Plant Physiology and Biochemistry 45 (2007) 657e664

www.elsevier.com/locate/plaphy

Research article

The ability of plants to secrete proteases by roots

Miros1aw Godlewski, Bartosz Adamczyk*
Laboratory of Plant Morphogenesis, Department of Plant Cytology and Cytochemistry, Institute of Plant Physiology,
Cytology and Cytogenetics, University of odz, 90-237 odz, Poland
Received 18 March 2007; accepted 14 June 2007
Available online 21 June 2007

Abstract
The aim of our study was to find out if the culture medium of aseptically cultivated seedlings exhibits proteolytic activity and if this event is
universal in angiospermous plants. Seedlings of 15 agricultural and wild-living plant species were cultivated for 14 days without any addition of
nutrients. Our studies showed that roots of higher plants could secrete proteases and that levels of proteolytic activity in the culture medium of
individual species (and cultivars of the same species) could be significantly different. The differences between quantities of the secreted
proteases were connected neither with the fresh weight of the growing seedlings nor with the surface of the root system. No proteins were
required to induce secretion of proteases. The culture medium of a few studied species (Allium porrum, Zea mays, Helianthus annuus) showed
the highest proteolytic activity at pH 7. Studies of the influence of standard protease inhibitors showed that examined proteases belong to the
cysteine protease family. The results suggest that the apical parts of roots exuded proteases more intensively than mature parts. Our studies
suggest that some plant species could develop a strategy to actively increase the level of free amino acids in the soil solution as a source of N.
Our results may contribute to studying plant N nutrition in natural ecosystems and to increasing yield after organic fertilization of agricultural
species.
2007 Elsevier Masson SAS. All rights reserved.
Keywords: Plant nitrogen nutrition; Protease activity; Roots; Secretion of proteases

1. Introduction
Nitrogen (N) is an important nutrient for plants and its
concentration in available forms in soils is one of the limiting
factors for plant growth [8,10]. Historically, it was assumed
that plants can uptake only the inorganic form of nitrogen

(NH
4 and NO3 ) originating from mineralization of soil organic matter via soil microbial biomass [44]. Now, it is well
known that plants of both natural and agricultural habitats
can take up amino acids from soil, and these amino acids
are potentially important N sources for plants (for review,
see [24]). The uptake of nitrogen in the form of amino acids
was shown in the case of plants growing in different natural
* Corresponding author at: Laboratory of Plant Morphogenesis, University
of qodz; Banacha 12/16, 90-237 qodz, Poland. Tel.: 48 042 635 44 27;
fax: 48 042 635 44 23.
E-mail address: bartek_adamczyk@op.pl (B. Adamczyk).
0981-9428/$ - see front matter 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.plaphy.2007.06.001

ecosystems ranging from arctic and alpine tundra to the subtropical rainforest and ephemeral pools in the Namibian desert
[1,22,29,35,38e40]. Amino acids as a source of nitrogen
could also play an important role for agricultural plant species
[19,30,44].
Species-specific preferences for using various sources of N
point out that plants developed several strategies of using
different forms of nitrogen, which are available in the soil
solution. It was shown that the quantity of taken up amino
acids depended on the concentration of free amino acids in
the soil solution (e.g. [13,22]), on the type of amino acid
[12,13,31,43], soil pH value [13], and competition between
plants and soil microbes [5,17,18,20,25,26,33]. In plants, multiple sets of amino acid transport proteins that are specific or
general in their transport of amino acids were identified
[11,32]. These transporters can be used to take up amino acids
from the soil and it is assumed that their expression pattern
could be tightly regulated and species-specific [11,13].

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M. Godlewski, B. Adamczyk / Plant Physiology and Biochemistry 45 (2007) 657e664

The release of free amino acids into the soil solution mainly
results from the action of proteolytic enzymes exuded by microorganisms into the environment; these enzymes are also
important factors promoting N cycle in the soil [4,28,42,45].
Plant root exudates include enzymes and other substances
which increase the availability of nutrients taken up by plants
from the soil [7,23]. Root exudates can also stimulate the development of rhizosphere microbes [8,14]. The question arises
if plants can increase the free amino acid pool only indirectly
by enlarging microbe populations in the rhizosphere, or if
plant roots are also able to exude proteolytic enzymes. Such
plant proteases would release amino acids independently
from the activity of rhizosphere proteases secreted by microbes and could be especially important for those plant
species which prefer amino acid N.
Exudation of significant quantities of proteases was
suggested by our previous observations, which originated
from the studies concerning a different problem. We observed
that immersion of a fragment of a photographic film (exposed,
fixed and developed) in the sterile medium of hydroponically
cultivated seedlings could result in the digestion of the film
emulsion after a few days of incubation. This effect was
observed for several plant species studied. The photographic
film method, which was used with over 30 agricultural plant
species and cultivars, demonstrated a species-specific ability
of plant root exudates to digest photographic film emulsion [2].
The aim of our present study was to test the ability of plant
roots to exude proteases and to verify the prevalence of this
event in angiospermous plants. We measured proteolytic activity in the culture medium of aseptically cultivated seedlings of
several agricultural and wild-living plant species using biochemical methods. In the case of the chosen plant species
we studied the correlation between the proteolytic activity
and the time of seedlings growth. Moreover, we also measured
the optimal pH value and the effect of protease inhibitors on
these enzymes.
2. Materials and methods
2.1. Plant material
For this study 12 agricultural plant species (including two
species in two cultivars) and 5 wild-living plant species
were used. The former being Allium cepa L. cv. Wolska, (Liliaceae), Allium porrum L. cv. Bartek (Liliaceae), Cucumis sativus L. cv. Hela and cv. Julian (Cucurbitaceae), Cucurbita
pepo L. cv. Bambino (Cucurbitaceae), Helianthus annuus L.
cv. Paskowany (Asteraceae), Lactuca sativa L. cv. Ewelina
(Asteraceae), Phaseolus vulgaris L. cv. Wiejska (Fabaceae),
Pisum sativum L. cv. I1owiecki (Fabaceae), Raphanus sativus
L. cv. Murzynka and cv. Krakowianka (Brassicaceae) and
Zea mays L. cv. Anawa (Poaceae) and the latter: Geranium
pusillum L. (Geraniaceae), Hippophae rhamnoides L. (Eleagnaceae), Ornithogalum umbellatum L. (Liliaceae), Ruta
graveolens L. (Rutaceae), and Vincetoxium hirundinaria L.
(Asclepiadaceae). For our studies we selected cultivated plant
species classified from various plant families, whose

cultivation demands soil varying in fertility and nitrogen content and wild-living species from different terrestrial
ecosystems.
2.2. Growth of plant material
The seeds were surface-sterilized by soaking in 70% ethanol for 2 min and then in 10% sodium hypochlorite with one
drop of Tween 20 (per 100 ml) for 15 min and rinsed five
times with sterile, deionised water. The seeds were germinated
at 23  C in autoclaved Petri dishes with two layers of moistened filter paper. The non-damaged seedlings of each plant
species with about 10-mm-long roots were used in sterile,
hydroponic cultures. The seedlings were separately placed
into small tubes (30 mm  80 mm) with 15 ml of autoclaved,
deionised water (pH 6.8) on absorbent gauze (cotton) in such
a way that only the bottom parts of the roots were immersed
in water with the top 5 mm above the water surface. Then,
each culture was placed into a bigger, covered tube
(35 mm  230 mm). All operations were done under aseptic
conditions in a laminar airflow cabinet. The seedlings were
cultivated in a controlled environment room at 23  1  C air
temperature, 70% relative humidity, and 16:8 h photoperiod
with 380 mmol m2 s1 light intensity at plant height.
Sterility in the culture medium after cultivation was verified
each time with microbiological tests (microcount combi;
Schulke-Mayr), which include medium suitable for growth
of microbes. Aliquots which were free of contamination
were used for analysis.
2.3. Purification of the culture medium
The culture medium was first centrifuged (3000  g,
20 min), the supernatant was brought to 70% saturation with
ammonium sulphate and then the aliquots were centrifuged
(3000  g, 30 min). Preliminary studies established that 70%
saturation with ammonium sulphate precipitated all proteases.
The precipitate was dissolved in 8 ml of 0.9% NaCl in 0.05 M
phosphate buffer (pH 6.8) and dialysed (Serva dialysis tubing)
at 4  C for 24 h against the same buffer. Then the dialysate
was brought to the initial volume (15 ml) with 0.9% NaCl in
0.05 M phosphate buffer (pH 6.8). These partially purified
aliquots were used to measure the proteolytic activity.
2.4. Casein-hydrolyzing activity
The caseinolytic activity was determined by the modified
Ansons method [3] in which the time-dependent release of
tyrosine from the substrate, casein, was monitored. The reaction mixture consisted of 0.5 ml of the partially purified culture medium and 0.5 ml of 2% (w/v) casein dissolved in
0.9% NaCl in 0.05 M phosphate buffer (pH 6.8). After 4 h
of incubation at 28  C the reaction was arrested by adding
2 ml of 20% (w/v) TCA (trichloroacetic acid). The protein
precipitation took place at 4  C and then aliquots were centrifuged (3000  g, 20 min). Then 0.5 ml of the supernatant was
first mixed with 2 ml of 6% (w/v) Na2CO3, and then 0.5 ml of

M. Godlewski, B. Adamczyk / Plant Physiology and Biochemistry 45 (2007) 657e664

FolineCiocolteau reagent (1/4, v/v diluted with deionized

water) was added. After 30 min, absorbance at 750 nm was
read. In the control, TCA was added to the culture medium
prior to casein. One unit of protease activity was defined as
the amount of the enzyme which released from casein the
amount of proteolysis products corresponding to 1 mmol of
tyrosine during 1 h of incubation at 28  C.
2.5. Azocasein-hydrolyzing activity
The azocaseinolytic activity was determined by the modified Tomarellis method [41], in which the time-dependent
release of azo-dye-coupled TCA-soluble peptide fragments
from the substrate, azocasein was monitored. The reaction
mixtures consisted of 2 ml of partially purified culture medium
and 1 ml of 0.5% (w/v) azocasein dissolved in 0.9% NaCl in
0.05 M phosphate buffer (pH 6.8). After 4 h of incubation at
28  C the reaction was arrested by adding 2 ml of 20% (w/
v) TCA. Precipitation and centrifugation (3000  g, 20 min)
took place at 4  C. Then 1.5 ml of the resulting supernatant
was mixed with 0.5 ml of 2 M NaOH, and after 30 min, absorbance at 440 nm was read. In the control TCA was added to
the culture medium prior to azocasein. One unit of protease
activity was defined as the amount of enzyme that increased
the absorbance by 0.1 at 440 nm per 1 h at 28  C.

659

28  C with azocasein in 0.9% NaCl in 0.05 M acetate buffer

(pH 4 and 5) or 0.05 M phosphate buffer (pH 6, 7 and 8).
2.7. Effect of class-specific protease inhibitor
The influence of inhibitors on the proteolytic activity in the
culture medium was measured after 14-day cultivation of A. porrum, Z. mays and H. annuus. The effect of inhibitors on the different classes of proteases was examined by the hydrolysis of
azocasein (see above) after 1 h preincubation with protease inhibitors (see Table 2). Control samples (without inhibitors)
were preincubated for 1 h with the appropriate solvents used
to prepare inhibitor stock solutions. Every determination was repeated four times and the mean values were reported.
2.8. Estimation of proteolytic activity in the culture
medium during growth of seedlings
The proteolytic activity of the culture medium after successive periods of cultivation (from 2 to 14 days) was measured
for Z. mays, H. annuus and A. porrum. Casein- and azocasein-hydrolyzing activities of the culture medium were measured every second day using the methods described above.
For these studies we used the whole volume (15 ml) of the
culture medium for each time period.

2.6. Optimal pH estimation

2.9. Root surface measurements
The optimal pH value for the proteolytic activity in the
culture medium was measured after 14-day cultivation of A. porrum, Z. mays, H. annuus using Tomarellis method (see above).
The proteolytic activity was measured after 4-h incubation at

Root surface was measured using a stereological method, as

described by Head [16]. In this method root surface is estimated on the basis of fresh weight and length of the root.

Table 1
Characteristics of the seedlings after 14-day cultivation and proteolytic activity of the culture medium extrapolated to the root system surface (means  SEM,
n 10)
Plant species

Allium porrum
Zea mays
Helianthus annuus
Cucurbita pepo
Allium cepa
Cucumis sativus cv. Hela
Cucumis sativus cv. Julian
Pisum sativum
Hippophae rhamnoides
Geranium pusillum
Raphanus sativus cv.
Murzynka
Raphanus sativus cv.
Krakowianka
Ruta graveolens
Phaseolus vulgaris
Vincetoxium hirundinaria
Lactuca sativa
Ornithogallum umbellatum

Age of seedling
used in culture (days)a

Proteolytic activity (U mm2 root surface)

Seedlings

Roots

Fresh weight
(mg)

Fresh weight
(mg)

Length
(mm)

Surface
(mm2)

Caseinolytic

Azocaseinolytic

5
4
4
8
5
7
6
5
7
6
4

13.8  1.1
363  20
374  8
1889  46
17  1.8
99.1  3.8
62.3  2
1068  100
19.3  4.6
11  0.7
37.4  1

0.83  0.11
157  19
67.4  4.4
366  21
5.4  0.85
38.7  0.8
13.4  1.3
178  13
2.7  0.6
1.3  0.01
9.5  0.9

58.2  6.5
301  48
348  28
334  26
50  5.3
135  2.8
81.4  8.7
89.1  6.9
49.9  3.1
60.4  2.8
113  28

22.4  2.5
760  99
532  21.5
1239  82
54.3  6.6
235  15.1
118  11
363  15
35.6  4.7
27  0.6
109  3.4

13  2.1
0.31  0.05
0.39  0.06
0.13  0.01
1.7  0.15
0.35  0.04
0.61  0.05
0.16  0.01
1.62  0.09
1.91  0.3
0.63  0.2

19.1  3.6
0.47  0.3
0.6  0.1
0.19  0.02
2.53  0.24
0.52  0.1
0.92  0.2
0.24  0.01
1.93  0.16
2.95  0.41
0.94  0.3

39.1  6.2

9.7  1.6

27.4  3.3

53  5.7

0.92  0.15

1.29  0.2

6
4
5
4
6

7.5  0.9
1172  150
26.9  5
11  0.3
19.6  3.1

0.61  0.09
240  33
6.4  2.2
0.8  0.1
2.2  0.2

26  1.7
323  43
32.2  3.6
65.3  7.7
32.6  3.2

18.1  1.2
984  129
49.6  7.2
25.3  2.7
28  2.8

1.91  0.3
0.03  0.003
0.51  0.16
0.49  0.14
negligible

3.31  0.18
0.05  0.006
0.89  0.27
0.72  0.19
0.38  0.16

Plant species were placed in order of decreasing proteolytic activity of the culture medium.
a
From seed imbibition.

M. Godlewski, B. Adamczyk / Plant Physiology and Biochemistry 45 (2007) 657e664

660

2.10. Statistics

The proteolytic activities of the culture medium of the seedlings similar in size, e.g. A. porrum and L. sativa, were several
fold different (Fig. 1 and Table 1). The culture medium of A.
porrum showed a comparable level of proteolytic activity to Z.
mays and H. annuus, whose seedlings were several fold larger
(Fig. 1 and Table 1). There was no correlation between the
fresh weight of the seedlings and the level of proteolytic
activity in the culture medium (azocaseinolytic activity,
r 0.38; P 0.14; caseinolytic activity r 0.34; P 0.19).
Correlation between the surface of the root system and the
level of proteolytic activity in the culture medium was weak
(azocaseinolytic activity, r 0.47; P 0.06; caseinolytic
activity, r 0.42; P 0.09).

Each experiment consisted of 10 replicates (n 10) for one

species or cultivar. Comparison of means between cultivars of
Raphanus and Cucumis was done using a t-test. Correlations
were determined using Spearman r test. All statistical analyses
were undertaken using the software Statistica (StatSoft, Inc.).
3. Results
3.1. Proteolytic activity in the culture medium
The studies of the proteolytic activity in the culture medium
showed that plant roots could exude enzymes which were able
to hydrolyze casein or azocasein. The levels of proteolytic
activity of various plant species after 14 days of cultivation
were significantly different (Fig. 1).
Among the species studied, the highest proteolytic activity
was found in the culture medium of Allium porrum, Zea mays
and Helianthus annuus. The values of casein- and azocazein-hydrolysing activity estimated for these species were around 0.02
and 0.04 U ml1, respectively (Fig. 1). In contrast, the proteolytic
activity in the culture medium of Ornithogalum umbellatum, Lactuca sativa, Vincetoxium hirundinaria, Phaseolus vulgaris and
Ruta graveolens was negligible or undetectable (Fig. 1).
In the case of Cucumis sativus and Raphanus sativus the
proteolytic activities for two cultivars of these species were
estimated (Table 1). The culture medium of C. sativus cultivar
Julian (A) showed a higher level of proteolytic activity per unit
of root surface system than cultivar Hela (B) (P < 0.003). No
statistically significant difference between the studied cultivars
of R. sativus was observed.
At the starting point all the roots were of equal length, but
after 14 days of cultivation significant differences appeared
(Table 1).

3.2. Proteolytic activity in the culture medium during

14 days of seedling cultivation
To investigate the level of the proteolytic activity in the
culture medium from the starting point to the 14th day of
cultivation we chose three plant species which showed the
highest level of proteolytic activity in the culture medium:
Z. mays, H. annuus, and A. porrum (Fig. 1). The measurements
were done every second day of cultivation. After the first
2 days of cultivation no proteolytic activity was detected in
any of the species studied, however successive measurements
revealed its increasing levels (Figs. 2e4).
A marked correlation between the proteolytic activity and
time of cultivation was observed (e.g. for Z. mays caseinolytic
activity, r 0.96, P 0.0001; azocaseinolytic activity,
r 0.97, P 0.0001). Analysing the curves shown in Figs.
2e4 one can put forward a hypothesis that after 14 days of
cultivation the levels of the proteolytic activity (U ml 1) in
the culture medium of none of the studied species reached
their maximum. Comparison of the level of proteolytic activity
in the culture medium with the surface of the root system

0.05
casein

Proteolytic activity, U/ml

0.045

azocasein

0.04
0.035
0.03
0.025
0.02
0.015
0.01
0.005
O. umbellatum

L. sativa

V. hirundinaria

P. vulgaris

R. graveolens

G. pusillum

R. sativus D

R. sativus C

H. rhamnoides

P. sativum

C. sativus B

C. sativus A

A. cepa

C. pepo

H. annuus

Z. mays

A. porrum

Fig. 1. Proteolytic activity of the culture medium after 14-day cultivation of the seedlings (means  SEM, n 10). Casein or azocasein were used as substrates.
Plant species were placed in the order of decreasing activity of the culture medium.

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661

Table 2
Effect of various protease inhibitors on secreted root proteases (means  SEM, n 4)
Inhibitor

Target proteases

Concentration (mM)

Residual activity (%)

A. porrum

H. annuus

Z. mays

AEBSF
SBTI
E-64
Iodoacetamide
EDTA
EGTA
Pepstatin

Serine proteases
Serine proteases
Cysteine proteases
Cysteine proteases
Metalloproteases
Metalloproteases
Aspartate proteases

1
1
0.05
0.1
1
1
0.01

100
100
30  2
40  3.5
100
100  1.9
73  2.5

100
100
33  3.3
45  2.9
100
100  2.5
90  4

100
98  1.3
35  6.5
50  4.1
98  1.3
100
85  1

Abbreviations: AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride; E-64, N-(trans-epoxysuccinyl)-L-leucine 4-guanidinobutylamide; SBTI, soybean trypsin inhibitor; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene-bis(oxyethylenenitrilo) tetraacetic acid.

throughout the experiment showed that the level of proteolytic

activity per root surface unit initially increased, and then
decreased (Figs. 2e4). In the case of all plant species studied,
the highest value of proteolytic activity per root surface unit
was observed after 6e8 days of cultivation and then a several-fold decrease followed in A. porrum with smaller
decreases in Z. mays and H. annuus.

3.4. Effect of class-specific protease inhibitor

The proteolytic activity was strongly decreased after preincubation with E-64 and iodoacetamide (Table 2) in case of all
species studied. A smaller decrease was observed after preincubation with pepstatin and no inhibition after using AEBSF,
SBTI and EDTA, EGTA.
4. Discussion

3.3. Optimal pH estimation

The proteolytic activity of the culture medium in the range
of pH 4e8 was measured after 14 days for A. porrum, Z. mays
and H. annuus. Azocasein (0.5%, w/v) in 0.05 M acetate
buffer (pH 4 and 5) or in 0.05 M phosphate buffer (pH 6, 7
and 8) was used as a substrate. In all three cases the proteolytic
activity was detectable between pH 4 and 8, the highest at pH
7 (Fig. 5). In the case of A. porrum an additional, less
pronounced peak was observed at pH 5.

It has long been a paradigm of plant and soil science that

soil heterotrophic microorganisms must decompose organic
matter, releasing inorganic N before nitrogen would become
available for plant uptake. Nowadays, it is well known that
dissolved organic nitrogen in the form of amino acids could
be a significant source of N in nitrogen nutrition [24]. It is
assumed that free amino acids in the soil solution result
from hydrolysis of proteins and peptides of soil organic matter
caused by proteases secreted by soil microbes.

casein, U/ml
azocasein, U/ml
casein, U/mm2
azocasein, U/mm2
0.04

casein, U/ml
azocasein,U/ml
casein, U/mm2
azocasein, U/mm2
25

15

0.02
10

0.015
0.01

5
0.005
0

0
2

10 12 14

Time of cultivation, days

Fig. 2. Proteolytic activity of the culture medium after successive periods of
A. porrum seedling cultivation (means  SEM, n 10).

0.03

Proteolytic activity, U/ml

Proteolytic activity, U/ml

0.025

0.025
4
0.02
3
0.015
2
0.01
1

0.005
0

Proteolytic activity, U/mm2 root surface

20
0.03

Proteolytic activity, U/mm2 root surface

0.035

0.035

0
2

10 12 14

Time of cultivation, days

Fig. 3. Proteolytic activity of the culture medium after successive periods of
H. annuus seedling cultivation (means  SEM, n 10).

M. Godlewski, B. Adamczyk / Plant Physiology and Biochemistry 45 (2007) 657e664

662

casein, U/ml
azocasein, U/ml
casein, U/mm2
azocasein, U/mm2
0.035

Proteolytic activity, U/ml

1.6
0.025
1.2

0.02

0.015

0.8

0.01
0.4
0.005

Proteolytic activity, U/mm2 root surface

0.03

0
2

10 12 14

Time of cultivation, days

Fig. 4. Proteolytic activity of the culture medium after successive periods of
Z. mays seedling cultivation (means  SEM, n 10).

The results presented in this work suggest that in many (if

not all) plant species intact roots are able to exude proteases.
We are sure that proteases detected in the culture medium
originated from plants because we always checked sterility
using microbial tests. The presence of these enzymes in the
culture medium was not due to leakage from cells. If such
an event existed there should be a strong correlation between
the root system surface and the proteolytic activity in the
culture medium. We did not find such correlation; for example
one of the highest proteolytic activities was found in the culture medium of A. porrum, which had a very small root system
surface. On the contrary in the case of P. vulgaris the root
0.05
Allium porrum
Helianthus annuus
Zea may s

Proteolytic activity, U/ml

0.045
0.04
0.035
0.03
0.025
0.02
0.015
0.01
0.005
0
4

pH
Fig. 5. Effect of pH variations on azocaseinolytic activity of the culture medium after 14-day cultivation of A. porrum, H. annuus and Z. mays
(means  SEM, n 10).

system surface was very large, but the proteolytic activity in

the culture medium was low.
Chang and Bandurski [7] observed a negligible level of
proteolytic activity in the culture medium of axenic corn
cultures. However, in our studies proteolytic activity was
relatively high. The reason for this divergence could be different pH conditions, age and cultivar of Z. mays seedlings.
Chang and Bandurski [7] used pH 7.5 for measurements of
the proteolytic activity and 4e5-day-old seedlings of Z.
mays var. Michigan hybrid 350. In our studies we used the
culture medium of 20-day-old seedlings (cv. Anawa) and
pH 6.8.
In our studies we observed that the level of proteolytic
activity differed significantly for various plant species
(Fig. 1). These results point out that the ability to secrete
proteases could be species-specific. This hypothesis may be
confirmed by the lack of correlation between the level of
proteolytic activity of the culture medium and the fresh weight
of seedlings (azocaseinolytic activity, r 0.38; P 0.14; caseinolytic activity r 0.34; P 0.19) and by the lack of
strong correlation between the level of proteolytic activity in
the culture medium and the surface of the root system (azocaseinolytic activity, r 0.47; P 0.06; caseinolytic activity,
r 0.42; P 0.09). Statistically significant differences between two cultivars of C. sativus (P < 0.003) suggested that
the levels of secreted proteases could be not only speciesspecific but also cultivar-specific.
The proteolytic activity in the culture medium of A. porrum, Z. mays, and H. annuus was measured for 14 days to
investigate its changes with time. These studies showed that
detectable proteolytic activity appeared after 4 days and was
increasing with time of cultivation (Figs. 2e4). Extrapolation
of the proteolytic activity to the root system surface showed
that the value of proteolytic activity after 8 days of cultivation
declined (Figs. 2e4). Decrease in the proteolytic activity per
one unit of the root surface system was especially marked in
the case of A. porrum (Fig. 3). Lack of correlation between
proteolytic activity per root surface and fresh weight (e.g.
for Z. mays, caseinolytic activity r 0.12, P 0.53; azocaseinolytic activity, r 0.12, P 0.54) in addition to the decline
of proteolytic activity per unit of the root system surface in the
case of older seedlings suggests that individual zones of growing roots could differ in effectiveness of secretion. It is probable that the apical parts of root (meristematic and elongation
zones), which demand larger quantities of nutrients and whose
sizes in the growing roots are almost constant, could secrete
proteases more effectively than mature parts of the root.
The mechanism of exudation of proteases is certainly
tightly regulated. Secretion of proteases by microbes could
be induced by the presence of proteins in the medium or by
the N status [9,15]. In these studies we measured the proteolytic activity in the culture medium of the seedlings cultivated
without any nutrients, so the secretion of proteases by the plant
roots could be regulated by endogenic mechanisms connected
with the level of endogenic sources of N, although it is possible that the presence of proteins in the medium could modify/
enhance protease secretion.

M. Godlewski, B. Adamczyk / Plant Physiology and Biochemistry 45 (2007) 657e664

It is well known that enzymatic activity depends on the pH

value. Soil microbes secrete multiple sets of proteases with
various optimum pH for activity (e.g. [9,21]). Analogically,
multiple sets of endogenous proteases with different pH optima of activity perform important functions in plants
[27,34,37]. We estimated the optimum pH for the proteases
secreted by A. porrum, Z. mays and H. annuus (Fig. 5). In
the case of all three plant species the maximum activity was
noted at pH 7, although in the case of A. porrum we also
observed a lower peak of activity at pH 5, which suggests
that this plant species could secrete a wider set of proteases.
The highest level of proteolytic activity at pH 7 is correlated
with the optimal soil pH value for plant growth. Plants usually
prefer pH values near to neutral or a little acidic (pH 6.0e6.5).
In addition this pH value is beneficial for the activity of amino
acid transporters, which were detected in plant roots [32].
Results after preincubation with standard protease inhibitors point to cysteine proteases as the main type of exuded
proteases by A. porrum, Z. mays and H. annuus (Table 2).
Small inhibition after using pepstatin suggests the additional
presence of aspartate proteases in the culture medium.
Our studies conducted in sterile conditions allowed us to
detect the exudation of proteases by plant roots. However, in
natural conditions plants are forced to compete with the soil
microorganisms for amino acids [5,20,26]. During this competition Phleum pratense and Deshampsia flexuosa incorporated
19% and 64% of labelled amino acids added to the soil, respectively [29,30]. Mycorrhiza is another important factor
for soil proteolysis and uptake of amino acids produced by
this reaction. Mycorrhizal fungi facilitate the uptake of amino
acids by plants. These fungi also exuded proteases accelerating rate of soil proteolysis [6,36].
Our studies showed that plant roots can secrete proteases.
Presence of proteins in the culture medium is not necessary
to induce exudation of proteases. Individual species and cultivars of the same plant species could differ in the levels of proteolytic activity in the culture medium, which could point to
species-specific or even cultivar-specific ability to secrete proteases. Measurement of the proteolytic activity in the culture
medium at different pHs showed that secreted proteases
were the most active at pH 7, although in the case of Allium
porrum the second, lower peak at pH 5 was also observed.
Acknowledgements
This work was supported by the University of qodz, grant
no. 505/381.
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