Plant Science 214 (2014) 88–98

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Endogenous cytokinin profiles of tissue-cultured and acclimatized ‘Williams’ bananas subjected to different aromatic cytokinin treatments Adeyemi O. Aremu a , Lenka Plaˇcková b,c , Michael W. Bairu a,1 , Ondˇrej Novák b,c , Lucie Szüˇcová b,c , Karel Doleˇzal b,c , Jeffrey F. Finnie a , Johannes Van Staden a,∗ a Research Centre for Plant Growth and Development, School of Life Sciences, University of KwaZulu-Natal, Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa b Laboratory of Growth Regulators, Palack´ y University and Institute of Experimental Botany AS CR, Sˇ lechtitel˚ u 11, 783 71 Olomouc, Czech Republic c Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palack´ y University, Sˇ lechtitel˚ u 11, 783 71 Olomouc, Czech Republic

a r t i c l e

i n f o

Article history: Received 23 August 2013 Received in revised form 24 September 2013 Accepted 25 September 2013 Available online 2 October 2013 Keywords: Micropropagation Musa spp. Phytohormones Physiological disorders Topolins

a b s t r a c t Endogenous cytokinin (CK) levels of in vitro-cultured and greenhouse-acclimatized ‘Williams’ bananas treated with six aromatic CKs were quantified using UPLC–MS/MS. The underground parts had higher endogenous CK levels than the aerial parts. Control plantlets had more isoprenoid CKs while the aromatictype CKs were predominant in all other regenerants. Following acclimatization of the control and 10 ␮M CK regenerants, there was a rapid decline in both isoprenoid and aromatic CK in the greenhousegrown plants. Apart from the control and 6-(3-Methoxybenzylamino)-9-tetrahydropyran-2-ylpurine (MemTTHP) treatment with higher level of isoprenoid CK, aromatic CK remain the predominant CKtype across all CK treatments. The most abundant CK forms were meta-topolin (mT) and benzyladenine (BA) in the micropropagated and acclimatized plants, respectively. Micropropagated plantlets had cisZeatin (cZ) as the major isoprenoid CK-type which was in turn replaced by isopentenyladenine (iP) upon acclimatization. On a structural and functional basis, 9-glucoside, a deactivation/detoxicification product was the most abundant and mainly located in the underground parts (micropropagation and acclimatization). The results establish the wide variation in metabolic products of the tested aromatic CKs during micropropagation and acclimatization. The findings are discussed with the possible physiological roles of the various CK constituents on the growth and development of banana plants. © 2013 Elsevier Ireland Ltd. All rights reserved.

Abbreviations: BA, N6 -Benzyladenine; BA9G, N6 -Benzyladenine-9-glucoside; BAR, N6 - Benzyladenine riboside; BAR5 MP, N6 -Benzyladenosine-5 -monophosphate; CDK, Cyclin-dependent kinase; CK, Cytokinin; cZ, cis-Zeatin; cZ9G, cis-Zeatin-9-glucoside; cZOG, cis-Zeatin-O-glucoside; cZR, cis-Zeatin riboside; cZR5 MP, cis-Zeatin riboside-5 -monophosphate; cZROG, cis-Zeatin-O-glucoside riboside; DHZ, Dihydrozeatin; DHZ9G, Dihydrozeatin-9-glucoside; DHZOG, Dihydrozeatin-O-glucoside; DHZR, Dihydrozeatin riboside; DHZR5 MP, Dihydrozeatin riboside-5 -monophosphate; DHZROG, Dihydrozeatin-O-glucoside riboside; IAC, Immunoaffinity chromatography; iP, N6 -Isopentenyladenine; iP9G, N6 -Isopentenyladenine-9-glucoside; iPR, N6 -Isopentenyladenosine; iPR5 MP, N6 -Isopentenyladenosine-5 -monophosphate; IPT, Isopentenyltransferase; KIN, Kinetin; KIN9G, Kinetin-9-glucoside; KINR, Kinetin riboside; KINR5 MP, Kinetin riboside-5 -monophosphate; MemT, meta-Methoxy topolin; MemTR, meta-Methoxy topolin riboside; MemTTHP, 6-(3-Methoxybenzylamino)-9-tetrahydropyran-2-ylpurine; MRM, Multiple reaction monitoring; MS, Murashige and Skoog medium; mT, meta-Topolin; mT9G, meta-Topolin-9-glucoside; mTOG, meta-Topolin-O-glucoside; mTR, meta-Topolin riboside; mTR5 MP, meta-Topolin-5 -monophosphate; mTROG, meta-Topolin-O-glucoside riboside; oT, ortho-Topolin; oT9G, ortho-Topolin-9-glucoside; oTOG, ortho-Topolin-O-glucoside; oTR, ortho-Topolin riboside; oTR5 MP, ortho-Topolin-5 -monophosphate; oTROG, ortho-Topolin-O-glucoside riboside; PGR, Plant growth regulator; PPF, Photosynthetic photon flux density; pT, para-Topolin; PTC, Plant tissue culture; pTOG, para-Topolin-O-glucoside; pTR, para-Topolin riboside; pTR5 MP, para-Topolin-5 -monophosphate; pTROG, para-Topolin-O-glucoside riboside; tZ, trans-Zeatin; tZ9G, trans-Zeatin-9-glucoside; tZOG, trans-Zeatin-O-glucoside; tZR, trans-Zeatin riboside; tZR5 MP, trans-Zeatin riboside-5 -monophosphate; tZROG, trans-Zeatin-O-glucoside riboside; UPLC, Ultra performance liquid chromatography. ∗ Corresponding author. Tel.: +27 33 2605130. E-mail address: [email protected] (J. Van Staden). 1 Current address: Institute for Commercial Forestry Research, P.O. Box 100281, Scottsville 3209, Pietermaritzburg, South Africa. 0168-9452/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.plantsci.2013.09.012

A.O. Aremu et al. / Plant Science 214 (2014) 88–98

1. Introduction Cytokinins (CKs) are naturally occurring N6 -substituted adenine compounds which are vital for the regulation of several developmental and physiological processes in plants [1]. These processes include shoot apical dominance, branching, chlorophyll production and root growth as well as contributing to nutritional signalling and formation of embryo vasculature [2,3]. Based on the side chain configuration, CKs are classified as of the isoprenoid [N6 2-isopentenyl adenine (iP), trans-Zeatin (tZ), cis-Zeatin (cZ) and dihydrozeatin (DHZ)] or aromatic [N6 -benzyladenine (BA), kinetin (KIN) and topolin] types. Together with auxins, CKs affect the basic mechanisms of cell proliferation and differentiation in plants [4]. In addition to the benefit of mass propagation, genetic improvement and conservation of several plant species, the plant tissue culture (PTC) technique is an avenue for exploring and understanding the often species-specific factors that control plant growth and development. In PTC, the use of exogenous CKs is necessary to stimulate various developmental and physiological processes [5]. Growth and development in vitro are regulated by the interaction and balance between the exogenously supplied and endogenously produced plant growth regulators (PGRs) [6,7]. In plant tissues, these exogenous supplied CKs are often metabolized to different forms such as products of ring substitution (ribosides, ribotides, Nglucosides) and side chain substitution (O-glucosides) or cleavage (adenine, adenosine, adenosine-5 -monophosphate) [3,8]. These metabolites and conjugates act as storage, transport or biologically inert forms of CKs which are responsible for the physiological and developmental plasticity observed in plants [3,9]. Changes in the levels of endogenous CKs alter the regulation of these physiological processes and influence plant architecture. Apart from stimulating shoot proliferation in vitro, many PTC problems such as shoot-tip necrosis [10], hyperhydricity [11] and rooting inhibition as well as acclimatization failure [12,13] have been partly associated with the level of endogenous CKs in plant tissue at any given time. In view of the increasing human population and associated problem of food security, the value and economic importance of bananas (Musa spp.) as a food and fruit crop cannot be overemphasized. The use of PTC has significantly improved the quality of planting materials and increased the productivity of bananas [14]. Even though considerable success has been achieved, banana remains one of the most highly prioritized research crops. Attempts at optimizing micropropagation protocols has prompted the use of the different CK derivatives, particularly topolins. We have evaluated the role of different aromatic CKs (BA in comparison to topolins) on shoot and root proliferation [15], photosynthetic pigment production [16] as well as on the phytochemical and subsequently ex vitro acclimatization competency of bananas [17]. Despite the recognized value of endogenous hormone levels on these aforementioned processes, limited studies have been reported in the case of bananas [18,19]. In the current study, the effect of six exogenous application of aromatic CKs on the endogenous CK profiles after micropropagation and greenhouse acclimatization of ‘Williams’ bananas (Musa spp. cv. ‘AAA’) was evaluated. The current study was aimed at understanding the previously reported growth and physiological data in relation to the quantified endogenous CK content. Details of our previous findings have been published [15–17] and the same plant materials were used for the current study.

89

topolin riboside (MemTR), and 6-(3-Methoxybenzylamino)9-tetrahydropyran-2-ylpurine (MemTTHP)] were prepared as described previously [20–22]. N6 -benzyladenine was obtained from Sigma-Aldrich (Steinheim, Germany). Gelrite was purchased from Labretoria, Pretoria, South Africa while 23 deuterium-labelled CK internal standards were obtained from Olchemim Ltd (Olomouc, Czech Republic). All chemicals used in the study were of analytical grade. 2.2. Micropropagation and greenhouse experiments The source of initial plant materials, explant initiation, medium composition and growth conditions were as described previously [15]. Aseptically-obtained explants were transferred onto modified Murashige and Skoog [23] medium supplemented with six aromatic CKs at 10, 20 or 30 ␮M. A control without any PGR was also included in the experiment. Cultures were incubated for 42 days in a growth room under 16 h light/8 h dark conditions and photosynthetic photon flux (PPF) of 45 ␮mol m−2 s−1 at 25 ± 2 ◦ C. Due to ease of rooting, 10 plantlets each from the 10 ␮M CK treatments and the control were washed and potted in 12.5 cm pots containing sand, soil, vermiculite (1:1:1, v/v/v) treated with 1% Benlate® (Du Pont de Nemours Int., South Africa). Plantlets were maintained in the mist-house with day/night temperatures of 30/12 ◦ C, relative humidity of 80–90% and a 10 s misting at 15 min intervals. After 3 months, the plantlets were transferred to a greenhouse with 30–40% relative humidity, day/night temperatures of 30/15 ◦ C with an average PPF of 450 ␮mol m−2 s−1 . Photoperiod during the experiment was that of prevailing natural conditions (summer-12 h). Plants were harvested after two months in the greenhouse for endogenous CK quantification. 2.3. Extraction, purification and quantification of endogenous cytokinins Harvested-micropropagated and greenhouse-maintained plants (five in each case) were washed and separated into aerial and underground sections. Plant material was immediately frozen in liquid nitrogen, freeze-dried and lyophilized. Prepared samples were extracted and purified using the methods [24,25] outlined for bananas in an earlier study [26]. The samples were analyzed by ultra-performance liquid chromatography (Acquity UPLCTM ; Waters) coupled to a XevoTM TQ MSTM ESI (Waters) triple quadrupole mass spectrometer equipped with an electro-spray interface. Further experimental details are as outlined previously [26]. Endogenous CK quantification was achieved by multiple reaction monitoring (MRM) of [M+H]+ and the appropriate product ion. For selective MRM experiments, optimal conditions (dwell time, cone voltage, and collision energy in the collision cell) corresponding to exact diagnostic transition were optimized for each CK [25]. Quantification was performed with Masslynx software using a standard isotope dilution method. The ratio of endogenous CK to appropriate labelled standard was determined and subsequently used to quantify the level of endogenous CKs in the original banana plant extract, based on the known concentration of internal standard added [24]. 3. Results 3.1. Endogenous CK content in micropropagated plantlets

2. Materials and methods 2.1. Chemicals The topolins used [meta-Topolin (mT), meta-Topolin riboside (mTR), meta-Methoxy topolins (MemT), meta-Methoxy

The total pool of all CKs quantified ranged from approximately 1.9–810 nmol g−1 FW as detected in control and the 10 ␮M mTR treatment, respectively (Table 1). With the exception of the control that had more isoprenoid CKs, aromatic CK were generally higher than the isoprenoid-type in all the treatments. In

1888.9 ± 649.72 649.4 ± 400.8 1239.4 ± 248.9 623.7 ± 391.5 25.8 ± 9.26 959.5 ± 224.70 0

279.9 ± 24.23 Control

30 ␮M

mT, meta-Topolin; mTR, meta-Topolin riboside; MemT, meta-Methoxy topolin; MemTR, meta-Methoxy topolin riboside; BA, Benzyladenine; MemTTHP, 6-(3-Methoxybenzylamino)-9-tetrahydropyran-2-ylpurine.

11,845.02 7965.73 2899.30 16,705.19 30,766.64 2596.53 ± ± ± ± ± ± 45,069.3 95,680.7 8746.6 216,106.4 127,089.6 55,751.8 10,305.4 7554.9 2305.5 14,713.1 27,673.5 2458.8 ± ± ± ± ± ± 42,407.1 93,976.0 6408.0 210,837.1 122,242.1 55,377.5 1539.6 410.8 593.8 1992.1 3093.1 137.7 ± ± ± ± ± ± 2662.2 1704.7 2338.6 5269.4 4847.4 374.2 10,186.8 6815.5 2248.0 12,661.7 26,766.5 1890.3 ± ± ± ± ± ± 37,550.5 91,142.4 6106.3 180,126.2 115,128.2 14,428.4 118.57 739.44 57.52 2051.36 907.02 568.51 ± ± ± ± ± ± 4856.6 2833.6 301.7 30,710.9 7114.0 40,949.1 1277.27 156.18 566.72 302.90 326.55 104.19 ± ± ± ± ± ± 262.36 254.64 27.10 1689.22 2766.56 33.52 ± ± ± ± ± ± 857.0 717.9 1104.3 4304.7 3728.9 164.5 mT mTR MemT MemTR BA MemTTHP

20 ␮M

46.16 33.68 30.25 24.80 22.93 15.96 215.7 190.2 264.2 299.6 280.0 256.9 mT mTR MemT MemTR BA MemTTHP

10 ␮M

± ± ± ± ± ±

1805.2 986.8 1234.3 964.6 1118.5 209.7

± ± ± ± ± ± 327,029.1 222,955.7 41,012.9 13,869.9 149,090.7 5115.1 41,605.1 21,557.1 27,879.7 6867.2 13,497.6 1884.5 ± ± ± ± ± ± 324,831.9 222,013.6 39,923.0 13,429.6 147,976.2 4567.4 1427.0 162.9 286.8 55.4 268.4 50.0 ± ± ± ± ± ± 2197.2 942.1 1089.9 440.2 1114.5 547.7 41,396.5 21,335.5 27,871.1 6850.6 13,072.2 1875.3 ± ± ± ± ± ± 321,764.8 220,674.7 39,853.7 13,313.1 142,347.9 4530.3 208.61 221.51 8.56 16.62 425.40 9.19 ± ± ± ± ± ± 3067.2 1338.9 69.3 116.5 5628.3 37.1 1380.88 129.21 256.57 30.62 245.47 34.00 ± ± ± ± ± ±

± ± ± ± ± ± 134,714.6 807,604.2 2984.0 3411.6 62,260.4 3329.2 331.0 633.3 121.1 58.7 509.6 187.7 ± ± ± ± ± ± 1591.9 2703.7 795.4 719.4 2018.3 2742.4 4199.8 97,736.7 583.9 258.7 6006.5 937.5 ± ± ± ± ± ± 134,288.0 807,284.1 2941.3 3322.9 62,085.7 3325.4

Underground part

21.54 34.82 3.29 11.20 7.94 0.71 ± ± ± ± ± ± 426.6 320.2 42.6 88.6 174.6 3.8

Aerial part

302.48 610.31 85.50 33.80 441.15 155.57 ± ± ± ± ± ±

Underground part

1378.2 2431.1 371.9 443.8 1525.3 2325.5 28.53 23.00 35.61 24.92 68.44 32.17 213.6 272.6 423.5 275.6 493.0 417.0 mT mTR MemT MemTR BA MemTTHP

± ± ± ± ± ±

Aerial part

1981.5 751.9 825.8 140.7 834.5 290.8

4221.3 97,771.5 587.2 269.9 6014.4 938.2

136,306.5 810,307.9 3779.4 4130.9 64,278.6 6071.6

± ± ± ± ± ±

Total cytokinin Total aromatic Total isoprenoid Aromatic Isoprenoid Cytokinins Conc.

Table 1 Endogenous isoprenoid and aromatic cytokinin content (pmol g−1 FW) of tissue-cultured ‘Williams’ bananas (Musa spp. cv. AAA) in relation to the applied cytokinin types and concentrations.

43,032.13 21,719.95 28,166.51 6922.65 13,765.98 1934.50

A.O. Aremu et al. / Plant Science 214 (2014) 88–98

4552.31 98,404.85 708.33 328.63 6524.03 1125.96

90

addition, the aromatic CK contents detected among the different treatments (with the exception of 30 ␮M MemTTHP) and control were considerably higher in the underground compared to the aerial parts. For instance, underground parts of 10 ␮M mTR treatment had about 2522-fold more aromatic CK than the aerial parts. With an increase in CK concentration during micropropagation, there was an increase in the level of quantified endogenous aromatic CKs in the aerial parts in all the treatments. In terms of the isoprenoid CK content, majority of the treatments and the control had higher concentrations in the underground parts. However, MemT (10 ␮M), MemTR (20 and 30 ␮M) and BA (30 ␮M) treatments had more isoprenoid CKs in their aerial parts. The quantified CKs consist of 47 CK-forms grouped into 9 classes. These include the four isoprenoid-types: tZ, cZ, DHZ, iP (Fig. 1) and five aromatic-types: BA, mT, oT, pT and KIN (Fig. 2). The aerial parts had lower amounts of tZ when compared to the underground parts (Fig. 1a and b). Highest level of cZ was detected in aerial parts of 30 ␮M MemTR treatment with 4062 pmol g−1 FW (Fig. 1c) and underground parts of 10 ␮M mTR with 2170 pmol g−1 FW (Fig. 1d). For the three tested CK concentrations, highest level of DHZ was found in 10 ␮M for BA and MemTTHP) and 30 ␮M (mT, mTR, MemT and MemTR) in the aerial parts (Fig. 1e). In the underground parts (Fig. 1f), 10 ␮M (mT, mTR, BA and MemTTHP) and 30 ␮M (MemT and MemTR) treatments had the highest level of DHZ. The level of iP ranged from 53 to 235 pmol g−1 FW as observed in aerial parts of 10 ␮M mT and 30 ␮M BA treatments, respectively (Fig. 1g). The control plants had the highest concentration (786 pmol g−1 FW) of iP and were considerably more than all the CK-treated plantlets (Fig. 1h). The aerial parts had low levels (≤ 20 pmol g−1 FW) of BA at all concentration of mT, mTR, MemT and MemTR treatments while 30 ␮M MemTTHP-treated plantlets (38,756 pmol g−1 FW) had the highest level (Fig. 2a). As depicted in Fig. 2b, 30 ␮M MemTR plantlets had the highest concentration (168,358 pmol g−1 FW) of BA in the underground parts. In both the aerial (Fig. 2c) and underground (Fig. 2d) parts, large amounts of mT was mainly observed in mT and mTR treatments. Even though 30 ␮M MemTTHP plantlets had the highest level (1210 pmol g−1 FW) of oT in the aerial parts, other treatments and the control generally had low (≤114 pmol g−1 FW) concentrations (Fig. 2e). In the underground parts, plantlets treated with the three concentrations of BA had the highest level of oT (Fig. 2f). There was no pT detected in the aerial parts of the control and majority of the treatments, however 30 ␮M MemTR plantlets had the highest concentration of pT (Fig. 2g). An increasing level of pT was observed with an increase in BA concentration in underground parts (Fig. 2h). Plantlets treated with BA had the highest concentration of KIN in both the aerial (Fig. 2i) and underground parts (Fig. 2j). The level of free bases in the aerial parts was lowest (2 pmol g−1 FW) in 10 ␮M MemTTHP and highest (16260 pmol g−1 FW) in 30 ␮M MemTR treatments (Fig. 3a). On the other hand, the underground parts of 20 ␮M BA-treated plantlets had the highest level (59,632 pmol g−1 FW) of free bases, which was approximately 95fold higher than in the control plantlets (Fig. 3b). In both aerial (Fig. 3c) and underground (Fig. 3d) parts, mT and mTR-treated plants had the highest level of O glucosides. The concentration of 9-glucosides ranged from 202 to 39,901 pmol g−1 FW (10 ␮M MemTR and 30 ␮M MemTTHP) in aerial parts (Fig. 3e) and 799–384,936 pmol g−1 FW (control and 10 ␮M mTR) in underground parts (Fig. 3f). High amounts of ribosides were quantified in the aerial part of all CK treatments including the control (Fig. 3g) while only mT and mTR treatment had higher concentrations among the underground parts (Fig. 3h). The majority of the treatments had more ribotides in the underground parts compared to the aerial parts (Fig. 3i and j). However, there was more ribotides in

A.O. Aremu et al. / Plant Science 214 (2014) 88–98

91

Fig. 1. Endogenous concentrations (pmol g−1 FW) of different forms of isoprenoid cytokinins in tissue-cultured ‘Williams’ bananas (Musa spp. cv. AAA) in relation to the applied cytokinin types and concentrations. a, b (tZ) = trans-Zeatin; c, d (cZ) = cis-Zeatin; e, d (DHZ) = Dihydrozeatin; g, h (iP) = Isopentenyladenine. For all graphs, dotted lines indicate the mean value for the control. mT, meta-Topolin; mTR, meta-Topolin riboside; MemT, meta-Methoxy topolin; MemTR, meta-Methoxy topolin riboside; BA, Benzyladenine; MemTTHP, 6-(3-Methoxybenzylamino)-9-tetrahydropyran-2-ylpurine.

the aerial parts of 10 ␮M MemT, BA and MemTTHP as well as 20 ␮M MemTTHP plantlets compared to the underground parts. 3.2. Endogenous CK content in greenhouse acclimatized plants Varying concentrations of isoprenoid and aromatic CKs were detected in the acclimatized ‘Williams’ bananas (Table 2). Among the treatments, the highest and lowest concentrations of total CKs were detected in BA and MemTTHP-derived plants, respectively. The plants from BA treatment had approximately 22-fold more endogenous CK than those of MemTTHP. Apart from MemTTHP and control plants with higher level of isoprenoid CK, aromatic CKs were generally higher than the isoprenoids in all the treatments. In fact, in BA-derived plants, aromatic CK constituted the bulk (97%) of the total CK content. In term of distribution/biosynthesis, aromatic CKs were more abundant in the underground parts in all treatments and the control. The underground parts also had higher level of isoprenoid CK in MemT, MemTR, MemTTHP and control plants. On the contrary, the isoprenoid-type CKs were higher in aerial parts of mT, mTR and BA treatments. Apart from MemT-derived plants with the lowest level of tZ, all the CK-treated plants yielded more tZ compared to control plants in the aerial parts (Fig. 4a). In the underground parts, all the CK-treated

plants had reduced level of tZ compared to the control (Fig. 4b). Plants-derived from mT had an estimated 8-fold higher cZ in the aerial parts than MemT treatment (Fig. 4c). The concentration of cZ ranged from 11 to 56 pmol g−1 FW in MemTTHP and MemT treatments, respectively in the underground parts (Fig. 4d). Both the aerial and underground parts of the acclimatized plants had a limited amount (≤6.5 pmol g−1 FW) of endogenous DHZ (Fig. 4e and f). Plants treated with mT and MemT showed the highest level of iP in the aerial and underground parts, respectively (Fig. 4g and h). While high levels of BA was distinctly present in both the aerial and underground parts of BA-treated plant (Fig. 5a and b), mTR treatment stimulated the highest level of mT in the acclimatized plants (Fig. 5c and d). As shown in Fig. 5e, almost similar concentration of oT was observed in aerial parts of the control and CK-treated plants (apart from MemT). Among the underground parts, BA had the highest concentration of oT which was about 11-fold higher than in the control plants (Fig. 5f). While pT was not detected in aerial parts of all plants, about 6 pmol g−1 FW of pT was recorded in the underground parts of BA-treated plants (Fig. 5g and h). With the exception of BA and MemTTHP treatments, there was a lower level of KIN in aerial parts compared to the underground parts (Fig. 5i and j).

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A.O. Aremu et al. / Plant Science 214 (2014) 88–98

Fig. 2. Endogenous concentrations (pmol g−1 FW) of different forms of aromatic cytokinins in tissue-cultured ‘Williams’ bananas (Musa spp. cv. AAA) in relation to the applied cytokinin types and concentrations. a, b (BA) = Benzyladenine; c, d (mT) = meta-topolin; e, d (oT) = ortho-topolin; g, h (pT) = para-topolin; i, j (KIN) = Kinetin. For all graphs, dotted lines indicate the mean value for the control. mT, meta-Topolin; mTR, meta-Topolin riboside; MemT, meta-Methoxy topolin; MemTR, meta-Methoxy topolin riboside; BA, Benzyladenine; MemTTHP, 6-(3-Methoxybenzylamino)-9-tetrahydropyran-2-ylpurine.

As depicted in Fig. 6a and b, BA-treated plants yielded more free bases compared to the control and other CK treatments. The levels of O-glucosides were generally more abundant in mT, mTR and BA in the aerial parts (Fig. 6c) as well as in mT and mTR for the underground parts (Fig. 6d). In terms of the amount of 9-glucosides,

mT and BA treatments had the highest concentration in the aerial (Fig. 6e) and underground (Fig. 6f) parts, respectively. The riboside concentration ranged from 8 to 29 pmol g−1 FW in aerial parts (Fig. 6g) and 59–285 pmol g−1 FW in underground parts (Fig. 6h). When compared to the control plants, MemT-treated (with highest

Table 2 Endogenous isoprenoid and aromatic cytokinin content (pmol g−1 FW) of greenhouse-acclimatized ‘Williams’ bananas (Musa spp. cv. AAA) in relation to the applied cytokinin types and concentrations. Cytokinins

Isoprenoid Aerial part

Control mT mTR MemT MemTR BA MemTTHP

261.6 557.1 363.6 88.2 201.2 465.9 210.9

± ± ± ± ± ± ±

15.02 58.61 15.54 2.76 28.41 51.46 89.95

Aromatic Underground part 478.8 302.3 272.7 1039.8 237.4 21.8 236.9

± ± ± ± ± ± ±

96.10 26.16 52.89 119.33 23.64 9.92 42.33

Aerial part 57.8 75.1 75.7 21.3 21.2 221.1 47.7

± ± ± ± ± ± ±

25.65 11.09 13.15 11.61 1.45 65.36 4.58

Total isoprenoid

Total aromatic

Total cytokinin

Underground part 513.5 2827.7 3516.0 1363.5 849.4 18,709.2 369.9

± ± ± ± ± ± ±

125.80 1054.82 1717.07 316.91 248.39 1181.12 75.61

740.4 859.4 636.3 1128.0 438.7 487.7 447.7

± ± ± ± ± ± ±

111.12 84.76 68.42 122.09 52.05 61.38 132.28

571.3 2902.9 3591.6 1384.8 870.7 18,930.3 417.5

± ± ± ± ± ± ±

151.45 1065.91 1730.22 328.52 249.84 1246.48 80.18

1311.7 3762.2 4227.9 2512.8 1309.3 19,418.0 865.3

± ± ± ± ± ± ±

262.57 1150.67 1798.64 450.60 301.90 1307.86 212.46

mT, meta-Topolin; mTR, meta-Topolin riboside; MemT, meta-Methoxy topolin; MemTR, meta-Methoxy topolin riboside; BA, Benzyladenine; MemTTHP, 6-(3Methoxybenzylamino)-9-tetrahydropyran-2-ylpurine.

A.O. Aremu et al. / Plant Science 214 (2014) 88–98

Aerial part 20000

Underground part 80000

(a) free bases

(b) free bases

10 µM 20 µM 30 µM

15000

10 µM 20 µM 30 µM

60000

10000

40000

5000

20000

0 4000

93

0 250000

(c) O glucosides

(d) O glucosides

200000

3000

150000

Cytokinin concentration (pmol g -1 FW)

1000 0 50000

(e) 9 glucosides

40000 30000 20000 10000 0

(g) ribosides

600

400

50000 0 500000

(f) 9 glucosides

400000 300000 200000 100000 0

(h) ribosides

300000

200000

100000

0 40000

A B

M

em TT

TR em M

em M

m

B A M em TT H P

0 em TR

0

T

10000

em

200

H P

20000

T

400

m TR

30000

T

600

(j) ribotides

M

(i) ribotides

m TR

0 800

M

200

100000

m T

Cytokinin concentration (pmol g -1 FW)

2000

Fig. 3. Endogenous levels (pmol g−1 FW) of the different structural and functional cytokinin forms in tissue-cultured ‘Williams’ bananas (Musa spp. cv. AAA) in relation to the applied cytokinin types and concentrations. For all graphs, dotted lines indicate the mean value for the control. mT, meta-Topolin; mTR, meta-Topolin riboside; MemT, meta-Methoxy topolin; MemTR, meta-Methoxy topolin riboside; BA, Benzyladenine; MemTTHP, 6-(3-Methoxybenzylamino)-9-tetrahydropyran-2-ylpurine.

level) plants had approximately 2.6- and 13-fold more ribotides in the aerial (Fig. 6i) and underground (Fig. 6j) parts, respectively. 4. Discussion 4.1. Effect of aromatic cytokinins (CKs) on quantified endogenous CK pool Numerous factors including the tissue culture conditions and exogenous PGRs (type and concentration) are known to affect

the biochemical pathways that regulate endogenous CK levels in plants [7,27]. As highlighted by Strnad [28], isoprenoid and aromatic CKs have an overlapping spectrum of biological activity but they are not considered as alternative forms of the same signals. While the isoprenoid CKs have a more potent effect on growth processes involving continuation of the cell cycle, aromatic CKs wield a greater influence on developmental processes such as morphogenesis and senescence [29]. In the control plant used in the present study, in the absence of exogenous CK, isoprenoid CKs were the predominant endogenous CK forms constituting about 65%

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Fig. 4. Endogenous concentrations (pmol g−1 FW) of different forms of isoprenoid cytokinins in greenhouse acclimatized ‘Williams’ bananas (Musa spp. cv. AAA) in relation to the applied cytokinin types at 10 ␮M. a, b (tZ) = trans-Zeatin; c, d (cZ) = cis-Zeatin; e, d (DHZ) = Dihydrozeatin; g, h (iP) = Isopentenyladenine. mT, meta-Topolin; mTR, metaTopolin riboside; MemT, meta-Methoxy topolin; MemTR, meta-Methoxy topolin riboside; BA, Benzyladenine; MemTTHP, 6-(3-Methoxybenzylamino)-9-tetrahydropyran-2ylpurine.

and 56% in micropropagated (Table 1) and greenhouse maintained (Table 2) ‘Williams’ bananas, respectively. The current observations are consistent with previous studies in several higher plants such as Cocos nucifera [30], Pinus species [7,31], Aloe polyphylla [11] and Harpagophytum procumbens [10] where isoprenoid CKs were the predominant forms. Even at the lowest tested concentration (10 ␮M), addition of aromatic CKs tilted the biosynthetic pathway and significantly reduced the endogenous isoprenoid CK concentration. In most cases e.g. mT and mTR, about 99% of the quantified endogenous CK content were aromatic-type CKs. On the contrary 10 ␮M MemTTHP-treated had approximately equal

portions of isoprenoid and aromatic CKs. However, there were rapid increases in the aromatic-type CKs at 20 and 30 ␮M MemTTHP. The developmental response of explants to exogenous PGRs is related to their concentration in the medium which in turn, is related to uptake, metabolism and transport of PGR within the explant [27]. The current findings possibly account for the beneficial (high number) rooting effect observed in MemTTHP-treated micropropagated and greenhouse-grown ‘Williams’ bananas in our previous studies [15,17]. Evidently, there is direct correlation between the level of endogenous isoprenoid CKs and the rooting tendency of the plants.

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tro

l

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em M

em M

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on C

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Fig. 5. Endogenous concentrations (pmol g−1 FW) of different forms of aromatic cytokinins in greenhouse acclimatized ‘Williams’ bananas (Musa spp. cv. AAA) in relation to the applied cytokinin types at 10 ␮M. a, b (BA) = Benzyladenine; c, d (mT) = meta-topolin; e, d (oT) = ortho-topolin; g, h (pT) = para-topolin; i, j (KIN) = Kinetin. mT, metaTopolin; mTR, meta-Topolin riboside; MemT, meta-Methoxy topolin; MemTR, meta-Methoxy topolin riboside; BA, Benzyladenine; MemTTHP, 6-(3-Methoxybenzylamino)9-tetrahydropyran-2-ylpurine.

Using Arabidopsis and tobacco as model plants, it has been established that CKs are synthesized in both shoots and roots, especially in actively dividing cells such as young leaves and root tips [32]. In the present study, significantly higher levels of endogenous CKs were detected in the underground region in the majority of the treatments (micropropagated and acclimatized plants). Increasing

evidence has strongly suggested the root region to be one of the primary sites of CK synthesis due to the presence of high levels of CK [10,26,33]. However, in relation to the concentration of the applied CK during micropropagation, there was a diverse response in quantity of endogenous aromatic CKs in the underground parts. While increasing concentrations of mTR had an inverse effect, increasing

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em

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Fig. 6. Endogenous levels (pmol g−1 FW) of the different structural and functional cytokinin forms in greenhouse-acclimatized ‘Williams’ bananas (Musa spp. cv. AAA) in relation to the applied cytokinin types at 10 ␮M. mT, meta-Topolin; mTR, meta-Topolin riboside; MemT, meta-Methoxy topolin; MemTR, meta-Methoxy topolin riboside; BA, Benzyladenine; MemTTHP, 6-(3-Methoxybenzylamino)-9-tetrahydropyran-2-ylpurine.

the concentration of MemTTHP and MemTR enhanced the level of quantified aromatic CKs. On the other hand, optimum levels of total endogenous CKs were obtained with 20 ␮M in mT, MemT and BA treatments (Table 1). The structural differences and/or similarities among the tested CKs may have accounted for the current observations. The presence of different classes of substituents (particularly on N9 position) on parent CK compounds could play diverse roles on the resultant biological activities [22,28,34,35]. Following

acclimatization of 10 ␮M CK treatments, BA-treated plants had the highest endogenous CK content which was approximately 15fold higher than the control plants. Conversely, MemTTHP had the lowest concentration, about 22-fold lower than the BA treatment (Table 2). It is apparent that the quality rather than the quantity of endogenous CK is important for successful acclimatization rates. A previous study showed that acclimatized MemTTHP-derived plants demonstrated superior morphological and physiological

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parameters compared to the BA-treated ones [17], with the 10 ␮M MemTTHP treatment generally having the best photosynthetic pigment stimulatory effect among all the tested CKs [16]. 4.2. Effect of aromatic cytokinins (CKs) on quantified endogenous isoprenoid and aromatic CK-types The aerial and underground parts of the micropropagated plantlets had cZ > iP > DHZ > tZ and cZ > iP > tZ > DHZ (Fig. 1), respectively in terms of abundance of isoprenoid CK. Following acclimatization, the trend was iP > cZ > tZ > DHZ in aerial parts and iP > tZ > cZ > DHZ in underground parts (Fig. 4). In terms of activity, iP and tZ which are often abundant in the phloem and xylem sap, respectively are considered to be the most active isoprenoid CKs. They play a vital role in the regulation of cell division, local acropetal and systemic long-distance signalling in planta [35]. Despite the increasing number of reports demonstrating the frequent occurrence and relatively high concentrations of cZ-type CK in both lower and higher plants [26,33,36,37], their precise role are still to be fully elucidated [37]. It has been hypothesized that cZ may play a role in the developmental stages associated with limited growth. The current findings show that in 10 ␮M mTR treatments, the level of cZ-type CK was about 250-fold higher than tZ in the underground parts. At higher concentrations, cZ elicit considerable biological activities which provide an indication of its potential to substitute for tZ [37]. In the presence of the increasing evidence of higher levels of cZ compared than tZ in micropropagated plants [10,26], it is logical to assume that cZ may play a vital role during micropropagation. It has also been established that cZ-type accounted for more than 50% of the total cytokinin pool in many monocotyledonous and dicotyledonous taxa [37] and is implicated in tuber dormancy regulation in potato [38]. The question thus remains, how do plants effectively cope with a high rate of cZ-type CK biosynthesis? Does the high level required to elicit biological activity drive their high biosynthesis rate in plants? Perhaps, cZ possess a longer life span compared to the tZ-type CKs. Further studies will be required to unravel physiological explanations for the intriguing traits of cZ-type CKs. The endogenous aromatic CK contents were grouped into five major CK-types. While similar abundance pattern of BA > mT > oT > KIN > pT was observed in aerial and underground parts of acclimatized plants (Fig. 5), the decreasing order was BA > mT > oT > KIN > pT in aerial parts and mT > BA > oT > pT > KIN was observed in the underground parts of the micropropagated plantlets (Fig. 2). The high accumulation of endogenous mT-type and BA-type CKs in response to exogenous application of mT (and derivatives) and BA, respectively is common [7,10,26,34,39]. In fact, the current findings have been postulated to be due to the existence of a unidirectional enzymatic pathway for the conversion of BA into topolins and their metabolites [7]. As demonstrated in the current study, high levels of endogenous oT and pT-type CKs associated with BA treatment have been observed in several plant species [7,10,26]. For some, yet unknown reasons, possibly related to CK metabolic pathways, the high endogenous oT and pT-type CK content are often absent in topolin treatments. More stringent experiments involving the use of radio-labelled CKs has been suggested as a possible approach to elucidate the biosynthetic pathways of oT and pT [26]. 4.3. Effect of aromatic cytokinins (CKs) on different endogenous CK-forms The levels of CKs are finely regulated by rates of biosynthesis, metabolic inter-conversions, inactivation, degradation and transport [40]. In plants, the concentrations of physiologically active CKs (free bases) are down-regulated mainly by side-chain

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cleavage with cytokinin oxidase/dehydrogenase, conjugation to glucose, either reversible (O- and 3N-glucosides) or irreversible (7N- and 9N-glucosides) forms [3,40,41]. When producing inactive CK storage forms, the deglucosylation reaction of O-glucosides is catalyzed by ␤-glucosidase when necessary [42]. The relative abundance of different CK-forms can vary greatly between plant species, tissues and developmental stages as well as environmental conditions [7,42]. In the current study, all the CK-forms detected were considerably lowered upon acclimatization. In both micropropagated (Fig. 3) and acclimatized (Fig. 6) plants, the deactivation/detoxicification CK-forms (9N-glucosides especially BA9G) were several fold higher than the active (free bases), precursor/transport (ribosides and ribotides) and storage (O-glucosides) forms. A possible explanation is that free bases and ribosides can be rapidly inactivated by conjugation [32]. These conjugates (9N-glucosides) are regarded as terminal products of the irreversible deactivation or a detoxification pathway and characterized by low bioactivity and high stability due to their resistance to de-glycosylating enzymes [3,41,43]. Recently, it was postulated that 9-glycosylation is the favoured deactivation pathway [44]. The authors reported that 9-glucosides were the prevalent (55% of the total CK pool) CK forms in non-treated germinating and thermo-inhibited Tagetes minuta achenes. As shown in the current study, the type and concentration of the applied CK contributed significantly to the concentration of 9-glucosides. Of significant physiological importance is the remarkable low level of 9-glucosides detected in underground parts of MemTTHP treatments during micropropagation and acclimatization. Conversely, as demonstrated in other studies, the use of BA for micropropagation is often linked to extremely high accumulation of 9-glucosides in (mainly in basal/underground region) regenerants [10,12,26,39]. 5. Conclusions The observed variation in the composition of quantified CK metabolites suggest that it is unlikely for all the CKs to be converted to a common metabolite. Better understanding of the effects of exogenous CKs on the endogenous CK pool and the resulting morphogenetic trends provides an avenue to manipulate the growth and development of regenerants. For instance, the use of lower concentration of MemTTHP readily elevates the levels of endogenous isoprenoid CK-types during micropropagation. The increased isoprenoid CK pool is possibly associated with enhanced in vitro rooting, phytochemicals and more efficient photosynthetic apparatus which allows for better acclimatization of micropropagated ‘Williams’ bananas. Findings from this study give valuable insights into the potential of the different aromatic CKs in micropropagation of ‘Williams’ bananas. Taken together, the current findings are useful for improving PTC protocols and controlling some PTC-induced physiological disorders related to endogenous CK concentrations. Nevertheless, further studies focusing on the enzymes involved in the CK metabolic processes will be required for the full elucidation of the physiology of micropropagated plants. It is necessary to highlight that by and large growth and development is a multifacet process which generally involves a whole lot of other complex factors beside the level of endogenous CKs. Acknowledgements AOA was supported by the Claude Leon Foundation, South Africa. We appreciate the financial support from the Centre of the Region Haná for Biotechnological and Agricultural Research, Palacky´ University (Grant no. ED0007/01/01) as well as the Operational Programme Education for Competitiveness - European Social Fund (project CZ.1.07/2.3.00/20.0165). This work was also co-financed by IGA of Palacky´ University (grant PrF 2013 012).

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