts may have resulted in diurnal but weak rhythms of ACC in shoots.