Plant Growth Regul (2011) 63:105–114 DOI 10.1007/s10725-010-9558-6
SI : TISSUE CULTURE
Changes in endogenous cytokinin profiles in micropropagated Harpagophytum procumbens in relation to shoot-tip necrosis and cytokinin treatments Michael W. Bairu • Ondrˇej Nova´k • Karel Dolezˇal • Johannes Van Staden
Received: 12 April 2010 / Accepted: 15 December 2010 / Published online: 1 January 2011 Ó Springer Science+Business Media B.V. 2010
Abstract Changes in cytokinin (CK) profiles and their physiological implications in micropropagated Harpagophytum procumbens [(Burch.) DC. ex Meisn.] tissues in relation to shoot-tip necrosis (STN) and CK treatments were studied. Total CK content was quantified in benzyladenine (BA)-treated necrotic and normal plantlets and in plantlets treated with the CKs BA, meta-topolin (mT) and meta-topolin riboside (mTR) with and without the auxin indole3-acetic acid (IAA). Generally necrotic shoots yielded more total CK compared to normal shoots. Cytokinin accumulation was higher at the basal section (basal [ middle [ top). Further analysis of the CKs based on structural and functional forms revealed excessive accumulation of 9-glucosides (deactivation products—toxic metabolites) and limited amounts of O-glucosides (storage forms—re-utilizable) in necrotic and BA-treated shoots compared to normal and topolin-treated cultures. The addition of IAA enhanced the formation of 9-glucosides in BA-treated cultures but reduced it in topolin-treated cultures. The symptom of STN could
M. W. Bairu J. Van Staden (&) Research Centre for Plant Growth and Development, School of Biological and Conservation Sciences, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa e-mail:
[email protected] M. W. Bairu e-mail:
[email protected] O. Nova´k K. Dolezˇal Laboratory of Growth Regulators, Palacky´ University & Institute of Experimental Botany AS CR, Sˇlechtitelu˚ 11, 783 71 Olomouc, Czech Republic e-mail:
[email protected] K. Dolezˇal e-mail:
[email protected]
therefore be attributed to conversion of active cytokinins to other forms such as 9-glucosides which are neither active nor reversibly sequestrated to active forms. Literature shows that metabolites like 9-glucosides of BA have a detrimental effect in plant tissue culture. Keywords Auxin cytokinin interaction Cytokinin purification and analysis Devil’s claw Glucosylation Harpagophytum procumbens Micropropagation Pedaliaceae Shoot-tip necrosis Abbreviations BA N6-benzyladenine BA9G N6-benzyladenine-9-glucoside BAR N6-benzyladenosine 0 BAR5 MP N6-benzyladenosine-50 -monophosphate CK Cytokinin cZ cis-zeatin cZ9G cis-zeatin-9-glucoside cZOG cis-zeatin-O-glucoside cZR cis-zeatin riboside cZR50 MP cis-zeatin riboside-50 -monophosphate cZROG cis-zeatin-O-glucoside riboside DHZ Dihydrozeatin DHZ9G Dihydrozeatin-9-glucoside DHZOG Dihydrozeatin-O-glucoside DHZR Dihydrozeatin riboside DHZR50 MP Dihydrozeatin riboside-50 -monophosphate DHZROG Dihydrozeatin-O-glucoside riboside iP N6-isopentenyladenine iP9G N6-isopentenyladenine-9-glucoside iPR N6-isopentenyladenosine 0 iPR5 MP N6-isopentenyladenosine-50 monophosphate
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IAC MRM MS medium mT mT9G mTOG mTR mTR50 MP mTROG oT oT9G oTOG oTR oTR50 MP oTROG pT pTOG pTR pTR50 MP pTROG STN tZ tZ9G tZOG tZR tZR50 MP tZROG UPLC
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Immunoaffinity chromatography Multiple reaction monitoring Murashige and Skoog (1962) basal medium meta-topolin meta-topolin-9-glucoside meta-topolin-O-glucoside meta-topolin riboside meta-topolin-50 -monophosphate meta-topolin-O-glucoside riboside ortho-topolin ortho-topolin-9-glucoside ortho-topolin-O-glucoside ortho-topolin riboside ortho-topolin-50 -monophosphate ortho-topolin-O-glucoside riboside para-topolin para-topolin-O-glucoside para-topolin riboside para-topolin-50 -monophosphate para-topolin-O-glucoside riboside Shoot-tip necrosis trans-zeatin trans-zeatin-9-glucoside trans-zeatin-O-glucoside trans-zeatin riboside trans-zeatin riboside-50 -monophosphate trans-zeatin-O-glucoside riboside Ultra performance liquid chromatography
Introduction Harpagophytum procumbens (Pedaliaceae), commonly known as Devil’s Claw, belong to some of the most studied medicinal plants with pharmacological activity analysis and clinical tests dating back to the 1960s. Extracts of tubers of Harpagophytum spp. are active in the treatments of degenerative rheumatoid arthritis, osteoarthritis, tendonitis, kidney inflammation and heart disease (Stewart and Cole 2005). Despite a wealth of knowledge on its pharmacology, very little published data is available on the growth and biology of this species. Attempts to propagate it from seeds failed due to low germination rates. Plants propagated by cuttings failed to produce primary roots resulting in a single harvest (Kathe et al. 2003). While attempting to develop a micropropagation protocol we encountered a serious problem of STN (Fig. 1). The development of plant tissue culture techniques and the utilization of plant hormones revolutionized the science of plant propagation. Among other things, the development of tissue culture (collective name used for in vitro culture of cells, tissues and organs) protocols relies largely on
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Fig. 1 Examples of normal (right) and severely affected necrotic shoots (left) of Harpagophytum procumbens plantlets. Note the basal callus-like tissue on the necrotic shoots
optimizing the type and concentrations of auxins and CKs. This is mainly due to the fact that plants respond differently to sub- or supra-optimal concentrations of plant growth regulators (PGR). This variation in response ranges from failure to grow, to various types of growth disorders (genetic and physiological) such as STN, hyperhydricity, diminished rooting, somaclonal variation and tissue browning. Shoot-tip necrosis is one of the most common problems in the micropropagation industry affecting a wide range of plant species. It is caused by many factors in the tissue culture system (Bairu et al. 2009a). Cytokinins are one of the main factors contributing to the problem of STN. There are, however, different opinions as to how they affect the problem which can be categorized into three groups. Some suggest that the cause is due to eliminating or reducing the concentration of CKs in the media (Kataeva et al. 1991; Piagnani et al. 1996). On the other hand there are reports to the contrary. Bairu et al. (2009b) for example, observed gradual recovery from necrotic symptoms when Harpagophytum procumbens cultures were transferred to cytokinin-free rooting medium. Others suggest that the effect of CKs on STN is influenced by the type and concentration of CKs used (Mackay et al. 1995; Bairu et al. 2009b) and genotype dependent responses (Grigoriadou et al. 2000). This investigation was aimed at providing a better explanation and understanding of the effect of CKs on STN. We undertook a metabolic study by analyzing CK profiles of micropropagated H. procumbens based on the following three hypotheses in two separate experiments; 1.
If STN is affected by CK metabolism, there must be variation in the CK profiles of normal and necrotic shoots of the various sections of the plant;
Plant Growth Regul (2011) 63:105–114
2.
3.
If STN is affected by the type of CK used, there must be variation in the CK profiles of plants treated with the different types of CK in relation to the untreated controls; and If the presence or absence of auxin in the multiplication medium affects STN, this might be reflected in a variation in CK profiles.
We acknowledge the fact that the cause and effect of the findings presented in this manuscript could have been better understood in conjunction with some growth and physiological data. Comprehensive growth and physiological data from the same experiment, however, can be found in our previous report (Bairu et al. 2009b). It is the observations of these experiments that led to the current analysis. Hence results are discussed aligned with previous findings on the same species.
Materials and methods Micropropagation conditions Maintenance cultures of H. procumbens, derived from nodal explants, were used as a source of samples for experiment one and as a source of explants for experiment two. Cultures were maintained by sub-culturing to fresh MS medium (Murashige and Skoog 1962) containing 2.5 lM of BA supplemented with 0.9% (w/v) agar and 3% (w/v) sucrose on a monthly basis. Cultures were kept in a growth room with cool white fluorescent tubes (Osram L75 W/20X) at a light intensity of 45 lmol m-2 s-1 and a temperature of 25 ± 1°C in a 16 h photoperiod. For details of the tissue culture protocol see Bairu et al. (2009b). Experiment one Jars containing one-month-old normal and necrotic cultures of H. procumbens were randomly taken from maintenance cultures. Whole plantlets were removed from the jars and the agar on the basal section of the plantlets removed. Plantlets for both normal and necrotic shoots were then cut into three sections (basal, medium and top). Sections were cut, frozen in liquid nitrogen and ground to fine powders using a mortar and pestle (one sample at a time). Ground samples (three replicates) were transferred to 1.5 ml Eppendorff tubes, frozen in liquid nitrogen and stored in -70°C until analysis. Experiment two To study the effects of CK types on CK profiles of H. procumbens, plants were cultured on MS medium containing 5 lM of BA, mT or mTR with and without IAA (2.5 lM). Two sets of controls, one with no PGR and one
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with 2.5 lM IAA were included. Medium supplements and growth conditions were the same as for the maintenance cultures. One-month-old whole-plant-samples were collected and prepared for CK analysis as above. Cytokinin purification and analysis For cytokinin purification, a modified method described by Faiss et al. (1997) was used. Deuterium-labelled CK internal standards (Olchemim Ltd, Czech Republic) were added, 1 pmol of each per sample, to check recovery during purification and to validate the determination (Nova´k et al. 2008). The samples were purified using a combination of cation (SCX-cartridge), anion [DEAE-Sephadex-C18-cartridge] exchangers and immunoaffinity chromatography (IAC) based on wide-range specific monoclonal antibodies against cytokinins (Nova´k et al. 2003). The eluates from the IAC columns were evaporated to dryness and dissolved in 20 ll of the mobile phase used for quantitative analysis. The samples were analysed by ultra performance liquid chromatography (UPLC) (Acquity UPLCTM; Waters, Milford, MA, USA) coupled to a Quatro microTM API (Waters, Milford, MA, USA) triple quadrupole mass spectrometer equipped with an electrospray interface. The purified samples were injected onto a C18 reversed-phase column (BEH C18; 1.7 lm; 2.1 9 50 mm; Waters). The column was eluted with a linear gradient (0 min, 10% B; 0–8 min, 50% B; flow-rate of 0.25 ml/min; column temperature of 40°C) of 15 mM ammonium formate (pH 4.0, A) and methanol (B). Quantification was obtained 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 cytokinin (Nova´k et al. 2008). Quantification was performed by Masslynx software using a standard isotope dilution method. The ratio of endogenous cytokinin to appropriate labeled standard was determined and used to quantify the level of endogenous compounds in the original extract, according to the known quantity of added internal standard (Nova´k et al. 2003).
Results and discussion Results of the CK analysis of the experiments are presented in Tables 1 and 2 respectively. Tables 3, 4, 5, 6, 7, and 8 are pooled from Tables 1 and 2 respectively to appraise the profiles of the specific CK groups and their metabolites (presented as the sum total) in the plant tissue in relation to STN and CK applications. To the best of our knowledge, this report is the first on endogenous CK analysis of H. procumbens. The type and
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Table 1 Cytokinins detected (pmol g-1 FW) after 4 weeks of growth in culture Cytokinins detected
Experiment one Nor. B
Nec. B
BA
121.32 ± 8.4
150.09 ± 9.2
BA9G
886.17 ± 104
1,223.6 ± 53.34
BAR
0.75 ± 0.04 0
1.65 ± 0.05
Nor. M
Nec. M
Nor. T
Nec. T
5.96 ± 0.79
6.55 ± 0.19
1.01 ± 0.04
8.01 ± 0.14
507.16 ± 41.12
544.65 ± 42.14
185.27 ± 13.54
203.26 ± 17.22
0.06 ± 0.008
0.05 ± 0.001
0.07 ± 0.004
0.13 ± 0.01
BAR5 MP
0.23 ± 0.08
0.22 ± 0.03
0.03 ± 0.006
0.03 ± 0.01
0.05 ± 0.01
0.02 ± 0.007
cZ
2.32 ± 0.1
2.72 ± 0.2
4.29 ± 0.07
5.26 ± 0.04
5.25 ± 0.24
7.13 ± 0.27
cZ9G
26.08 ± 2.09
32.19 ± 0.8
51.48 ± 2.53
46.85 ± 3.56
44.67 ± 1.96
cZOG cZR
2.03 ± 0.1 6.32 ± 0.3
4.05 ± 0.5 7.22 ± 0.3
4.06 ± 0.12 26.62 ± 0.15
5.87 ± 0.67 50.28 ± 3.59
4.07 ± 0.48 54.9 ± 0.74 14.26 ± 1.53
55.54 ± 4.7 3.13 ± 0.08 25.6 ± 0.75
cZR50 MP
2.01 ± 0.61
2.27 ± 0.58
11 ± 3.18
9.33 ± 2.24
19.66 ± 7.29
cZROG
3.57 ± 0.13
5.86 ± 0.72
6.2 ± 0.19
7.23 ± 0.2
9.35 ± 1.23
8.64 ± 0.91
DHZ
0.02 ± 0.002
0.03 ± 0.003
0.01 ± 0.001
0.01 ± 0.001
0.05 ± 0.002
DHZ9G
0.28 ± 0.03
DHZOG
0.12 ± 0.03
0.34 ± 0.04 \LOD
\LOD 0.37 ± 0.02 0.1 ± 0.02
0.36 ± 0.01 \LOD
0.28 ± 0.01
0.43 ± 0.02
0.03 ± 0.009
0.04 ± 0.01 0.45 ± 0.02
DHZR
0.12 ± 0.01
0.15 ± 0.01
0.17 ± 0.003
0.19 ± 0.005
0.33 ± 0.02
DHZR50 MP
0.04 ± 0.04
0.06 ± 0.05
0.04 ± 0.01
0.07 ± 0.009
0.13 ± 0.03
0.15 ± 0.04
DHZROG
0.26 ± 0.01
0.28 ± 0.001
0.14 ± 0.008
0.17 ± 0.01
0.38 ± 0.02
iP
0.13 ± 0.003
0.72 ± 0.01
0.32 ± 0.01
0.75 ± 0.03
iP9G
45.36 ± 2.61
49.11 ± 1.96
93.34 ± 0.15
105.95 ± 2.76
38.54 ± 1.33
81.93 ± 2.38
iPR
0.23 ± 0.02
0.15 ± 0.01
3.94 ± 0.09
4.27 ± 0.04
6.15 ± 0.32
7.92 ± 0.15
0
iPR5 MP
0.51 ± 0.007
0.03 ± 0.02
0.41 ± 0.12
0.26 ± 0.07
0.86 ± 0.19
0.58 ± 0.04
0.1 ± 0.004
0.19 ± 0.06
0.33 ± 0.01
0.05 ± 0.008
0.12 ± 0.01
0.09 ± 0.02
mT9G mTOG
3.82 ± 1.07 0.05 ± 0.007
5.09 ± 03 0.07 ± 0.001
0.6 ± 0.05 0.03 ± 0.009
0.06 ± 0.02 0.02 ± 0.004
0.11 ± 0.003 0.02 ± 0.005
0.18 ± 0.005 0.05 ± 0.007
mTR
0.05 ± 0.002
0.09 ± 0.002
0.03 ± 0.003
0.04 ± 0.004
0.02 ± 0.002
0.05 ± 0.001
mT
0
mTR5 MP
0.07 ± 0.02
0.13 ± 0.008
0.1 ± 0.006
\LOD
\LOD
\LOD
\LOD
mTROG
0.04 ± 0.007
0.07 ± 0.002
0.03 ± 0.004
0.05 ± 0.006
oT
0.26 ± 0.01
0.62 ± 0.04
0.51 ± 0.007
0.06 ± 0.01
13.45 ± 1.27
25.67 ± 1.82
1.57 ± 0.1
1.86 ± 0.15
oTOG
oT9G
0.03 ± 0.003
0.07 ± 0.02
0.01 ± 0.003
0.01 ± 0.001
oTR
0.17 ± 0.007
0.42 ± 0.03
0.08 ± 0.007
oTR50 MP
0.03 ± 0.007
0.04 ± 0.008
0.01 ± 0.008
oTROG pT pTOG pTR pTR50 MP
\LOD
\LOD
\LOD
0.04 ± 0.008
\LOD \LOD 0.03 ± 0.008 0.2 ± 0.009 \LOD 0.02 ± 0.002
\LOD 0.02 ± 0.001 0.06 ± 0.02 0.36 ± 0.02 \LOD 0.03 ± 0.007
\LOD
\LOD
\LOD
\LOD
\LOD
\LOD
0.13 ± 0.01
0.2 ± 0.04
0.09 ± 0.005
0.17 ± 0.02
0.05 ± 0.006
0.05 ± 0.006
0.7 ± 0.02
1.52 ± 0.28
0.43 ± 0.14
0.63 ± 0.13
0.17 ± 0.02
0.21 ± 0.03
0.4 ± 0.01 \LOD
0.72 ± 0.11 \LOD
0.27 ± 0.03 \LOD
0.53 ± 0.01 \LOD
0.15 ± 0.003 \LOD
0.21 ± 0.02 \LOD
pTROG
0.63 ± 0.05
1.1 ± 0.05
0.26 ± 0.03
1.01 ± 0.04
0.14 ± 0.008
0.26 ± 0.006
tZ tZ9G
0.1 ± 0 1.93 ± 0.1
0.11 ± 0 0.89 ± 0.05
0.67 ± 0 2.42 ± 0.05
1.68 ± 0.1 5.41 ± 0.05
1.31 ± 0.1 2.71 ± 0.15
2.95 ± 0 7.45 ± 0.03
tZOG
1.84 ± 0.04
1.81 ± 0.08
4.91 ± 0.16
7.06 ± 0.13
7.56 ± 0.5
9.4 ± 0.26 5.46 ± 0.06
tZR
0.36 ± 0.05
0.3 ± 0.06
1.11 ± 0.01
2.29 ± 0.07
2.8 ± 0.3
tZR50 MP
0.94 ± 0.2
2.5 ± 0.8
2.28 ± 0.7
1.58 ± 0.2
9.16 ± 2.9
1.97 ± 0.69
tZROG
1.27 ± 0.05
1.06 ± 0.03
1.07 ± 0.03
2.44 ± 0.04
1.34 ± 0.12
3.46 ± 0.11
Samples were taken from maintenance cultures grown on media containing 2.5 lM BA. Nor normal shoots, Nec necrotic shoots, B bottom part, M middle part, T top part of the plant. Results are mean ± SD, n = 3. \ LOD indicates values below the detection limit
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\LOD \LOD \LOD \LOD
\LOD
\LOD
\LOD
\LOD
mT
mT9G
mTOG
mTR
\LOD \LOD \LOD \LOD \LOD \LOD \LOD \LOD \LOD
\LOD
\LOD
\LOD \LOD
\LOD
\LOD
\LOD
\LOD
\LOD
oT
oTOG oTR
oTR50 MP
oTROG
pT
pTOG
pTR
mTROG
oT9G
\LOD \LOD
\LOD
\LOD
mTR5 MP
0
\LOD
\LOD
iPR50 MP
126.45 ± 8 7.88 ± 0.9
115.87 ± 1.6
6.66 ± 0.2
iP9G
0.76 ± 0.05
0.27 ± 0.02
0.043 ± 0.01 \LOD
0.035 ± 0.01
iPR
0.15 ± 0.04
0.04 ± 0.0 \LOD
DHZR DHZR50 MP
1.09 ± 0.07
\LOD
DHZOG
DHZROG
\LOD
0.44 ± 0.03
DHZ9G
iP
0.95 ± 0.1
0.04 ± 0.01
DHZ
42.03 ± 10.6 12.22 ± 2.7
30.14 ± 3.6
7.61 ± 1.8
cZROG
44.54 ± 6.6
7.44 ± 1.8
79.72 ± 14.7
5.27 ± 0.5
cZR50 MP
2.52 ± 0.6
35.24 ± 0.8
33.41 ± 3.8
cZ9G
cZOG
3.88 ± 0.2
cZ
cZR
\LOD
BAR5 MP
\LOD
\LOD
\LOD
0
BAR
2.46 ± 0.08 116.7 ± 13.8
1.31 ± 0.2
30.68 ± 0.9
BA
Control ? IAA
Control
Experiment two
BA9G
Cytokinins detected
0.69 ± 0.07
6.09 ± 0.28
0.34 ± 0.04
\LOD
\LOD
\LOD 0.24 ± 0.05
6.64 ± 0.8
0.22 ± 0.06
\LOD
\LOD
\LOD
\LOD
\LOD
\LOD
\LOD
4.76 ± 0.11
71.44 ± 0.9
0.39 ± 0.02
0.13 ± 0.02
0.34 ± 0.004 \LOD
\LOD
0.99 ± 0.04
19.40 ± 3.95
0.85 ± 0.23
\LOD
\LOD
\LOD 0.24 ± 0.06
9.13 ± 1.6
0.28 ± 0.03
\LOD
\LOD
\LOD
\LOD
\LOD
\LOD
\LOD
3.97 ± 0.1
53.88 ± 4
0.30 ± 0.01
0.09 ± 0.004
0.027 ± 0.003 \LOD
\LOD
\LOD
\LOD
\LOD
\LOD
\LOD \LOD
\LOD
\LOD
235.48 ± 17.7
32.63 ± 8
25.61 ± 2.3
830.54 ± 213.7
744.43 ± 111.4
81.51 ± 5.6
\LOD
3.97 ± 0.05
79.5 ± 7.8
0.36 ± 0.04
0.26 ± 0.05
0.044 ± 0.01 \LOD
\LOD
0.79 ± 0.2
\LOD
\LOD
\LOD \LOD
0.26 ± 0.08
\LOD
13 ± 1.7
24.85 ± 6.4
44.4 ± 2.36
7.39 ± 0.7
75.71 ± 13.7
3.13 ± 0.3
\LOD
\LOD
306.64 ± 23.5
1.55 ± 0.12
mT
11.45 ± 0.4
22.87 ± 4.2
33.67 ± 1.8
6.27 ± 0.5
24.04 ± 2.9
2.32 ± 0.1
\LOD
3.69 ± 0.03
6,948.43 ± 202.6
58.03 ± 3.9
BA ? IAA
12.20 ± 1.8
32.89 ± 9.8
40.46 ± 1.3
6.87 ± 0.4
43.72 ± 11.4
2.91 ± 0.02
\LOD
2.73 ± 0.08
5,478.66 ± 209.2
61.06 ± 2.7
BA
Table 2 Cytokinins detected (pmol g-1 FW) after 4 weeks of growth in culture
\LOD
\LOD
\LOD
\LOD
\LOD
\LOD \LOD
\LOD
\LOD
206.21 ± 24.4
39.73 ± 1.7
27.14 ± 2.8
816.93 ± 65.2
513.9 ± 9.5
85.65 ± 3.2
\LOD
3.35 ± 0.22
28.53 ± 1.5
018 ± 0.005
0.07 ± 0.02
0.03 ± 0.003 \LOD
\LOD
\LOD
\LOD
9.4 ± 1.2
19.87 ± 5.9
32.69 ± 2.08
5.22 ± 0.5
14.58 ± 1.8
2.07 ± 0.05
\LOD
\LOD
69.12 ± 5.6
0.54 ± 0.1
mT ? IAA
\LOD
\LOD
\LOD
\LOD
\LOD
\LOD \LOD
\LOD
\LOD
379.9 ± 17
71.12 ± 11.6
45.16 ± 4.4
1,262.4 ± 227
550.5 ± 31.5
114.27 ± 4.4
\LOD
5.6 ± 0.13
152.18 ± 10.5
0.41 ± 0.01
0.36 ± 0.08
0.07 ± 0.01 \LOD
\LOD
0.83 ± 0.2
\LOD
17.12 ± 2.5
31.88 ± 7.4
53.75 ± 2.05
11.37 ± 0.6
56.81 ± 9.9
3.82 ± 0.06
\LOD
\LOD
167.5 ± 7.08
1.40 ± 0.1
mTR
\LOD
\LOD
\LOD
\LOD
\LOD
\LOD \LOD
2.93 ± 0.8
\LOD
181.82 ± 19.4
31.08 ± 2.29
25.93 ± 2.3
664.73 ± 185.3
242.2 ± 20.6
53.34 ± 1
\LOD
5.34 ± 0.2
47.53 ± 3
0.31 ± 0.02
0.15 ± 0.003
0.04 ± 0.01 \LOD
\LOD
\LOD
\LOD
12.76 ± 1.1
22.05 ± 6.4
38.45 ± 1.02
7.41 ± 1.4
29.52 ± 4.4
2.33 ± 0.05
\LOD
\LOD
243.74 ± 20.9
2.52 ± 0.2
mTR ? IAA
Plant Growth Regul (2011) 63:105–114 109
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Whole plant samples were taken from the various cytokinin (5 lM) treatments with (2.5 lM) or without IAA as indicated. The control samples were from cytokinin-free media. Results are mean ± SD, n = 3. \ LOD indicates values below the detection limit
\LOD
2.51 ± 0.2 6.57 ± 0.9
\LOD \LOD
1.39 ± 0.1 5.01 ± 0.2
\LOD \LOD
1.79 ± 0.2 2.73 ± 0.3
\LOD
4.86 ± 0.3 tZROG
4.61 ± 0.4
\LOD tZR50 MP
\LOD
3.84 ± 0.2
1.99 ± 0.1 2.17 ± 0.2
11.16 ± 0.5 1.26 ± 0.3
0.77 ± 0.02 3.1 ± 0.1
7.1 ± 0.6 5.22 ± 0.6
1.38 ± 0.1 2.34 ± 0.1
12.32 ± 0.6 14.65 ± 4.2
2.82 ± 0.5
5.33 ± 1.02
2.45 ± 0.1
tZOG
tZR
0.73 ± 0.14
2.28 ± 0.5 7.06 ± 1.8
0.98 ± 0.14 0.32 ± 0.03
0.55 ± 0.1 7.24 ± 1.8
0.78 ± 0.09 0.75 ± 0.07
1.48 ± 0.5 2.93 ± 0.8
0.7 ± 0.06 1.28 ± 0.15 1.14 ± 0.04
5.65 ± 0.4
tZ
tZ9G
8.33 ± 1
\LOD \LOD
\LOD \LOD
\LOD \LOD
\LOD \LOD
\LOD \LOD \LOD \LOD pTROG
\LOD \LOD \LOD pTR50 MP
mTR mT ? IAA mT BA ? IAA BA Control ? IAA Control
Experiment two Cytokinins detected
Table 2 continued
123
\LOD
Plant Growth Regul (2011) 63:105–114
mTR ? IAA
110
Table 3 Total cytokinin pool (pm g-1 FW) on the different sections of normal (Nor.) and necrotic (Nec.) shoots Treatments
Aromatic CKs
Total CK
95.36
1,028.32
1,123.68
Nec. B
111.27
1,411.42
1,522.69
Nor. M
217.2
517.46
734.66
Nec. M
236.43
556.46
792.89
Nor. T
208.97
187.44
396.41
Nec. T
257.04
212.91
469.95
Nor. B
Isoprenoid CKs
B basal section, M middle section, T top section
Table 4 Total aromatic cytokinin pool (pm g-1 FW) on the different sections of normal (Nor.) and necrotic (Nec.) shoots Treatments
BA
mT
oT
pT
Total
Nor. B
1,008.47
4.05
13.94
1.86
1,028.32
Nec. B
1,375.56
5.5
26.83
3.53
1,411.42
Nor. M
513.21
1.01
2.18
1.05
517.45
Nec. M
551.36
0.79
1.97
2.34
556.46
Nor. T
186.39
0.27
0.25
0.52
187.43
Nec. T
211.33
0.39
0.43
0.73
212.88
B basal section, M middle section, T top section
concentration of the CKs detected varied among the treatments (Tables 1, 2). Samples from the maintenance cultures (cultured in 2.5 lM BA) yielded smaller total CK pools compared to samples from the CK treatments (cultured in 5.0 lM CKs). This indicates that exogenous application of CK does have an effect on the endogenous CK pool. The positive effects of applied CK on increasing the endogenous CK pool has been reported (Auer et al. 1999; Vandemoortele et al. 2001; Ivanova et al. 2006). This increase in total CK pool was mainly due to high levels of 9-glucosides and to a lesser extent on O-glucosides in topolin-treated cultures (Tables 5, 8). Of the isoprenoid type of CKs detected, cZ types were found in relatively high levels. Little or no ribotides were detected except for cZR50 MP in both experiments and mTR50 MP in experiment two. The variation in the cytokinin types detected could be due to the presence of more than one CK biosynthetic pathway (Taylor et al. 2003) being affected by the various CK applications. Higher levels of CKs were detected in necrotic tissues compared to the normal tissues in all three plant parts analysed. One would presume that this is due to the drier nature of necrotic shoots since quantification was made as pmol g-1 FW. This, however, was not the case as indicated by the basal section of the samples where there was no difference in moisture content (data not presented) but significant difference in the CK pool size. Cytokinin accumulation was significantly higher at the basal section
Plant Growth Regul (2011) 63:105–114 Table 5 Total cytokinin pool (pm g-1 FW) based on structural and functional forms on the different sections of normal (Nor.) and necrotic (Nec.) shoots
B basal section, M middle section, T top section
111
Treatments
Free base
Ribosides
Isoprenoid CKs
Control
256.52
124.38
8.4
10.54
977.09
3.32
1,123.73
10.7
15.89
1,336.89
5.12
1,522.69
Nor. M
12.36
31.27
16.27
661
11.77
732.67
Nec. M
14.5
34.11
22.65
710.34
11.01
792.61
Nor. T
8.1
59.81
24.97
273.96
29.86
396.7
Nec. T
19.09
69.07
26.53
338.28
16.98
469.95
Aromatic CKs 31.99
Total 288.51
359.3
119.16
478.46
237.39
5,556.72
5,794.11
BA ? IAA
169.92
7,041.15
7,211.07
mT
276.65
2,258.2
2,534.85
mT ? IAA
129.1
1,758.66
1,887.76
mTR mTR ? IAA
362.2 177.24
2,591.9 1,448.29
2,954.1 1,625.53
Table 7 Whole-plant aromatic cytokinin pool (pm g-1 FW) detected from the various treatments
Control
31.99
mT
oT
pT
\LOD
\LOD
\LOD
Total 31.99
\LOD
\LOD
\LOD
BA
5,542.5
\LOD
7.1
7.12
BA ? IAA
7,010
\LOD
9.65
21.2
7,040.85
1,950
\LOD
\LOD
2,258.2
1,689
\LOD
\LOD
1,758.66
Control ? IAA
mT mT ? IAA
119.16
308.2 69.66
Total
154.09
BA
BA
Ribotides
Nec. B
Control ? IAA
Treatments
9-Glucosides
Nor. B
Table 6 Whole-plant total cytokinin pool (pm g-1 FW) detected from the various treatments Treatments
O-Glucosides
119.16 5,556.72
mTR
168.94
2,423
\LOD
\LOD
2,591.94
mTR ? IAA
246.26
1,199.1
2.93
\LOD
1,448.29
\LOD indicates values below the detection limit
followed by the middle and top sections (Table 3). The presence of a larger CK pool in necrotic plants explains partly that the problem of STN in H. procumbens is associated with distribution rather than the availability of CKs in the shoots and/or in the medium. Shoot-tip necrosis in chestnut was controlled by localized application of CK to the shoot-tip (Vieitez et al. 1989; Piagnani et al. 1996) suggesting CK transport from the site of synthesis or storage to the meristematic tissues in necrotic plants as the limiting factor. Measures improving the uptake and transport of CK may therefore help alleviate the problem. In necrotic grape cultures for instance, axillary branching and vigorous rooting rendered STN relatively harmless (Thomas 2000). We have also observed (Bairu et al. 2009b) recovery from necrotic symptoms in H. procumbens
cultures in the presence of roots. These reports suggest that the development of new shoots and roots might have triggered some physiological processes in plants lacking roots and shoot-tips due to STN thereby reinstating the physiological cross talk including CK transport, metabolism and signaling between the different plant parts. Positive correlation between an higher CK pool and hyperhydricity has been reported (Kataeva et al. 1991; Ivanova et al. 2006). These authors attributed the effect to either overproduction of CK and/or their accumulation resulting from an inability to use or metabolize them due to failed physiological processes such as CK signaling. The presence of elevated levels of 9-glucosides in this study shows the latter to be a more likely explanation. The relatively high level of aromatic CKs detected (Tables 3, 6) could be the result of the exogenous application of CKs. The presence of the isoprenoid type of CKs (Tables 3, 6) in the samples indicates the presence of de novo CK synthesis by the plantlets. This was more evident in samples derived from media without exogenous CKs (Table 6) where the isoprenoid type of CKs was higher than the aromatic types. In the absence of roots, the presence of isoprenoid CKs suggests CK synthesis in shoots. Compared to the isoprenoid CKs, the amounts of non-BA aromatic CKs detected in the samples were very low (Tables 4, 7) which indicates a very low level of endogenous synthesis of topolins. Further observation of the CKs detected based on the various structural and functional groups revealed that necrotic samples consistently yielded more 9-glucosides compared to their normal counterparts in the three sections of the plants analysed (Table 5). Abnormalities in BAtreated tissue cultures have often been associated with a relatively high level of BA9G (Werbrouck et al. 1995; Bairu et al. 2007). This metabolite, the result of glucosylation of the adenine ring at the N9-position, is neither active nor readily convertible to other usable forms of CKs (Sakakibara 2006). Similar to Werbrouck et al. (1995) we detected an extremely high level of this metabolite in the basal portion of the plant. The amounts detected in the middle and upper sections of the plant were also consistently higher than the other CK forms viz free bases, ribosides, O-glucosides and ribotides (Table 5). Werbrouck
123
112
Plant Growth Regul (2011) 63:105–114
Table 8 Whole-plant total cytokinin pool (p mg-1 FW) based on structural and functional forms detected from the various treatments Treatments
Free bases
Ribosides
O-Glucosides
9-Glucosides
Ribotides
Total
Control
7.46
44.39
20.47
186.05
30.14
Control ? IAA
9.81
55.28
39.19
332.15
42.03
478.46
BA
65.62
51.32
40.34
5,603.69
32.89
5,793.86
BA ? IAA
62.5
44
44.25
7,036.51
22.9
7,210.16
mT
87.33
77.12
1,099.02
1,214.24
57.48
2,535.19
mT ? IAA
106.58
60.63
1,040.48
626.68
59.6
1,893.97
mTR
120.91
106.75
1,688.48
934.9
59.23
71.75
873.22
568.2
mTR ? IAA
et al. (1995, 1996) highlighted that the excessive presence of this metabolite coupled with its pronounced stability in plant tissue causes various disorders during acclimatization and rooting. The same could be said for STN in H. procumbens cultures with slightly different physiological processes. We assume that when plants were cultured in BAcontaining media, the majority of the BA undergoes Nglucosylation as part of detoxification/inactivation efforts. With time, when the plantlets need CK to elicit various physiological processes such as cell division, in the absence of O-glucosides, the available active forms (free bases and ribosides) may not be sufficient leading to cessation of cell division and necrosis of meristematic regions, resulting in STN. In a previous report (Bairu et al. 2009b) it was noted that plants treated with mTR showed reduced incidence of STN. To test one of our hypotheses, we analysed plants treated with various types of CKs to assess the various metabolites formed. Plants treated with topolins (mT and mTR) consistently yielded higher amounts of both the active forms (free bases and ribosides) and the storage forms (O-glucosides) compared to their BA-treated counterparts (Table 8). This would ensure sufficient active CK when needed. This result explains why the topolins consistently out-performed BA in our tissue culture experiments (Bairu et al. 2007, 2008, 2009b, c). Furthermore, high amounts of BA9G still persisted in topolin-cultivated explants even after 1 month of cultivation on BA-free media (Table 2). On the contrary, these explants contained only very low levels of BA itself and no BAR and/or BAR50 MP, highlighting the stability and/or extended effect of BA9G in plant culture systems. This scenario is in agreement with a previous report (Werbrouck et al. 1996). To ascertain this phenomenon experiments are in progress on cultures with no previous exposure to BA. It has been 52 years since Skoog and Miller (1957) demonstrated the significance of auxin:cytokinin ratios in organogenesis. Since then various experiments were done on the interaction of these two hormones. Coenen and Lomax (1997) concluded that these interactions could be
123
103 53.13
288.51
2,954.04 1,625.53
synergistic, antagonistic and/or additive. In this study the addition of IAA enhanced the formation of 9-glucosides in necrotic and BA-treated cultures but reduced it in topolintreated cultures. Better O-glucosylation was also observed in topolin-treated cultures when IAA was omitted (Table 8). The presence of an –OH group gives the topolins a structural advantage over BA since this allows them to form O-glucosides which can not occur with BA. The generally low levels of the free bases and ribosides can be attributed to them being actively utilized in the regulation of the cell cycle and various developmental processes (Schmu¨lling 2004). The addition of IAA also showed a marked differential effect on the total cytokinin pool; IAA-treated samples showed an increase in total cytokinin in BA-treated and the control samples but had an opposite effect on topolintreated samples (Table 8). Furthermore, there was a significant decrease of the isoprenoid cytokinin pool in all IAA-treated samples (grown on BA as well as on topolins) in comparison with non IAA-treated samples (Tables 2, 6). Auxin-mediated negative control of the CK pool size, by suppressing isopentenyladenosine-50 -monophosphate-independent biosynthetic pathways, was reported (Nordstro¨m et al. 2004). These authors also indicated the lateral roots to be the likely sites of isopentenyladenosine-50 -monophosphate-dependent CK synthesis. The ability of auxin to impose regulation of CK biosynthesis and the ability of these hormones to interact at metabolic level (Nordstro¨m et al. 2004) could also have attributed to the change in total CK pools between the treatments. In view of this report the increase in total cytokinin by the IAA-control plants compared to the controls without IAA, could likely be attributed to root development. Likewise a reduction in total CK by the topolin-treated samples when IAA was included could be due to this auxin-mediated negative control on the CK pool size. What caused an increase in total CK when IAA was included in BA-treated samples is subject to speculation. In our previous experiments on the same plant (Bairu et al. 2009b) an increase in basal-callus-like tissue (Fig. 1) was
Plant Growth Regul (2011) 63:105–114
observed when IAA was added to the multiplication medium. This phenomenon and STN were also more pronounced in BA-treated cultures in general and in BA-treated cultures containing IAA in particular. Based on growth parameters, we suspected that this callus-like tissue, by virtue of being a sink to essential growth requirements and/or an obstruction to root development might have caused physiological disorders. It was these observations that led us to the current study and it is on these grounds that we speculate that the physiological effect of IAA on BA-treated plants might have been altered due to other factors which are not known at the moment. Alternatively, it is possible that the following sequence of events could have occurred; the inability of BA to form O-glucosides coupled with the excessive deactivation of the active forms to BA9G caused a CK deficiency in the growing tissue unlike in the topolin-treated plants. This CK deficiency could have caused up-regulation of CK synthesis. But, since the presence of IAA aggravates the formation of basal callus-like tissue (a sink), the total CK (both the applied and synthesized) might have been trapped in this callus-like tissue making it unavailable to the plant. A possible repeat of these events could be the cause for the larger CK pool in BA-treated samples from IAA containing medium. This was more apparent from the relatively large quantity of BA9G in BA-IAA-treated samples compared to BA-treated samples (Table 2). However, increased CK biosynthesis is a less likely scenario since there was a consistent decrease in the isoprenoid CK pool size in the presence of IAA (Table 6). Instead, an increase in the CK pool of exogenous CKs and their direct metabolites in IAA-treated plants (Table 7) may suggest that the high CK pool size in BA-IAA-treated plants is the result of the sink effect of the basal callus-like tissue.
Conclusions Exogenously applied CKs affected STN by altering the endogenous CK pool. The change in endogenous CK pool affects physiological processes related to CK metabolism, signaling and transport. In H. procumbens the problem is aggravated by the formation by the plant of basal calluslike tissue which acts as a sink. This was evident by the over-accumulation of CKs in these tissues. The type of CK applied had an effect on the total CK pool. The presence of an hydroxyl group in topolins gives them a structural advantage over BA. This was reflected by the presence of a generous amount of O-glucosides in topolin-treated samples and hence little or no CK shortage. The contrasting effects observed when IAA was used shows the complexity of auxin-cytokinin interaction. Molecular characterization and explanation of these interactions are largely based on
113
Arabidopsis mutants. The application of such findings on plants with a more complex genome and physiology are a subject for debate. In the absence of roots, presence of a sink and poorly developed anatomical structures of in vitro plants, STN in H. procumbens may be attributed to a poor CK transport system in topolin-treated cultures and both CK metabolism and transport in BA-treated cultures. The presence of oT and pT and their derivatives on BA treated cultures needs further investigation. Moreover, the role of mineral elements such as calcium and boron which play an important role in auxin and CK transport and signaling should not be overlooked. Acknowledgments The financial support of the National Research Foundation-South Africa, the Grant Agency of the Czech Republic (GA 206/07/0570) and Czech Ministry of Education (MSM 6198959216, 1M06030). We also wish to thank Hana Martı´nkova´ for technical assistance.
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