An unusual minor protein appearing in embryonic axis cells of haricot bean seeds following germination process stimulated by 6-methylthiouracil
Using the two-dimensional polyacrylamide gel electrophoresis approach, an unusual – 30 kDa protein was found in embryonic axis cells of haricot bean seeds following seed germination process stimulated by 6-methyluracil. No similar protein was found both in control and lutidine N-oxide stimulated see...
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| Дата: | 1998 |
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Інститут молекулярної біології і генетики НАН України
1998
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| Назва видання: | Биополимеры и клетка |
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| Цитувати: | An unusual minor protein appearing in embryonic axis cells of haricot bean seeds following germination process stimulated by 6-methylthiouracil / V. A. Tsygankova, V.N. Zayetz, L.A. Galkina, L.P. Prikazchikova, Y.B. Blume // Биополимеры и клетка. — 1998. — Т. 14, № 5. — С. 438-448. — Бібліогр.: 31 назв. — англ. |
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irk-123456789-1555952019-07-04T21:57:47Z An unusual minor protein appearing in embryonic axis cells of haricot bean seeds following germination process stimulated by 6-methylthiouracil Tsygankova, V.A. Zayetz, V.N. Galkina, L.A. Prikazchikova, L.P. Blume, Y.B. Структура и функции биополимеров Using the two-dimensional polyacrylamide gel electrophoresis approach, an unusual – 30 kDa protein was found in embryonic axis cells of haricot bean seeds following seed germination process stimulated by 6-methyluracil. No similar protein was found both in control and lutidine N-oxide stimulated seeds. The synthesis of an additional low molecular weight protein was also detected in a cell-free system prepared from rabbit reticulocytes in the presence of poly(A)⁺RNA isolated from 6-methylthiouracil stimulated embryonic axes of haricot been seeds. At the same time the lutidine N-oxide was found to stimulate drastically the total polypeptide synthesis in an in vitro system prepared from wheat embryo in the presence of a standards poly(A)⁺RNA preparation, no similar effect of the 6-melhylthiouracil having been seen. The ratio of informosomes, free and incorporated into polyribosomes, was investigated following RNP-particles fractionation in a preformed CsCl gradient; the 6-methylthiouracil seed stimulation was shown to induce the development of an additional peak of synthetically active informosomes, their buoyant density being 1.46 g/cm . The 6-methylthiouracil stimulated seed germination causes a significant shortening of haricot plant ontogenesis period without any harmful changes of plant phenotype, the lutidine N-oxide stimulation leads, however, to deformed accelerated vegetative organ appearance accompanied by no reproductive organ development. Nature of 30 kDa protein as well as some problems concerning the correlation between different stimulator-induced cellular gene expression changes taking place during early postembryogenesis and further processes of haricot bean plant growth and development are discussed; some possible practical consequences of our exoeriments arc also mentioned. За допомогою двомірного електрофорезу білків у поліакриламідному гелі виявлено незвичайний мінорний білок з молекулярною масою ~ 30 кДа у клітинах зародкової осі при стимуляції проростання насіння квасолі (Phaseolus vulgaris L.) 6-метилтіоурацилом. Появу цього білка не зафіксовано в нормі та при проростанні, стимульованому N-оксидом лутидина. Синтез додаткового низькомолекулярного білка спостерігався також у безклітинній системі з ретикулоцитів кроля на матриці полі(А)⁺РНК, одержаній з клітин зародкової осі квасолевого насіння, обробленого 6-метилтіоурацилом. У той же час у безклітинній системі з проростків пшениці при використанні «стандартного» препарату полі(А)⁺РНК показано, що N-оксид лутидина різко стимулює синтез поліпептидів, а метилтіоурацил прямо не впливає на процес трансляції. Вивчаючи співвідношення рибосом, вільних та включених у полірибосоми in vivo, за допомогою фракціонування РНП-часточок у преформованому градієнті густини CsCl, встановлено присутність додаткового піка (1,46 г/см ) у фракції полірибосом. з зародкових осей насіння, обробленого 6-метилтіоурацилом. Така стимуляція суттєво скорочує період онтогенезу, ніяк не пошкоджуючи фенотипу рослини. При цьому обробка пророщуваного насіння N-оксидом лутидина призводить до деформованого прискореного розвитку вегетативних органів без розвитку органів розмноження рослини. Обговорюється природа білка 30 кДа і взаємозалежність між змінами в експресії генів, шр викликані ростовими стимуляторами у клітинах зародкової осі під час раннього постембріогенезу, та наступними різнонаправленими процесами росту та розвитку рослин квасолі. Розглянуто також деякі практичні напрямки застосування ростових стимуляторів, пов'язані з результатами проведених експериментів. С помощью двухмерного электрофореза белков в полиакриламидном геле показано появление в клетках зародышевой оси при стимулируемом 6-метилтиоурацилом прорастании семян фасоли (Phaseolus vulgaris L.) необычного минорного белка с молекулярной массой около 30 кДа. Этот белок не обнаруживался в норме и при стимулируемом N-окисью лутидина прорастании семян растений. Синтез дополнительного низкомолекулярного белка отмечен и в бесклеточной системе белкового синтеза из ретикулоцитов кролика на матрице поли(А)⁺РНК из клеток зародышевых осей со стимулируемым 6-метилтиоурацилом прорастанием семян фасоли. В то же время с помощью бесклеточной системы белкового синтези из проростков пшеницы с использованием в качестве матрицы стандартного препарата поли(А)⁺РНК установлено, что N окись лутидина резко стимулирует синтез гюлипвптидов, а 6-метилтиоурацил не оказывает прямого влияния на процесс трансляции. Изучение соотношения свободных и включенных в полирибосомы информосом in vivo методом фракционирования РНП-частиц в преформированном градиенте плотности CsCl выявило присутствие во фракции полирибосом из зародышевых осей при стимулируемом 6-ме.тилтиоурацилом прорастании семян дополнительного пика активных в белковом синтезе информосом с плавучей плотностью 1,46 г/см . Показано, что стимуляция прорастания семян фасоли 6-метилтиоурацилом приводит к существенному сокращению сроков онтогенеза растения фасоли без каких-либо нарушений фенотипа растения, а стимуляция N-окисью лутидина – к деформированному ускоренному развитию вегетативных органов без развития репродуктивных органов растения. Обсуждается, природа белка 30 кДа и связь между различиями в изменении ростстимуляторами экспрессии генов в клетках зародышевых осей в раннем постэмбриогенезе и последующими разнонаправленными процессами роста и развития растений фасоли, а также некоторые практические аспекты, вытекающие из этого 1998 Article An unusual minor protein appearing in embryonic axis cells of haricot bean seeds following germination process stimulated by 6-methylthiouracil / V. A. Tsygankova, V.N. Zayetz, L.A. Galkina, L.P. Prikazchikova, Y.B. Blume // Биополимеры и клетка. — 1998. — Т. 14, № 5. — С. 438-448. — Бібліогр.: 31 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.0004E9 http://dspace.nbuv.gov.ua/handle/123456789/155595 en Биополимеры и клетка Інститут молекулярної біології і генетики НАН України |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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DSpace DC |
| language |
English |
| topic |
Структура и функции биополимеров Структура и функции биополимеров |
| spellingShingle |
Структура и функции биополимеров Структура и функции биополимеров Tsygankova, V.A. Zayetz, V.N. Galkina, L.A. Prikazchikova, L.P. Blume, Y.B. An unusual minor protein appearing in embryonic axis cells of haricot bean seeds following germination process stimulated by 6-methylthiouracil Биополимеры и клетка |
| description |
Using the two-dimensional polyacrylamide gel electrophoresis approach, an unusual – 30 kDa protein was found in embryonic axis cells of haricot bean seeds following seed germination process stimulated by 6-methyluracil. No similar protein was found both in control and lutidine N-oxide stimulated seeds. The synthesis of an additional low molecular weight protein was also detected in a cell-free system prepared from rabbit reticulocytes in the presence of poly(A)⁺RNA isolated from 6-methylthiouracil stimulated embryonic axes of haricot been seeds. At the same time the lutidine N-oxide was found to stimulate drastically the total polypeptide synthesis in an in vitro system prepared from wheat embryo in the presence of a standards poly(A)⁺RNA preparation, no similar effect of the 6-melhylthiouracil having been seen. The ratio of informosomes, free and incorporated into polyribosomes, was investigated following RNP-particles fractionation in a preformed CsCl gradient; the 6-methylthiouracil seed stimulation was shown to induce the development of an additional peak of synthetically active informosomes, their buoyant density being 1.46 g/cm . The 6-methylthiouracil stimulated seed germination causes a significant shortening of haricot plant ontogenesis period without any harmful changes of plant phenotype, the lutidine N-oxide stimulation leads, however, to deformed accelerated vegetative organ appearance accompanied by no reproductive organ development. Nature of 30 kDa protein as well as some problems concerning the correlation between different stimulator-induced cellular gene expression changes taking place during early postembryogenesis and further processes of haricot bean plant growth and development are discussed; some possible practical consequences of our exoeriments arc also mentioned. |
| format |
Article |
| author |
Tsygankova, V.A. Zayetz, V.N. Galkina, L.A. Prikazchikova, L.P. Blume, Y.B. |
| author_facet |
Tsygankova, V.A. Zayetz, V.N. Galkina, L.A. Prikazchikova, L.P. Blume, Y.B. |
| author_sort |
Tsygankova, V.A. |
| title |
An unusual minor protein appearing in embryonic axis cells of haricot bean seeds following germination process stimulated by 6-methylthiouracil |
| title_short |
An unusual minor protein appearing in embryonic axis cells of haricot bean seeds following germination process stimulated by 6-methylthiouracil |
| title_full |
An unusual minor protein appearing in embryonic axis cells of haricot bean seeds following germination process stimulated by 6-methylthiouracil |
| title_fullStr |
An unusual minor protein appearing in embryonic axis cells of haricot bean seeds following germination process stimulated by 6-methylthiouracil |
| title_full_unstemmed |
An unusual minor protein appearing in embryonic axis cells of haricot bean seeds following germination process stimulated by 6-methylthiouracil |
| title_sort |
unusual minor protein appearing in embryonic axis cells of haricot bean seeds following germination process stimulated by 6-methylthiouracil |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| publishDate |
1998 |
| topic_facet |
Структура и функции биополимеров |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/155595 |
| citation_txt |
An unusual minor protein appearing in embryonic axis cells of haricot bean seeds following germination process stimulated by 6-methylthiouracil / V. A. Tsygankova, V.N. Zayetz, L.A. Galkina, L.P. Prikazchikova, Y.B. Blume // Биополимеры и клетка. — 1998. — Т. 14, № 5. — С. 438-448. — Бібліогр.: 31 назв. — англ. |
| series |
Биополимеры и клетка |
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ISSN 0233-7657. Биополимеры и клетка. 1998. Т. 14. № 5
An unusual minor protein appearing in embryonic
axis cells of haricot bean seeds following
germination process stimulated by 6-methylthiouracil
Victoria A. Tsygankova, Vladimir N. Zayetz, Larisa A. Galkina 1,
Ludmila P. Prikazchikova 1, Yaroslav B. Blume
Institute of Cell Biology and Genetic Engineering, National Academy of Sciences of Ukraine
Acad. Zabolotny str., 148, Kyiv, 252143
Institute of Bioorganic Chemistry and Petrochemistry, National Academy of Sciences of Ukraine
Murmanska str., 1, Kyiv, 252600
Using the two-dimensional poly aery lamide gel electrophoresis approach, an unusual » 30 kDa protein was
found in embryonic axis cells of haricot bean seeds following seed germination process stimulated by
6-methylthiouracil. No similar protein was found both in control and lutidine N-oxide stimulated seeds.
The synthesis of an additional low molecular weight protein was also detected in a cell-free system prepared
from rabbit reticulocytes in the presence of poly(A)+RNA isolated from 6-methylthiouracil stimulated
embryonic axes of haricot been seeds. At the same time the lutidine N-oxide was found to stimulate
drastically the total polypeptide synthesis in an in vitro system prepared from wheal embryo in the presence
of a «standard» poly(A)+RNA preparation, no similar effect of the 6-methylthiouracil having been seen.
The mtio of informosomes, free and incorporated into polyribosomes, was investigated following
RNP-particles fractionation in a preformed CsCl gradient; the 6-methylthiouracil seed stimulation was
shown to induce the development of an additional peak of synthetically active informosomes, their buoyant
density being J.46 glcm . The 6-methylthiouracil stimulated seed germination causes a significant
shortening of haricot plant ontogenesis period without any harmful changes of plant phenotype, the lutidine
N-oxide stimulation leads, however, to deformed accelerated vegetative organ appearance accompanied by-
no reproductive organ development. Nature of 30 kDa protein as well as some problems concerning the
correlation between different stimulator-induced cellular gene expression changes taking place during early
postetnbryogenesis and further processes of haricot bean plant growth and development are discussed; some
possible practical consequences of our experiments arc also mentioned.
Introduction. Due to vast biological screening we have
earlier shown [1 ] such substances as lutidine N-oxide
(LNO, ivin-yan) and 6-methylthiouracil (methyur,
6-MTU) accelerate sharply haricot bean seed ger
mination. Our conviction is an embryonic axis of
haricot bean plant (morpho-physiological and bio
chemical characteristics of embryonic axis set, for
mation and germination, its growth as well as deve
lopment during embryogenesis and early postem-
bryogenesis, i. e. during haricot bean seeds matu
ration and germination were studied in detail [2—4])
© V. A. T S Y G A N K O V A , V. N. ZAYETZ, L. A. GALKINA,
L. P. PRIKAZCHIKOVA, Y. В BLUME, 1'598
to be a suitable model permitting to investigate several
ac tua l p rob lems of p lan t physiology and
biotechnology, namely:
(i) to answer some questions concerning the
mechanism of plant growth stimulator effect (these
compounds are strongly different from natural plant
hormones in their chemical structure), to understand
if synthetic stimulators act through the cellular re
ceptor systems similarly to plant hormones or interact
directly with some target molecules on the level of
cellular regulatory system (i. e. at the level of genetic
control or phytohormonal regulation of plant growth
and morphogenesis);
Ш) to understand if the artificially stimulated
438
plaint growth causes any harmful changes affecting the
sequence of normal morphogenetical events during the
early postembryogenesis (mature fully differentiated
organs from primary non-differentiated embryonic
axis organs — root, hypocotyl and leaf, beginning to
form in this period) and if any drastic growth
stimulation realized during the first developmental
stages is able to impair the balanced growth and
development of plant vegetative and reproductive
organs during further plant ontogenesis stages (for
example, acceleration of vegetative organ growth and
inhibition set as well as formation of plant repro
ductive organs).
To solve such problems as fully as possible we
decided to get at first two principal goals:
(i) to understand the mechanisms concerning the
effect of two synthetic growth stimulators — 6-MTU
and LNO changing cellular gene expression in em
bryonic axis cells at early postembryogenesis stage
and programming plant growth and development;
(a) to study the consequences of artificial haricot
seed germination on following plant vegetation.
Materials and Methods. Seeds, In our expe
riments seeds of haricot beans (Phaseolus vulgaris L.)
of the variety «Bielozernaya» were used. Ethanol-
sterilized seeds were put for germination in a thermo
stat at 26 °С; they were incubated between layers of
filter paper moistened by distilled water or by 2 %
solutions containing plant growth regulators. After the
seed incubation, embryonic axes were separated from
cotyledons, washed by distilled water and divided
into three parts (each containing 100 axes) aimed for
isolation of proteins, RNA, and RNP-particles.
Protein extraction. The tissue samples of em
bryonic axes were frozen in liquid nitrogen and
powdered carefully in a china mortar. 10 volumes of
preliminary cooled to 0 °С extracting buffer consisting
of 30 mM tris-HCl, pH 8.7, 1 mM di thiothreitol
(DTT), 1 mM ethylenediaminetetraacetic acid (diso-
dium salt), 1 mM ascorbic acid, 5 rnM MgCl2, and
10 img polyvinylpyrrolidone were added to the tri
turated material. The extract obtained v/as twice run
(during 10 min and 15 min) in a centrifuge at
35.000 £. The 3/4 v acidified acetone (pH 4.5),
containing 0.07 % 2-mercaptoethanol was added to
supernatant fluid. This mixture was strongly shaken
and incubated at -20 °С during 1 h. The denatured
proteins were then pelleted by centrifugation at
35.000 g during 10 min. The pellets obtained were
dried using a vacuum evaporator and dissolved in a
buffer containing (50 jn) per I mg of dry precipitate):
8 M urea, 5 mM K 2 C0 3 , 0.5 % DTT, 2 % ara-
phollynes solution — carrier ampholytes, their pH
range being 3.5—9.5, 2 % Triton X-100 (MKSD-
AN U N U S U A L MINOR PROTEIN A P P E A R I N G IN EMBRYONIC AXIS
buffer). The soluble protein extract was separated
from insoluble material by probes centrifugation
(3 min at 15.000 g) and then kept at -20 °С until use.
Two-dimensional gel electrophoresis of proteins
was realized according to the method described by
O'Farrell [5] with several modifications. The protein
fractionation in the first direction was made in a 5 %
poly aery lamide laminar gel (C = 4 % w/w). The gel
was polymerized in a 8 M urea solution supplemented
by 2 % Triton X-100 and 2 % ampholines mixture
(containing pharmalytes with pH ranges 3.5—9.5 and
5.0—7.0, their ratio being 3:1) to stabilize the central
pH gradient zone. The gel was 1 mm wide, its size
being 140 x 80 mm. 0.02 M NaOH was used as a
cathode electrode solution, and 0,01 M H 3 P 0 4 was
taken as an anode one. The gel pre-focusing was
made at 400 V during 30 min. The protein elec
trophoresis was realized during 2 h at 1200 V in a
cooled chamber. A gel strip with marker proteins was
cut out and stained for marking gel lanes, all other
ones containing protein fractions were polymerized in
a polyacrylamide gel with a 5—15 % concentration
gradient to fractionate the proteins in the second
direction realized in a buffer system described by
Laemmli [6]. This gel was 1.5 mm wide, its size being
145 x 145 mm. As an electrode buffer a tris-glycme
solution (pH 8.3) supplemented with SDS was used.
The proteins entered to the concentrating gel at 70 V
during 1 h, the protein fractionation in a separating
gel was run during 6 h at 160 V. Following separation
the gel strips containing protein fractions were fixed
by a mixture containing 30 % isopropanol and 20 %
acetic acid and stained by a Coomassie brilliant blue
solution. The stain excess was washed out by a
mixture containing 5 % methanol and 7 % acetic
acid. The gels stained were then photographed on a
glass plate lit from the opposite side.
The isolation of total RNA preparations was
realized after embryonic axis tissue destruction using
a buffer solution (buffer I) containing 0.05 M tris-
HCl, pH 7.6, 0.01 M MgCL, 0.06 M KC1, 1 % SDS,
and 4 M guanidine isothiocyanate. The lysate ob
tained was twice treated by a mixture of hot water-
saturated phenol and chloroform; the RNA was pre
cipitated from a water phase by ethanol, treated by
proteinase K, deproteinized again by the same phe
nol-chloroform mixture and precipitated by ethanol.
Some polysaccharide contaminants present in RNA
preparations (preventing fractionation molecular RNA
and manifestation biological activity mRNA in vitro)
were extracted by methoxyethanol; the RNA mole
cules were then precipitated by cetylthrimethylam-
monium bromide; this last reagent was eliminated by
multiple re-dissolving of RNA preparations in a
439
TSYGANKOVA V. A. ET Л1
0.01 M sodium acetate solution followed by sodium
acetate saturated ethanol precipitation.
Poly(A)+RNA separation from poly(A)~ RNA mo
lecules was made using total RNA chromatography on
the oligo(dT)-cellulose columns [7]. Total RNA pre
parations were analysed using electrophoresis in a
1.5 % agarose gel containing 7 M urea according to
Locker [8 ]; the gels obtained had been saturated by
ethidium bromide solution before RNA fractions pho
tographing using an UV lamp. The poly(A)+RNA was
analysed using a Northern-blot approach [91. cDNA
was synthesized on poly(A)+RNA template according
to the protocol described by Bueli et al. [10] using
reverse transcriptase (revertase) and [a- 3 2 P ]-con-
taining deoxy-CTP as a label. A poly(A)+RNA pre
paration was fractionated by electrophoresis in an
agarose gel in the presence of formalyn; electro-
phoretically pure poly(A)+RNA fractions were trans
ferred on nitrocellulose filters and hybridized with
cDNA. The hybridization mixture contained 50 %
formamide, 5X Upper buffer (pH 7.0), 5X Denhardt's
solution [ 1 1 ] , dena tu red calf thymus DNA
(lOO^g/ml), poly(A)*RNA (1 //g/ml), 0.1 % SDS,
and [a - 3 2 P]-cDNA (1.5-10 8 cpm) . Following
hybridization, filters were carefully washed from
exogenous label and exposed during 24 h to a film
PM-1 with an accelerating screen (at -70 °С). The
limits of poiy(A)+RNA sizes were evaluated according
to radioautograph distribution of labelled po
ly (A)+RNA-cDNA hybrids along the gel lanes (with
regard to marker polynucleotides).
The experiments with polyfAf RNA translation
were carried out in cell-free systems derived from
wheat embryos [12] for quantitative determination
incorporation of radioactive label into polypeptide
material and rabbit reticulocytes [9 ] for definition
fractional composition of full-length new synthesizing
polypeptides, using [3'*S [-methionine as a labelling
compound. The polypeptides synthesized de novo in
this last in vitro system were analyzed using the
one-dimensional PAGE approach as mentioned above.
The gels obtained were dried using a heating vacuum-
dryer («LKB», Sweden). The gel fluorography was
made using a protocol described in [13], the gels
having been saturated by a fluorescent reagent,
2,5-diphenyloxazole (PPO) [14].
Radioactive labelling of RNP-particles. The em
bryonic axis samples (100 axes) were thoroughly
washed by distilled water and incubated in a [ 3 H]-
uridine solution (3.7-10* Bq/ml) containing also pe
nicillin and streptomycin (50 /ig/ml) to prevent the
label incorporation by contaminating bacterial cells
present in incubation mixture. The material was
incubated during 1 h in a thermostat at 28 °С in a
Petri dish and then washed by distilled water to
remove all the non-incorporated label.
Isolation of labelled cytoplasmic RNP-particles. A
sample of embryonic axis tissue was homogenized in
the buffer II (containing 20 mM TEA-HC1, pH 7.6,
25 mM KC1, 3 mM MgCl2, 10 mM 2-mercaptoethanol,
250 mM sucrose, heparin (100 fig/ml) as an RNAse
inhibitor, phenylmethylsulfonyl fluoride (50 /ug/ml)
as a trypsin-like protease inhibitor, and cyclohexi-
mide (100 /ug/ml) as an inhibitor of mRNA trans
location on cytoplasmic ribosomes, the corellation
weighed tissue sample to volume of this buffer being
1:10. The homogenate obtained was then filtered and
run at 18.000 g (20 min). A cytoplasmic post-
mitochondrial fraction was incubated with a 0.5 %
Triton X-100 solution (20 min), layered on 0.5 ml
0.5 M sucrose, prepared on the buffer II solution
containing no sucrose and centrifugated in a SW 60
Ті rotor (Beckman centrifuge L5-50) (3 h at 49.000
rpm).
Investigation of RNP-particles in a. CsCl density
gradient. A pellet of cytoplasmic RNP-particles was
suspended in the sucrose-free buffer II and divided
by two; a half of this suspension was a control
sample, its second portion was treated by a 15 mM
EDTA-Na 2 solution to cause polyribosome disso
ciation. Both preparations containing RNP-particles
were then fixed (during 24 h) in a 4 % formaldehyde
solution, all the aggregates formed being removed by
pelleting at 10.000 g (10 min). The soluble material
was then centrifuged in a preformed CsCl density
gradient (1.33—1.65 g/cm 3) (12 h, 45.000 rpm, 2 °С)
using the same SW 60 Ті rotor.
The fractions of this soluble material were then
obtained using a siphon device; each fraction refrac
tive index was determined and compared to density
values of a calibration curve. The fraction optical
density was taken in a spectrophotometer at 260 nm,
Buoyant density (p) of RNP-particles in CsCl was
calculated according to an equation given in [15]:
p = D-10.8601 - 13.4979;
D is a refractive index, the values 10.8601 and
13.4979 are experimentally found corrections ne
cessary because of polarizing buffer properties.
Radioactivity of proteins and RNP fractions was
determined in a LS 100C scintillation counter («Bec-
kman») using Millipore AP-15 fibrous glass filters and
scintillator dissolved in toluene.
Results and Discussion. While elaborating our
approaches concerning mechanisms of action some
synthetic compounds regulating the plant growth we
took into account a lot of data suggesting the gene
expression changes caused by phytohormones to
440
belong to the principal mechanisms of plant growth
and development [16—18] (however, the question
concerning regulation expression of concrete genes by
each from phytohormones are not yet correctly ans
werable). We proposed that the synthetic stimulators
of seed germination (similarly to natural plant hor
mones) increasing drastically the embryonic organism
size and its organ development, cannot mediate their
action without some expression changes of genes
coding synthesis of structural and functional proteins;
the increasing of the pliant mass (by cell enlargement
and cell division) being impossible-without intensive
protein synthesis switching on a lot of reactions
leading to cell and organ differentiation and spe
cialization.
According to [19], the protein «spectrum» in any
cell is to be changed during cell differentiation and
specialisation, the protein synthesis process being a
stage- and organospecific one. We proposed the
protein assortment caused by any stimulated embryo
growth (in view of accelerated development stages) is
to be different from the assortment appeared in
normally developed embryos during the same period;
this difference was proposed to be nearly the same as
between embryo protein «spectrum» determined at an
car у and at a later postembryonic stages during
natural plant development.
The main protein mass in eucaryotic cells is
known to be presented by structural proteins and by
enzymes of the main metabolic pathway (named also
mu ticopied, constitutive, abundant or major pro
teins), these compounds being present at all develop
mental stages and detected without any difficulty
using any one-dimensional gel electrophoresis ap
proach. However, this approach does not permit to
find «гаге» (so-called minor) stage- and organo
specific enzymes and regulatory proteins, presented
in some few copies and «hidden» on «one~dimen~
sional» electrophoregrams by major protein bands. So
we fractionated a total protein preparation isolated
from embryonic axes using a two-dimensional poly-
acrylamide gel electrophoresis (TD-PAGE) protocol.
The stained gel photographs obtained after our
TD-PAGE experiment are present in the Fig. 1 (a, b,
c), the total protein preparations having been pre
viously focused following the first one-dimensional
separation according to their isoelectric point (pi) in
a pH-gradienl gel.
Our results demonstrate the absence of any
differences in embryonic axis protein preparations of
haricot seeds after 12 h of postembryonic develop
ment, both normal and LNO-stimuiated (Fig. 1, a
and b). Simultaneously, the 6-MTU-stimulated ha-
ricct bean germination causes the appearing of a
AN U N U S U A L MINOR P R O T E I N A P P E A R I N G IN EMBRYONIC A;
Fig. 1. Two-dimensional polyacrylamide gel electrophoresis of <
toplasraic proteins isolated from embryonic axis cells in 12 h post
beginning of haricot bean seed germination; the proteins invesliga
are taken from: a --- control (non-stimulated) seeds; /; — LN
stimulated seeds; с — 6-MTU-stimulated seeds
«blurred-bordered» spot formed by an about 30 kl
protein in a zone containing positively charged pi
teins (Fig, 1, c). This spot (although of decreas
intensity) is also seen in photographs obtained frc
TD-PAGE after minimal quantities of total protc
(4 jug per lane) having been loaded into gel pocket
To determine this protein belonging to major
minor ones, we realized in addition an experime
with a one-dimensional PAGE approach in the pi
sence of the SDS, total proteins of embryonic a>
after 12 h of postembryonic development having be
loaded on the gel. However, in this experiment
failed to detect any separate band of the 30 ki
4
protein or to find any staining band differences
between control and 6-MTU-stimulated protein sam
ples. So we concluded this protein to be a minor one,
its blurred spot in the TD-PAGE permits to suppose
it to be also a short-lived compound. This 30 kDa
protein was not detected in embryonic axis pre
parations in 24 h after the beginning of non-sti
mulated germination process, the embryonic axis
sizes being the same as their sizes in 6-MTU-
stimulated seeds in 12 h of postembryonic deve
lopment (no photograph is presented).
The 30 kDa protein detected by the TD-PAGE
approach appears only as a result of the 6-MTU-
stimulated seed germination, no its traces having been
found both in control and LNO-stimulated samples;
our aim was to gain some data explaining such
results. Some physiological consequences of seed tre
atment by LNO and 6-MTU are similar, the ger
mination periods becoming twice shorter (and fi
nishing in 2 days) with both stimulators. However, a
lot of intracellular events followed by these substances
treatment are to be quite different. There are some
facts confirming this point of view. The LNO-treat-
ment stimulates the total cellular protein synthesis
proved in our one-dimensional electrophoresis ex
periment using a 136S J-methionine labelled in vivo
total proteins isolated from embryonic axes and also
following a gel fluorography approach [20 J. At the
same time, the 6-MTU, according to our data,
stimulates the only unusual 30 kDa protein synthesis.
To explain such a marked difference concerning
the effect of two growth stimulating compounds, we
suppose they action through different mechanisms
influencing on certain stages of gene expression
(transcription, formation of synthetically active RNP
complexes, mRNA translation, etc.). The LNO was
already proved to possess no action specificity on the
level of gene expression regulation [20—221 acti
vating both transcription and active RNP formation
and increasing the synthesis of all the cellular pro
teins without changing their assortment.
We thought the appearance of the 30 kDa protein
«поп-typical» for a given developmental stage of
embryonic axis cells might have been a result of some
simultaneous changes at the translation level or at
any precursor stage of the gene expression regulation
(during transcription and/or transcript maturation
levels). To answer this question, we realized some
experiments concerning:
1) the comparative investigation of the LNO and
6-MTU effect on the translation process in an in vitro
system of protein synthesis using a template of
poly(A)*RNA as standard isolated from non-stimu
lated embryonic axis cells;
2) the study of biological activities of po~
ly(A)+RNA preparations isolated from control em
bryonic axes as well as from those ones stimulated
and non-stimulated by LNO and 6-MTU using tin
same in vitro system of protein synthesis;
3) the evaluation of activity protein-synthesizing
apparatus (polyribosomes) in vivo, i. a. proportion of
free and incorporated into polyribosomes (H-uridine
labelling in vivo) of mRNP and rRNP-particles in
embryo axis haricot bean seeds germinated withoul
any treatment and following LNO and 6-MTU sti
mulation.
In the Fig. 2 (a, b) the results of summary
embryonic axes RNA preparations (using agarose gel
electrophoresis) obtained with control and 6-MTU
stimulated material; these data demonstrate the RNA
preparations to contain non-degraded discrete frac
tions of both high and low molecular masses (hnRNA
including preRNAs, preRNAs «wasie products* origi
nated due to processing rRNAs, mRNAs and 1RN As
a h с d e f' g
big. 2. Agarose gel electrophoresis of RNA preparations isolated і'гош
embryonic eell axes in 12 h post the beginning of haricot bean seed
germination; a and b — total RNA preparations from control and
6-М'Ги-stimulated seeds, respectively; с and d — poly (A) RNA and
poly(A) + RNA preparations, respectively, separated on an ol igoidTr
cellulose column; e and / - radioautographs of hybrid molecules
containing [ 3 2 P ] - c D N A and poly(A) + RNA fractions immobilized on
nitrocellulose filter: poly(A) + RNA and | 3 'TJ cDNA isolated from
embryonic axis cells of non-stimulated (e) and 6 MTU stimulated (/)
haricot bean seeds, respectively; g electrophoretie distribution of
marker polynucleotides on a parallel gel lane
442
«mature» forms these RNAs of nuclear and cyto
plasm, regulatory RNA molecules). The ratios
E 2 6 0 / E 2 M
a n d E 2 ( , 0 /E 2 3 0 for our preparations were
> 2.0 and > 2.3, respectively, confirming a good
degree of isolated RNA purification being practically
free from protein and polysaccharide contaminations.
We present also our electrophoregrams of a poly (A) "
RNA preparation (Fig. 2, c) and of a poly(A)^RNA
one (Fig. 2, d) demonstrating the rRNA to be
practically absent.
In the Fig. 2 (e, f) our radioautographs are given
obtained as a result of a Northern blot-hybridization
procedure of poly (A)~RNA fractions with a [ 3 2P J-
cDNA preparation; our results demonstrate these
fraction to contain highly heterogeneous poly(A)+RNA
molecules of different molecular masses (due to
differences the lengths of mRNA coding regions as
well as of its regulatory ones, apparently); so there is
no contradiction to other data [23] concerning the
existence of a marked discretion (in the same size
ranges) for eucaryotic mRNAs (from 8.0 up to 0.24
kbs); a series of spots fused along gel lanes can be
seen on our photographs because of radioactive track
autographs being overlapped on the X-ray film due to
labelled highly heterogeneous hybrid mRNA-cDNA
molecules localized on the filter too near from each
other. An electrophoregram presenting marker poly
nucleotides is also shown (see Fig. 2, g). So it is clear
the poly (A )+RNA preparations studied here to be
non-degraded ones and to keep their high molecular
components.
However, the principal nativity criterion for any
poly (A) "RNA molecules is their messenger activity
directing the polypeptide synthesis process in in vitro
(cell-free systems); the evaluation of RNA messenger
activity was a principal moment in our investigation.
In the Fig. 3 (a, b) the kinetics of [ 3 5 S]-
methionine incorporation into the TCA-insoluble ma
terial is presented; these data were obtained in a
well-known wheat embryo cell-free system using a
template poly(A)"RNA as standard isolated from
embryonic axes of non-stimulated haricot bean seeds.
Our control data concerning TCA-insoluble fraction
radioactivity were obtained with the same in vitro
system containing no growth activator (see Fig. 3, a).
We evaluated also the effect of the LNO and 6-MTU
added to the incubated mixture (Fig. 3, b and c,
respectively). We would like first of all to note the
label incorporation increase during incubation in all
the experiment materials (including also control ones)
suggesting the nativity of poly(A)+RNA preparations
used. Our second finding is that the 6-MTU inhibits
slightly the level of protein label incorporation during
all the incubation period comparing to the control
AN U N U S U A L MINOR PROTEIN A P P E A R I N G IN RMRRYONIC AXIS
L # " i 1 1 —1 г г
0 JO 20 ЗО 40 50
Fig. 3. Kinetics of [ SI-methionine incorporation into the TCA-
insoluble material in a wheat embryo in vitro system with using as
template poly(A) + RNA: a— a control sample (no growth stimulators
were used); b and с—label incorporation into peptides in the
presence of the 6-MTU and LNO, respectively
incorporation level. On the contrary, the LNO sti
mulates the poly (A)"RNA directed polypeptide syn
thesis in our in vitro system, the synthesis level
becoming almost twice higher.
Thus our results prove directly that the LNO
activates the translation processes as well as the
transcription one [20, 21 ]; while the 6-MTU have no
regulatory effect on this crucial stage of gene exp
ression.
So it became of great interest to compare some
functional properties of embryonic axes poly(A)+RNA
preparations isolated from 6-MTU-stimulated and
control haricot bean seeds. In the Fig. 4 (a, b) there
are fluorograms demonstrating the PAGE distribution
of polypeptide fractions. They had been previously
labelled by [3 5S ]-methionine in cell-free system from
rabbit reticulocytes using of poly (A) "RNA as template
RNAs isolated from embryonic axes of haricot been
443
Fig. 4. Fluorographic analysis of electrophoretic polypeptide «spec-
ІГШП» i n the course of poly (A) + RNA-directed in vitro synthesis
(rabbit reticulocyte cell-free system): polypeptide synthesis in the
presence of poly (A)'RNA preparations from 6-MTU stimulated (a)
and non-stimulated (/>) embryonic axis cells, respectively; с — label
incorporation in vitro synthesized peptides in the absence of any
added poly(A) T R.\A preparation (control sample)
seeds following germination stimulated and non-
stimulated by 6-MTU. The in vitro synthesized
polypeptide fractions are very similar with both RNA
preparations used with the only exception: an ad
ditional polypeptide was detected in a gel zone of low
molecular weight proteins while analyzing the labelled
material from the incubation mixture containing the
poly(A)4RNA preparation isolated from 6-MTU-sti-
muiated embryonic axes. Our data do not irrep
roachably prove this additional peptide to be identical
to the one detected in our previous in vivo studies.
However, we have a fact of some 6-MTU-induced
changes of an active poly(A)+RNA pool, i. e. of the
6-MTU participation in the regulatory transcription
mechanisms.
However, contrary to in vitro experiments, any
regulative events having taken place at the
transcription level in vivo cannot be realized а і
translation level because of several following cir
cumstances: mRNA molecules exist and function in
any plant cell as informosomes, i. e. mRN P-pai tides
[24 ], no «naked» RNA is there present; at any stage
of plant development there are bolh a reserved pool
of functionally inactive mRNP being activated ai
following stages and a pool of «working» mRNP
taking part in the translation process.
The isolation of any poly (A) RNA preparation
using phenol deproteinization of mRNP abolishes
these mentioned above differences of mRNA pools,
both types of them becoming equally able to be
translated. So we needed to answer the question
concerning the correlation between the appearance of
the 30 kDa minor protein in vivo and the changes ai
the translation level due to the 6-МTU-induced
regulatory changes of transcription process. So we
studied the correlation of active (incorporated into
polyribosome complexes) and free, inactive in protein
synthesis mRNPs in embryonic axis cells following
stimulated and non-stimulated haricot bean seed
germination.
The data concerning the CsCl gradient frac
tionation of formaldehyde-fixed RNP-particles iso
lated from stimulated and control seeds are given in
the Fig. 5 ia—d). In both cases the obtained ra
dioactive profiles of RNP-particles in the CsCl gra
dient are presented by two radioactivity maxima,
their densities being 1.39 and 1.42 g /cm\ respectively
(small «peaks» of free informosomes), and two large
peaks with their densities 1.52 g/cnr* (for the 6~
MTU-stimulated material) and 1.54 g/cm 1 (for the
control sample), respectively, presenting d e novo
synthesized mRNP and rRNP-particles incorporated
into polyribosomes. While EDTA treating of RNP
preparations only a small part of radioactivity (fol
lowing pulse RNP labelling during I h) remains in the
region of ribosome subparticles localization, the main
radioactive pool in both control and experimental
samples being transported to I he region of free
informosomes; its peak in control is localized ai
1.45 g/cm 3; however, the pulse-labelled RNPs of
stimulated material form two peaks, the lesser one
being concentrated in a region of the buoyant density
l.39 g/cm 3, the last main labelled material being
found in a narrow de novo appeared peak at
1.46 g/cm 3.
So we note a clear correlation between the
6-MTU-induced changes of transcription process as
well as of poly (АГ RNA messenger activity (inducing
the additional protein synthesis) influencing the
444
AN U N U S U A L MINOR P R O T E I N A P P E A R I N G IN EMBRYONIC AXIS
2 6 10 14 18 22 2 6 10 14 18 22
Fraction number
Fig. 5. Fractionation of RNP-particles in a CsCl density gradient: a and b — RNP-particle preparations from embryonic axes of
MTL-stimulated seeds, untreated by EDTA and treated ones, respectively; с and d — RNP-particle preparations from embryonic axes of
non-stimulated seeds, untreated by EDTA and treated ones, respectively
qualitative changes of acting mRNPs (included into
polyribosomes) and the appearance of the minor
protein in embryonic axis cells in vivo; however, to
prove the identity of protein molecules produced in
vitro and in vivo, immunological approaches are
necessary as well as the study of in vitro obtained
protein fractions whose synthesis is directed by
mRNAs isolated from polyribosomes.
It is also evident the changes being realized by
synthetic plant growth activators on the gene exp
ression level and triggering most probably the ac
celeration of plant growth and development are to be
a result of natural developmental process modi
fications due to the activator effect.
Today two tightly interconnected forms of plant
growth and regulation are known — genetic control
445
TSYGANK.OVA V А ЦТ ЛЬ
and phytohormonal regulation; the results of their
interaction may be summarized as follows:
(i) the plant genome codes the synthesis of
cellular constitutive (i. e. common for all the deve
lopment stages) structural and. functional compounds
as well as of stage- and organospecific ones to assure
all the successive events of cell differentiation and
specialization accompanying the formation of new
tissues and organs;
Ш) phytohormones being derivative of gene func
tions (through a series of protein-enzyme molecules)
as well as some other factors according to the
feed-back regulation realize the re-programming of
the cell genome by switching on and off conditionally
«еагіу», «middie», and «late» genes [2.5] controlling
the formation of stage-specific cell homeostasis and,
besides, participating in the regulation of «peripheral»
intracellular metabolic processes.
The question to be now answered is how the
synthetic plant growth regulators are wedged (in
scribed) in such a well-coordinated multi-step regu
lation hierarchy?
This problem was earlier discussed in detail in
our previous paper [1 ]; we propose that the synthetic
compounds with their unusual structures having been
never found in any plant cell are hardly able to
realize their effect through the cell receptor system
specific for a lot of natural compounds. It is more
probably they mediate their effect by changing the
active endogenic phytohormonal pool. An alternative
explanation may also be proposed — a non-specific
effect of growth regulators due to, apparently, of their
higher binding (affinjty) with cellular effector systems
(being their specific «binding sites» or «targets»)
realized more quickly comparing to natural phy
tohormones, the last ones being forced out from their
own receptors. The combined action of growth regu
lators (observed in our experiments with the LNO)
cannot be also ruled out; it may include a phy-
tohormone-mediated effect at the transcription level
and a direct regulator effect at the translation one (in
addition to mentioned above the taking down of
inhibitory action ABA by growth activators is pos
sible) .
Discussing a possible 30 kE)a protein function one
may suppose that the plants possess some genes
coding some «obscure» protein products being nor
mally absent; the promoters of these «silent», «cryp-
tic», «hidden» genes («archaeological signs») are able
[26 ), however, to be switched on by certain stress
factors; in our experiment the 6-MTU was shown to
be such a factor. We suppose that the 30 kDa protein
is one from enzymes transforming the «unsuitable»
(«strange») for cells 6-MTU structure into a natural
substance possessing a phytohormone activity; this
phytohormone is able to accelerate drastically the
plant cell growth by enlargement because in early
postembryogenesis embryonic axis growth due to
enlargement of hypocotyl. The «ephemeral» 30 kDa
protein appears and disappears very quickly, ac
cording to entering into cells and disappearance from
cells the 6-MTU. The data of work [27 J witness in
behalf of such possibility. Authors discovered the
timing changes in enzymes synthesis in elicitor-
treated cell suspension cultures of Parsley.
It should be noted that this 30 kDa polypeptide
is highly similar to polypeptide, forming the base of
hydroxyproline-rich glycoprotein extensin (the mole
cular mass of glycoprotein is 86 kDa, while its
polypeptide without carbohydrate part forms precisely
30 kDa) [28—30], which is the major protein com
ponents of the cell wall of dicotyledon plants. Ex
tensin comprises 5—10 % from all proteins of cell
wall and executes plural functions for cell. According
with these works 30 kDa polypeptide is positively
charged (pi of 9.9) and soluble in water medium
precursor of extensin. This polypeptide is synthesised
and glycosylated by posttranslational modifications in
cytoplasm and then integrated into the cell wall space.
The expression one of the extensin gene family
(SbHRGP3) increases with seedling maturation, and
its expression is relatively high in the mature regions
of the hypocotyl and in the root of soybean seedlings
(it is possibly that the intensification of expression
some from extensin genes under the of 6-MTU
stimulated haricot bean seed germination is noted).
The finale step of our work consisted of expe
riments concerning the effects of the LNO and
6-MTU (possessing quite different mechanisms) on
the following haricot bean ontogenesis. So the plants
originated from germinated seeds (with or without
growth stimulators) were cultivated further on the
minimal nutrient media. It was shown the 6-MTU-
stimulation of seed germination to cause a significant
plant development acceleration comparing to control
plant ontogenesis duration (it is about 40 days in
laboratory conditions); a plant originated from a
6-MTU-stimulated seed is able to complete its deve
lopment in 25 days having passed all the ontogenesis
steps including also flowering, ovary and even pod
formation, its root network being also well developed
(see Fig. 6, a> b). At the same time the LNO-induced
seed germination leads to the plant growth defor
mations — accelerated growth of stems carrying un
derdeveloped foliage, no reproductive organs having
been formed (not shown).
It is also noteworthy the similar effect of vege
tative organs predominance is usually seen in plants
446
AN U N U S U A L MINOR PROTEIN A P P E A R I N G IN EMBRYONIC' AXIS
Fig. 6. Plants grown from haricot bean seeds germinated without any
stimulation (a) and following the 6-MTU directed stimulation (b)
growing on soils containing superfluous organic fer
tilizers; the abundance of organic compounds is
known to cause the nitrogen metabolism activation in
plant cells, this process being somewhat analogous to
the LNO-induced total protein synthesis.
It should be also taken into account the LNO-
induced increased total protein synthesis and the
6-MTU-induced accelerated minor protein synthesis
(these phenomena are somewhat similar to synthesis
with protective functions of heat and cold shock
proteins in plants to response on critical or extremely
higin or low temperatures, respectively, for example
[31 j) may be genetic «markcrs» determining the
following plant development (vegetative organs pre
dominance or complete although shortened onto
genesis) already at the early postembryogenesis sta
ge.
Concluding this paper we would like to note the
selective 6-MTU-induced high level synthesis of the
only minor protein suggests the gene coding this
protein to belong to a family of unique genomic DNA
sequences, its function being controlled by a strong
promoter. The cloning of this markedly inducible
promoter is to be perspective in the field of plant
genetic engineering, namely, for recombinant gene
constructions whose expression in transgenic plants is
to be induced by 6-MTU.
Acknowledgement. Authors thank professor V. M.
Kavsan for letting enzyme reverse transcriptase.
В. А. Цыганкова, В. M. Заєць, Л. О. Галкіна,
Л. П. Приказчикова, Я. Б. Блюм
Поява незвичайного мінорного білка в клітинах зародкової осі
при стимуляції проростання насіння квасолі 6-метилтіоурацилом
Резюме
За допомогою двомірного електрофорезу білків у поліакрил-
амідному гелі виявлено незвичайний мінорний білок з молеку
лярною масою ~ ЗО кДа у клітинах зародкової осі при стиму
ляції проростання насіння квасолі (Phaseolus vulgaris L.) 6-ме-
тилтіоурацилом. Появу цього білка не зафіксовано в нормі та
при проростанні, стимульованому N-оксидом лутидина. Син
тез додаткового низькомолекулярного білка спостерігався та
кож у безклітинній системі з ретикулоцитів кроля на мат
риці полі(А) РНК, одержаній з клітин зародкової осі квасоле
вого насіння, обробленого 6-метилтіоурацилом. У той же час
у безклітинній системі з проростків пшениці при викори
станні «стандартного» препарату полі(А) РНК показано, що
N-оксид лутидина різко стимулює синтез поліпептидів, а
метилтіоурацил прямо не впливає на процес трансляції.
Вивчаючи співвідношення рибосом, вільних та включених у
полірибосоми in vivo, за допомогою фракціонування РИП-час
точок у преформованому градієнті густини <JsC7, встановлено
присутність додаткового піка (1,46 г/см ) у фракції по-
лірибосом. з зародкових осей насіння, обробленого Ь-метил-
тіоурацилом. Така стимуляція суттєво скорочує період онто
генезу, ніяк не пошкоджуючи фенотипу рослини. При цьому
обробка пророщуваного насіння N-оксидом лутидина призво
дить до деформованого прискореного розвитку вегетативних
органів без розвитку органів розмноження рослини. Обгово
рюється природа білка ЗО кДа і взаємозалежність між змі
нами в експресії генів, шр викликані ростовими стимулятора
ми у клітинах зародкової осі під час раннього постемб-
ріогенезу, та наступними різнонаправленими процесами росту
та розвитку рослин квасолі. Розглянуто також деякі прак
тичні напрямки застосування ростових стимуляторів, по
в'язані з результатами проведених експериментів.
В. А. Цыганкова, В. 77. Заец, Л. А. Галкина,
Л. 77. Приказчикова, Я. Б. Блюм
Появление в клетках зародышевой оси необычного белка
при стимуляции прорастания семян фасоли
6-метилтиоурацилом
Резюме
С помощью двухмерного электрофореза белков в полиакрила-
мидном геле показано появление в клетках зародышевой оси
при стимулируемом 6-метилтиоурацилом прорастании семян
фасоли (Phaseolus vulgaris L.) необычного минорного белка с
молекулярной массой около 30 кДа. Этот белок не обнаружи
вался в норме и при стимулируемом N-окисью лутидина
прорастании семян растений. Синтез дополнительного низко
молекулярного белка отмечен и в бесклеточной системе бел
кового синтеза из ретикулоцитов кролика на матрице по
ли(А) РНК из клеток зародышевых осей со стимулируемым
6-метилтиоурацилом прорастанием семян фасоли. В то же
время с помощью бесклеточной системы белкового синтези из
проростков пшеницы с использованием в качестве матрицы
стандартного препарата. поли(А) РНК установлено, что N
окись лутидина резко стимулирует синтез гюлипвптидов, а
6-метилтиоурацил не оказывает прямого влияния на процесс
трансляции. Изучение соотношения свободных и включенных в
полирибосомы информосом in vivo методом фракционирования
РНП-частиц в преформированном градиенте плотности CsCl
447
TSYCANKOVA V. Л. ВТ AL.
выявило присутствие во фракции полирибосом из зародышевых
осей при стимулируемом 6-ме.тилтиоурацилом прорастании
семян дополнительного пика активных в белковом синтезе
информосом с плавучей плотностью 1,46 г/см . Показано,
что стимуляция прорастания семян фасоли 6-метилтиоура-
цилом приводит к существенному сокращению сроков онтоге
неза растения фасоли без каких-либо нарушений фенотипа
растения, а стимуляция N-окисью лутидина — к деформиро
ванному ускоренному развитию вегетативных органов без
развития репродуктивных органов растения. Обсуждается,
природа белка 30 кДа и связь между различиями в изменении
ростстимуляторами экспрессии генов в клетках зародышевых
осей в раннем постэмбриогенезе и последующими разнонаправ
ленными процессами роста и развития растений фасоли, а
также некоторые практические аспекты, вытекающие из
это а о.
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