The Morphoregulatory Role of Thidiazuron: Metabolomics-Guided Hypothesis Generation for Mechanisms of Activity

Abstract

Thidiazuron (TDZ) is a diphenylurea synthetic herbicide and plant growth regulator used to defoliate cotton crops and to induce regeneration of recalcitrant species in plant tissue culture. In vitro cultures of African violet thin petiole sections are an ideal model system for studies of TDZ-induced morphogenesis. TDZ induces de novo shoot organogenesis at low concentrations and somatic embryogenesis at higher concentrations of exposure. We used an untargeted metabolomics approach to identify metabolites in control and TDZ-treated tissues. Statistical analysis including metabolite clustering, pattern and pathway tools, logical algorithms, synthetic biotransformations and hormonomics identified TDZ-induced changes in metabolism. A total of 18,602 putative metabolites with extracted masses and predicted formulae were identified with 1412 features that were found only in TDZ-treated tissues and 312 that increased in response to TDZ. The monomer of TDZ was not detected intact in the tissues but putative oligomers were found in the database and we hypothesize that these may form by a Diels-Alder reaction. Accumulation oligomers in the tissue may act as a reservoir, slowly releasing the active TDZ monomer over time. Cleavage of the amide bridge released TDZ-metabolites into the tissues including organic nitrogen and sulfur containing compounds. Metabolomics data analysis generated six novel hypotheses that can be summarized as an overall increase in uptake of sugars from the culture media, increase in primary metabolism, redirection of terpene metabolism and mediation of stress metabolism via indoleamine and phenylpropanoid metabolism. Further research into the specific mechanisms hypothesized is likely to unravel the mode of action of TDZ and to provide new insights into the control of plant morphogenesis.

Keywords: herbicide; mechanism of action; metabolomics; morphongenesis; phytohormone; plant growth and development; plant growth regulator; thidiazuron.

Figure 1

Thidiazuron (TDZ) metabolism in plant tissues. Orange is the TDZ parent. Blue represents features found in both low and high TDZ treatments. Green represents features found only in the low TDZ treatment. Black compounds are predicted but were not detected in the dataset. Dashed lines indicate predicted bonds consistent with the molecular features.

Figure 2

Relative expression of metabolite putatively identified as glutahione in African violet petioles treated with 0, 2 or 20 µM thidiazuron. Bars represent mean, with error bars represent standard error margins.

Figure 3

Effects of thidiazuron (TDZ) treatment on gibberellins (GA; (a,b)), jasmonates (12-oxo-phytodienoic acid; OPDA; (c,d)), abscisic acid (ABA; (e)) and tyrosine (Tyr; (f)) levels, identified by hormonomics analysis in African violet petioles. Bars represent mean, error bars span standard error margin. Where no error bars are displayed, the feature was present in only one replicate.

Figure 4

Effects of thidiazuron (TDZ) exposure on brassinosteroids (a) 28-norcastasterone, (b) epicastasterone or castasterone, (c) dolicholide and (d) teasterone or typhasterol levels in African violet petioles. Bars represent mean, error bars span standard error margin. Where no error bars are displayed, the feature was present in only one replicate.

Figure 5

Effects of thidiazuron (TDZ) exposure on tryptophan (Trp) and auxin (inole-3-acetic acid; IAA) metabolism in African violet petioles. NMN, nicotinic acid mononucleotide. Bars represent mean, error bars span standard error margin. Where no error bars are displayed, the feature was present in only one replicate. Arrows indicate the normal flow of the pathways. Yellow indicates the IAA biosynthetic pathway, purple Trp degradation pathways.

Figure 6

Effects of thidiazuron (TDZ) exposure on indoleamine biosynthesis and metabolism in African violet petioles. Bars represent mean, error bars span standard error margin. Where no error bars are displayed, the feature was present in only one replicate. Arrows indicate the normal flow of the pathways.

Figure 7

Effects of thidiazuron (TDZ) exposure on proposed indoleamine (serotonin, 5HT and melatonin, MEL) amino acid conjugates in African violet petioles. Purple denotes 5HT conjugates (a–f), blue MEL conjugates (g–i). Bars represent mean, error bars span standard error margin. Where no error bars are displayed, the feature was present in only one replicate.

Figure 8

Effects of thidiazuron (TDZ) exposure on proposed indoleamine (serotonin (5HT) and melatonin (MEL)) phenolic conjugates in African violet petioles. Purple denotes 5HT conjugates (a), blue MEL conjugates (b–d). Bars represent mean, error bars span standard error margin. Where no error bars are displayed, the feature was present in only one replicate.

Figure 9

Thidiazuron (TDZ) mediation of glycolysis and shikimic acid derived metabolism. 5-HT, serotonin; ABA, abscisic acid; CS, coumaroylserotonin; DMAPP, dimethylallyl diphosphate; FPP, farnesyl diphosphate; GA, gibberellic acid; GGPP, geranyl geraniol diphosphate; IPP, isopentenyl diphosphate; MEL, melatonin; NMN, nicotinic acid mononucleotide; PPP, pentose phosphate pathway. Figure created in BioRender.

Figure 10

Proposed model for thidiazuron-induced morphogenesis in African violet. Figure created in BioRender.