Download Protein reutilisation in corms of Colchicum autumnale

Biologia, Bratislava, 61/1: 97—102, 2006
97
Protein reutilisation in corms of Colchicum autumnale
Lenka Franková*1, Katarína Cibírová2, Károly Bóka3, Otília Gašparíková1
& Mikuláš Pšenák2
1
Institute of Botany, Slovak Academy of Sciences, Dúbravská cesta 14, SK–84523 Bratislava, Slovakia; tel.: ++421-259426119; *e-mail: [email protected]
2
Comenius University, Faculty of Pharmacy, Department of Cell and Molecular Biology of Drugs, Kalinčiakova 8, SK–83232
Bratislava, Slovakia; tel.: ++421-2-50117308
3
Department of Anatomy, ELTE, Pázmany Péter Sétany 1/C, H–1117 Budapest, Hungary
Abstract: Colchicum autumnale L. is a monocotyledonous geophyte with hysteranthous leaves, i.e. flowering and leaf growth
occur in different time periods. Because after the starch, the second prominent storage compound of corm is represented
by proteins, we were interested in nitrogen remobilisation during the annual life cycle of C. autumnale. In this context the
content of soluble and insoluble proteins were measured in parallel with determination of some exo- and endopeptidase
activities. Our results indicate that the continual proteolysis occurs in both mother and new daughter corms during the
whole life-cycle of the plant. L-Ala-aminopeptidase and trypsin-like endopeptidase were the most active peptidases in both
mother and daughter corms. As the protein level of mother corm did not change significantly during the development of
the future above-ground part under the soil surface (the first, autumnal developmental stage), the developmental profile
of nitrate reductase activity was estimated followed by evaluation of total nitrogen and amino acid contents. Significant
activity of root nitrate reductase was detected in the roots only in the second, vernal stage. Our results showed that the
stored proteins constituted a relevant nitrogen source partly required by the growth processes of the late autumnal stage,
but mainly by the intensive growth of leaves and reproductive structures during the second, photosynthetically active stage
of the life-cycle.
Key words: Colchicum, corm, geophyte, peptidases, proteolysis, nitrogen storage
Abbreviations: d.m. – dry mass; L-Ala-AP – L-alanine-aminopeptidase; BAPA-ase – (N-benzoyl-arginine-hydrolase)
trypsin-like endopeptidase; Z-Glu-Phe-CP – Z-(Z-benzyloxycarbonyl)-glutamyl-phenylalanine-carboxypeptidase; Z-GluTyr-CP – Z-glutamyl-tyrosine-carboxypeptidase; Gly-L-Pro-DPP – glycyl-L-proline-dipeptidylpeptidase; L-Leu-AP – Lleucine-aminopeptidase; PMSF – phenylmethylsulfonyl fluoride; Suc-Phe-EP – (N-succinyl-phenylalanine-hydrolase) –
chymotrypsin-like endopeptidase; TDG – thiodiglycol
Introduction
Colchicum autumnale L. is monocotyledonous geophyte
with underground basal storage stem, called corm. Because this perennial medicinal plant flowers without
leaves, it belongs to the group of hysteranthous geophytes (Franková, 2005). Geophyte species are often
considered as a source of carbohydrates. However, they
do contain proteins the content of which is substantially lower than that of starch (Monte-Neshich et al.,
1995; Shewry, 2003). Usually the underground storage
organs, such as tubers, storage roots and corms, accumulate less than 10% of the stored proteins (Pate &
Dixon, 1982). Unlike seed storage proteins, very little
is known about the reserve proteins of the underground
storage organs of geophytes (Guimarães et al., 2001).
The importance and some characteristics of proteins of plant storage tubers, bulbs and corms have been
reported by Shewry (2003). Vegetative storage organs
contain a large amount of storage proteins, which are
mobilized during seed filling, foliar (Rossato et al.,
2002) and floral (Wagstaff et al., 2002) senescence,
regrowth (Gomez & Faurobert, 2002) or sprouting
(Shewry, 2003). In addition to their storage function,
they can play additional roles in the plant, e.g. enzymatic (Strickland et al., 1995; Tonón et al., 2001;
Rosahl et al., 1987), inhibitory (Yeh et al., 1997; Hou
et al., 1999), antioxidant (Hou et al., 2001), antibacterial (Flores et al., 2002) or antifungal ones (Flores
et al., 2002, Terras et al., 1993). The storage proteins
can be defined as proteins whose major role is to act as
stores of nitrogen, sulphur and carbon (Shewry, 2003).
In plant cells protein breakdown is mediated by
different proteolytic systems: vacuolar proteolysis, selective nuclear and cytosolic proteolysis, and organellar
proteolysis. Because each cellular compartment is af-
98
fected by sugar starvation, these different proteolytic
systems might be involved in starvation-induced proteolysis (Brouquisse et al., 1998). Proteins are hydrolysed to their amino acids by the sequential action of
three types of enzymes: endo-, carboxy-, and aminopeptidases (Callis, 1995).
Although a few papers aimed at storage proteins in
tuberous geophytes have already been published (Pate
& Dixon, 1982; Shewry 2003), nothing is known
about the proteins and their major role in life strategy of hysteranthous geophytes generally and that of
cormous ones especially. In case of C. autumnale, as
a typical representative of hysteranthous cormous geophytes, neither protein content, nor protein breakdown
have been studied so far.
In the present study, the changes in the protein
amount in the mother and daughter corms during the
annual life-cycle of C. autumnale are reported and correlated with some proteolytic activities of these tissues.
Potential contribution of nitrate assimilation (taken up
by roots) to the protein synthesis in the daughter corm
is also evaluated. To better understand the protein
changes during the first part of the life cycle, the content of total nitrogen and that of total amino acids have
also been determined. The results obtained were interpreted in compliance with the individual developmental
stages of the life cycle of autumn crocus.
Material and methods
Plant material
Colchicum autumnale L. was collected in a natural population (Malacky, Slovak Republic – 48◦ 26 N, 17◦ 02 E).
Protein extraction and content
Soluble proteins were extracted from fresh corms by 0.05 M
citrate buffer pH 6.6 in presence of 0.02 M KCl, 0.002 M
MgCl2 , 0.001 M dithiotreithol, 0.002 M Na2 S2 O3 and 0.5%
Polyclar AT (MOHAN-KUMAR et al., 1999) for 1 h. The
extract was centrifuged (10 000 g) and the sediment two
times re-extracted. After centrifugation the supernatants
were pooled and used for determination of soluble protein
content. The sediment was washed and used for extraction of cell wall-bound proteins. To determine insoluble proteins Triton X 100 was added to extraction medium (final
concentration of 0.05%) and extracted as described before.
The protein content was estimated according to BRADFORD
(1976) using bovine serum albumin as a standard.
Enzyme preparation
Corms were pulverised in presence of liquid nitrogen, washed
with excess of cooled acetone (–50 ◦C, delipidization) and
dried at laboratory temperature. 1 g of acetone-powder was
extracted with 0.1 M Na-phosphate buffer, pH 7.5 (proteolytic enzymes) in presence of Polyclar AT. To extract
nitrate reductase 0.1 M Na-phosphate buffer, pH 8.0 containing 5 mM MgCl2 , 0.5 mM EDTA, 0.5 mM PMSF, 1%
glycerol and 0.05% Triton X 100 were used (FRANKOVÁ et
al., 2005). After extraction (3 × 30 min) and centrifugation
(10 000 g, 30 min) the proteins in supernatants were pooled
and precipitated by ammonium sulphate (70% saturation).
L. Franková et al.
After centrifugation (10 000 g, 45 min) the sediment was
dissolved in buffer used and dialysed. All operations were
carried out at 4 ◦C.
Determination of proteolytic activities
To determine aminopeptidase activity the L-alanine pnitroanilide and L-leucine p-nitroanilide (Sigma, Richmond,
USA) were used as substrates. Standard reaction mixture
at final volume of 1.5 mL contained 0.1 mL of enzyme
solution and 1.4 mL of substrate solution (1.4 µmol Lalanine p-nitroanilide or L-leucine p-nitroanilide) in 0.01 M
Tris-HCl buffer, pH 8.0. After incubation at 30 ◦C for 30
min the reaction was stopped by adding 0.25 mL of 30%
acetic acid and absorbance of released p-nitroaniline was
measured at 400 nm (KOVÁCS et al., 1985). For calculation of enzyme activity the molar absorption coefficient of
9950 M−1 cm−1 was taken. In case of trypsin-like endopeptidase, chymotrypsin-like endopeptidase and dipeptidylpeptidase assay 1.4 µmol of BAPA (α-N-benzoyl-D,L-arginine
p-nitroanilide), 1.4 µmol of N-succinyl-phenylalanine pnitroanilide or 1.29 µmol of glycyl-L-proline p-nitroanilide
were added to the reaction mixture and assayed as described
before. Carboxypeptidase activities were estimated using Zglutamine-thyrosine and Z-glutamine-phenylalanine as substrates. Reaction mixture (1 mL) contained 1.2 µmol of substrate in 0.02 M Tris-HCl, pH 7.2 and enzyme solution. After
incubation (37 ◦C, 30 min) the rate of liberated Phe or Tyr
was evaluated by ninhydrine method [formation of Schiffbase and detection of blueviolette dye at 570 nm, ROSEN
(1957)] using Phe and Tyr as standards. All enzyme activities were expressed as µmol s−1 g−1 d.m.
Nitrate reductase assay
Reaction mixture at final volume of 0.5 mL included enzyme solution and 0.05 M Na-phosphate buffer pH 8.0 containing 10 mM KNO3 , 1 mM EDTA, 0.2 mM NADH and
0.01 mM FAD. After 5 min incubation at 30 ◦C the reaction
was stopped by adding 50 µL of 0.5 mM Zn(CH3 COO)2
and 50 µL of 10 mM phenazine methosulphate [oxidation of
NADH excess; DRUART et al. (2000)]. NO−
2 produced was
determined by pipetting 1 mL of sulfanillamide (1% in 1.5
M HCl) and 1 mL of N-(1-Naphthyl)-ethylendiamine dihydrochloride (0.02% in distilled water). The absorbance of
azo dye was measured at 540 nm.
Total nitrogen and total amino acid content
Total nitrogen assay was estimated using Kjeldahl method
(FERENČÍK et al., 1981). The spectrum and level of total amino acids (free and protein bound) were analysed by
HPLC. The proteins present in 0.5 g of fresh corms were
suspended in 30 mL of 6 M HCl followed by nitrogen bubbling. After hydrolyzation (110 ◦C, 23 h), the sample was
neutralized with 30 mL of 12% NaOH and diluted up to
100 mL with dilution buffer pH 2.2 containing 1.4% citric acid, 1.25% NaCl, 0.01% (w/w) caprylic acid and 0.5%
TDG. Amino components were identified using an AAA 400
amino acid analyzer (Ingos, Prague) equipped with Ostion
LG AN B cation-exchange column (0.37 × 45 cm) and with
two channel photometer 440 and 570 nm. In the assay the
following phosphate buffer systems were used: 0.2 M (pH
2.7), 0.4 M (pH 4.25), 1.12 M (pH 7.9), 0.2 M (pH 3.0) and
0.2 M NaOH.
Protein reutilisation in corms of Colchicum autumnale
99
B
A
Insoluble proteins
Soluble proteins
200
80
150
60
100
40
50
0
0
1.IX
15.IX
1.X
8.X
22.X
29.X
6.XI
13.XI
20.XI
27.XI
4.XII
15.I
1.II
28.II
10.III
24.III
31.III
7.IV
14.IV
21.IV
28.IV
12.V
19.V
26.V
2.V
9.VI
22.VI
20
Protein content (mg.g-1 d.m.)
100
250
Insoluble proteins
Soluble proteins
15.VIII
1.IX
15.IX
1.X
8.X
22.X
29.X
6.XI
13.XI
20.XI
27.XI
4.XII
15.I
1.II
28.II
10.III
24.III
31.III
7.IV
14.IV
21.IV
28.IV
12.V
19.V
26.V
2.V
9.VI
22.VI
Protein content (mg.g-1 d.m.)
120
Fig. 1. Total protein content in old mother corm (A) and new daughter corm (B). (Means ± SE, SE ≤ 7%, n = 6).
13
4
8
11
10
6
5
4
1
2
12
7
3
1
2
9
5
6
3
7
8
A
B
Statistics
The data are means of three independent experiments performed each in two replicates.
Results and discussion
Generally, the polysaccharides are the most common
storage materials in perennial plants (Buckeridge et
al., 2000; Pate & Dixon, 1982). Only a limited group
of geophytes accumulates proteins as a main storage
material. Starch appeared to be the main storage component of corms of C. autumnale (Franková et al.,
2003, 2004). The results in this paper revealed that
the proteins formed the second major component of
corm’s storages. In the mother corm the protein fraction represents only 6–11% of dry mass while that in
new daughter corm forms at least 10–22% of dry mass.
Fig. 2. The first developmental (autumnal) stage
of C. autumnale: A – Formation of root system
from the root disk after flowering: 1 – new daughter corm, 2 – root disk, 3 – root system, 4 – stem,
5 – peduncles, 6 – ovaries, 7 – sieve-like structure
(connection between old and new corm), 8 – style
lobes. B – Development of new shoot (regular)
under the soil surface – the whole future aboveground part of the plant: 1 – old mother corm, 2 –
protuberance, 3 – non-developing irregular bud,
4 – new shoot, 5 – new daughter corm, 6 – root
disk, 7 – next, new regular bud for future generation, 8 – roots, 9 – stem, 10 – peduncles, 11 –
young capsules with unmature seeds, 12 – rest of
perianth tubes, 13 – leaves. The arrows indicate
the position of soil surface.
The amount of total proteins varies in dependence on
the individual developmental phases of the life cycle of
perennial autumn crocus.
The continuity of autumn crocus is provided by the
annual corm replacement, i.e. the new daughter corm
replaces old mother one each year. The annual life cycle consists of two developmental stages: the first – autumnal one (September-December) followed by winter
season, and the second – photosynthetically active one
(March–May), which tends to senescence (June) and
summer dormancy (July–August, Franková et al.,
2003, 2004). The developmental profile of total proteins
in mother corm (Fig. 1A) shows that their level does
not change significantly during the autumnal stage. For
this period the differentiation of new shoot (from regular bud ensuring the plant continuity) is characteristic (Franková et al., 2004). The appearance of ir-
100
L. Franková et al.
2
0,35
A
mother corm
daughter corm
C
1,6
0,28
1,2
0,21
0,8
0,14
0,4
0,07
0
0
0,5
B
mother corm
daughter corm
0,4
0,5
mother corm
daughter corm
D
0,4
0,3
0,3
0,2
0,2
0,1
0,1
Jun
Apr
May
Mar
Jan
Feb
Dec
Oct
Nov
Sep
Jul
Aug
Jun
Apr
May
Mar
Jan
Feb
Dec
Oct
Nov
Sep
Jul
0
Aug
0
Activity (µkat.g-1 d.m.)
Activity ( µkat.g-1 d.m.)
mother corm
daughter corm
Fig. 3. Developmental profile of some exo- and endopeptidases in old mother and new daughter corm: L-Ala-AP (A), L-Leu-AP (B),
N-Suc-Phe-EP (C) and BAPA-ase (D). (Means ± SE, n = 6).
regular shoot (from irregular bud serving for vegetative propagation, Fig. 2B) depends on both environmental conditions and level of storage reserves in the
mother corm. The regular bud gives rise to new daughter corm and its shoot regularly at the end of August.
The new shoot (yet without roots) is in flower in the
middle of September. After flowering the differentiation of roots and the complete future above-ground part
(stem, leaves and capsules) takes place in the soil (Figs
2A,B). These events proceed without any photosynthesis and are energetically covered up by intensive reutilisation of starch reserves of mother corm (Franková
et al., 2003, 2004). In the course of autumnal stage
the amount of total proteins (soluble and insoluble) in
the new developing corm increases (Fig. 1B), but the
corresponding decrease of proteinous nitrogen was not
observed in the mother corm (Fig. 1A). Thus, it seems
that the proteins in new corm are formed independently
from the proteins stored in the mother corm. Looking
for details what happens in the new corm at the time
of root (middle of October, Fig. 2A) and shoot (future above-ground part, October-November, Fig. 2B)
development, we obtained following results: the levels
of total nitrogen and that of total amino acids in the
new corm slowly decreased during shoot development,
but the amount of total proteins increased by the end
of November. The opposite phenomenon was observed
after complete shoot development, i.e. an increase of
amino acid amount, and decrease of protein content and
that of total nitrogen (Figs 1B, 4). Total protein content in mother corm remains almost constant (Fig. 4).
It seems that during the first part of the autumnal stage
the proteins in the new developing daughter corm (Fig
2B) are synthesised from the pool of free amino acids.
This pool may be maintained by a) free amino acids
from the mother corm, b) proteolytic activities in both
corms, and c) nitrate assimilation in root system directly connected with new corm. Because the protein
content of the mother corm almost did not change, it is
difficult to find the origin of nitrogen used for metabolic
processes connected with root and new shoot formation (Figs 2A,B). On the other hand, the remarkable
proteolytic process was observed in both mother and
daughter corms. Of the proteolytic enzymes five exopeptidases and two endopeptidases were detected having maximum activity during winter season. There were
L-Ala-AP (Fig. 3A), L-Leu-AP (Fig. 3B), Z-Glu-TyrCP, Z-Glu-Phe-CP, Gly-L-Pro-DPP (data not shown),
Suc-Phe-EP (Fig. 3C) and BAPA-ase (Fig. 3D). LAla-AP and BAPA-ase appeared to be the most active peptidases in both mother and daughter corms.
The importance of L-Ala-AP and L-Leu-AP in protein turnover has been reported by Mikola & Mikola
(1986). The highest proteolytic activities were detected
in the mother corm during late autumn and winter
period and at the end of the life cycle of the plant.
These findings indicate that an active process of protein turnover takes place during autumnal stage in both
corms. The level of free amino acids in the new daughter
corm might be renewed exactly at the cost of protein
degradation during the late autumnal developmental
stage (end of November-December, Fig. 4).
The results of amino acid analyses in the new
corm presented in Fig. 5 show that N-rich amino acids
Protein reutilisation in corms of Colchicum autumnale
content (mg g-1 d.m.)
350
total nitrogen NC
total proteins NC
f ree amino acids NC
total proteins OC
280
210
140
70
14.XII
7.XII
21.XI
14.XI
7.XI
31.X
0
Fig. 4. Changes in the different nitrogen fractions in new daughter
corm during root and shoot formation of C. autumnale. OC – old,
mother corm, NC – new, daughter corm. (Means ± SE, n = 6).
2N
Content (mg g-1 d.m.)
30
4N
2N
24
18
2N
12
3N
6
Asn
Arg
Gln
Tyr
Leu
Ala
Ser
Lys
Val
Pro
Gly
Thr
Ile
His
Phe
0
Fig. 5. The spectrum and content of amino acids in the new
daughter corm (the early autumnal stage); N – nitrogen rich
amino acids.
Activity (nkat g -1 d.m.)
30
25
20
15
10
5
30.IX
15.X
30.X
15.XI
30.XI
15.I
30.I
15.II
28.II
15.III
30.III
15.IV
30.IV
15.V
30.V
15.VI
0
Fig. 6. Seasonal variation of nitrate reductase activity in the root
system of C. autumnale. (Means ± SE, n = 6).
Asn, Gln, Arg are present at the highest levels. It
means that not only proteinous nitrogen but also Nrich units can serve as immediately accessible form of
reduced nitrogen. Low activity of nitrate reductase (3–
4 nkat g−1 d.m.) at the first, autumnal stage indicates that free amino acids are de novo formed only
101
at limited amounts. Paradoxically the nitrate reductase reached maximum activity during the second developmental stage (March-June, Fig. 6). For the second, photosynthetically active period following developmental processes are typical: a) the intensive growth
of leaves and reproductive structures (capsules with
seeds), b) formation of reserves in daughter corm (the
new corm ripening), c) physical disappearance of old
mother corm, d) dying back of the above-ground part.
The total reutilization of the rest of protein content in
the mother corm went in parallel with its physical destruction. A certain correlation between the decline in
protein content (Fig. 1A) and relatively high activities
of N-Suc-Phe-EP, BAPA-ase (Figs 3C,D) and Z-GluPhe-CP confirms this. The amino acids released in the
new and old corms as well as those formed de novo may
saturate their demands for growth and differentiation
of vegetative tissues, as well as the development of reproductive structures during the second developmental
stage. In addition, the level of proteinous nitrogen in the
new corm also decreased to the end of spring period. So
at the end of May the amount of total proteins in the
new ripe corm was almost the same as it was in the old
mother one at the beginning of the life-cycle (August).
To maintain the critical level of storage reserves in the
ripe corm at the end of the annual bioprogram seems
to be the specific feature of C. autumnale.
We have found that proteolytic activities are
present in both corms during the whole life cycle of
the plant. In addition, the activities are varying significantly depending on the individual developmental
stages. These findings evoke the question what was the
reason for the presence of proteolytic activities in both
corms of C. autumnale during the whole annual lifecycle. It is known that one of the crucial functions
of proteolysis is to help regulate metabolism. Therefore, the presence of proteolytic activities in corm tissues might be combined with the immediate degradation of enzymes and the others, less essential proteins,
which are not required by the basic metabolism at a
given developmental stage. This phenomenon was observed in case of developmental profile of polyphenoloxidase (Bilecová et al., 1996) and β-glucosidase (unpublished results) during the annual life-cycle of mother
and daughter corms of autumn crocus. Together with
protein synthesis, degradation is essential to maintain
appropriate enzyme levels (Scheurwater et al., 2000).
For C. autumnale it means that the levels of the most
abundant enzymes with a high specific activity (such
as amylases, glucosidases, polyphenoloxidases and peptidases) could also be modulated through their selective
breakdown. The main significance of remarkable proteolytic activities is probably to ensure the turnover of
proteins and their trade-off in distribution between the
mother corm, new developing plant and its new corm.
Although starch represents 50% of corm’s dry
mass, the storage proteins still remain the required
component of storage material of C. autumnale. Our
102
results show that the stored proteins constituted a relevant nitrogen source being required by the growth processes of the late autumnal stage, but mainly by the
metabolic events taking place during the second, photosynthetically active stage, when the individual organs
of vegetative part are intensively growing and the storage compounds are formed in the ripening daughter
corm. The elucidation of nitrogen metabolism and its
remobilization in corms of autumn crocus contributes
to our knowledge of the basic features of hysteranthous
geophytes.
Acknowledgements
This work was supported by Grant Agency VEGA under
contract No. 1/1275/04.
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Received Aug. 20, 2005
Accepted Nov. 10, 2005