Molecular Characterization of Photomixotrophic Tobacco Cells Resistant to Protoporphyrinogen Oxidase-Inhibiting Herbicides

Abstract

Peroxidizing herbicides inhibit protoporphyrinogen oxidase (Pro-tox

Dual Targeting of Spinach Protoporphyrinogen Oxidase II to Mitochondria and Chloroplasts by Alternative Use of Two In-frame Initiation Codons

“…Tobacco Mitochondrial Protox Homolog-To isolate cDNA encoding the spinach homolog of tobacco mitochondrial Protox, one set of specific oligonucleotide primers (SMT-1F and SMT-1R) was synthesized based on the PPX-II sequence from N. tabacum (tobacco) (13,21). PCR amplification of cDNA from spinach produced a 565-bp product.…”

“…Several lines of data provide strong evidence that the herbicide resistance of YZI-1S cells is due to the overproduction of mitochondrial Protox, even though the primary target of the herbicide is chloroplast Protox. In the resistant cells, excess Protogen generated by inhibition of chloroplast Protox is rapidly utilized for heme synthesis in mitochondria by the abnormally high level of mitochondrial Protox, thus preventing the accumulation of Proto IX (21). This suggests that mitochondrial Protox is also important in preventing the accumulation of the photosensitizer, Proto IX, and in maintaining the flow of tetrapyrrole precursors for heme and chlorophyll biosynthesis.…”

“…First-strand cDNA was synthesized from total RNA using a ReadyTo-Go T-primed first-strand kit (Amersham Pharmacia Biotech). For polymerase chain reaction (PCR) isolation of mitochondrial Protox cDNA, one set of oligonucleotide primers was synthesized on the basis of the nucleotide sequences of tobacco PPX-II cDNA (13,21). The primer sequences were as follows: 5Ј-GAAGG(A/G)GCAAA(C/T)AC(C/T)AT-GACT-3Ј (SMT-1F) and 5Ј-ATCAGGAAG(A/T)(A/G)T(C/T)TGCAT-TCC-3Ј (SMT-1R).…”

Resistance to Protoporphyrinogen Oxidase-inhibiting Compound S23142 from Overproduction of Mitochondrial Protoporphyrinogen Oxidase by Gene Amplification in Photomixotrophic Tobacco Cells

“…19) An analysis of the Protox cDNA sequences in YZI-1S and wild-type cells shown that there was no mutation in either the plastidal or mitochondrial Protox-encoding gene in YZI-1S cells. 20) A northern blot analysis has revealed that the expression level of plastid Protox mRNA was almost the same in both wild-type and YZI-1S cells, whereas mitochondrial Protox mRNA was expressed by up to 10 times more in YZI-1S cells. These data led us to speculate that the S23142 resistance of YZI-1S cells was due to the overproduction of mitochondrial Protox, even though the primary target of S23142 was plastidal Protox.…”

“…These data led us to speculate that the S23142 resistance of YZI-1S cells was due to the overproduction of mitochondrial Protox, even though the primary target of S23142 was plastidal Protox. 20) However, it remained unknown whether the mitochondrial Protox protein levels and W or activity were actually increased in YZI-1S cells, and whether this was truly responsible for S23142 resistance. Furthermore, the mechanism by which mitochondrial Protox was overexpressed also remained unresolved.…”