Modulation of root branching by a coumarin derivative

Abstract

A healthy root system is crucial to plant growth and survival. To maintain efficiency of root function, plants have to dynamically modulate root system architecture through various adaptive mechanisms such as lateral root formation to respond to a changing and diversified soil environment. Exogenous application of a coumarin derivative, 4-methylumbelliferone (4-MU), in Arabidopsis thaliana inhibits seed germination by mainly reducing primary root growth. UDP-glycosyltransferases play an integral role in the biochemical mechanism of 4-MU detoxification in plant roots.1 However, 4-MU treatment also dramatically led to increased lateral root initiation, elongation and density. Moreover, marked root bending at the root-hypocotyl junction and auxin redistribution appeared to contribute to the 4-MU-mediated lateral root formation. We propose that 4-MU would serve as a useful chemical tool to study auxin-mediated root branching.

Plant roots are required for the acquisition of water and nutrients, for response to abiotic and biotic factors in the soil, and to anchor the plant in the ground.² To maintain efficiency of root function, plants have to dynamically modulate root system architecture (RSA) by regulation of primary root growth, lateral root (LR) formation and elongation and root hair increase.² Recent studies on root patterning have made significant progress toward understanding the molecular and physiological basis of RSA.³ For example, auxin synthesis, transport and distribution are required for LR initiation and primordium development.² However, determination of the underlying RSA patterning mechanism remains to be elucidated.

Coumarins are a group of natural products in plants that originate from the general phenylpropanoid pathway.⁴ They are often found to accumulate in the root tissues⁵ and are involved in plant defense, root development and nitrogen uptake and metabolism.^1,6-8 Some coumarins also receive attention for their pharmacological properties. For example, 4-MU is a potent apoptotic agent with strong anti-invasive and antiangiogenic properties against prostate cancer cells.⁹

We have demonstrated that exogenous 4-MU was accumulated in the root system in a concentration-dependent manner. After continuous exposure to 4-MU, growth of the primary roots exhibited a dosage-dependent inhibition of root length, whereas the growth of cotyledon and hypocotyls was not significantly changed. Moreover, 4-MU was found to be glycosylated to 4-methylumbelliferyl-β-D-glucoside (4-MU-Glc) by UDP-glycosyltransferases (UGTs) for detoxification.¹ Here we report that marked bending of the primary roots and auxin redistribution in root system contributes to 4-MU-induced root branching. After exposure to 125 µM 4-MU for 6 d, the primary root length was reduced by 25% compared with the untreated seedlings, but the first LR emerged at the root-hypocotyl junction 3 d earlier in the Arabidopsis DR5::GUS lines compared with untreated seedlings. The GUS activity and distribution in the primary roots of DR5::GUS seedlings were coordinately regulated in response to 4-MU treatment (Fig. 1A-D). Interestingly, primary root shape was also affected upon 4-MU treatment as evidenced by marked bending of the primary roots followed by emergence of lateral roots at the root-hypocotyl junctions. As the roots grew, the bend continued to develop and a hook formed at the root-hypocotyl junction (Fig. 1F). After exposure to 125 μM 4-MU for 22 d, abundant lateral roots formed from the bent region (Fig. 1F). We also observed that auxin accumulation in the bent region was significantly reduced after root branching was well established, compared with the untreated plants (Fig. 1E and F). It has been demonstrated that LR formation can be induced mechanically by either gravitropic curvature or by transient bending.^10,11 We suggest that 4-MU-induced LR proliferation is triggered by both mechanical bending of the primary roots at the root-hypocotyl junctions and the local auxin redistribution.

Figure 1.

Changes of auxin distribution in response to 4-MU as observed using DR5::GUS reporter fusion. (A) Auxin accumulation in root-hypocotyl junction after exposure to 125 µM 4-MU for 6 d. (B-D) Detection of 4-MU accumulation in root under UV (325 nm). (B) Brightfield; (C) UV channel (325 nm); (D) Merge of (B) and (C). (E) An untreated root system of 22-d-old DR5::GUS seedling. (F) A root system of 22-d-old DR5::GUS seedling in the presence of 125 µM 4-MU. Asterisks indicate the localization of auxin accumulation. It was noted that LR formation upon 4-MU treatment was closely associated with auxin distribution and 4-MU accumulation in roots.

Our finding of 4-MU-dependent root patterning is intriguing in light of the important role of RSA in plant physiology. Given that LR initiation is stimulated by 4-MU and that this compound is effectively detoxified in plant roots by glycosylation, a new way to augment root function could be provided through applying 4-MU to modulate RSA. In addition, 4-MU could serve as a useful chemical tool for understanding auxin-mediated root branching, for example, by screening Arabidopsis mutants in the presence of this compound.

Coumarins synthesis from phenylpropanoid precursors occurs with an especially high number of structural variations in higher plants via numerous possible modifications at specific positions of the benzene ring.^4,5 For example, hydroxylation of coumarins at 6-position catalyzed by a 2-oxoglutarate-dependant dioxygenase (F6'H1) is important for the biosynthesis of scopoletin.¹² Coumarin synthesis in Arabidopsis plants can result in the accumulation of umbelliferone and its derivative skimmin but not 4-MU⁵ in which 4-MU possesses a pivotal methyl group at the 4-position of the benzene ring. Our results suggest that 4-MU uptake does not benefit plant growth as it is a phytotoxic compound found to inhibit primary root growth and seed germination. This finding explains why Arabidopsis plants do not naturally accumulate 4-MU and its derivatives. Nevertheless, 4-MU has been found and isolated from other higher plants such as Dalbergia volubilis and Eupatorium pauciflorum, indicating the existence of a biosynthetic pathway leading to the formation of 4-MU in nature.¹³