Plant Cell Tiss Organ Cult (2014) 117:401–409

DOI 10.1007/s11240-014-0449-9

ORIGINAL PAPER

Effect of strigolactones and auxins on growth and metabolite

content of Sutherlandia frutescens (L.) R. Br. microplants in vitro
Maria C. Grobbelaar • Nokwanda P. Makunga •

Marietjie A. Stander • Jens Kossmann •

Paul N. Hills

Received: 13 September 2013 / Accepted: 9 February 2014 / Published online: 21 February 2014
Ó Springer Science+Business Media Dordrecht 2014

Abstract The influence of strigolactones as hormones in
plants is not fully characterised even though they are
known to affect plant architecture, both above ground and
in the roots. Using an in vitro system, the effects of the
synthetic auxins 1-naphthalene acetic acid and indole-3-
butyric acid (NAA and IBA) and synthetic strigolactones
(GR24 and Nijmegen-1) were tested on microplant devel-
opment of Sutherlandia frutescens, a leguminous medicinal
plant native to South Africa. Considerable phytochemical
variation in wild populations has led to the proposal of
using micropropagation for this species. This will assist
with domestication and provide plants with a more pre-
dictable chemistry for the phytopharmaceuticals industry.
Nodal explants with an axillary bud were grown on Mu-
rashige and Skoog (Plant Physiol 15:473–497, 1962)
medium [0.8 % (m/v) agar (pH 5.8), 3 % (m/v) sucrose and
0.1 g/L myo-inositol] supplemented with NAA, IBA, GR24
and Nijmegen-1, either singly or in combination. The
Electronic supplementary material The online version of this
article (doi:10.1007/s11240-014-0449-9) contains supplementary
material, which is available to authorized users.
M. C. Grobbelaar  N. P. Makunga  J. Kossmann 
P. N. Hills (&)
Institute for Plant Biotechnology, Department of Genetics,
Stellenbosch University, Private Bag X1, Matieland 7600, South
Africa
e-mail: phills@sun.ac.za
N. P. Makunga (&)
Department of Botany and Zoology, Stellenbosch University,
Private Bag X1, Matieland 7600, South Africa
e-mail: makunga@sun.ac.za
M. A. Stander
Department of Biochemistry, Stellenbosch University,
Matieland 7600, South Africa
amino acid profile and secondary metabolite pool was
monitored using LC–MS-profiling. Treatment with NAA
promoted mass shoot production, whilst a combination of
NAA and Nijmegen-1 also positively influenced the
accumulation of amino acids, flavonoids (sutherlandins)
and terpenoids (sutherlandiosides) that S. frutescens pro-
duces. Since these compounds represent the presumed
active compounds in this species and the biomarkers used
in quality control assessment of S. frutescens tissues har-
vested for the pharmaceutical industry, this treatment holds
promise for the commercial production of Sutherlandia
extracts and herbal medications.
Keywords Amino acids  GR24  IBA  NAA 
Nijmegen-1  Polyamines  Sutherlandiosides
Abbreviations
GABA c-Aminobutyric acid
IBA Indole-3-butyric acid
LC–MS Liquid chromatography–mass spectrometry
MS Murashige and Skoog (1962)
NAA 1-Naphthalene acetic acid
SU1 Sutherlandioside B
Introduction
Sutherlandia frutescens (syn. Lessertia frutescens; Legu-
minosae) has historically been used to treat a broad spec-
trum of ailments in folk medicine including certain
cancers; hence the common name of ‘kankerbos’ (in
Afrikaans) or cancer bush. Van Wyk and Albrecht (2008)
reviewed the history and ethnobotanical importance of this
plant, noting that it is also used as a supportive therapy for

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those suffering from HIV/AIDS to boost the immune sys-
tem and prevent muscle wasting. Extracts have shown
antiproliferative effects on human breast and leukemia
tumor cell lines in vitro (Tai et al. 2004). Several other
studies have focused on the effects of plant extracts on
cancer cell lines, including work on apoptosis of Chinese
Hamster Ovary (CHO) cells and neoplastic cells (cervical
carcinoma) (Chinkwo 2005), an oesophageal cancer cell
line (Skerman et al. 2011) and tumorigenic breast adeno-
carcinoma cells (Stander et al. 2009). The precise com-
pounds responsible for the antiproliferative effects are not
clear. Sutherlandia contains a wide range of potentially
therapeutic primary and secondary metabolites, which are
generally produced and stored in the leaves. Both free and
non-proteinogenic amino acids, particularly arginine,
asparagine, proline, canavanine and c-aminobutyric acid
(GABA), have been implicated in the health-benefitting
effects of traditional extracts. Mncwangi and Viljoen
(2012), for example, emphasised the importance of
assessing the amino acids of Sutherlandia extracts for
quality assurance purposes. Recently, however, this activ-
ity has been suggested to be attributed mostly to the unique
terpenoids and flavonoids (sutherlandiosides and suther-
landins; respectively) that are solely produced by Suther-
landia (Van Wyk and Albrecht 2008; Albrecht et al. 2012).
A variety of metabolomic platforms have been used, in a
growing body of literature, to monitor the phytochemical
constituents of Sutherlandia populations. Mncwangi (2009)
used high performance liquid chromatography (HPLC),
high performance thin layer chromatography (HPTLC),
near infrared spectroscopy (NIRS) and Fourier transform
infrared (FT-IR) spectrometry to assess wild Sutherlandia
populations for chemotypic variation. Considerable varia-
tion amongst bioactive compounds is evident within and
between populations (Mncwangi and Viljoen 2012). Suth-
erlandia is regarded as an adaptogen and chemical heter-
ogeneity of populations is expressed in a wide range of
metabolites including amino acids, vitamins, flavonoids
and terpenoids, which may alter considerably under dif-
ferent environmental conditions. The amino acid and
polyamine content of plants can give an indication of the
physiological status of the plant. This includes amino acids
such as proline, arginine, alanine, asparagine, aspartate,
glutamate and serine (Rai 2002). Ornithine, too, is involved
in the production of polyamines, which are important in
growth and development of plants (Hunter and Burritt
2012) as well as stress tolerance (Edreva 1996; Kuznetsov
and Shevyakova 2007). This emphasises the need to grow
Sutherlandia plants that are to be used for therapeutic
extracts under controlled conditions to limit the influence
of the environment on the metabolic pool, in order to
obtain a more predictable phytochemical content. For the
development of quality medicines and consumer products
123
Plant Cell Tiss Organ Cult (2014) 117:401–409
from this species, it should become imperative and routine
to assess and quantify biomarker compounds after propa-
gation for commercial purposes. This is also important as a
measure of the safety and efficacy of preparations made
from cultivated Sutherlandia plants.
Similar changes in metabolites can be induced through
the application of phytohormones and these shifts can be
exploited using metabolomics tools. In this study, the
effects of exogenously applied synthetic strigolactones
(GR24 and Nijmegen-1) and auxins [indole-3-butyric acid
(IBA) and 1-naphthalene acetic acid (NAA)] on S. frutes-
cens microplants were investigated to monitor possible
changes in plant growth and metabolism using a tissue
culture system. Strigolactones present a new class of
commercially useable growth regulating substances that
may be applied in the farming of medicinal plants. Gen-
erally, strigolactones control above-ground growth through
their involvement in shoot apical dominance via the inhi-
bition of shoot branching of a variety of plant species
(Gomez-Roldan et al. 2008; Umehara et al. 2008). Below
ground, strigolactones stimulate interactions with symbi-
otic arbuscular mycorrhizal fungi (Besserer et al. 2006) as
well as the germination of parasitic seeds (Matusova et al.
2005). Root architecture is also affected by strigolactones,
although the effects may vary under different conditions.
Under optimal conditions, strigolactones generally reduce
lateral root formation and increase the length of root hairs
in Arabidopsis (Kapulnik et al. 2011a, b). Under subopti-
mal conditions, however, strigolactone levels rise (Kohlen
et al. 2011) and positively affect root growth, including
lateral roots (Ruyter-Spira et al. 2011) and root hairs
(Mayzlish-Gati et al. 2012), thereby increasing the root
area for phosphate absorption. Exudation of strigolactones
into the soil further aids in phosphate acquisition through
the promotion of mycorrhizal associations (Yoneyama
et al. 2012).
This study aimed at testing the effects of auxins and
synthetic strigolactones on explant growth and metabolite
production of Sutherlandia microplants in a tissue culture
environment. Auxin and strigolactones were tested in
conjunction and alone, because it is believed that strigo-
lactones act as a secondary messenger to auxin to inhibit
axillary bud outgrowth of plants (Brewer et al. 2009). We
further hypothesised that strigolactones, since they can
adapt plant growth to suit specific conditions such as
phosphate deficiency, may also be able to cause modifi-
cations in the levels of medicinally important metabolites.
Tissue culture systems offer the potential of producing
high-quality plant material (with a predictable metabolite
profile) in large volumes. Metabolites generally used as
quality assessment biomarkers, namely sutherlandioside B
(SU1), amino acids and polyamines, were analysed via a
broadscale metabolomic profiling approach.
Plant Cell Tiss Organ Cult (2014) 117:401–409 403

a
Stem length (mm) vessels (90 9 50 mm) containing MS medium. Various
70 de e e de combinations of auxins and strigolactones were tested for
cde
60 bcd bcd
50 ab ab ab a
abc
a abc ab their effects (Fig. 1). All media were autoclaved at 121 °C
40
30 and 103 kPa for 20 min. Auxins (IBA and NAA, Sigma,
20 Germany) were added to the relevant media at 0.1 mg/L
10
0 (0.49 lM IBA; 0.54 lM NAA) and 1 mg/L (4.9 lM IBA;
5.4 lM NAA) prior to autoclaving, whilst the strigolac-
tones, 0.1 lM GR24 or Nijmegen-1, were filter sterilised
and added to their respective media after the media had
Treatment (mg/L) been autoclaved. Both synthetic strigolactones were pur-
b chased from Prof. Binne Zwanenburg, Radboud University,
Number of leaves

7 g
6 defg bcde efg fg cdefg defg abcd bcdefg Nijmegen, The Netherlands. Control media were devoid of
ab abcde abcd
5 a ab abc plant growth regulators. All cultures were maintained for
4
3 28 days under a 18/6 h light–dark photoperiod (light
2
1 intensity of 50 lmol/m2/s using two Osram L58 W/640
0
Energy Saver cold fluorescent lamps) at 25 ± 3 °C. Mi-
croplants were then removed from the treatments and the
stem elongation, fresh and dry mass were measured
(Fig. 2). The number of new leaves produced per explant
Treatment (mg/L)
was recorded. Plant material was frozen in liquid nitrogen
c (-196 °C) and freeze-dried for 24 h, then stored at -80 °C
30 gh h
Dry mass (mg)

25 efg cdef fgh until metabolite analysis.

20 abcd bcde abcde bcde defg abcde
abcab a a
15
10 Extraction of metabolites
5
0
Freeze-dried material was ground to a fine powder with a
mortar and pestle and 0.05 g tissue used for metabolite
extraction. Three different extraction solvents for the
Treatment (mg/L) amino acids were tested in triplicate: (1) acetoni-
trile:water:formic acid (50:50:0.1 v/v); (2) methanol or (3)
Fig. 1 Effect of strigolactones and/or auxin on in vitro growth of 0.1 % formic acid and 1 mL of each solvent was used.
Sutherlandia frutescens after 4 weeks. The auxins were added at
either 0.1 mg/L (0.49 lM IBA; 0.54 lM NAA) or 1 mg/L (4.9 lM
After sonication using an ultrasonic bath (Branson B220H)
IBA; 5.4 lM NAA), strigolactones GR24 and Nijmegen-1 (NM-1) at for 60 min, the tubes were centrifuged for 10 min at
0.1 lM each. Controls (untreated) were grown in medium without maximum speed in a desktop microfuge at room temper-
hormones. The bars represent the mean ± standard error (n = 30) ature. The supernatant was then transferred to a clean
and different letters indicate a statistical significant difference at the
95 % confidence level. a Stem length (mm); b number of leaves;
microfuge tube and the samples stored at -20 °C until
c dry mass (mg) analysis.

Sample preparation for amino acid and polyamine

Materials and methods analysis

Plant material and in vitro culture conditions
Nodal explants with an axillary bud from a continuous
in vitro S. frutescens plantlet culture established by Colling
et al. (2010) were placed in full strength Murashige and
Skoog (1962) medium solidified with 0.8 % (m/v) agar–
agar (pH 5.8, Biolab, Merck, South Africa) containing 3 %
(m/v) sucrose and 0.1 g/L myo-inositol (Sigma, Germany).
Conditions were kept similar to those described by Colling
et al. (2010) for the continuous culture system.
Briefly, ten nodal explants of approximately 2 cm in
length and containing an axillary bud were placed in glass
Methanol was by far the least effective of the three solvents
tested for extraction efficiency; methanol, 0.1 % formic
acid and 50 % acetonitrile, 0.1 % formic acid, with the
latter two solvents producing similar results. For further
amino acid analysis, the combination of acetonitrile and
formic acid was used as the extractant. Samples were
derivatised using the Waters AccQ Tag Ultra Derivatiza-
tion Kit, according to manufacturer’s instructions (Cohen
and Michaud 1993) (Waters, Milford, MA, USA). All
samples were analysed twice, initially undiluted and then
diluted 100 times, in order to quantify the amino acids that
occurred at higher abundance. Norvaline was added as

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404 Plant Cell Tiss Organ Cult (2014) 117:401–409

a 200 c b 4.50
180 4.00 a
160 ac a a

µg SU1 / explant
a 3.50
a
µg SU1/ g DM

140 3.00
120 ab
2.50
100 b c
2.00
80 bc
60 1.50 b
40 1.00
20 0.50
0 0.00
Untreated GR24 NM 1 1NAA 1NAA + 1NAA+ Untreated GR24 NM 1 1NAA 1NAA + 1NAA+
GR24 NM 1 GR24 NM 1
Treatment (mg/L) Treatment (mg/L)

Fig. 2 a Amount of sutherlandioside B (SU1) represented as micrograms per gram of dry mass plant material (data are mean ± SE; N = 5);
b Amount of sutherlandioside B (SU1) in an average 4 week old tissue culture plant. Data are mean ± SE; N = 24. NM-1- Nijmegen-1

internal standard to a final concentration of 2 ppm. The had a linear range of 0–50 ppm (R2 = 0.998) with a
relative standard deviations (RSD) for the different amino detection limit of 10 ppb.
acids were all below 7 %, with the lower abundance amino
acids having slightly higher RSDs. The response of the
General amino acid and polyamine analysis
different derivatised amino acids were very similar on the
PDA detector and the limit of detection was 1 pmol on
LC–MS analysis was performed on a Waters API Quattro
column, although the lowest standard was 10 pmol and it
Micro triple quadrupole mass spectrometer (Milford, MA,
was therefore used as limit of quantitation.
USA linked to a Waters Acquity UPLC and Acquity PDA
detector. Electrospray ionisation was applied in the positive
mode, cone voltage of 15 V and capillary voltage of
3.5 kV. Nitrogen was employed as the desolvation gas (rate
General metabolite and sutherlandioside B analysis
of 350 L/h; desolvation temperature of 350 °C). An
injection volume of 1 lL was introduced into a Waters
Filtered samples were diluted ten times and an injection
AccQ Tag C18 column (2.1 9 100 mm, 1.7 lm particle
volume of 3 lL was injected into the LC–MS system. The
size). The derivatives were eluted with dilutions of Waters
LC–MS analysis was conducted on a Waters Synapt G2
AccQ Tag Ultra Eluent A and Waters AccQ Tag Ultra
quadrupole time of flight mass spectrometer (Milford, MA,
Eluent B prior to them being quantitated using UV detec-
USA), connected to a Waters Acquity ultraperformance
tion (255 nm). The mass spectrometer was set to scan from
liquid chromatograph (UPLC) and Acquity photo diode
m/z 200 to 600 and was used as a confirmation tool to
array (PDA) detector. Ionisation was achieved using elec-
verify the molecular weights of the various amino acids
trospray ionisation in the positive mode source, cone
and to ensure that the UV detector was selective for the
voltage of 15 V and capillary voltage of 2.5 kV. Nitrogen
various analytes and there was no co-elution.
was used as desolvation gas at 650 L/h and the desolvation
A gradient was set up and initiated by combining
temperature was set to 275 °C. A Waters UPLC BEH C18
99.9 % eluent A and 0.1 % eluent B (holding time of
column (2.1 9 50 mm, 1.7 lm particle size) was used. The
0.54 min). A linear gradient to 21.2 % eluent B over
gradient started with 100 %, 0.1 % formic acid (solvent A),
7.2 min, to 90 % eluent B over 0.31 min, to 100 % eluent
holding at 100 % solvent A for 0.5 min, followed by a
B over 0.45 min then followed. The column was kept at
linear gradient to 22 % acetonitrile (solvent B) over
100 % eluent B for another 1 min to yield a total run time
2.5 min, to 44 % solvent B over 4 min and to 100 % sol-
of 9.5 min. Solvent flow rate was maintained at 0.7 mL/
vent B over 5 min. The column was kept at 100 % solvent
min throughout the run.
B for another 2 min, followed by re-equilibration over
1 min for a total run time of 15 min. A flow rate of 0.4 mL/
min was applied. Sutherlandioside B (SU1) was quantified Data collection and statistical analysis
for each sample using an SU1 standard (kind gift from
Professor Albrecht; Cancer Association of South Africa). All growth experiments were repeated three times. Quan-
This standard was isolated by V. Gabrielse (refer also to titative analysis is represented as mean values and the
Van Wyk and Albrecht 2008). The SU1 calibration curve reproducibility of the results is conveyed as standard error.

123
Plant Cell Tiss Organ Cult (2014) 117:401–409
The normality of the data was determined using a Shapiro–
Wilk’s W test. A Post Hoc Fisher LSD test was performed
for normally distributed data and a Kruskal–Wallis test was
performed for all data that were not normally distributed.
Differences between means that reached a minimal confi-
dence level of 95 % were considered as being statistically
significant. All data were analysed using Statistica Release
8 (Statsoft Inc. 2007).
For the metabolite analysis, five samples were used to
measure the SU1 content in the shoots. The MZmine 2
(version 2.9.1) software package was used for the pro-
cessing, visualisation and analysis of raw LC–MS data
(Pluskal et al. 2010). A principal component analysis
(PCA, Smith 2002) was performed to identify patterns in
the LC–MS data and to highlight the differences and
similarities of this data. A PCA-analysis was also per-
formed on the amino acid content of the samples using the
LatentiX data analytical software version 2.11 (Latent5
2012).
Results and discussion
The effects of strigolactones in plants remain poorly
defined and it is not known whether observed effects are
universal across plant species. In this study, strigolactones
alone had no significant effect on explant elongation
(Fig. 1a). Treatment with 1 mg/L IBA, 0.1 mg/L NAA and
1 mg/L NAA caused significant elongation of the micro-
plants compared with the control (Fig. 1a). The addition of
strigolactones to the auxin generally reversed this elonga-
tion to control levels, except in the presence of 1 mg/L
NAA, where elongation was equivalent to samples treated
with only the NAA. Achieving stem elongation with auxin
application depends on the amount of auxin that is supplied
to the plant (Yang et al. 1993) as supra-optimal exoge-
nously-applied synthetic auxins may negatively influence
cell elongation. Dose–response curves of auxin have been
developed and differ slightly between plant species (Yang
et al. 1993).
Only GR24 had a significant effect on the production of
leaves by the explants. Whether applied alone or in com-
bination with auxins, GR24 significantly altered the leaf
number to less than that of the control (Fig. 1b). Only in
the presence of 1 mg/L NAA was the inhibitory effect of
GR24 ameliorated. Nijmegen-1 caused no effects regarding
leaf number, either when this growth regulator was used
alone or when combined with auxins (Fig. 1b). Unlike
GR24 and all known naturally-occurring strigolactones,
Nijmegen-1 has an open C-ring. In studies on the induction
of suicidal germination of Striga seeds by various strigo-
lactones, Nijmegen-1 was shown to be much less effective
than GR24 at promoting germination (Wigchert et al.
405
1999). This reduced biological activity may also explain
why Nijmegen-1 had less of an effect on leaf production
than GR24.
Neither GR24 nor Nijmegen-1 had any significant effect
on dry biomass of the microplants (Fig. 1c). Significantly
higher fresh and dry masses were recorded when plants
were grown in medium supplemented with 1 mg/L IBA or
1 mg/L NAA. The addition of either strigolactone to the
IBA medium reversed the mass increases to control levels,
whilst 1 mg/L NAA promoted biomass production whether
applied alone or in combination with either GR24 or Nij-
megen-1 (Fig. 1c). Measurements of fresh biomass showed
similar trends (data not shown).
By themselves, strigolactones had minimal effects on
growth, with only GR24 having a negative effect on leaf
number. In the presence of IBA, however, the strigolac-
tones tended to reverse the promontory effects of the auxin,
although growth was not reduced below that of the control
explants. Strigolactones are known to inhibit auxin trans-
porting PIN proteins (Lazar and Goodman 2006). NAA is
able to move more easily through the cell wall via diffusion
than IBA, which is converted in vivo to indole acetic acid
(IAA) that moves only via PIN-directed auxin transport and
that appears to use different auxin influx and efflux carriers
than NAA (Normanly et al. 1995; Rashotte et al. 2003;
Campanoni and Nick 2005). This therefore suggests a
possible explanation for the negative effects of the strigo-
lactones on IBA-induced growth, whereby the strigolac-
tones inhibit IBA uptake to a greater extent than NAA
uptake, which may still proceed via diffusion in the
absence of PIN-directed auxin transport.
Sutherlandioside B (SU1) is an important terpenoid
compound in S. frutescens and is now postulated to be one
of the sutherlandiosides responsible for the anti-cancer
properties of Sutherlandia extracts (Van Wyk and Albrecht
2008). The strigolactones tested had different effects on
SU1 levels in the microplants. GR24 treatment resulted in
lower SU1 levels than the control, whilst Nijmegen-1
treated microplants had similar levels to the controls. NAA
treatment alone was also similar to the controls, but in
combination with Nijmegen-1 resulted in a significant
increase in SU1 levels (Fig. 2). In vitro plants growing in
medium with NAA and Nijmegen-1 had 3.6 lg SU1 per
plant, indicating a doubling of the SU content compared to
control plants (1.8 lg SU1 per plant; Fig. 2b). In order to
obtain higher levels of SU1, supplementation of the in vitro
growth medium with Nijmegen-1 and NAA proved the
most promising for the development of plants that may be
used for the production of Sutherlandia phytopharmaceu-
ticals. This, however, remains to be tested in an ex situ
environment.
Using NAA in conjunction with Nijmegen-1 not only
increased the amount of SU1 (Fig. 2) but also increased the
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123
Table 1 Amino acid content per gram dry mass of Sutherlandia frutescens in vitro-grown leaf and stem explant material
Amino acid Untreated GR24 Nijmegen-1 NAA NAA ? GR24 NAA ? Nijmegen-1

Alanine 1,674.94 ± 262.32 ab 782.48 ± 84.55a 1,223.23 ± 394.82ab 1,688.12 ± 480.27ab 1,617.90 ± 415.17ab 2,072.62 ± 420.25b
L-Arginine 49,457.19 ± 6,941.46a 69,617.66 ± 12,065.04a 68,642.52 ± 10,568.77a 57,786.74 ± 8,129.30a 57,144.15 ± 6,803.72a 137,646.18 ± 16,363.50b
L-Asparagine 8,034.00 ± 674.11c 9,124.00 ± 489.78ac 9,687.50 ± 389.77ab 9,802.00 ± 420.72ab 9,970.00 ± 384.90ab 10,522.00 ± 424.56b
Aspartate 1,336.85 ± 140.08a 1,709.48 ± 246.63a 1,839.55 ± 250.75a 1,368.33 ± 172.89a 1,127.08 ± 79.95a 3,505.25 ± 403.14b
L-Canavanine 12,742.00 ± 2,495.62ab 12,272.00 ± 2,506.68ab 17,577.50 ± 891.48b 9,284.00 ± 1,325.84a 11,322.00 ± 1,226.04a 10,398.00 ± 1,707.33a
Cysteine 27.11 ± 4.45b 80.47 ± 23.00a 84.86 ± 16.20a 48.47 ± 8.50ab 55.47 ± 10.09ab 144.85 ± 25.56c
GABA 840.00 ± 137.62a 786.00 ± 222.21a 1,240.00 ± 876.86a 650.00 ± 102.76a 762.00 ± 237.71a 548.00 ± 117.19a
Glutamate 4,427.60 ± 280.62a 4,670.16 ± 469.41a 4,738.21 ± 250.28a 4,542.56 ± 438.75a 3,480.15 ± 207.03a 10,066.99 ± 854.41b
Glycine 63.65 ± 12.84a 88.44 ± 8.15ab 173.06 ± 92.89bc 84.35 ± 8.53ab 67.12 ± 5.00a 200.58 ± 15.35c
Histidine 1,617.52 ± 227.32a 1,841.63 ± 366.10a 2,130.38 ± 248.07a 1,829.25 ± 276.38a 2,176.23 ± 334.15a 4,291.14 ± 453.34b
Isoleucine 245.67 ± 23.66a 262.01 ± 57.38a 494.32 ± 145.89b 223.96 ± 20.29a 215.62 ± 20.22a 643.99 ± 73.63b
Leucine 1,137.67 ± 157.22ab 1,247.13 ± 206.46b 1,160.57 ± 112.49ab 1,079.42 ± 114.50ab 764.34 ± 131.55a 2,623.14 ± 125.93c
Lysine 134.33 ± 11.45a 192.22 ± 38.75ab 404.54 ± 227.84b 121.63 ± 13.46a 123.96 ± 10.64a 339.66 ± 30.63ab
Methionine 11.97 ± 4.69a 12.44 ± 3.10a 11.30 ± 2.90a 3.04 ± 1.95a 7.54 ± 3.52a 26.47 ± 2.56b
Phenylalanine 256.40 ± 44.45a 284.59 ± 55.06a 555.31 ± 222.27bc 309.26 ± 27.45ab 248.34 ± 29.96a 667.65 ± 44.69c
Proline 8,324.99 ± 1,727.30a 12,147.05 ± 1,503.02a 10,898.66 ± 1,555.59a 12,725.00 ± 1,316.91a 13,251.95 ± 1,410.53a 31,413.12 ± 3,675.79b
Serine 2,265.54 ± 141.59a 2,647.90 ± 510.87a 3,208.74 ± 287.85a 2,393.68 ± 227.73a 2,394.64 ± 233.26a 5,887.76 ± 500.49b
Threonine 3,224.92 ± 693.77a 3,468.07 ± 1,105.83a 5,191.39 ± 809.08ab 2,777.34 ± 398.54a 2,789.10 ± 372.49a 7,046.54 ± 1,355.66b
Tyrosine 11.62 ± 2.49a 16.78 ± 3.73ab 18.72 ± 3.50ab 12.27 ± 3.16a 14.26 ± 5.65ab 31.46 ± 11.75b
Valine 462.70 ± 42.64a 426.47 ± 75.44a 777.39 ± 239.62bc 486.96 ± 51.74ab 484.59 ± 46.21ab 1,045.49 ± 69.03c
Strigolactones GR24 and Nijmegen-1 were added at 0.1 lM each, whilst NAA was added at 1 mg/L (5.4 lM). Within rows, treatments marked with the same letters are not significantly
different from one other (Fisher’s Least Significant Difference). Data are mean ± SE; N = 5
GABA c-aminobutyric acid, NAA 1-naphthalene acetic acid
Plant Cell Tiss Organ Cult (2014) 117:401–409
Plant Cell Tiss Organ Cult (2014) 117:401–409
a 1.E+6
* 90%
1.E+5
*
* *
µg amino acid / g DM
1.E+4
1.E+3
1.E+2
1.E+1
1.E+0
Treatment (mg/L)
GABA L Canavanine
Fig. 3 a Micrograms of bioactive amino acids per gram dry mass;
b Relative percentage of amino acids per treatment of Sutherlandia
frutescens cultures. Strigolactones GR24 and Nijmegen-1 (NM-1)
were added at 0.1 lM each, whilst NAA was added at 1 mg/L
concentration of many of the amino acids tested in this
study (Table 1; Suppl. Fig. 1). Treatment with NAA and
Nijmegen-1 increased levels of histidine, serine, arginine,
aspartate, glutamate, threonine, alanine, proline, cysteine,
tyrosine, methionine, valine, isoleucine, leucine, phenyl-
alanine and asparagine (Table 1). Interestingly, SlCCD7
antisense tomato plants, that lack CCD7 (Carotenoid
Cleavage Dioxygenase7) and are thus unable to produce
strigolactones, showed a reduction in the amino acid con-
tent of the stem (Vogel et al. 2010). There is also a con-
siderable overlap between the most affected amino acids in
that study and those most upregulated by Nijmegen-1 and
NAA treatment, including arginine and asparagine.
The amino acids proline, arginine, aspartate, alanine,
asparagine, glutamate and serine have been shown to
increase under various plant stresses (Rai 2002). Treatment
with Nijmegen-1 and NAA thus induces amino acid
changes resembling a stress response, which indirectly
gives an explanation of the increased secondary metabolite
SU1 (Fig. 2a), since secondary metabolites are mainly
accumulated under stress conditions (Ramakrishna and
Ravishankar 2011).
In addition to SU1, the health benefitting effects of
Sutherlandia are also ascribed to a wide variety of amino
acids, including the L-arginine, L-asparagine, L-canavanine
and GABA (Fig. 3) that are regarded as biomarker
407
b 100%
Relative % of bioactive amino acids
80%
70%
60%
50%
40%
30%
20%
10%
0%
Treatment (mg/L)
L Asparagine L Arginine
(5.4 lM). Asterisks indicate significant differences in amino acid
content compared to control of specific amino acid. Data are
mean ± SE; N = 5. GABA c-aminobutyric acid
molecules for assessing the quality of Sutherlandia-based
products (Mncwangi and Viljoen 2012). Nijmegen-1 alone
increased the amount of asparagine and canavanine sig-
nificantly whereas a combination of both NAA and Nij-
megen-1 in addition promoted asparagine (10.52 mg/g)
and arginine (137.64 mg/g) accumulation significantly
(Fig. 3a). Of the amino acids recorded, GABA accumu-
lated at the lowest relative abundance whereas arginine
levels were always high in both control and in propagules
exposed to NAA and Nijmegen-1 (Fig. 3b). Canavanine
levels were lower when plants were grown in auxin and/or
strigolactones (Fig. 3b). Both arginine and canavanine are
synthesised from the same precursor, making canavanine
an arginine antagonist (Rosenthal 1977). The increase in
arginine, and not in canavanine after treatment with NAA
and Nijmegen-1, can therefore be explained since they
compete for the same precursor.
Arginine and canavanine can then be degraded to the
polyamine putrescine, which can be converted to spermi-
dine and spermine. The polyamines putrescine and sper-
midine were detected via LC–MS (Fig. 4a), whilst
spermine could not be detected in any of the microplants.
Putrescine contributed 70–80 % of the measured poly-
amine content with spermidine contributing the remaining
20–30 % (Fig. 4b). The untreated in vitro plants contained
approximately 77 % (3.33 mg/g) putrescine and only 23 %
123
408
6 100%
a b
5
mg polyamines / gDM
4
3 50%
2
1
0 0%
Treatment (mg/L)
Spermine
Fig. 4 a Micrograms of polyamines per gram dry mass; b relative
percentage of polyamines per treatment of Sutherlandia frutescens
cultures. Strigolactones GR24 and Nijmegen-1 (NM-1) were added at
(0.99 mg/g) spermidine (Fig. 4). No spermine could be
detected in any of the samples with the LC–MS profiling
technique used in this study.
Conclusion
The use of NAA to increase the synthesis of important
compounds in S. frutescens seems promising, as it appears
to stimulate production of a range of medicinally important
metabolites. Strigolactones generally had a minimal effect
on explant growth. However, the combination of NAA and
Nijmegen-1 proved favourable for metabolite production
and enhanced the positive effects of NAA on metabolite
content. This combination is thus promising for commer-
cialisation when higher yields of specific amino acids, such
as arginine and asparagine, and SU1 content are desirable.
This study forms an important part in metabolite discovery
and investigation of medicinal products produced by S.
frutescens.
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