Download Biologically active cis-cinnamic acid occurs naturally in Brassica

REPORTS
Chinese Science Bulletin 2003 Vol. 48 No.6 555 558
Biologically active
cis-cinnamic acid occurs
naturally in Brassica
parachinensis
YIN Zhiqi1, WONG Weishing1, YE Wenchai2,3
& LI Ning1
1. Department of Biology, The Hong Kong University of Science and
Technology, Clear Water Bay, Hong Kong SAR, China;
2. Department of Chemistry, The Hong Kong University of Science and
Technology, Clear Water Bay, Hong Kong SAR, China;
3. Department of Phytochemistry, China Pharmaceutical University,
Nanjing 210009, China
Correspondence should be addressed to Li Ning, (e-mail: boningli@
ust.hk)
Abstract The biologically active cis-cinnamic acid (cisCA) has been perceived as a synthetic plant growth regulator
for decades. However, in the present study, we found that cisCA actually exists as a naturally occurring compound in a
Brassica plant. This natural growthregulating substance presents in both the sunlight-irradiated leaf tissue and the
non-irradiated root tissue. The concentrations of cis-CA in
both tissues are comparable to the biologically effective levels
of those major plant hormones. The presence of cis-CA in
root tissue suggests that it may be produced through both
light-dependent and -independent path- ways or it can be
transported from a plant organ to another.
Keywords: cis-cinnamic acid, Brassica, HPLC, mass spectrometry,
plant growth regulator, natural product, phenylpropanoid.
The biological activity of cis-cinnamic acid (cis-CA)
was first reported in 1935[1,2]. This synthetic cis-CA was
found not only to promote the growth in the pea split stem
curvature test, the pea segment test and the Avena straight
growth and curvature tests[2 4] but also to induce epinastic
response in tomato plant [5,6]. In a recent study, cis-CA was
discovered to act through an ethylene-receptor independent pathway to inhibit hypocotyls elongation and stem
gravicurvature, to promote leaf petiole epinastic response
and to delay climacteric fruit ripening [7]. As cis-CA was
believed to be extremely scarce in nature and the trace
amount of free cis-CA found in the oil of Alpinia
malacensis[8] was considered to be insufficient to have any
physiological implications, this unique compound has
therefore been perceived as a synthetic plant growth
regulator for decades. For this very reason, little effort has
been devoted to study the production and function of this
plant growth regulator in higher plants in the past years
and few researches have been conducted to show the
physiological roles of cis-CA in plants. In this paper, we
report an unexpected finding that a Brassica plant contains the naturally occurring cis-CA. More importantly,
Chinese Science Bulletin Vol. 48 No. 6 March 2003
the level of cis-CA found in this Brassica plant is comparable to those effective levels of known plant hormones[9
11]
.
1 Materials and methods
( ) Plant material. Brassica parachinensis Bail
used for cinnamic acid extraction was grown in the open
experimental field in Guangzhou Vegetable Research
Institute (Guangzhou, China). The whole plant was harvested at the flowering stage. Plant leaves and roots were
excised out after the whole plants were cleaned with double-distilled water. These tissues were briefly air-dried
before they were frozen in liquid nitrogen and stored for
later experimental analysis.
( ) Preparation of cis-CA crystals and establishment of cis-CA standard curve. Trans-cinnamic acid (Eisomer) was purchased from SIGMA (C-6004). Approximately 1.5 g of trans-CA was dissolved in 96% ethanol
and irradiated under UV-light for overnight. The mixture
of E (trans-isoform) and Z (cis-isoform) isomers of cinnamic acid was re-dissolved in ethanol and separated on
HPLC. Crystals of cis-CA were precipitated out of a cisCA solution under vacuum. 1H nuclear magnetic resonance (NMR) was used to authenticate the structure of
cis-CA and trans-CA as previously described[7]. To establish the cis-CA standard curve, 1.04 mg of cis-CA crystals
were dissolved in the pure methanol to make a final concentration of 1 mg/mL. The standard curve of cis-CA was
established with 3, 5, 10, 30, 60 and 100 ng of cis-CA
standards versus each of the corresponding area numbers
of mass spectra (m/z 147) obtained from LC-MS analysis.
The regression equation for quantitation of cis-CA was
calculated in the following (cis-CA amount, ng) = [(m/z
147 Peak Area) - 886622.0004] /1409853.577, R2 =
0.9961.
( ) Preparation of cis-cinnamic acid sample from
Brassica. The frozen Brassica tissues were first ground
to fine powders with cold mortar and pestle. The powder
was dissolved in two volumes of 96% ethanol and then
sonicated for three times. The ethanol extract was filtered
through a filter paper. The filtrate was dried for several
hours in a rotary evaporator to remove the ethanol/water
mixture. Solid extraction materials were re-dissolved into
10% ethanol and passed through a macro-porous resin
column (D 101, 250 g, column size 6 cm 40 cm). The
cinnamic acid-enriched compounds were then eluted out
with the ethanol step gradients. 55% and 70% ethanol
eluates were combined and dried in a rotary evaporator to
reduce the aqueous extraction volume. The resulting natural compounds were re-dissolved in 600 µL of methanol
and stored for the next step of HPLC separation.
( ) High performance liquid chromatography. The
high performance liquid chromatography (HPLC) used to
purify cis-CA consists of Waters 600 controller, 600 pump
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and the 486 Tunable Absorbance Detector. Separation of
cis-CA from other natural compounds was achieved on a
reversed-phase C18, 4 µmol/L, 3.9 mm 330 mm column
(Nova-Pak, Waters). The mobile phase was a mixture of
acetonitrile (buffer A) and acetic acid (buffer B). The ratio
of buffer A to B was set to 3︰7 at the initial stage and
changed gradually to 4︰6 by 8 min and finally to 4.5︰
5.5 by 30 min. The flow rate was set at 1.0 mL/min. The
wavelength of UV-light in the Waters 486 Tunable Absorbance Detector was set at 261 nm because of the cisCA absorption peaks at this wavelength.
( ) LC-MS. Mass spectra was acquired using
Thermo Finnigan LCQ (San Jose, USA) ion-trap mass
spectrometer equipped with an electrospray ionization
source. Both positive and negative-ion mass spectra were
acquired. Negative-ion electrospray ionization was performed using an ion source voltage of 4.0 kV. Nebulization was aided with coaxial high purity grade nitrogen
sheath gas provided at a pressure of 482.63 kPa. Desolvation was aided using a counter current high purity grade
nitrogen flow set at a pressure of 68.947 kPa and a capillary temperature of 200 . Mass spectra were recorded
over the range of 100 300 m/z. The retention time of
authentic cis-CA standard on LC-MS was determined to
be 20 ± 0.25 min. The semi-purified cis-CA sample was
eluted with a buffer gradient which consists of acetic acid
buffer and acetonitrile on a reversed-phase, C18, 5
µmol/L, 4.6 mm 150 mm column. The mobile phase is
the same as the above described. The flow rate was set at
0.8 mL/min.
30 µL of which was then taken for LC-MS analysis. The
chromatographic results of leaves and roots extracts were
recorded in both mass spectrum and UV-light absorbance
(Figs. 1(a), 1(b) and 2(a), 2(b)). Although UV-light absorbance data indicated that both leaf and root extracts
collected from HPLC separation were still a mixture of
many natural compounds (Fig. 1(b) and Fig. 2(b)), the
mass spectra data of both samples showed (Figs. 1(a), 1(c)
and 2(a), 2(c)) that there existed a single ion peak of m/z
147 at retention time of 20 ± 0.25 min, which was identical to the retention time of authentic cis-CA standard.
Therefore, LC-MS data of Brassica extracts, as shown in
Figs. 1 and 2, have unequivocally demonstrated that cisCA indeed exists as a naturally occurring compound in
both leaf and root.
2 Results and discussion
In contrast to the biosynthesis of trans-CA, which is
produced from L-phenylalanine by phenylalanine ammonia lyase (PAL, E.C.4.3.1.5), cis-CA is believed to be a
product of sunlight-mediated conversion from trans-CA[6].
Thus, when selecting the experimental materials for study
of the naturally occurring cis-CA, the open field-grown
Brassica plants were considered to be ideal for this investigation because these plants should have been exposed to
sunlight for many weeks before being harvested. Hence,
nearly 10 kg of the field-grown Brassica plants were harvested for cis-CA extraction. Leaves and roots were separated from each other and frozen separately in liquid nitrogen. Following the organic solvent extraction, the resulting soluble cis-CA extract, either from leaves or roots,
has passed through a silicon gel column to enrich the cisCA content. The resulting eluate was then subjected to
HPLC separation. Because the retention time of cis-CA
standard analyzed on HPLC was around 11 min, cis- CAcontaining fractions were collected between 9.5 to 12.5
min starting from the sample injection. The cis- CAcontaining eluate was concentrated again to 0.2 mL, 10
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Fig. 1. Liquid chromatography mass spectrometery analysis of Brassica leaf extract. (a) Ion chromatograms for m/z 147 of leaf extract; (b)
UV-light traces (absorbance wavelength, 261 nm) of leaf extract; (c) the
selected mass spectra of m/z 147 ion peak.
According to the cis-CA standard curve, the concentration of cis-CA in leaf and root was determined to be
135 and 112 ng per gram of fresh weight (gfw), respe ctively. The concentration of cis-CA in Brassica plant appears to be comparable to the physiologically effective
concentrations of major plant hormones. For examples,
IAA in pea vegetative tissue ranges from 42.7 to 53.5 ng
/gfw[9], whereas Rumex palustris root has an IAA range of
49 72 ng /gfw[12]. The concentration of GA in rice seedlings varies from 0.45 to 93 ng /gfw[13]. In terms of ABA
content, tomato plant has 79.3 ng/gfw[14] and Arabidopsis
6.5 ng/gfw under unstressed conditions [15]. In fact, the
Chinese Science Bulletin Vol. 48 No. 6 March 2003
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amount of cis-CA in Brassica tissues determined by LC-
Fig. 2. Liquid chromatography mass spectrometery analysis of Brassica root extract. (a) Ion chromatograms for m/z 147 of root extract; (b)
UV-light traces (absorbance wavelength, 261 nm) of root extract; (c) the
selected mass spectra of m/z 147 ion peak.
MS should have been underestimated because a certain
percentage of cis-CA might have been lost during extraction. The radioactive cis-CA tracer should have been
added to the initial ethanol extraction solution to measure
the recovery rate at the end of cis-CA extraction process.
Furthermore, because cis-CA is able to undergo spontaneous conversion back into trans-CA gradually at room
temperature (the s-cis-CA is less stable than s-cis-CA), a
minute portion of cis-CA identified from Brassica tissue
could also be converted into trans-CA in solution spontaneously. Taken together, it is conceivable that the actual
cis-CA’s level in plant tissues should be higher, instead of
lower, than what we report here.
Cis-CA has long been perceived as a biologically
active compound that elicits an array of physiological or
cellular responses in plant. It was considered to be auxinlike but to lack a polar transport property because it possesses the unique structural characteristics of auxin-like
substances[16]. Secondly, it was once believed to be an
ethylene analog because it also has a double bond formed
between Cα and Cβ and the vapor of cis-CA causes epinasty of tomato seedling as ethylene does[6]. Thirdly, cisCA is also speculated to be coumarin-like also because of
its structural similarity[17]. Lastly, The photo-isomerization
of cell wall-bound CA derivatives and their esters from
Chinese Science Bulletin Vol. 48 No. 6 March 2003
trans- to cis-isoform or vice versa might serve as a
mechanism for transduction of light energy leading to
changes in cell wall, then to changes in water flux and
turgor pressure and eventually to phototropism[18] To elucidate the actual functions of cis-CA, more research need
to be performed. Our preliminary results from the study of
cis-CA-responsive genes using genechip technology have
revealed that cis-CA is indeed capable of inducing a group
of genes in Arabidopsis that are distinctly different from
what trans-CA can induce (data not shown).
Trans-CA is a key intermediate in the upstream of
plant phenylpropanoid metabolic pathways [19]. These
pathways lead to the synthesis of an array of secondary
metabolites and signaling molecules such as tannin, flavonoids, anthocyanins, coumarins, lignin, subrin and salicylic acid. It will be interesting to find out if cis-CA, the
biologically active cinnamate isomer, also contributes to
the biosynthesis of these metabolites. Does cis-CA negatively feedback the activity of PAL[20] or it positively
regulate the PAL activity? Clearly, the production, metabolism and action of cinnamic acid await further investigation.
As cis-CA exists as a naturally occurring compound
in the Brassica plant, the next immediate and relevant
question is how cis-CA is produced. We hereby propose
four possible pathways for production of cis-CA (Fig. 3).
They are: (1) a sunlight-mediated conversion from transCA; (2) a spontaneous conversion from trans-CA in the
presence of electron-transfer sensitizer; (3) a product of
isomerase catalysis from trans-CA; and (4) a product of
enzyme catalysis from L-phenylalanine. From the reports
documented in the literature, it seems that the sunlightmediated conversion pathway is highly likely. For instance, many cell wall-bound cis-isoforms of hydroxycinnamic acids can be found only in the light-grown
grasses[21]. The irradiation of the hypocotyls of etiolated
gherkin seedlings generated a cis-isoform of 4-hydroxycinnamic acid, which becomes less inhibitory to the
activity of PAL in seedlings[22]. When the etiolated barley
seedlings were irradiated with UV-A light, cis-ferulic acid
appeared immediately[23]. Sunlight induced isomerization
and dimerization of CA derivatives have also been found
in cell walls of barley straw[24]. However, these dada do
not exclude a possible enzyme-mediated cis-CA production pathway. Our data presented here show that cis-CA
exists in root tissue. The question is where the root cis-CA
comes from if the root has never been exposed to sunlight.
A plausible explanation could be that cis-CA in root is
produced either through an enzyme-dependent or through
a non-enzymatic and light-independent pathway. Alternatively, we can speculate that cis-CA is transported to root
from leaf tissue.
For more than half a century, cis-CA has been widely
perceived as a synthetic plant growth regulator because no
report has yet been documented to show the universal
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7.
8.
9.
10.
11.
Fig. 3. Possible pathways for in vivo production of cis-CA in higher
plants. a, sunlight-dependent pathway; b, spontaneous conversion pathway; c, trans-CA isomerase; d, an unknown enzyme that converts Lphenylalanine to cis-CA; PAL stands for phenylalanine ammonia lyase.
The dashed and solid arrows indicate the hypothetical and the known
cinnamic acid production pathways, respectively.
existence of cis-CA in plants nor the endogenous level of
this biologically active compound has been determined.
However, this work has changed our view on cis-CA. It is
clearly evident that cis-CA exists as a naturally occurring
compound in a higher plant and it may be universally
occurring in higher plants too. It is also highly likely that
the unique sunlight-activated cinnamic acid may serve as
a non-proteinaceous photoreceptor for UV-A & UV-B
light in plants. Because little is known about its production and the molecular mechanism of its action in higher
plants, it is deemed necessary to conduct a thorough investigation of the production and physiological and cellular functions of cis-CA in plant tropism, growth and development.
12.
13.
14.
15.
16.
17.
Acknowledgements Authors want to express their sincere thanks to
Mr. Bobby Chen from Biotechnology Institute of HKUST for his assistance in LC-MS analysis. This work was supported by a RGC grant
HKUST6105/00M awarded to Li Ning.
18.
References
19.
1.
2.
3.
4.
5.
6.
558
Hitchcock, A. E., Indole-3-n-propionic acid as a growth hormone
and quantitative measurement of plant response, Contributions
from Boyce Thompson Institute, 1935, 7: 87 95.
Haagen-Smit, S. A. J., Went, F. W., A physiological analysis of
the growth substance, Proceedings Koninklijke Akademie van
Weten schappen te Amsterdam, 1935, 38: 852 857.
Koepfli, J. B., Thimann, K. V., Went, F. W., Plant hormones:
Structure and physiological activity, Journal of Biological Chemistry, 1938, 122: 763 779
Went, F. W., Analysis and integration of various auxin effects,
&
, Proceedings: Koninklijke Akademie van Wetenschappen te
Amsterdam, 1939, 42: 581 739.
Hitchcock, A. E., Zimmerman, P. W., The use of green tissue test
objects for determining the physiological activity of growth substances, Contributions from Boyce Thompson Institute, 1938, 9:
463 518.
Zimmerman, P. W., Hitchcock, A. E., Activation of cinnamic acid
20.
21.
22.
23.
24.
by ultraviolet light and the physiological activity of its emanation,
Contributions from Boyce Thompson, 1939, 10: 197 200.
Yang, X. X., Choi, H. W., Yang, S. F. et al., A UV-light activated
cinnamic acid isomer regulates plant growth and gravitropism via
an ethylene receptor-independent pathway, Australian Journal of
Plant Physiology, 1999, 26: 325 335.
Buch, M. L., Bibliography of organic acids in higher plants, United States Department of Agriculture, Agricultural Research Service, 1957, 73-18: 1 136.
Zhu, Y. X., Davies, P. J., The control of apical bud growth and
senescence by auxin and gibberellin in genetic lines of peas, Plant
Physiol., 1997, 113: 631 637.
Zhang, Z. P., Baldwin, I. T., Transport of [2-14C]jasmonic acid
from leaves to roots mimics wound-induced changes in endogenous jasmonic acid pools in Nicotiana sylvestris, Planta, 1997,
203: 436 441.
Clarke, S. F., McKenzie, M. J., Burritt, D. J. et al., Influence of
white clover mosaic potexvirus infection on the endogenous cytokinin content of bean, Plant Physiol., 1999, 120: 547 552.
Visser, E. J. W., Cohen, J. D., Barendse, G. W. M. et al., An ethylene-mediated increase in sensitivity to auxin induces adventitious root formation in flooded Rumex palustris Sm, Plant Physiol.,
1996, 112: 1687 1692.
van der Straeten, D., Zhou, Z., Prinsen, E. et al., A comparative
molecular-physiological study of submergence response in lowland and deepwater rice, Plant Physiol., 2001, 125: 955 968.
Herde, O., Peña Cortés, H., Wasternack, C. et al., Electric signaling and pin2 gene expression on different abiotic stimuli depend
on a distinct threshold level of endogenous abscisic acid in several
abscisic acid-deficient tomato mutants, Plant Physiol., 1999, 119:
213 218.
Lang, V., Mantyla, E., Welin, B. et al., Alterations in water status,
endogenous abscisic acid content, and expression of rab18 gene
during the development of freezing tolerance in Arabidopsis thaliana, Plant Physiol., 1994, 104: 1341 1349.
Veldstra, H., The relation of chemical structure to biological activity in growth substances, Annual Review of Plant Physiology,
1953, 4: 151 198.
Letham, D. S., Naturally-occurring plant growth regulations other
than the principal hormones of higher plants, Phytohormones and
Related Compounds-A Comprehensive Treatise (eds. Letham, D.
S., Goodwin, P. B. Higgins, T. J. V.), Biomedical Press Elsevier:
North-Holland, 1978, 349 403.
Towers, G. H. N., Abeysekera, B., Cell wall hydroxycinnamate
esters as UV [ultraviolet]-A, receptors in phototropic responses of
a new hypothesis, Phytochemistry, 1984, 23:
higher plants
951 952.
Dixon, R. A., Paiva, N. L., Stress-induced phenylpropanoid metabolism, Plant Cell, 1995, 7: 1085 1097.
Blount, J. W., Korth, K. L., Masoud, S. A. et al., Altering expression of cinnamic acid 4-hydroxylase in transgenic plants provides
evidence for a feedback loop at the entry point into the phenylpropanoid pathway, Plant Physiology, 2000, 122: 107 116.
Hartley, R. D., Jones, E. C., Phenolic components and degradability of cell walls of grass and legume species, Phytochemistry,
1977, 16: 1531 1534
Engelsma, G., On the mechanism of the changes in phenylalanine
ammonia-lyase activity induced by ultraviolet and blue light in
gherkin hypocotyls, Plant Physiol., 1974, 54: 702 705.
Yamamoto, E., Towers, G. H. N., Cell wall bound ferulic acid in
barley seedlings during developments and its photoisomerization,
Journal of Plant Physiology, 1985, 117: 441 449.
Turner, L. B., Mueller-Harvey, I., McAllan, A. B., Light-induced
isomerization and dimerization of cinnamic acid derivatives in
cell walls, Phytochemistry, 1993, 33: 791 796.
(Rceived October 31, 2002; accepted December 13, 2002)
Chinese Science Bulletin Vol. 48 No. 6 March 2003