Phenyl-adenine, identified in a LIGHT-DEPENDENT SHORT HYPOCOTYLS4-assisted chemical screen, is a potent compound for shoot regeneration through the inhibition of CYTOKININ OXIDASE/DEHYDROGENASE activity

Abstract

In vitro shoot regeneration is implemented in basic plant research and commercial plant production, but for some plant species, it is still difficult to achieve by means of the currently available cytokinins and auxins. To identify novel compounds that promote shoot regeneration, we screened a library of 10,000 small molecules. The bioassay consisted of a two-step regeneration protocol adjusted and optimized for high-throughput manipulations of root explants of Arabidopsis (Arabidopsis thaliana) carrying the shoot regeneration marker LIGHT-DEPENDENT SHORT HYPOCOTYLS4. The screen revealed a single compound, the cytokinin-like phenyl-adenine (Phe-Ade), as a potent inducer of adventitious shoots. Although Phe-Ade triggered diverse cytokinin-dependent phenotypical responses, it did not inhibit shoot growth and was not cytotoxic at high concentrations. Transcript profiling of cytokinin-related genes revealed that Phe-Ade treatment established a typical cytokinin response. Moreover, Phe-Ade activated the cytokinin receptors ARABIDOPSIS HISTIDINE KINASE3 and ARABIDOPSIS HISTIDINE KINASE4 in a bacterial receptor assay, albeit at relatively high concentrations, illustrating that it exerts genuine but weak cytokinin activity. In addition, we demonstrated that Phe-Ade is a strong competitive inhibitor of CYTOKININ OXIDASE/DEHYDROGENASE enzymes, leading to an accumulation of endogenous cytokinins. Collectively, Phe-Ade exhibits a dual mode of action that results in a strong shoot-inducing activity.

Figure 1.

Chemical structure and activity of Phe-Ade. A, Chemical structure. B, LSH4 expression after 12 d (top panel) and shoot induction after 19 d (bottom panel) of SIM incubation containing 10 µm Phe-Ade. Bars = 1 mm.

Figure 2.

Phe-Ade is an efficient inducer of shoot formation on C24 and Col-0 root explants. A, Regeneration of C24 root explants after 15 d on SIM with Phe-Ade or 2-iP using shoot area as a quantitative measure (see “Materials and Methods”). B, Regeneration rate of Col-0 root explants expressed as a percentage of responsiveness (i.e. the average number of root explants forming at least one shoot). Responsive explants were counted 14 d after transfer to SIM. Data represent averages of three biological repeats each with 19 root explants. Different letters indicate statistical differences evaluated with Duncan’s multiple range test in conjunction with ANOVA. Error bars represent se.

Figure 3.

Phe-Ade exhibits several cytokinin-like activities. A, Phenotype of Col-0 plants grown for 1, 2, or 3 weeks in the presence or absence of 10 µm Phe-Ade or 2-iP. Notice the stunted growth, anthocyanin accumulation, serrated leaf margins, and inhibited root growth. Bars = 1 mm. B, Overview of plants grown for 3 weeks in the presence or absence of 10 µm Phe-Ade or 2-iP. In general, shoot growth was only inhibited by 2-iP. Bars = 10 mm. C, Root elongation inhibition assay on Col-0 seedlings grown for 6 d on different concentrations of 2-iP or Phe-Ade. Error bars represent se (n ≥ 20). D, Root growth over a period of 20 d on 10 µm Phe-Ade or 2-iP. Error bars represent se (n ≥ 20). E, Tobacco callus assay. Phe-Ade is not cytotoxic at high concentrations, in contrast to 2-iP. Error bars indicate se (n = 6).

Figure 4.

Transcript profiling of cytokinin-related genes in Col-0 shoot tissues at different time points of 10 µm Phe-Ade or 2-iP treatment. Different letters indicate statistical differences between the samples evaluated with the Tukey’s range test in conjunction with ANOVA. Asterisks indicate statistical differences from the hormone-free control, evaluated with a two-tailed Student’s t test and adjusted with the Benjamini-Hochberg false discovery rate method. Error bars represent se (n = 4).

Figure 5.

Interaction between Phe-Ade and the cytokinin receptors. A, ARR5:GUS expression in 14-d-old plants of Col-0 and the cytokinin receptor double mutants ahk2 ahk3, ahk2 ahk4, and ahk3 ahk4 treated for 5 d with 10 µm Phe-Ade or 2-iP. B, Root elongation inhibition assay on Col-0 and on double ahk mutant seedlings grown for 7 d on 10 µm 2-iP or Phe-Ade. Error bars represent se (n ≥ 10). C, In silico binding of Phe-Ade in the active site of AHK4. D, Effect of Phe-Ade, adenine (Ade), and tZ on the specific binding of 2 nm [³H]tZ in a live-cell hormone-binding assay employing E. coli cells expressing AHK4. NS, Nonspecific binding in the presence of a 5,000-fold excess of unlabeled tZ. Error bars indicate se (n = 3). E, Activation of the cytokinin receptors AHK3 and AHK4 by Phe-Ade and tZ in the E. coli receptor assay. Error bars indicate se (n = 3). F, Regeneration rate of root explants of Col-0 and the double ahk mutants on 10 µm Phe-Ade or 2-iP. Error bars indicate se. The data are averages of three biological repeats with 10 explants each. WT, Wild type.

Figure 6.

Phe-Ade is an inhibitor of CKX activity. A, CKX2-inhibiting activity of Phe-Ade in dehydrogenase (DCPIP and Q₀) and oxidase (O₂) mode. The degradation of 2-iP (83 µm) was determined by measuring the absorbance of the Schiff base at 352 nm (see “Materials and Methods”). B, Phe-Ade is an inhibitor of CKX1, CKX3, and CKX5. 2-iP (60 µm) degradation was determined by subtracting the concentration after 45 min from the concentration before the reaction and is expressed relative to the amount of degraded 2-iP in the absence of inhibitor. C, CKX2 degradation of 2-iP, BA, and Phe-Ade (166 µm). 2-iP and BA are, respectively, a strong and a weak substrate, while Phe-Ade is not a substrate of CKX2. Degradation was determined as bleaching of the absorbance of the electron acceptor DCPIP at 600 nm and is expressed relative to the same reaction without substrate. D to G, Raw data (D and F) and Lineweaver-Burk plots (E and G) of CKX2 (D and E) and CKX7 (F and G) activity in the absence or presence of Phe-Ade. Degradation rates were determined by continuously measuring the bleaching of DCPIP for different concentrations of 2-iP and Phe-Ade. Each combination was repeated at least three times.