HPLC and in vivo Spectrophotometric Detection of Porphyrins in Plant Tissues Treated with Porphyrinogenic Herbicides

Protoporphyrinogen oxidase (Protox) inhibitors and other com pounds which block o r stim­ ulate the porphyrin pathway can cause sufficient levels o f porphyrins to accum ulate in plant tissues for severe photodynam ic dam age to occur. The gross sym ptom ology for all o f these p o r­ phyrinogenic herbicides is similar. Porphyrin accum ulation induced by three porphyrinogenic herbicides acifluorfen (AF), 5-aminolevulinic acid (ALA), and 2,2'-dipyridyl (D Y ) was deter­ mined by in vivo spectrophotom etry and H PLC m ethods. The averaged in vivo difference spec­ tra between untreated and A F-treated (30 hm for 20 h in darkness) yellow cucum ber cotyledon discs approxim ated the absorption spectra o f p ro toporphyrin IX (Proto IX). There was also an enhanced peak near 503 nm. Treatm ent o f cotyledon discs with A LA alone generated a differ­ ence spectrum o f protochlorophyllide (PChlide) in com bination with M g-Proto IX or M g-Proto IX m onom ethyl ester (M g-Proto IX ME). W ith ALA and A F in com bination, the PChlide and M g-Proto IX portions o f the difference spectrum were reduced and the Proto IX peak and peak near 503 nm were increased. DY treatm ent yielded a difference spectrum with peaks approxim ating those o f Proto IX and M g-Proto IX M E, along with a peak near 503 nm. The presence o f all porphyrins detected by in vivo spectrophotom etry except for the 503 nm peak was confirm ed with H PLC. Proto IX monom ethyl ester was found by H PLC to be espe­ cially elevated in treatm ents with AF. The in vivo 503 nm peak and in vitro studies with Protoxcontain ing barley etioplast preparations suggest that proto tetrahydroporphyrin IX (an oxida­ tion state interm ediate between protoporphyrinogen IX and Proto IX) may accum ulate under some conditions. These data dem onstrate that rapid in vivo spectrophotom etric studies can provide m uch o f the qualitative results o f H PLC studies and can confirm that in vitro results correspond with the in vivo situation.