Download Identification of Enzymatic Properties in Crocus sativus Roots

Identification of Enzymatic Properties in Crocus sativus Roots
E. Keyhani, J. Keyhani
Laboratory for Life Sciences
19979 Tehran
Tehran
Iran
S. Saeidian, F. Attar, S. Oveissi
Institute of Biochemistry and Biophysics
University of Tehran
13145 Tehran
Tehran
Iran
Keywords: catalase, dianisidine peroxidase, lignin peroxidase, polyphenol oxidase,
superoxide dismutase
Abstract
Even though saffron is the most expensive spice in the world, knowledge
on the biology of the producing plant (Crocus sativus L.) is still limited. In this
research we report the identification and kinetics properties of some enzymes
active in the plant’s roots. Dormant corms planted in potted soil were collected
after 20 days. The roots had grown at an average rate of 3.8 mm/day for the
first ten days and of 1 mm/day thereafter; they were 5 to 6 cm long at day 20.
The roots were separated from the corms, washed several times in doubledistilled water, homogenized in phosphate buffer 0.1 M, pH 7.0 and centrifuged
at 10,000 g for 10 min, then at 35,000 g for 30 min. The supernatant, termed
“crude extract”, was used for enzymatic assays. 265 units (u) catalase, 0.9 u
lignin peroxidase, 3.3 u o-dianisidine peroxidase, 16 u superoxide dismutase
(SOD) and 1.4 u polyphenol oxidase (PPO) were detectable per mg protein in the
extract; catalytic efficiency, calculated per mg protein, was 9, 30, 28 and 0.0052,
respectively for catalase, lignin peroxidase, o-dianisidine peroxidase, and PPO.
Catalase, lignin peroxidase and o-dianisidine peroxidase were sensitive to KCN
with IC50 of 0.32, 0.07 and 0.02 mM, respectively; PPO was sensitive to kojic
acid with IC50 of 0.03 mM. Thus, quantitatively, the enzymes could be classified
as: catalase > SOD > o-dianisidine peroxidase > PPO > lignin peroxidase. In
terms of catalytic efficiency, the classification went from lignin peroxidase > odianisidine peroxidase > catalase > PPO. Thus the most efficient enzyme studied
was lignin peroxidase, using ferulic acid as substrate, while the most abundant
enzyme was catalase in C. sativus roots.
INTRODUCTION
Root is the principal organ providing mechanical support to the plant and
allowing water and nutrient uptake (Walker et al., 2003). Moreover, roots have the
ability to synthesize, accumulate, and secrete into the rhizosphere, a vast array of
compounds (Flores et al., 1999; Walker et al., 2003). The ability to regulate the soil
microbial community in their vicinity is one of the most remarkable metabolic
features of plant roots (Walker et al., 2003). Approximately 5 to 21 % of all
photosynthetically fixed carbon is transferred to the rhizosphere through root exudate
(Marschner, 1995). As a consequence of normal growth and development, a large
range of organic acids and inorganic substances are secreted by roots into the soil
(Rougier, 1981; Walker et al., 2003), which inevitably leads to changes in its
biochemical and physical properties. For example, the compounds secreted by plant
roots play important roles as chemical attractant and repellants in the rhizosphere
(Estabrook and Yoder, 1998; Bais et al., 2001; Walker et al., 2003).
Root growth and root elongation are dependent of numerous factors such as
oxygen availability and temperature (Dowers, 1991; Drew and Stolzy, 1991).
Waterlogging and hypoxia/anoxia decrease the level of oxygen and change the
chemical and microbiological transformations as soil becomes reduced and hostile to
plants and their roots (Drew and Stolzy, 1991; Walker et al., 2003). The minimum
temperature for root growth is about 5 °C and the maxima are 35 to 40 °C, with an
optimum at 20 to 25 °C (Dowers, 1991). Root length can be a more sensitive indicator
of the effects of soil temperature than root weight, and root diameter is inversely
related to soil temperature (Dowers, 1991).
In vitro studies conducted in our laboratory showed that Crocus sativus L.
roots are very sensitive to changes in oxygen concentration and ionic environment
(Keyhani and Keyhani, 2004; Keyhani et al., 2004). Thus the purpose of this research
was the determination of key enzymatic activities in Crocus sativus roots. Emphasis
was put on enzymes involved in the plant defense such as superoxide dismutase,
catalase and peroxidases, and also on enzymes involved in cell wall synthesis such as
lignin peroxidase (Ward et al., 2001) and in wound-induced rootings and wound
healings such as polyphenol oxidase (Constabel et al., 1995; Ho, 1999). The
conclusion drawn was that the most efficient enzyme studied was lignin peroxidase,
while the most abundant enzyme was catalase.
MATERIALS AND METHODS
Roots
Crocus sativus corms were obtained from the University of Tehran farm
located in Karaj, near Tehran. They were planted in plastic pots measuring 24 cm in
diameter and 20 cm in depth that contained soil similar to that in the farm. Seven to
eight corms were planted per pot. After 20 days, corms were collected and the roots
were separated, cleaned from the soil, washed 3 times with distilled water to remove
any remaining extraneous material, and then washed twice in phosphate buffer 0.01
M, pH 7. Extracts were prepared according to the method described in Attar et al.
(2006). Briefly, 100 g cleaned roots were homogenized in phosphate buffer 0.01 M,
pH of 7, containing 0.02 % phenylmethanesulfonyl fluoride (PMSF) as protease
inhibitor. After centrifugation at 10,000 g for 10 minutes, then at 35,000 g for 30
minutes, a clear, transparent supernatant termed “crude extract” was obtained and
used for our studies. Protein concentrations were determined by the Lowry method.
Enzyme Activity Assays
All assays were carried out at room temperature (~22-25 °C). The specific
procedure followed for each enzyme assayed is described below. Results are
averages of at least three assays.
1. Catalase. Catalase activity was measured as described in Keyhani et al. (2002) by
following the dismutation of H2O2 spectrophotometrically using an extinction
coefficient of 27 M-1 cm-1 for H2O2 at 240 nm. One unit was defined as the amount of
enzyme decomposing 1 μmol H2O2 per minute.
2. Peroxidases. Peroxidase activity was determined by following the H2O2-mediated
oxidation of o-dianisidine at 460 nm (o-dianisidine peroxidase) and ferulic acid at 310
nm (lignin peroxidase), with extinction coefficients 11.3 mM-1cm-1 and 8.68 mM-1cm1
, respectively. One unit was defined as the amount of enzyme needed for the
oxidation of 1 μmol of substrate per minute.
3. Superoxide Dismutase. The superoxide dismutase (SOD) activity present in root
extract was assayed by a method based on the inhibition of pyrogallol autoxidation in
alkaline solution as described by Attar et al. (2006). Briefly, the reaction mixture
consisted of 1 ml Tris-HCL buffer 50 mM, pH of 8.2, 1 mM EDTA, 1 μM Aspergillus
niger catalase, 0.2 mM pyrogallol, and the extract. The rate of autoxidation was
measured by monitoring the increase in absorbance at 420 nm One unit of SOD
activity corresponded to the amount required to inhibit pyrogaloll autoxidation by 50
%.
4. Polyphenol Oxidase. Polyphenol oxidase (PPO) activity was determined
spectrophoto-metrically by following the increase in absorbance at 400 nm due to the
oxidation of catechol to its corresponding o-quinone. Assays were conducted in a 3ml reaction mixture containing 0.1 M phosphate buffer at pH 6.7, the substrate, and an
aliquot of root extract; absorbancies were measured using an Aminco DW2
spectrophotometer in the split beam mode. In order to correct for substrate
autoxidation, the reaction mixture, as described, was placed in the sample cuvette
while the reference cuvette contained buffer and the substrate. One unit of PPO was
defined as the amount of enzyme producing a change in absorbance of 0.001 per
minute.
RESULTS
Root Elongation
Gross morphological studies show that Crocus sativus corm can be divided
into 3 distinctive regions: a) a ventral region with a central depression corresponding
to the attachment to the mother corm (umbilical scar); the umbilical scar is at the
center of a circumference of points from where the roots emerge (Fig. 1Aa); b) a
circular lateral region surrounding the corm (Fig. 1Ab); c) a dorsal region, region of
emergence of shoots (Fig. 1Ac). The roots are unbranched. Figure 1B shows the
increase in root length during 30 days of cultivation under our experimental
conditions. The root growth rate remained roughly constant at 3.8 mm per day for the
first ten days when the average root length reached 38 mm; thereafter the growth rate
decreased to 1mm per day so that after cultivation for 30 days, the average root length
was 60 mm.
Enzymatic Activities
For all enzymes studied, the relationship between the rate of substrate
oxidation, or dismutation (in the case of catalase), varied as a function of substrate
concentration according to the Michaelis-Menten equation. As examples, the curves
shown in Figure 2 were obtained when the oxidation rate of three substrates, each
pertaining to a different enzyme, was plotted against substrate concentration. Figure
2A shows the rate of H2O2-mediated oxidation of ferulic acid, a substrate for lignin
peroxidase (LIP), plotted as a function of substrate concentration. Figure 2B show the
rate of oxidation of catechol to its corresponding o-quinone plotted as a function of
substrate concentration; catechol is a substrate for PPO under our experimental
conditions. Figure 2C shows the rate of H2O2-mediated oxidation of o-dianisidine,
substrate for o-dianisidine peroxidase, plotted as a function of substrate concentration.
In all cases, a rectangular hyperbola was obtained as described by the MichaelisMenten equation. Kinetics parameters such as Km and Vmax were deduced from the
plots and catalytic efficiencies were calculated.
In addition to the kinetics parameters, the sensitivity to inhibitors such as KCN
for catalase and peroxidases and kojic acid for PPO was investigated and the IC50
determined. As an example, the decrease in PPO activity as a function of kojic acid
concentration is illustrated in Fig. 3.
Table 1 shows the kinetics parameters, including the IC50 for inhibitors such as
KCN and kojic acid, for all five enzymes studied. The highest Km was found for PPO
with catechol as a substrate (90 mM) and the next highest Km was found for catalase
(30 mM) while the lowest Km was found for LIP with ferulic acid as the reducing
substrate (0.03 mM). The catalytic constant calculated per mg extract protein
(mM.min-1.mg prot-1) was maximum for catalase (265) and minimum for PPO
(0.047). The highest amounts of u per mg prot. were 265 and 16, respectively for
catalase and SOD, and the lowest amount was 0.8, for LIP. However, LIP exhibited
the highest catalytic efficiency (30 min-1.mg prot.-1) while PPO exhibited the lowest
(0.0052 min-1 mg prot.-1). Among the enzymes studied, o-dianisidine peroxidase was
the most sensitive to KCN (IC50 = 0.02 mM). As expected, PPO was sensitive to kojic
acid (IC50 = 0.03 mM).
DISCUSSION
The results obtained in this study indicated that a battery of enzymes
specialized in detoxification and protection against oxidative stress, such as SOD,
catalase and peroxidases were active in Crocus sativus L. roots and that catalase was
the most abundant one. Given their function in a plant, roots are likely to encounter a
variety of stresses, including oxidative stress and they may be the site of accumulation
of reactive oxygen species (ROS) in excess of those produced by physiological
processes. Indeed, it is now well established that root take up and transfer nutrients to
xylem for onward transport to shoot (Tester and Leigh, 2001). Moreover, most roots
develop a highly branched structure that provides a larger surface for nutrient and
water. From the base to the tip, the root can be subdivided into root tip, elongation
zone, maturation zone, and matured zone. The root tip itself includes the root cap and
the meristematic region. Beside mechanical support and water and nutrients uptake,
roots exudates a wide variety of compounds (Walker et al., 2003). Root cap
exudation has various functions including root-soil contact lubrication of the root tip,
protection of roots from dessication and selective absorption and storage of ions
(Bengough and McKenzie, 1997; Hawes et al., 2000 and references therein).
Although the general structure and various cell layers of Crocus sativus L.
root are similar to other roots, they do not develop a branched structure. They do
however fulfill the same functions and are exposed to the same potential stresses as
other roots. The first line of defense against oxidative stress is provided by SOD that
catalyzes the dismutation of the superoxide radical anion into O2 and H2O2, thus
generating another ROS, namely H2O2. The latter is disposed of by catalase.
Interestingly, the two most abundant enzymes found in Crocus sativus L. roots were
catalase and SOD, indicative of an active defense system against ROS. In addition to
enzymes directly involved into the defense against oxidative stress, the activity of
enzymes such as LIP and PPO, involved, respectively, in cell wall synthesis and
wound-induced rootings and wound healings, was also detectable. The activity of
those two enzymes in roots was not unexpected and although the amount detectable
for both enzymes was lower than for the other enzymes studied, the catalytic
efficiency found for lignin peroxidase with ferulic acid as the reducing substrate was
particularly elevated, in line with the utilization of that substrate in cell wall
strengthening (Ward et al., 2001).
Studies on enzymatic activities detectable in extracts obtained from Crocus
sativus L. roots revealed the presence of enzymes involved in the defense against
oxidative stress as well as enzymes involved in cell wall synthesis and wound-induced
rootings and wound healings. The anti-oxidative stress enzymes included SOD,
catalase and peroxidases, with catalase being the most abundant, the next most
abundant being SOD. On the other hand, LIP, an enzyme participating in cell wall
synthesis, was the least abundant but it exhibited the highest catalytic efficiency.
Other enzymatic activities remain to be identified in Crocus sativus L. roots.
ACKNOWLEDGEMENTS
This work was supported in part by the University of Tehran (Interuniversities
Grant # 31303371), Tehran, Iran, and in part by the J. and E. Research Foundation,
Tehran, Iran.
Literature Cited
Attar, F., Keyhani, E. and Keyhani, J. 2006. A comparative study of superoxide
dismutase activity assays in Crocus sativus L. corms. Appl. Biochem. Microbiol.
42:101-106.
Bais, H.P., Loyola-Vargas, V.M., Flores, H.E. and Vivanco, J.M. 2001. Root specific
metabolism: their biology and biochemistry of underground organs. In vitro Cell
Dev. Biol. Plant 37:730-741.
Bengough, A.G. and McKenzie, B.M. 1997. Sloughing of root cap cells decreases the
frictional resistance to maize (Zea mays L.) root growth. J. Exp. Bot. 48:885-893.
Constabel, C.P., Bergey, D.R.and Ryan, C.A. 1995. Systemin activates synthesis of
wound-inducible tomato leaf polyphenol oxidase via the octadecanoid defense
signaling pathway. Proc. Natl. Acad. Sci. U.S.A. 22:407-411.
Dowers, G.D. 1991. Soil temperature, root growth, and plant function. p. 309-330.
In: Y. Waisel, A. Eschel and V. Kafkafi (eds.), Plant Roots the Hidden Half.
Marcel Dekker, Inc., NewYork.
Drew, M.C. and Stolzy, L.H. 1991. Growth under oxygen stress. p. 331-350. In: Y.
Waisel, A. Eschel and V. Kafkafi (eds.), Plant Roots the Hidden Half. Marcel
Dekker, Inc., New York.
Estabrook, E.M. and Yoder, J.I. 1998. Plant-plant communications: Rhizosphere
signaling between parasitic angiosperms and their host. Plant Physiol.116:1-7.
Flores, H.E., Vivanco, J.M. and Loyala-Vargas, V.M. 1999. “Radicle” biochemistry:
the biology of root specific metabolism. Trend Plant Sci. 4:220-226.
Hawes, M.C., Gunawardena, U., Mijasaka, S. and Zhao, X. 2000. The role of root
border cells in plant defense. Trends in Plant Sci. 5:128-133.
Ho, K.K. 1999. Characterization of polyphenol oxidase from aerial roots of an
orchid, Aranda “Christine 130”. Plant Physiol. Biochem. 37:841-848.
Keyhani, E., Keyhani, J., Hadizadeh, M., Ghamsari, L. and Attar, F. 2004. Cultivation
techniques, morphology and enzymatic properties of Crocus sativus L. Acta Hort.
650:227-246.
Keyhani, E. and Keyhani, J. 2004. Hypoxia/anoxia as signaling for increased alcohol
dehydrogenase activity in saffron (Crocus sativus L.) corm. Ann. N.Y. Acad. Sci.
1030:449-457.
Keyhani, J., Keyhani, E. and Kamali, J. 2002. Thermal stability of catalase active in
dormant saffron (Crocus sativus L.) corms. Mol. Biol. Rep. 29:125-128.
Marschner, H. 1995. Mineral nutrition of higher plants. Academic Press, London.
Rougier, M. 1981. Secretory activity of the root cap. p. 542-574. In: W. Tanner and
F.A. Loewus (eds.), Encyclopedia of Plant Physiology, New Series, Vol. 13B,
Plant Carbohydrate II. Springer Verlag, Berlin.
Tester, M. and Leigh, R.A. 2001. Partitioning of nutrient transport processes in roots.
J. Exptl. Botany 52:445-457.
Walker, T.S., Bais, H.P., Grotewold, E. and Vivanco, M. 2003. Root exudation and
rhizosphere biology. Plant Physiol. 132:44-91.
Ward, G., Hadar, Y., Bilkis, I., Konstantinovsky, L. and Dosoretz, C.G. 2001. Initial
steps of ferulic acid polymerization by lignin peroxidase. J. Biol. Chem.
276:18734-18741.
Table 1. Kinetics parameters for enzymatic activities detectable in Crocus sativus
roots
Enzyme
Vmax
(mM.min-1)
SOD(1)
Catalase
12
o-dianisidine 0.036
peroxidase
LIP
0.036
PPO
0.014
(1)
The assay for SOD
(2)
KCN; (3)Kojic acid
Vmax/mg prot
(mM.min-1.mg prot.-1)
265
3.33
Km u/mg Catalytic efficiency
(mM) prot. (min-1.mg prot.-1)
16
30
265 9
0.12 3.33 28
IC50
(mM)
0.32(2)
0.02(2)
0.9
0.03 0.8
30
0.07(2)
0.47
90
1.4
0.0052
0.03(3)
activity does not allow for the determination of K m and Vmax;
A
(a)
(b)
(c)
Average root length (mm)
70
70
B
60
60
50
50
40
40
30
30
20
20
10
10
00
00
10
10
20
20
30
30
Days
Fig. 1. A: In Crocus sativus L., roots are produced at a point around the
circumference of the umbilical scar of the corm and they are unbranched. (a):
Ventral view of dormant corm; (b): Lateral view of cultivated corm; (c):
Dorsal view of cultivated corm. B: Increase in root length at various times
during cultivation under our experimental conditions. The root elongation rate
was 3.8 mm per day for the first ten days, and 1 mm per day for the next
twenty days.
0.04
0.04
1515
B
0.03
0.03
Vi (μM.min-1)
Vi (mM.min-1)
A
0.02
0.02
0.01
0.01
00
00
0.05
0.1
0.15
0.2
0.05
0.1
0.15
0.2
[Ferulic acid] (mM)
1010
55
00 0
0.05
0.1
0.15
0.2
0.25
0 0.05
0.1
0.15
0.2
0.25
[Catechol] (M)
0.045
C
Vi (mM.min-1)
0.04
0.04
0.035
0.03
0.03
0.025
0.02
0.02
0.015
0.01
0.01
0.005
Activity (% control)
00
00
1
1
22
33
44
[o-dianisidine] (mM)
Fig. 2. A: Effect of ferulic acid
concentration on LIP activity in Crocus
sativus root extract; B: Effect of catechol
concentration on PPO activity in Crocus
sativus root extract; C: Effect of odianisidine concentration on dianisidine
activity in Crocus sativus root extract.
All three curves obtained are rectangular hyperbolas as described by the
Michaelis-Menten equation. Substrate
inhibition is observed for LIP with
ferulic acid as substrate.
100
100
8080
6060
4040
Fig. 3. Effect of increasing concentrations
of kojic acid on PPO activity in
Crocus sativus root extract.
2020
00
00
0.5
0.5
11
1.5
1.5
[Kojic acid] (mM)
22