Hplc and in vivo spectrophotometric detection of porphyrins in plant tissues treated with porphyrinogenic herbicides

Abstract

Protoporphyrinogen oxidase (Protox) inhibitors and other compounds which block or stimulate the porphyrin pathway can cause sufficient levels of porphyrins to accumulate in plant tissues for severe photodynamic damage to occur. The gross symptomology for all of these porphyrinogenic herbicides is similar. Porphyrin accumulation induced by three porphyrinogenic herbicides acifluorfen (AF), δ-aminolevulinic acid (ALA), and 2,2′-dipyridyl (DY) was determined by in vivo spectrophotom etry and HPLC methods. The averaged in vivo difference spectra between untreated and AF-treated (30 μm for 20 h in darkness) yellow cucumber cotyledon discs approximated the absorption spectra of protoporphyrin IX (Proto IX). There was also an enhanced peaknear 503 nm. Treatment of cotyledon discs with ALA alone generated a difference spectrum of protochlorophyllide (PChlide) in combination with Mg-Proto IX or Mg-Proto IX monom ethyl ester (Mg-Proto IX ME). With ALA and AF in combination, the PChlide and Mg-Proto IX portions of the difference spectrum were reduced and the Proto IX peak and peak near 503 nm were increased. DY treatment yielded a difference spectrum with peaks approximating those of Proto IX and Mg-Proto IX ME, along with a peak near 503 nm. The presence of all porphyrins detected by in vivo spectrophotometry except for the 503 nm peak was confirmed with HPLC. Proto IX monom ethyl ester was found by HPLC to be especially elevated in treatments with AF. The in vivo 503 nm peak and in vitro studies with Protoxcontaining barley etioplast preparations suggest that prototetrahydroporphyrin IX (an oxidation state interm ediate between protoporphyrinogen IX and Proto IX) may accumulate under some conditions. These data demonstrate that rapid in vivo spectrophotometric studies can provide much of the qualitative results of HPLC studies and can confirm that in vitro results correspond with the in vivo situation. © 1993, Walter de Gruyter. All rights reserved.