Dopamine, Chlorogenic Acid, and Quinones as Possible Cofactors of Increasing Adventitious Rooting Potential of In Vitro Krymsk 5 Cherry Rootstock Explants

Abstract

In the present study, the effect of some not commonly used phenolic compounds was evaluated during the in vitro rooting stage of the cherry rootstock ‘Krymsk 5′ (P. fruticosa × P. lannesiana), in the absence or presence of auxin. Two sets of experiments were conducted. In the first set, the following substances were tested: the o-diphenol chlorogenic acid, in five concentrations (0 μΜ, 0.5 μΜ, 1 μΜ, 5 μΜ, and 50 μΜ) in the presence of a suboptimal indolebutyric acid (IBA) concentration (5 μΜ), the catecholamine dopamine in five concentrations (0 μΜ, 0.5 μΜ, 1 μΜ, 5 μΜ, and 50 μΜ), and the quinone 5-hydroxy-1,4-naphthoquinone in four concentrations (0 μΜ, 0.25 μΜ, 1 μΜ, and 5 μΜ) in the absence or presence of 5 μΜ IBA. In the second experiment, the quinones p-benzoquinone; 1,4-napthoquinone; and 2-hydroxy-1,4-naphthoquinone were tested in four concentrations (0 μΜ, 5 μΜ, 50 μΜ, and 100 μΜ) in the presence of 5 μΜ IBA. An application of 5 μΜ of 5-hydroxy-1,4-naphthoquinone in the auxin-free medium increased rooting potential almost 1.7 times. Rooting percentage was also enhanced up to 4.2 times by dopamine; chlorogenic acid; 5-hydroxy-1,4-naphthoquinone; p-benzoquinone; and 1,4 napthoquinone in the presence of IBA. The present results indicate a possible promotive role of quinones and dopamine during in vitro rooting, at least for Prunus species, and their potential use as rooting cofactors. Moreover, a possible mode of action of the compounds studied related to IAA-oxidase is discussed.

1. Introduction

In the adventitious root formation (ARF) of cuttings and microcuttings, auxin (either its endogenous content or exogenously applied) has a crucial role, especially during the early stages of rhizogenesis [1], but in many cases, it is not adequate for sufficient rooting [2].

Phenolic compounds are known to play an ambiguous role in ARF, acting either as cofactors or inhibitors [3,4,5]. In general, o-diphenols, p-diphenols, coumarins, and polyphenols are thought to have a promotive role, whereas monophenols and m-diphenols can have inhibitory effects [1,6,7]. Despite the role of the phenolic compounds per se, it has also been reported that the products of their oxidation by polyphenoloxidase (PPO) may be necessary for successful rooting [8,9].

The enhancing effects of phenolics in ARF may be attributed to the “protection” of endogenous auxin from oxidation and decarboxylation [6,7,10], to the improvement of basipetal transportation of 3-indoleactetic acid (IAA) to the rooting zone [11] or to the conjugation with auxin, forming a complex more active than the auxin per se [12]. Reduced forms of phenolic compounds, such as chlorogenic acid, caffeic acid, catechol, ferrulic acid, salicylic acid, and others, have been tested for improving rooting potential, with various results [1,2,13,14] depending on the species or variety [2,15,16], the physiological condition of the cutting [16,17], and the type of compound used [2]. On the other hand, the oxidized products of phenolics (i.e., quinones) are not well studied for their role in ARF.

The aim of this preliminary study is to investigate the effect of some unprecedented (in ARF experiments) phenolic compounds as rooting co-factors in in-vitro rooting of the Prunus hybrid ‘Krymsk 5^®’ (Prunus fruticosa × Prunus lannesiana), and to postulate their possible role.

2. Materials and Methods

2.1. Explant Source, Plant Material and Culture Conditions

In-vitro-proliferated ‘Krymsk 5’ mother culture, cultivated according to [18], acted as the microshoot donor. Nodal microshoots approximately 1 to 1.5 cm in length were planted in Driver and Kuniyuki for Walnut (DKW) nutrient medium supplemented with 20 g L⁻¹ sucrose and 9 g L⁻¹ Agar-agar. The pH was adjusted to 5.8 prior to autoclaving and explants were grown in a growth chamber at 22 ± 1 °C, under a photoperiod and light intensity of 16 h and 3000 lux, respectively. Explants were cultivated in tubes containing 10 mL of rooting medium. All phenolic compounds used were filter-sterilized.

2.2. Effect of Chlorogenic Acid, Dopamine, and Quinones on Rooting in the Absence or Presence of a Suboptimal Concentration of Auxin

Two experiments were conducted in order to assess the effect of the phenolic acid chlorogenic acid, the catecholamine dopamine, and some quinones in the absence or presence of 5 μΜ IBA, which constituted a suboptimal auxin concentration based on the findings of [18].

In the first set of experiments, the effect of (a) chlorogenic acid in the presence of a suboptimal concentration of IBA and (b) dopamine and 5-hydroxy-1,4-naphthoquinone in the presence or absence of the suboptimal concentration of IBA (5 μΜ) in in-vitro rooting of ‘Krymsk 5’ explants was evaluated. Thus, microshoots were transplanted in rooting medium supplemented with chlorogenic acid at 0 μΜ (control), 0.5 μΜ, 1 μΜ, 5 μΜ, and 50 μΜ plus 5 μΜ IBA; dopamine at 0 μΜ (control), 0.5 μΜ, 1 μΜ, 5 μΜ, and 50 μΜ; or 5-hydroxy-1,4-naphthoquinone at 0 μΜ (control), 0.25 μΜ, 1 μΜ, and 5 μΜ with or without 5 μΜ IBA.

In the second experiment, the effect of the quinones 1,4-napthoquinone; 2-hydroxy-1,4-napthoquinone; and p-benzoquinone in the presence of 5 μΜ IBA was evaluated. The concentrations tested for all the three quinones were 0 μΜ (control), 5 μΜ, 50 μΜ, and 100 μΜ.

2.3. Experimental Design and Statistical Analysis

Eighteen to twenty explants per treatment were measured after a period of six weeks of culture for the estimation of morphogenic responses in terms of rooting percentage as well as number and length of the formed roots. Each experiment was repeated twice. The experiments were arranged according to a completely randomized design (CRD) with five replications of three to four explants. The raw data were analyzed by analysis of variance (ANOVA) using JMP 10.0 (SAS, Cary, NC, USA) software. Statistically significant differences among means were detected using the Tukey HSD at p ≤ 0.05.

3. Results

Compared to the control, 5-hydroxy-1,4-naphthoquinone increased the rooting percentage of explants from 33% to 58% (

Table 1. Effect of dopamine and 5-hydroxy-1,4-naphthoquinone on rooting variables of explants cultivated in rooting medium without IBA.

	Con (μM)	Rt (%)	No	Lgth (cm)
5-hydroxy-1,4-naphthoquinone	0	33 b	6.3 a	0.3 a
	0.25	40 ab	2.8 a	0.22 a
	1	28 b	5.3 a	0.21 a
	5	58 a	4.2 a	0.23 a
Dopamine	0	33 a	6.3 a	0.3 a
	0.5	36.4 a	3.8 a	0.3 a
	1	26.4 a	4.2 a	0.24 a
	5	45 a	5.6 a	0.28 a
	50	40 a	1.9 a	0.18 a

Con, Concentration; Rt, Rooted explants; No, number of formed roots; Lgth, length of formed roots. Means within the same column per phenolic compound followed by the same letter do not differ significantly according to the Tukey HSD (α = 0.05) multiple range test (p ≤ 0.05) (n = 5).

) in the absence of auxin, whereas dopamine inclusion into the rooting medium did not affect any rooting parameter studied (Table 1; Figure 1).

On the contrary, in the presence of a suboptimal concentration of auxin, all compounds increased the rooting potential (

Table 2. Effect of dopamine; 5-hydroxy-1,4-naphthoquinone; and chlorogenic acid on rooting variables of explants cultivated in rooting medium in the presence of IBA.

	Con (μM)	Rt (%)	No	Lgth (cm)
5-hydroxy-1,4-naphthoquinone	0	15 c	3 a	0.1 b
	1	45 ab	4.3 a	0.25 ab
	5	30 bc	1.8 a	0.17 ab
	50	45 ab	4.3 a	0.19 ab
	100	63 a	5 a	0.36 a
Dopamine	0	15 b	3 a	0.1 a
	1	45 a	3.7 a	0.36 a
	5	35 ab	5.8 a	0.14 a
	50	35 ab	2 a	0.19 a
	100	15 b	1.6 a	0.23 a
Chlorogenic acid	0	15 b	3 a	0.1 a
	1	45 a	4.1 a	0.3 a
	5	35 ab	5.5 a	0.33 a
	50	30 ab	5.4 a	0.26 a
	100	40 a	3.9 a	0.17 a

Con, Concentration; Rt, Rooted explants; No, number of formed roots; Lgth, length of formed roots. Means within the same column per phenolic compound followed by the same letter do not differ significantly according to the Tukey HSD (α = 0.05) multiple range test (p ≤ 0.05) (n = 5).

). Dopamine and chlorogenic acid increased rooted explants from 15% to 45% (three times) under 1 μΜ, whereas 5-hydroxy-1,4-naphthoquinone increased rooted explants from 15% to 63% (more than four times) under 100 μM concentration (Table 2). Moreover, the application of 100 μΜ 5-hydroxy-1,4-naphthoquinone resulted in greater root length (0.36 cm) than that of the control (0.1 cm) (Table 2; Figure 2).

The inclusion of p-benzoquinone at 50 and 100 μΜ and 1,4-napthoquinone at 50 μΜ in the rooting medium increased the rooting percentage of ‘Krymsk 5^®’ microcuttings, compared to the control, from 20% to 45% (i.e., approximately 2 times) and to 68.3% (i.e., 3.4 times) for p-benzoquinone, and to 55% (i.e., 2.7 times) for 1,4-napthoquinone, respectively (

Table 3. Effect of 1,4-napthoquinone; 2-hydroxy-1,4-naphthoquinone; and p-benzoquinone on rooting variables of explants cultivated in rooting medium in the presence of IBA.

	Con (μM)	Rt (%)	No	Lgth (cm)
Control	0	20 de	3 a	0.1 a
1,4-napthoquinone	5	30 cd	2.6 a	0.15 a
	50	55 ab	5.5 a	0.29 a
	100	25 cd	4.8 a	0.20 a
2-hydroxy-1,4-napthoquinone	5	0 e	---	---
	50	0 e	---	---
	100	11.6 de	2.5 a	0.1 a
p-benzoquinone	5	23.3 de	2 a	0.19 a
	50	45 bc	3.9 a	0.20 a
	100	68.3 a	4.9 a	0.19 a

Con, Concentration; Rt, Rooted explants; No, number of formed roots; Lgth, length of formed roots. Means within the same column followed by the same letter do not differ significantly according to the Tukey HSD (α = 0.05) multiple range test (p ≤ 0.05) (n = 5).

). On the contrary, 2-hydroxy-1,4-napthoquinone had no effect at the concentrations tested (Table 3; Figure 3).

4. Discussion

The role of chlorogenic acid as a rooting cofactor in the presence of auxin in several species and physiological conditions is well established [1,2,19,20]. However, the effect of dopamine in rooting has not been elucidated yet. Dopamine increased the rooting of ‘Krymsk 5′ explants only in the presence of auxin (Table 1 and Table 2), highlighting auxin’s key role [1,5], as well as the synergism between auxin and o-diphenols [5]. Similarly to our observations on Krymsk 5, the application of dopamine to the softwood cuttings of other Prunus rootstocks had the same promotive effect (unpublished data), strengthening its role as a rooting cofactor. Furthermore, as dopamine is not ubiquitously present in Prunus species (to our knowledge), this indicates that it could be effective in the induction of ARF in other species too. Thus, the possible role of dopamine, and possibly of other catecholamines, is now emerging.

Both chlorogenic acid and dopamine are characterized by ortho-diphenolic moiety and therefore, a similar mode of action could be assumed. Indeed, chlorogenic acid is known as a “protector” of IAA enzymatic degradation by indoleacetic acid oxidase (IAA-ox) [21]. Similarly, dopamine has been reported to prevent IAA oxidation [22,23] through the specific inhibition of IAA-ox activity [23], indirectly improving the availability of IAA for rooting. Moreover, both compounds consist PPO substrates [24], indicating a possible increase in PPO activity due to their application. An increase in PPO activity can favor rooting, since PPO is positively related to ARF [9] and may produce cofactors of rooting [25], driving [26] to suggest PPO as a rooting marker.

As already quoted, mainly reduced phenolic compounds, such as o-diphenols, have been tested and studied for their effect on rooting. On the other hand, [27] implied that the oxidized products of phenolics (i.e., quinones) are responsible for IAA-ox inhibition. Despite their obvious relation with PPO, i.e., quinones consist of the oxidized product of phenolics by the enzyme [28], and their possible effect on IAA-ox [27], the role of quinones in rooting is neglected and has not been sufficiently studied.

In the present study, the majority of the quinones tested increased the in vitro rooting potential of ‘Krymsk 5′explants compared to the control (Table 2 and Table 3), suggesting a promotive effect of quinones in the ARF of Prunus species. According to [29], quinones can enhance the rooting of phaseolus cuttings and may be associated with dedifferentiation processes in plant tissues. A high quinone content during the early phases of rhizogenesis has been found in Prunus cuttings and microcuttings characterized by high rooting potential (unpublished data), supporting their involvement in the rooting process. In agreement with the present results, 5-hydroxy-1,4-naphthoquinone induced ARF in Morus sp. shoots in vitro [30] and was related to in vitro rooting of walnut [31], suggesting a consistent favorable effect for several species. Nevertheless, 5-hydroxy-1,4-naphthoquinone is a known allelopathetic factor that can induce the production of reactive oxygen species (ROS), such as H₂O₂ [32], the application of which can increase rooting in Prunus cuttings [33], whereas its scavenging can inhibit rooting [34]. Thus, the effect of the quinones on ARF may rely on direct and/or indirect action, through the production of rooting promoting molecules, such as H₂O₂, or the regulation of hormonal levels, such as auxin. Indeed, 1,4-napthoquinone [29] and p-benzoquinone [27] have been reported to inhibit IAA-ox, and thus increase auxin levels, indicating a mode of action similar to the one of the reduced phenolics, such as dopamine and chlorogenic acid.

In conclusion, through these preliminary results, the role of the easily oxidized o-diphenol dopamine and the quinones 5-hydroxy-1,4-naphthoquinone; 1,4-napthoquinone; and p-benzoquinone as cofactors of rooting is emerging. Moreover, a possible mode of their action is discussed, indicating that both reduced and oxidized phenolic compounds may enhance rooting through the inhibition of IAA-ox. Further research is deemed necessary in order to fully elucidate the role and possible mode of action of these compounds on ARF.