Isolation of protoplasts from loblolly pine needles and their flow-cytometric analysis for air pollution effects

Abstract

Flow-cytometric analysis was used for the first time to determine the effects of air pollutants on plant biochemistry. The methods used were exploratory, but the results indicate that the technique is a valuable tool for researchers interested in the effects of air pollutants on vegetation. Evidence of carryover effects was seen in protoplasts isolated from new needles of 2-year-old loblolly pines ( Pinus taeda L.) which had been treated with ozone and simulated acidic rain the preceding year. Overall, staining of the protoplasts with constituent-specific fluorescent dyes showed decreased levels of fluorescence for esterase activity, protein, neutral lipids, and RNA in the ozone-treated samples compared with the charcoal-filtered or ambient samples. However, within pollutant treatments, low-level shifts in biochemistry were seen; for instance, simulated acidic rain at pH 5.0 ameliorated the effects of ozone on neutral lipids and RNA, and at pH 4.3 ameliorated the effects of ozone on protein. This research provides evidence at the biochemical level that pollutant treatments given in one year may be reflected in the succeeding year's growth. Image analysis of cross sections of fresh young pine needles supported the biochemical data.