Three genes encoding flavonoid 3'-hydroxylase (F3'H) in apple (Malus x domestica), designated MdF3'HI, MdF3'HIIa, and MdF3'HIIb, have been identified. MdF3'HIIa and MdF3'HIIb are almost identical in amino acid sequences, and they are allelic, whereas MdF3'HI has 91% nucleotide sequence identity in the coding region to both MdF3'HIIa and MdF3'HIIb. MdF3'HI and MdF3'HII genes are mapped onto linkage groups 14 and 6, respectively, of the apple genome. Throughout the development of apple fruit, transcriptional levels of MdF3'H genes along with other anthocyanin biosynthesis genes are higher in the red-skinned cv Red Delicious than that in the yellow-skinned cv Golden Delicious. Moreover, patterns of MdF3'H gene expression correspond to accumulation patterns of flavonoids in apple fruit. These findings suggest that MdF3'H genes are coordinately expressed with other genes in the anthocyanin biosynthetic pathway in apple. The functionality of these apple F3'H genes has been demonstrated via their ectopic expression in both the Arabidopsis (Arabidopsis thaliana) transparent testa7-1 (tt7) mutant and tobacco (Nicotiana tabacum). When grown under nitrogen-deficient conditions, transgenic Arabidopsis tt7 seedlings expressing apple F3'H regained red color pigmentation and significantly accumulated both 4'-hydrylated pelargonidin and 3',4'-hydrylated cyanidin. When compared with wild-type plants, flowers of transgenic tobacco lines overexpressing apple F3'H genes exhibited enhanced red color pigmentation. This suggests that the F3'H enzyme may coordinately interact with other flavonoid enzymes in the anthocyanin biosynthesis pathway.

To date, the only known mechanism conferring protoporphyrinogen IX oxidase (PPO)-inhibitor resistance in waterhemp ( Amaranthus tuberculatus ) is a glycine deletion in PPO2 (ΔG210), which results in cross-resistance to foliar PPO-inhibiting herbicides. However, a metabolism-based, HPPD-inhibitor resistant waterhemp population from Illinois (named SIR) was suspected of having a non-target site resistance (NTSR) mechanism due to its resistance to carfentrazone-ethyl (CE) but sensitivity to diphenylethers (DPEs). In greenhouse experiments, SIR sustained less injury than two PPO inhibitor-sensitive populations (WCS and SEN) after applying a field-use rate of CE, and after initial rapid necrosis, regrowth of SIR plants was comparable to a known PPO inhibitor-resistant population (ACR) possessing the ΔG210 mutation. Dose-response analysis determined 50% growth reduction rates in CE-resistant (SIR and ACR) and sensitive (SEN) waterhemp populations, which showed SIR was 30-fold resistant compared to SEN and two-fold more resistant than ACR. Deduced amino acid sequences derived from SIR PPX2 partial cDNAs did not contain the ΔG210 mutation found in ACR or other target-site mutations that confer PPO-inhibitor resistance previously reported in Palmer amaranth ( Amaranthus palmeri ). Although several SIR cDNAs contained amino acid substitutions, none were uniform among samples. Additionally, SIR plants treated with malathion and CE showed a significant reduction in biomass accumulation compared to CE alone. These results indicate robust CE resistance in SIR is not mediated by amino acid changes in the PPO2 protein, but instead resistance may be conferred through a NTSR mechanism such as enhanced herbicide metabolism.

Waterhemp [Amaranthus tuberculatus (Moq.) Sauer] is a problematic dicot weed in maize, soybean, and cotton production in the United States. Waterhemp has evolved resistance to several commercial herbicides that inhibit the 4-hydroxyphenylpyruvate-dioxygenase (HPPD) enzyme in sensitive dicots, and research to date has shown that HPPD-inhibitor resistance is conferred by rapid oxidative metabolism of the parent compound in resistant populations. Mesotrione and tembotrione (both triketones) have been used exclusively to study HPPD-inhibitor resistance mechanisms in waterhemp and a related species, A. palmeri (S. Wats.), but the commercial HPPD inhibitor topramezone (a pyrazolone) has not been investigated from a mechanistic standpoint despite numerous reports of cross-resistance in the field and greenhouse. The first objective of our research was to determine if two multiple herbicide-resistant (MHR) waterhemp populations (named NEB and SIR) metabolize topramezone more rapidly than two HPPD inhibitor-sensitive waterhemp populations (named SEN and ACR). Our second objective was to determine if initial topramezone metabolite(s) detected in MHR waterhemp are qualitatively different than those formed in maize. An excised leaf assay and whole-plant study investigated initial rates of topramezone metabolism (<24 h) and identified topramezone metabolites at 48 hours after treatment (HAT), respectively, in the four waterhemp populations and maize. Results indicated both MHR waterhemp populations metabolized more topramezone than the sensitive (SEN) population at 6 HAT, while only the SIR population metabolized more topramezone than SEN at 24 HAT. Maize metabolized more topramezone than any waterhemp population at each time point examined. LC-MS analysis of topramezone metabolites at 48 HAT showed maize primarily formed desmethyl and benzoic acid metabolites, as expected based on published reports, whereas SIR formed two putative hydroxylated metabolites. Subsequent LC-MS/MS analyses identified both hydroxytopramezone metabolites in SIR as different hydroxylation products of the isoxazole ring, which were also present in maize 48 HAT but at very low levels. These results indicate that SIR initially metabolizes and detoxifies topramezone in a different manner than tolerant maize.

Summary Metabolic resistance to 4‐hydroxyphenylpyruvate dioxygenase (HPPD)‐inhibiting herbicides is a threat in controlling waterhemp (Amaranthus tuberculatus) in the USA. We investigated resistance mechanisms to syncarpic acid‐3 (SA3), a nonselective, noncommercial HPPD‐inhibiting herbicide metabolically robust to Phase I oxidation, in multiple‐herbicide‐resistant (MHR) waterhemp populations (SIR and NEB) and HPPD inhibitor‐sensitive populations (ACR and SEN). Dose–response experiments with SA3 provided ED50‐based resistant : sensitive ratios of at least 18‐fold. Metabolism experiments quantifying parent SA3 remaining in excised leaves during a time course indicated MHR populations displayed faster rates of SA3 metabolism compared to HPPD inhibitor‐sensitive populations. SA3 metabolites were identified via LC‐MS‐based untargeted metabolomics in whole plants. A Phase I metabolite, likely generated by cytochrome P450‐mediated alkyl hydroxylation, was detected but was not associated with resistance. A Phase I metabolite consistent with ketone reduction followed by water elimination was detected, creating a putative α,β‐unsaturated carbonyl resembling a Michael acceptor site. A Phase II glutathione–SA3 conjugate was associated with resistance. Our results revealed a novel reduction–dehydration–GSH conjugation detoxification mechanism. SA3 metabolism in MHR waterhemp is thus atypical compared to commercial HPPD‐inhibiting herbicides. This previously uncharacterized detoxification mechanism presents a unique opportunity for future biorational design by blocking known sites of herbicide metabolism in weeds.