ORIGINAL PAPER Role of ethylene and jasmonic acid on rhizome induction and growth in rhubarb (Rheum rhabarbarum L.) Usha P. Rayirath • Rajasekaran R. Lada • Claude D. Caldwell • Samuel K. Asiedu • Kevin J. Sibley Received: 31 May 2010 / Accepted: 30 September 2010 / Published online: 20 October 2010 Ó Her Majesty the Queen in Right of Canada 2010 Abstract Experiments were conducted to elucidate the hormonal induction and regulation of rhizome growth in rhubarb (Rheum rhabarbarum L.). It was found that eth- ylene is the key regulator of rhizome induction and development. The role of jasmonic acid (JA) and its interaction with ethylene in rhizome induction and growth were also examined. Both ethylene and JA have a signifi- cant effect on promoting rhizome formation in vitro. Conversely, the ethylene biosynthesis inhibitor aminoeth- oxyvinylglycine (AVG) (1.5 lM) inhibited rhizome induction in multiple-shoot clumps in vitro, and suppressed the stimulatory effects of exogenously applied ethephon (1 mg l -1 ) and JA (10 ng l -1 ) in promoting mini-rhizome formation, further confirming the role of endogenous eth- ylene in the process. In addition, rhizome growth was significantly enhanced in the presence of both ethylene and JA (ethephon 1 mg l -1 and JA 10 ng l -1 ) compared to JA alone. These results suggest that endogenous ethylene is the key regulator of rhizome growth in rhubarb and JA promotes rhizome formation, possibly through inducing endogenous ethylene synthesis. Keywords Ethylene Jasmonic acid Hormonal induction Growth Mini-rhizome Rhubarb Abbreviations JA Jasmonic acid ACC 1-Aminocyclopropane-1-carboxylic acid AVG Aminoethoxyvinylglycine PGRs Plant growth regulators Introduction Rhubarb (Rheum rhabarbarum L.) is a herbaceous cool- season perennial vegetable having high market potential for expansion as a commercial vegetable industry in North America and Europe. The current method of propagation by rhizome division often results in a very low rate of propagule production; thus, enhancing rhizome growth is critical to generate more propagules. The physiological mechanisms and hormonal relationships that determine the growth and enlargement of rhizomes in rhubarb are not known (Rayirath et al. 2009). On the basis of the infor- mation currently available on the role of plant growth regulators (PGRs) in promoting vegetative storage organs, it is expected that ethylene, jasmonic acid (JA), ethylene gibberellic acid (GA 3 ) and abscisic acid (ABA) can all modulate the growth and development of these plant organs (Ja ´sik and de Klerk 2006; Bhatia et al. 1992; Kim et al. 2005; Zaib-Un-Nissa and Rafiq 1980). Determining the role(s) of these PGRs in manipulating rhizome growth in rhubarb will help to identify the key regulators of the rhizome formation process in rhubarb. Plant hormones are the key factors that elicit various physiological changes and modulate plant growth and development under varying environmental conditions (Ecker 1995; Etheridge et al. 2005). In general, exogenous U. P. Rayirath R. R. Lada (&) C. D. Caldwell S. K. Asiedu Department of Plant and Animal Sciences, Nova Scotia Agricultural College, 50 Pictou Road, P.O. Box 550, Truro, NS B2N 5E3, Canada e-mail: [email protected]K. J. Sibley Department of Environmental Sciences, Nova Scotia Agricultural College, Truro, NS B2N 5E3, Canada 123 Plant Cell Tiss Organ Cult (2011) 105:253–263 DOI 10.1007/s11240-010-9861-y
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Role of ethylene and jasmonic acid on rhizome induction and growth in rhubarb ( Rheum rhabarbarum L.)
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ORIGINAL PAPER
Role of ethylene and jasmonic acid on rhizome inductionand growth in rhubarb (Rheum rhabarbarum L.)
Usha P. Rayirath • Rajasekaran R. Lada •
Claude D. Caldwell • Samuel K. Asiedu •
Kevin J. Sibley
Received: 31 May 2010 / Accepted: 30 September 2010 / Published online: 20 October 2010
� Her Majesty the Queen in Right of Canada 2010
Abstract Experiments were conducted to elucidate the
hormonal induction and regulation of rhizome growth in
rhubarb (Rheum rhabarbarum L.). It was found that eth-
ylene is the key regulator of rhizome induction and
development. The role of jasmonic acid (JA) and its
interaction with ethylene in rhizome induction and growth
were also examined. Both ethylene and JA have a signifi-
cant effect on promoting rhizome formation in vitro.
Conversely, the ethylene biosynthesis inhibitor aminoeth-
oxyvinylglycine (AVG) (1.5 lM) inhibited rhizome
induction in multiple-shoot clumps in vitro, and suppressed
the stimulatory effects of exogenously applied ethephon
(1 mg l-1) and JA (10 ng l-1) in promoting mini-rhizome
formation, further confirming the role of endogenous eth-
ylene in the process. In addition, rhizome growth was
significantly enhanced in the presence of both ethylene and
JA (ethephon 1 mg l-1 and JA 10 ng l-1) compared to JA
alone. These results suggest that endogenous ethylene is
the key regulator of rhizome growth in rhubarb and JA
promotes rhizome formation, possibly through inducing
endogenous ethylene synthesis.
Keywords Ethylene � Jasmonic acid � Hormonal
induction � Growth � Mini-rhizome � Rhubarb
Abbreviations
JA Jasmonic acid
ACC 1-Aminocyclopropane-1-carboxylic acid
AVG Aminoethoxyvinylglycine
PGRs Plant growth regulators
Introduction
Rhubarb (Rheum rhabarbarum L.) is a herbaceous cool-
season perennial vegetable having high market potential
for expansion as a commercial vegetable industry in North
America and Europe. The current method of propagation
by rhizome division often results in a very low rate of
propagule production; thus, enhancing rhizome growth is
critical to generate more propagules. The physiological
mechanisms and hormonal relationships that determine the
growth and enlargement of rhizomes in rhubarb are not
known (Rayirath et al. 2009). On the basis of the infor-
mation currently available on the role of plant growth
regulators (PGRs) in promoting vegetative storage organs,
it is expected that ethylene, jasmonic acid (JA), ethylene
gibberellic acid (GA3) and abscisic acid (ABA) can all
modulate the growth and development of these plant
organs (Jasik and de Klerk 2006; Bhatia et al. 1992; Kim
et al. 2005; Zaib-Un-Nissa and Rafiq 1980). Determining
the role(s) of these PGRs in manipulating rhizome growth
in rhubarb will help to identify the key regulators of the
rhizome formation process in rhubarb.
Plant hormones are the key factors that elicit various
physiological changes and modulate plant growth and
development under varying environmental conditions
(Ecker 1995; Etheridge et al. 2005). In general, exogenous
U. P. Rayirath � R. R. Lada (&) � C. D. Caldwell � S. K. Asiedu
Department of Plant and Animal Sciences, Nova Scotia
elongation through inhibiting endogenous ethylene. The
regulation of the expression of ACO genes, thus, has
functional significance (Prescott and John 1996) and it
Fig. 3 a Mini-rhizome width in
plantlets grown in JA, ethephon
and their most effective
combinations in the presence
and absence of AVG. The
explants were incubated in the
culture for 6 weeks. The valuesrepresent the means of ten
replicates and the error barsrepresent the SE at a = 0.05.
b Plants after 6 weeks growth in
1 mg l-1 ethephon and
10 ng l-1 JA combination and
1 mg l-1 ethephon (top), and
the mini-rhizome growth in
1 mg l-1 ethephon and 10 ng
l-1 JA combination and 1 mg
l-1 ethephon (bottom). The
explants were cultured in MS
basal medium with PGRs for
6 weeks (pictures taken from
the bottom of the culture jars).
c Left to right: mini-rhizome
growth inhibited in explants
incubated with 10 ng l-1 JA and
1.5 lM AVG combination;
1 mg l-1 ethephon and 1.5 lM
AVG combination; 1 mg l-1
ethephon, 10 ng l-1 JA and
1.5 lM AVG combination
treatment
Plant Cell Tiss Organ Cult (2011) 105:253–263 259
123
has also been proved that at least two ACO genes in
Arabidopsis thaliana are ethylene-inducible (Alonso et al.
2003; Van Zhong and Burns 2003). Hence, in this study,
exogenous ethylene (ethephon) appears to induce mini-
rhizomes in treated cultures through enhanced ethylene
biosynthesis, which is inhibited by AVG, resulting in the
suppression of mini-rhizome growth in the combination
cultures. However, the present investigation, being purely
physiological, cannot address the molecular mechanisms
which result in these differential responses to the biosyn-
thetic inhibitors. The results presented in Fig. 1a suggest
that GA3 and ABA did not have any significant role in the
process as supported by previous studies (Obasi and Atanu
2004; Zheng et al. 2005). Even though ABA induces tuber
formation and growth in potato by counteracting the effect
of GA3 (Xu et al. 1998), scientific evidence on its role in
inducing rhizome growth in vegetatively propagated
perennial crops is not available thus far.
The comparison of microscopic sections of in-vitro-
generated mini-rhizomes and mother rhizomes of a normal
plant grown in the greenhouse clearly revealed some
structural similarities (Fig. 2b). These structural similari-
ties strongly support that the growth obtained at the base of
the explants treated with effective concentrations of ethe-
phon and JA is a miniature rhizome. A rhizome is a stem
modification that is a common storage organ in vegeta-
tively propagated crops (Hartmann et al. 2002). In some
herbaceous perennials which over-winter (like rhubarb),
rhizomes serve as carbon reserves for survival (Moore et al.
1998). Anatomically, rhizomes mostly lack a proper stele
pattern and form shoot and root buds from the nodes
(Raven et al. 2005; Hartmann et al. 2002). The presence of
structures, which are more likely chloroplasts (seen as
green-coloured structures), further confirms that it is not a
Fig. 4 Mini-rhizome weight (g) of in vitro plantlets treated with
1 mg l-1 ethephon, 10 ng l-1 JA and their combination (1 mg l-1
ethephon, 10 ng l-1 JA) after 4 months in pot culture. The valuesrepresent the means of nine replicates and the error bars represent the
SE at a = 0.05
Table 1 Growth of in-vitro-induced mini-rhizomes after transplantation to pots
Treatments Rhizome
width (cm)
Rhizome
length (cm)
Rhizome
weight (g)
No. of
rhizome branches
No. of buds
Ethephon 1 mg l-1 6.6a 6.9a 53.5a 5.1a 19.1a
JA 10 ng l-1 4.8b 5.0b 38.2b 3.4b 10.2c
JA 1 lg l-1 5.5b 4.9b 36.2b 3.8b 7.4d
Ethephon 1 mg l-1 and JA 10 ng l-1 5.4b 5.4b 49.4a 3.1b 11.7b
Control DW 2.7c 4.8bc 11.1c 1.4c 3.2e
Control EtOH 1.6c 2.8c 6.9c 0.8c 2.4e
The in-vitro-produced rhubarb plantlets with the induced mini-rhizomes were grown under controlled conditions in the greenhouse for 4 months
and then uprooted and observations were made
EtOH ethanol, DW distilled watera–e Means with the same letters indicate no significant difference based on Tukey’s test at a = 0.05; n = 9
Table 2 Foliar treatment with ethephon on plants in pot culture
Treatments Rhizome width (cm) Rhizome weight (g) No. of buds
Ethephon 1 mg l-1 8.6a 117.6a 22.2a
Control 7.5b 110.9a 11.6b
Measurements were made on the rhizome growth of pot-grown plants 5 months after foliar treatment with ethephona, b Means with the same letters indicate no significant difference based on Tukey’s test at a = 0.05; n = 10
260 Plant Cell Tiss Organ Cult (2011) 105:253–263
123
root structure and the presence of vascular structures also
supports that it is not a mere callus growth but a well-
differentiated plant organ.
To further support the results of the in vitro studies,
exogenously applied ethephon in the form of foliar spray
on in situ rhubarb plants during active vegetative growth (8
and 12 weeks after shoot emergence) significantly
enhanced the number of potential buds on the rhizome.
Being a gaseous hormone, ethylene is not translocated
through the vascular system. Ethylene is neither actively
transported nor degraded (Bleecker and Kende 2000). The
induction of ethylene synthesis by signals such as auxin or
wounding usually occurs through the activation of ACC
synthase through increased gene expression (Bleecker and
Kende 2000). However, the exact molecular mechanism of
ethephon- and JA-induced rhizome growth is yet to be
uncovered. Here, exogenous ethylene appeared to trigger
adventitious bud development in the sprayed plants, pos-
sibly through breaking lateral bud dormancy. Previous
reports indicate that ethylene (and ethylene-releasing
compounds) could positively regulate developmental pro-
cesses like adventitious root formation (Liu et al. 1990),
trigger adventitious bud development releasing lateral bud
dormancy (Reid 1987), enhance cambial activity and
induce radial swelling of storage organs (Reid 1987; Ne-
uteboom et al. 2002).
Our study clearly indicates the specific role of ethylene
in the induction, growth and development of rhizomes in
rhubarb. Although ethylene has a pleiotropic role in various
processes of plant growth and development, specific roles
of this hormonal signal in plant morphogenesis, such as the
formation and development of vegetative storage organs or
vegetative reproductive structures, has not been reported so
far.
In the present study, JA at lower concentrations
(10 ng l-1 and 1 lg l-1) significantly induced mini-rhi-
zomes in rhubarb plantlets (Fig. 1a). However, the rhizome-
inducing effect of JA was suppressed in the presence of
ethylene synthesis inhibitor, AVG, and, at the same time,
further enhanced by ethylene (ethephon) when they were
combined and applied exogenously in the growing media.
This implies that, while JA could promote rhizome induc-
tion, it requires the presence of endogenous ethylene as
AVG inhibited JA-induced rhizome growth. Also, it is
possible that, since JA induces ethylene synthesis (Tang
et al. 2008), the JA-induced mini-rhizome synthesis may be,
perhaps, due to JA-induced ethylene. Though synergistic
interactions between ethylene and JA have been proposed to
contribute to diverse plant responses to abiotic and biotic
stresses (Zhu et al. 2006; Feys and Parker 2000; Glazebrook
2001; Lorenzo et al. 2003), the relationships between JA and
ethylene in regulating developmental processes, other than
plant defence responses, has not been studied intensively.
The exogenous application of JA rapidly enhances ethylene
emission in tomato and supports the indication that they act
synergistically in the expression of proteinase inhibitors
(PIN) genes in response to wound stress (O’Donnell et al.
1996). Ethylene is found to act downstream of JA in the
wound signal transduction pathway (O’Donnell et al. 1996).
Similarly, methyl jasmonate acts synergistically with ACC
in enhancing ethylene production and promoting gum for-
mation in tulips through stimulated ACC oxidase activity
(Saniewski et al. 2003). The interactive role of these hor-
monal signals in developmental processes such as root hair
formation has been clearly demonstrated in recent studies
(Zhu et al. 2006). Studies on the promoter activities of
Arabidopsis ACC synthase genes have recently shown that
exogenous JA could enhance the activity of Arabidopsis
ACC synthase (the rate-limiting step in ethylene biosyn-
thesis) promoter AtACS4 in the wild-type genotype (Tang
et al. 2008). The above explanations indicated that ethylene
is the key player in the process of rhizome induction and
development and JA might act through ethylene induction in
the process. All of these results support a significant role of
the PGRs ethylene and JA in the induction and growth of
rhizomes in Rheum rhabarbarum.
Taken together, the findings of this study strongly suggest
that ethylene is the rhizome induction signal in rhubarb. The
significant antagonistic effect of AVG on rhizome growth
even in the presence of exogenous ethylene and JA further
confirms this and suggests that JA’s role in the process is
possibly through stimulating endogenous ethylene levels
(Zhu et al. 2006; Tang et al. 2008). Further experiments
focusing on the interaction of ethylene and JA utilising
respective biosynthesis and action inhibitors would certainly
disclose the cross-talk between the two hormones in the
regulation of rhizome induction and growth in rhubarb. The
findings of such investigations would improve our under-
standing of how these two hormones regulate growth and
development processes in different crop species. However,
unlike in model plant species like Arabidopsis, in the
absence of hormonal signalling mutants of this plant, it is
very challenging to unveil the hormonal signalling pathway
involved in this developmental process.
Acknowledgements Financial support from AgriFocus 2000—
Technology Development Program (Nova Scotia Department of
Agriculture and Fisheries) and Knol Farms Ltd., Nova Scotia, Can-
ada, to Dr. Lada is gratefully acknowledged.
Appendix: details of the plant growth regulator
application
Plant-cell-tested commercial formulations of growth regu-
lators from Sigma-Aldrich, Canada, were used. Stock
solutions of GA3 (1,000 mg l-1), ABA (100 mg l-1) and
Plant Cell Tiss Organ Cult (2011) 105:253–263 261
123
JA (1,000 mg l-1) were prepared by dissolving them first
in four drops of 95% ethanol and diluted with distilled
water to the final stock concentrations. ACC, AVG and
ethephon (Sigma-Aldrich, Canada) were dissolved in dis-
tilled water to make the stock solutions of concentrations