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VARIABILITY OF THE INHIBITORS OF SERINE, CYSTEINE PROTEINASES
AND INSECT
AMYLASES IN VIGNA AND PHASEOLUS
1
2
3
AL.V.KONAREV , N.TOMOOKA , M. ISHIMOTO , AND
2
DUNCAN A.VAUGHAN
1
All-Russian Institute of Plant Protection
Podbelskogo 3, St. Petersburg, 189620, Russia; [email protected]
2
National Institute of Agrobiological Resources
2-1-2, Kannondai, Tsukuba, Ibaraki 305-8602, Japan
3
National Institute of Agro-Environmental Sciences,
3-1-3, Kannondai, Tsukuba, Ibaraki 305, Japan
1. Introduction
Proteinase and -amylase inhibitors are factors involved with plant resistance to insects and
microorganisms [2, 7, 21, 27]. Many inhibitors in legumes, cereals and other plants are well
studied biochemically and genetically [5, 27]. Our research focuses on the study of natural
inhibitor variability in plants. Spectra of inhibitors provide biochemical and genetic
information. In wheat, levels of amylase and proteinase inhibitory activity in seeds are related
to the spectra of inhibitors [17, 21].
Combination of various inhibitor systems cause
synergistic effects increasing plant resistance to pests [25] or to pathogens [1]. Proteinase
inhibitors theoretically can protect amylase inhibitors from destruction by gut proteinases and
preserve them as anti-nutritional factors. Inhibitors can also be useful as genetic markers in
the study of plant diversity and evolution [12, 16, 18, 20, 21] in combination with
conventional methods [9, 11, 22], in particular, for elucidation the origin of resistant plant
forms. Consequently, parallel analysis of variability of all main inhibitor systems in species
and genera can be highly informative.
The objectives of the present work were to study proteinase and -amylase inhibitor
polymorphism in Vigna and Phaseolus and estimate the variation in inhibitors in both genera.
2
AL.V.KONAREV et al.
Although inhibitors in both these economically important genera are well studied, inhibitor
variation in diverse germplasm, especally, in Vigna species, is still unclear. In addition,
taxonomy of both genera is imperfectly understood [23, 24, 29]. The methods used here are
based on a set of methods for inhibitor identification which have been efficient in studies of
inhibitors in wheat and related cereals [16, 15, 18]. Analysis of digestive enzymes of several
Coleoptera and Hemiptera insect species in relation to inhibitors from cereals revealed amylases, trypsin-like , chymotrypsin-like and cystein proteinases [13, 15, 17, 18]. Results
from wheat studies confirm the validity of conducting most of our research on inhibitor
variability using standard enzymes, such as bovine trypsin. In case of legumes we worked
with insect amylases and did not work with insect proteinases, but is known that many legume
pests have cysteine and serine digestive proteinases [7, 10].
2. Materials and Methods
Inhibitor variability was studied in 93 accessions of 24 species in the 6 subgenera of Vigna
and 55 accessions of 14 Phaseolus species collected by authors or obtained from MAFF,
Japan and USDA, USA gene banks (TABLE 1). Species names and taxonomy were used
according to Marechal et al. (1978) and Tomooka et al. (1996) [24, 29]. Seed and leaf proteins
were extracted with 20 % glycerin (1:10 and 1:2 correspondingly) and were separated by
isoelectric focusing in Servalyt precotes gels (Serva) pH 3-10. Proteinase inhibitors were
detected by gelatin replicas method [14, 15]. -Amylase inhibitors were detected by
starch/amylase/PAG replicas [13, 19].
3. Variability in Hydrolase Inhibitors.
3.1. PROTEINASE INHIBITORS IN SEEDS
Trypsin-like enzymes are common in guts of many insect species and in extracellular liquids
of some fungi. Inhibitors of trypsin-like enzymes are the most active and complex inhibitor
system in legumes studied and they are of Bowman-Birk type [6]. Variability of seed trypsin
inhibitors (TI) was studied in accessions of six subgenera of Vigna and in the genus Phaseolus
(arranged according to taxonomy [24] in Fig. 1). Not all, but majority of TI components in
VARIABILITY OF THE INHIBITORS
3
Vigna and in Phaseolus species were active to chymotrypsin as well (not shown). This is
characteristic of Bowman-Birk type inhibitors.
Vigna subgenus Vigna species (Fig. 1: 2 & 3, V.
luteola; 4,
V. marina; 5&6, V.
oblongifolia; 7, V. membranacea and, 9-11, V. unguiculata) had, in general, species specific
TI spectra. V. oblongifolia and V. unguiculata were polymorphic by inhibitor spectra. Some
bands were common for two or three species (for example, bands with high pI in patterns 2, 6
and 10). For most V. vexillata accessions (12-14, subgenus Plectotropis) TI bands possessed
pI below 5,3.
Some results, perhaps reflected species misidentification in analyzed
accessions. For example, sample 13 called V. vexillata had TI spectra similar to V.
oblongifolia (6) and has been now been reidentified as this species.
Main types of spectra found in Vigna subgenus Ceratotropis are shown (Fig. 1). We found
that the majority of Ceratotropis species examined had species specific spectra for TI and
chymotrypsin inhibitors (CI). In addition, considerable intraspecific variability was detected
for trypsin inhibitors in most species, for example, V. angularis (16-19 in Fig. 1), V. hirtella
complex (24-27) and V. radiata (33-35). TI and CI in seeds of some V. hirtella accessions (g
& h) were not detected (27). However, intraspecific variability for inhibitors was very low in,
for example, V. minima and V. mungo.
Most species in the subgenus Ceratotropis are diploid except for two closely related
tetraploids V. reflexo-pilosa subsp. glabra and V. reflexo-pilosa subsp. reflexo-pilosa, which
are considered to be amphidiploids [3]. Both tetraploid species are resistant to bruchid
beetles [28] and diseases [4], thus the origin of their genomes, which has not been clearly
established, is of particular interest to plant breeders. TI of diploid V. trinervia (30) were
closer to those of the tetraploids V. reflexo-pilosa subsp. reflexo-pilosa (31) and subsp. glabra
(32) than other species. This result corresponds to data on izoenzymes [3]. Several TI and CI
bands in tetraploid species were similar to inhibitors of accessions “a” and “b” (Table 1) with
unclear taxonomy included to V. hirtella complex. It is possible that forms related to these
accessions and V. trinervia were donors of the genomes in the allotetraploid species [14].
TI spectrum of V. lasiocarpa (45) differed significantly from that of V. longifolia (46)
(subgenus Lasiocarpa). V. caracalla accessions 47 and 48 (subgenus Sigmoidotropis) had
only two similar TI bands. Three single seeds of the accession “a” of V. adenantha (49-51)
differed strongly by TI (Fig.1), morphology and color. Two TI components of seed 49 were
similar to those of V. caracalla (48). In seeds 50 and 52 (b) TI bands were very faint.
4
AL.V.KONAREV et al.
Figure 1. Variability of trypsin inhibitors in Vigna and Phaseolus species. Seed proteins extracted with water were separated in Servalyt precotes pH 3-10 gel and trypsin
inhibitors were detected by gelatin replicas method. V-M, subgenera Vigna. a,b,... designation of accessions listed in TABLE 1.
V, s/g Vigna:
20-22, V. umbellata (a-c)
40-41,V. aconitifolia (a&b)
Phaseolus :
72, P. leptostachyus (a)
2, 3& 63, V. luteola (a&b)
23, V. minima (c)
42&43, V. stipulacea (a&b)
56-59 &79-81, P. vulgaris (f-h, 73, P. maculatus
4, V. marina
24-27, V. hirtella (b, c , e&h)
L, s/g Lasiocarpa:
q, a&c )
74, P. microcarpus (a)
5&6, V. oblongifolia (a&b)
28, V. nepalensis (b)
45, V. lasiocarpa
60-61& 64-65, P. acutifolius (f, g, 75, P. parvulus
7, V. membranacea;, 8, 30, V. trinervia
46, V. longifolia
a&d)
76, P. polystachios (a)
9-11, V. unguiculata (a-c)
31, V. reflexo-pilosa (a)
S, s/g Sigmoidotropis
66, P. angustissimus (a)
77, P. ritensis (a)
P, s/g Plectotropis:
32, V. glabrescens (a)
47&48, V. caracalla (a&b)
67, P. coccineus (a)
78, P. stenolobus (a)
12-14, V. vexillata (a, b&i)
33-35, V. radiata (a, c&h )
49-51 & 52, V. adenantha (a&b) 68, P. filiformis (a)
“*”, marker mixture of samples
C, s/g Ceratotropis:
36-37, V. mungo (a&d)
H, s/g Haydonia:
69, P. glabellus (b)
10 and 21
16-19, V. angularis (a-d)
38-39, V. grandiflora (a&b)
53, V. schimperi
71, P. hybrid (b)
VARIABILITY OF THE INHIBITORS
5
High intra-accession variability for TI may be a result of outcrossing. We did not find any
relationship between the TI of V. schimperi (53) (subgenus Haydonia) and TI of other Vigna
representatives.
Most Phaseolus species possessed distinctive TI spectra, though some of them showed
intraspecific variability. Some P. vulgaris lines (56-59) varied by TI. P. acutifolius accessions
(60, 61, 64 and 65) differed by TI spectra as well. Some TI bands had similar pI in two or more
species (for example,
P. acutifolius, 65, and
P. angustissimus, 66; P. coccineus, 67, P.
glabellus, 69, and P. hybrid, 71). ). In the genus Phaseolus the same TI variants occur in
several species which enables relationships between inhibitor systems within Phaseolus to be
analysed. As to relationship between TI systems of Vigna and Phaseolus, there are some
similarities in structure of TI spectra of representatives of Vigna subgenus Lasiocarpa (V.
caracalla, acc. 47) , and P. vulgaris (57, 79). Both Vigna subgenus Lasiocarpa and Phaseolus
are of America [24] which may explain their seemingly close evolutionary relationship based on
TI spectra.
Subtilisin inhibitors (SI) are possible plant protective factors to fungi, because subtilisin-like
extracellular proteinases are common for many plant pathogens. SI spectra are less complex and
less variable than those of TI (not shown). Spectra of SI were specific for species and groups of
species within subgenus Ceratotropis [14], other subgenera of Vigna and for Phaseolus species.
Some species had polymorphic SI.
Cysteine proteinase inhibitors (CPI) are poorly studied in Vigna compared to other inhibitors.
Cysteine proteinases are very important in protein digestion of bruchid beetles which are a major
pests of Vigna and Phaseolus. In this work we used the plant cysteine proteinase papain for
detection of inhibitors. Papain inhibitors generally showed variation at the species level in both
Vigna and Phaseolus.
Though all Ceratotropis species had species specific spectra of TI and other inhibitors, the
majority of inhibitor components were common for two or more species. Combined data
inhibitor bands in all accessions were scored (present or absent) in order to estimate evolutionary
relationships between species (74 band positions were taken into account) [14]. It was much
more difficult to find common TI components between species of Ceratotropis and other Vigna
subgenera. Only in V. vexillata (s/g Plectotropis) some TI bands were similar to inhibitors of V.
hirtella accessions “a” and “b” and both tetraploid species. The results of evaluation of
evolutionary relationships between Ceratotropis species are shown (Fig. 2) (data
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AL.V.KONAREV et al.
on all analysed inhibitor systems: TI, CI, SI and CPI for all accessions of each species, or
groups of accessions with characteristic spectra were combined).
Figure 2. Relationships between Vigna subgenus Ceratotropis species on the basis of analysis of polymorphism of
four inhibitor systems (TI + CI + SI +CPI). Results were estimated by UPGMA method (NTSYS 1.80). Data on all
accessions of each species were combined except for clearly distinctive forms. hirtella*, acc. c-f and i-j (TABLE 1)
combined; hirtella**, acc. g and h combined; hirtella*** , acc. a and b combined.. Tetra-trin*, hypothetical Vigna
form constructed by subtraction of inhibitor bands characteristic of V. trinervia only from inhibitor set of tetraploid
species. radiata*, acc. h (35 in Fig. 1).
The inhibitor systems of the tetraploid and diploid forms possibly related to their putative
genome donors were similar (Fig. 2). This scheme coincides, in general, to schemes of
evolutionary
relationships
between
Ceratotropis
species
based
on
morphological
characteristics and data of RAPD and isoenzyme analysis [3, 29], however in the case of
diploid–polyploid interrelations inhibitor analysis is more informative than RAPD analysis
[29].
3.2. INSECT -AMYLASE INHIBITORS IN SEEDS
Seed proteins of Phaseolus and Vigna accessions were separated by isoelectric focusing and
inhibitors of
mexican bean weevil, Zabrotes subfasciatus, and azuki and bean weevil,
Callosobruchus chinensis, -amylases were detected on replicas (Fig. 3). Active and
polymorphic insect -amylase inhibitors were found only in some Phaseolus species (P.
acutifolius, P. coccineus, P. hybrid, and P. vulgaris) not Vigna, that corresponded with data
on inhibitors of Tenebrio molitor amylase [26]. A weak inhibitor of C. chinensis -amylase
VARIABILITY OF THE INHIBITORS
7
was found in one accession of P. ritensis (26). We detected inhibitors by their specific
activity, while previous reports have identified inhibitors in gels only as low molecular
glycoproteins or by immunochemical methods [8, 26]. Sometimes the presence of
glycoproteins does not correspond to a protein with inhibitory activity. The main forms of amylase inhibitors characteristic of P. vulgaris [8] (34, AI-1; 35, AI-2; 36, AI-3),
including some bands of specific Z. subfasciatus amylase inhibitors (arrowed) which are
absent among azuki bean weevil inhibitors, are present in analyzed accessions.
Figure 3. Insect -amylase inhibitors in Vigna and Phaseolus species. Seed proteins extracted with water were
separated by isoelectric focusing in Servalyt precotes pH 3-10. Z.subfasciatus and C. chinensis -amylase inhibitors
were detected in replicas A and B correspondingly. a, b, ..., designations of accessions in TABLE 1.
1-5&37, P. acutifolius (a-e, f)
6&7, P. angustissimus (a&b)
8-10, P. coccineus (a&b)
11-12, P. filiformis (a&b)
13-14 , P. glabellus (a&b)
15&16, P. hybrid (a&b)
17-18, P. leptostachyus (a&b)
19&20, P. maculatus (a&b)
20&21, P. microcarpus (a&b)
22, P. parvulus
23&24, P. polystachios (a&b)
25&26, P. ritensis (a&b)
27&28, P. stenolobus
(a&b)
29-36, P. vulgaris (a-e,
k, q, t)
38, V. unguiculata (b)
39, V. lasiocarpa (a)
40, V. caracalla (a)
41, V. schimperi
42, V. angularis (a)
43, V. radiata (a)
3.3. PROTEINASE INHIBITORS IN LEAVES
Proteinase inhibitors in leaves of various plants are less studied than inhibitors in seeds.
Wheat seeds (endosperm) and leaves contain different inhibitor systems [21]. In Vigna and
Phaseolus species leaf TI and CI did not correspond to those in the seed and had lower pI
values. In both genera species differed by spectra of TI and CI. Obtaining two gelatin replicas
from the same separating gel facilitated comparison of inhibitors of both proteinases (Fig. 4).
In V. angularis (1-3), three groups of accessions of V. hirtella complex (4-9), V. mungo
(16&17) and V. stipulacea (18) most of TI bands coincided in replicas with CI (1-9). In V.
reflexo-pilosa (10), V. radiata (11-14) and V. oblongifolia (22-23) only a few bands of both
inhibitor types coincided. In one of V. radiata accessions (13-14) CI bands had higher pI than
8
AL.V.KONAREV et al.
TI. In V. luteola (20&21), V. unguiculata (24-26), V. lasiocarpa (28-29), V. vexillata (30-32)
and V. schimperi (33-34) TI were detected but not CI. In normal undamaged leaves SI were
not found. However, V. angularis seedlings damaged by mites contained active SI band with
pI near 4.5 (not shown) while TI and CI spectra did not change. Thus, SI may be induced by
damaging Vigna leaves.
Figure 4. Variability of leaf trypsin and chymotrypsin inhibitors in Vigna species. Leaf proteins extracted with water
were separated in Servalyt precotes pH 3-10 and two gelatin replicas were obtained consequently from the same gel.
Replicas A and B were developed by trypsin and chymotrypsin correspondingly. a, b, ..., accessions listed in
TABLE 1; 2 p., two plants of the same accession.
1-3, V. angularis (a, c, o )
4&5, V. hirtella (a&b) 2 p.
6, V. hirtella (b, 25 in Fig.1)
7&8, V. hirtella (h, 27 in Fig.1) 2 p.
10, V. reflexo-pilosa (a)
11&12, V.radiata, (a, 33 in Fig.1) 2 p.
13&14, V.radiata (c, 34 in Fig.1) 2 p.
16&17, V. mungo (a) 2 p.
18, V. stipulacea (a)
20&21, V. luteola (a) 2 p.
22&23, V. oblongifolia (a) 2 p.
24&25, V. unguiculata (a) 2 p.
26, V. unguiculata (b)
28&29, V. lasiocarpa 2 p.
30, V. vexillata (k)
31&32, V. vexillata (l) 2 p.
33&34, V. schimperi 2 p.
4. Conclusions
Proteinase and amylase inhibitor composition is generally specific for Vigna and Phaseolus
species but in some species inhibitors are highly variable. In both genera seeds and leaves
contain different proteinase inhibitor systems. Variation revealed and approaches elaborated in
this study can be useful in relation to breeding legumes for pest and pathogen resistance.
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10
AL.V.KONAREV et al.
APPENDIX
TABLE 1. Accessions of Vigna and Phaseolus used in the study.
Species
Accessions
V. luteola
a&b
V. marina
V. oblongifolia a&b
V. membranacea
V. unguiculata a-c
V. vexillata
a-l
V. angularis
a-d
V. umbellata
V. minima
e-o
a-c
a-f
V. hirtella
a-j
V. nepalensis a&b
V. trinervia
V. reflexo-pilosa ssp.
reflexo-pilosa a&b
ssp. glabra
a&b
V. radiata
a-g
V. mungo
a-h
V. grandiflora a&b
V. aconitifolia a&b
V. stipulacea
a&b
PI: 354915 & 406329
PI: 360072
PI: 406352 & 406354
PI: 292867
PI: 406365, 427087 & 292883
PI: 406391, 406387, 280132,
406400, 406390, 406393, 365095365097, 406383, 406401, 225934
03031366, 03013592, 03028830,
PI 527686
11 populations collected in Japan
03025780, 03031356, PI 220249
03028839, 03030456, 03028854,
03028850, 03028849, 03028840
03030505, 03031362, NI1377,
NI1394, NI 970, NI 971, 03031363,
03031364, 03031369, 03031370
03028851 & 03031368
03028858
03028860 & 03030466
03031365 & NI1185
03025800, 00035043, 03028846,
03028847, 03028845, NI876,
NI1135
03031349, 03019941, 03027092,
03026841, 03028842, 03028843,
03028844, 03028848
03028832 & 03031348
03020037 & 03020041
NI251 & NI1030
Species
V. lasiocarpa
V. longifolia
V. caracalla
V. adenantha
V. schimperi
Phaseolus :
P. vulgaris
Accessions
a&b
a&b
a&b
a&b
PI 306376
PI 310294
PI 195333 & 322586
PI 312898 & 319444
PI 337032
a-e
W6 17000, PI 319441, PI:
326054, 535411 & PI 535414
G02771,G06388, G12866,
G12888,TO
G12882, TS,G:10009, 12915A,
19892,Taishou-kintoki
G12932,TAr4, G12923
G01221,G02744, G03453,
G04270,G04287, G09998
W6 15625, PI: 535200, 535201,
535233, 535240
PI 3111897, Nayarit
PI: 535272, 535273
PI: 311165, 311171 & 313417
PI: 535293 & 535294
W6 15688 & PI 535311
PI: 378974 & 390535
PI: 535315 & 535329
PI 535350
PI 535353 & 535358
PI 511998
PI: 264607 & 264608
PI: 494138 & 535372
W6 15587 & W6 15704
Lines with AI-0 f-j
AI-1 k-p
AI-2 q-s
AI-3 t-y
P. acutifolius
a-e
f&g
P. angustissimus
P. coccineus
a-c
P. filiformis
a-b
P. glabellus
a-b
P. hybrid
a-b
P. leptostachyus a-b
P. maculatus
P. microcarpus a-b
P. parvulus
P. polystachios a-b
P. ritensis
a-b
P. stenolobus a-b
a, b,..., designation of accessions; PI and W6 – accessions from USDA gene bank system; lines of Phaseolus differing
in -amylases were provided by the National Agricultural Research Institute, Japan including accessions with G prefix
originally from CIAT,Colombia; NI accessions from the Phaseolinae collection, Jardin Botanique National de Belgium;
eight-digit numbered materials from Japanese MAFF gene bank.