ISSN 10227954, Russian Journal of Genetics, 2014, Vol. 50, No. 1, pp. 37–44. © Pleiades Publishing, Inc., 2014. Original Russian Text © B.R. Kuluev, A.V. Knyazev, B.N. Postrigan, A.V. Chemeris, 2014, published in Genetika, 2014, Vol. 50, No. 1, pp. 44–51.
PLANT GENETICS
The Creation of Transgenic Tobacco Plants Expressing Fragments of the ARGOS and NtEXPA4 Genes in Antisense Orientation B. R. Kuluev, A. V. Knyazev, B. N. Postrigan, and A. V. Chemeris Institute of Biochemistry and Genetics of Ufa Scientific Centre, Russian Academy of Sciences, Ufa, 450054 Russia email:
[email protected] Received June 5, 2013
Abstract—Transgenic tobacco plants expressing the fragments of the ARGOS and NtEXPA4 genes in antisense orientation have been created. Eleven lines of transgenic plants were investigated and five of them were char acterized by a decrease in the sizes of the leaves and flowers as compared to control. Stem sizes decreased when only the NtEXPA4 gene fragment was used. The organ size of the experimental plants decreased because of a reduction in the level of both cell division and cell expansion. Two lines of transgenic tobacco plants expressing the part of the ARGOS gene in antisense orientation were characterized by a reduction in the level of the NtEXPA1 and NtEXPA4 gene expression. DOI: 10.1134/S1022795414010074
According to data in the literature, an increase in the level of expansin expression, for example, can lead to an increase in organ size. The A. thaliana plants that are transgenic in the AtEХРA10 gene are characterized by an increase in both the length of the petiole and the area of the leaf plate [5]. At the same time, the trans genic plants expressing a part of the AtEXPA10 gene in antisense orientation are characterized by a reduction of organ size. In plants, expansins are coded by a mul tigene family of genes. At present, in the tobacco plant, for example, six genes of expansins (named NtЕХРA, with serial numbers from one to six) were identified, and the nucleotide sequences of these genes are available in GеnВаnk (AF049350–АF049355) [6].
INTRODUCTION One of the perspective trends in modern phytode sign is the cultivation of large plants in miniature. However, the cultivation of such plants usually requires much time and effort. Modern technologies for the geneengineering of plants can reduce the time period required for the creation of dwarf plants. According to the data in the literature, a reduction of organ size was observed when the expression level of some plant genes was changed [1, 2]. The organ size of plants depends on the regulation of cell division and cell expansion. In plants, such genes as AINTEGUMENTA, ARGOS, CYCLIND3; 1, WUSCHEL, СLАVАТАЗ and many others participate in the regulation of cell division [3]. The ARGOS gene of Arabidopsis thaliana codes a transmembrane pro tein, which participates in signal transduction from phytohormones to transcription factors [1]. It is known that the signals from the ARGOS protein are transduced to the gene of the AINТЕGUМЕNТA transcription factor, which in turn controls cell divi sion in the germs of overground organs [4]. It was shown that an increase in the level of expression of both the ARGOS gene, and the АINТЕGUМЕNTА gene promotes an increase in the sizes of all over ground organs because of the prolonged meristematic competence of cells in the anlages of organs, which leads to an increase in the quantity of cells per organ. A decrease in the level of the expression of these genes promotes the reduction of the sizes of Arabidopsis thaliana overground organs [1, 4]. In plant tissues, expansins, endogluconases, xylo glucan endotransglycosylases, and a number of trans membrane proteins and transcription factors also par ticipate in the regulation of cell expansion [3].
These data allow us to assume that a decrease in the level of the expression of the ARGOS and the expansin genes will promote a reduction of the organ size of transgenic plants. In the first case, this is due to the reduction of cell quantity, and in the second it is because of a decrease in cell size. One of the most effective strategies for the suppres sion of plant gene expression at the posttranscriptional level is RNAinterference [7]. For this purpose, a short part of the target gene, either in sense orientation or in antisense orientation in relation to the promoter, is cloned, and the plant is exposed to transformation. Such works are performed mainly with such a model object as A. thaliana. There is little literature data about the functioning of the geneengineered con structions in other plants. Because the genomes of many plants are not sequenced and the cloning of the genes of each plant takes some time, the creation of universal geneengineering constructions that will function in many dicotyledons is of special interest. The created geneengineering constructions have to 37
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be approved not only with A. thaliana, but also using other model vegetative objects, for example, tobacco plants. The purpose of this work was to clone short, con servative parts of the ARGOS and expansin genes in antisense orientation and to create transgenic tobacco plants on the basis of these geneengineering con structions. MATERIALS AND METHODS Bacterial cells, cultures, plasmids, and gene engi
neering manipulations. Еsherichia coli bacteria from the ХL1 В1uе culture and Agrobacterium tumefaciens of the AGL culture were used. To create gene engi
neering constructions, we used the T vector рКRХ and the binary vector рСambiа 1301 with 35S pro
moter, with the gene for resistance to hygromycin and the GUS as the reporter gene (САМВIА, Australia). A part of the АRGOS gene with of a size 171 bp was iso
lated from A. thaliana genomic DNA by the АRGiF primer CGGССААТТАТТТСАСТТТАGАG and the АRGiR primer GАGААGААGGСАТGААG
GСААGАА. For the amplification of the NtЕХРА4 part of the gene with a size of 227 bp, we used the ЕХРiF primer AАТАСТGСАGСТТТААGСАСАGС and the ЕХРiR primer GСААТGТGТТGGА
АGАСАGGCTG. In the search for target clones for ligation in the рCambia 1301 vector, the 35SСаmbF primer АGАGGАССТААСАGААСТСG was used along with the 1301R primer ТGСТСТАGСА
ТТСGССАТТG. Sequencing was performed using an ABI РRISМ 310 Genetic Analyzer automatic ana
lyzer of nucleic acids (Applied Вiosystems, United States). A search and alignment for nucleotide sequences of genes was performed using the МegAlign program from the Lasergene I pack, (DNASTAR, United States) and MegaBlast program (http://www.ncbi.nlm.nih.gov). Obtaining transgenic tobacco plants, morpho physio
logical description and conditions of plant growth. Transgenic tobacco Nicotiana tabacum L. var. Рetit Havana SR1 plants were obtained by the method of agrobacterial transformation of leaf disks, which were cut from the leaves of 3 month old plants [8]. A quali
tative estimation of the activity of the reporter GUS gene in the leaves of Т0 shoots was performed histochemi
cally. For this purpose the samples of leaf tissue were incubated for a night at 37°C in a 0.1% solution of Х Gluc (sodium salt of 5 bromine 4 chlorine 3
indolyl β D glucuronic acid, (Fermentas, Lithua
nia), containing 0.1 M sodium phosphate buffer (pH 7.0), 10 mm Na2EDTA, and a 0.1% (w/v) Triton Х
100. The samples were incubated with the histochem
ical reagent for a night, whereupon the green tissues were discolored by 70% ethanol and investigated using a stereomicroscope to reveal the presence of dark blue color [9]. To check the inheritance of the DNA parts of the ARGOS and the NtEXPA4 genes and to detect
the quantity of insertions, some of the Ò1 seeds from each of the obtained lines were couched in МS medium supplemented with hygromicin in a Binder climate chamber (Germany). After three weeks, the calculation of the seedlings resistant and nonresistant to the selective agent was performed and splitting in the inheritance of the selective marker gene was detected. The results were calculated by the χ2 method using the standard procedure; the lines possessing 3 : 1 splitting were selected for further study. The plants of transgenic lines and control plants were cultivated in 450 mL vegetation vessels filled with universal soil (Terra vita, Russia) on an open pho
toplate at 25–27°С, a light exposure of ~4000 lux and a photoperiod of 16–8 h of light and dark. The plants of the Т1 generation were under observation from the stage of adaptation to cultivation (in soil in conditions of a greenhouse) to seed harvesting, which took from four to six months. The untransgenic tobacco plants var. Petit Havana SR1 grown in МS medium without antibiotics and adapted to soil conditions (SR1), as well transgenic plants that contain T DNA of the binary рCаmbia 1301 vector without the target gene (1301), were used as a control. The length and the area of leaves were determined in flowering. From each of the plant lines, five plants were selected, the lengths of the first, second and third large lower leaves of which were measured along the central rib from the begin
ning to the end of the leaf plate. The stem length of each plant was measured in flowering. The lengths of five flowers from the pedicel to the border of the corona flute were also measured, and the mean value was calculated. Five days after the beginning of the fructification of each plant, the lengths of three seed boxes were measured and mean value was calculated. To determine the mean area of the cells of the lower leaf epidermis and that of the external epidermis of the corona, a layer of epidermis with an area of 0.5–1 cm2 was detached by tweezers from the average part of three lower leaves and the average part of three corona of flowers, placed on an object plate, and, after the addition of a physiological solution, the epidermal layer was covered with cover glass. The measurements were performed by an Аxio Imagег М1 universal fluo
rescent microscope (Carl Zeiss, Germany) using orig
inal software. Total RNA from investigated plants was isolated with trizol, the first strand of cDNA was made using the oligo(dТ)primer (Sileks, Russia) and ММuLV
revertase (NEB, United States). The primers ААСАТТGGСАТТТАСАGАGGТG and AAGGGT
TGGSSATTGАGАТА were used for RT PCR of the NtEXPA1 gene. For RT PCR of the NtEXPA4 gene were used the ТGТААТССТССССТТСАССАТ and АТААТТGТТGТТТТGССАGTTTTG primers. The gene of tobacco α tubuline was used as a reference gene, for which RT PCR was performed using the ССААGGТGСАААGGGСТGТАТ and ТТССТС
GТТАТСАТСGТСТТСТСС primers.
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(a) AtARGOS 1 AtARL 1 B. divaricarpa 1 B. rapa 1 P. trichocarpa 1 R. communis 1 V. vinifera 1
153 171 168 174 168 156 180
AtARGOS 154 AtARL 172 B. divaricarpa 169 B. rapa 175 P. trichocarpa 169 R. communis 157 V. vinifera 181
321 336 333 339 324 312 336
(b) AtEXPA10 127 NtEXPA4 127 PttEXPA3 127 NtEXPA5 1
265 265 265 139
AtEXPA10 266 NtEXPA4 266 PttEXPA3 266 NtEXPA5 140
404 404 404 278
Fig. 1. Search for conservative parts in the nucleotide sequences of the ARGOS and the NtEXPA4 genes; (a) results of the alignment of open reading frames of the ARGOS and the ARL genes, as well as these of the homologous DNA parts of Boechera divaricarpa, Brassica rapa, Ricinus communis, Vitis vinifera, and Populus trichocarpa; (b) results of the alignment of open reading frames of the AtEXPA10, the NtEXPA4, the PttEXPA3, and the NtEXPA5. The fragments of the target genes, which were amplified and cloned in the pCambia 1301 binary vector, are marked in grey.
RESULTS Search and Cloning of Conservative Fragments of the АRGOS gene of A. thaliаnа and the NtЕХPА4 gene of N. tabaсum The nucleotide sequences of the АRGOS gene of many plants such as: A. thaliana (NМ115853.4), Аrabi
dopsis lyrata (ХМ002880036.1), Boechera divaricarра (DQ226826.1), Вrassica rapa (FJ71724.1), Vitis vinifera (АМ475052.1), Ricinus соmmunis (ХМ002533293.1), Solanum lусореrsiсum (NM001247750.1), Populus triсhосаrра (ХМ002310336.1, ХМ002331606.1), Рicea sitchensis (ЕF678316.1), Оryzа sativa (DQ641272.1), Zea mays (AEQ59626.1), Hordeum vulgare (АК369743.1) are presented in GеnВаnk. The ARGOS LIKE gene of A. thaliana (NM180078.3) has also high level of similarity to the ARGOS gene. Earlier we have shown that the homologous parts of DNA of B. divaricarpa, B. rapa, R. соmmunis, V. vinifera, and P. trichocarpa are the most similar in nucleotide com
position to the ARGOS gene of A. thaliana [8]. The open reading frames of the ARGOS genes of these plants were aligned among themselves and with the АRGOS LIKE gene for the search for conservative parts. However, such a part of DNA in the АRGOS gene was not successfully found, therefore we decided to select the fragment of DNA with 171 bp in size, which is the most conservative (Fig. 1a). This part of DNA was amplified from the A. thaliana genome DNA and cloned in the рСаmbia 1301 binary vector in antisense orientation. Then the nucleotide sequences of the open reading frames of the genes of various expansins were aligned. It was shown that the NtЕХРА4 gene of tobacco, the РttЕХРА3 gene of poplar, and the NtЕХРА5 gene of tobacco are the most similar to the АtЕХРА10 gene in RUSSIAN JOURNAL OF GENETICS
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nucleotide composition. It is supposed that exactly expansin NtЕХРА4 first of all takes part in the growth regulation of leaves, because of the similarity to the АtEХРА10 gene [5]. The nucleotide sequences of these genes were aligned again for search for a conservative part. During the study we decided to select the frag
ment of the NtЕХРА4 gene of tobacco plant with 227 bp in size (Fig. 1b). This DNA fragment was amplified and cloned in the рСаmbia 1301 binary vector in anti
sense orientation. The gene engineering construc
tions created during the study were introduced into the cells of A. tumefaciens. Comparative Morpho Physiological Description of Transgenic Tobacco Plants that Express the Part of the АRGOS Gene of A. thaliаnа and the Part of the NtЕХРА4 Gene of N. tabасum in Antisense Orientation By means of the gene engineering construction of the fragment of the АRGOS gene of A. thaliana, 17 shoots were obtained, 12 of which were deep
rooted on the selective medium. Five plants were rejected because of the negative results of the PCR analysis and the analysis of the reporter GUS gene expression. As a whole, seven plants (2, 3, 9, 12, 13, 14 and 17) were adapted for soil conditions. The splitting of 3 : 1 was shown only for lines 3, 9, 12, 14, and 17, which were used in further experiments for the mor
phologic description of these lines. The selection of the seedlings, which were without splitting on the selective medium, was not performed in this study because of the prolonged vegetative period of the tobacco plant, i.e., both homozygous, and heterozy
gous plants were used for morphological analysis. When the рСаmbiа 1301 vector with the fragment of the NtЕХРА4 gene was used, 43 shoots were 2014
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obtained after transformation, 20 of which were deep
rooted on the selective medium. From these 20 plants, only 12 were selected after the PCR analysis and the analysis of the reporter GUS gene expression. Of them, 10 plants (1, 5, 15, 19, 27, 29, 30, 31, 35 and 36) sur
vived and were adapted to conditions of soil. The seeds of these plants were sown on the selective medium with hygromicin, and the splitting as 3 : 1 was shown for the lines 5, 15, 19, 27, 29, and 30. These plants were adapted for cultivation in soil in the hothouse, and the morphological analysis of the plants was performed during flowering. When construction of the fragment of the АRGOS gene in antisense orientation was used, only two plant lines, 3 and 12, from five lines of plants were charac
terized by a reliable reduction of organ size; other lines of transgenic plants did not differ from control (Fig. 2). The length of leaves in plant lines 3 and 12 was on aver
age 13% less than in control plants (Fig. 2a) and the area of leaves was 16% less as compared to control (Fig. 2b). The control and experimental plants statis
tically did not differ among themselves in stem height (Fig. 2c). The length of the flower of some experimen
tal plants was statistically reliably reduced. The flower length of the plants of line 3 were 6% less, those of line 9 were 2% less, and those of plant lines 12 and 14 were 4% less, as compared to control. The flower length of line 17 was different from that of control (Fig. 2d). The length of boxes was reduced only in the plants of lines 3 and 12 (by 17 and 13% respectively) (Fig. 2e). The part of transgenic tobacco plants expressing the fragment of the NtЕХРА4 gene in antisense orien
tation also was characterized by a statistically reliable reduction of organ size (Fig. 2), and it was typical for lines 15, 19, and 29. The length of leaves of experi
mental plants of line 15 was reduced by 12%, that of line 19 was reduced by 13%, and the leaf length of plant line 29 was 14% less as compared to control (Fig. 2a). The leaf area of the same plant lines was reduced on average by 19, 11, and 16% respectively, as compared to control (Fig. 2b). The stem height was decreased by 24 and 33% only in plant lines 15 and 27, respectively (Fig. 2c). The length of the flowers of plant line 5 was decreased by 5%, that of plant line 15 was decreased by 6%, that of plant line 19 was decreased by 4%. The flower length of plant line 27 was decreased by 2%, that of line 29 was decreased by 5%, and the flower length of the plants of line 30 was 3% less than in con
trol plants (Fig. 2d). It was noted that the flowers of some experimental plants decreased not only in length, but also in diameter (Fig. 3). The length of the boxes of experimental plants statistically did not differ from that of control plants (Fig. 2e). In each line of both transgenic and control plants, the distinctions in the sizes of leaves and stem between different variants of sample never exceeded 10%, and those of flower sizes did not exceed 1%. At the same time, the introduction of the binary vector T DNA
also did not influence considerably the morphological parameters of the investigated plants (Figs. 2, 4). This means that the decrease in the organ size of transgenic plants most likely was caused by the specific influence of the gene engineering constructions that were received during the study. Reduction of the plant organ size may be caused either by a decrease in the level of cell division or par
tial inhibition of cell expansion. To reveal the reasons of the reduction of the organ size, we evaluated the sizes of the epidermal cells of leaves and flowers of the investigated plants. It was shown that the sizes of the leaf epidermal cells of experimental plants expressing the fragment of the АRGOS gene vary depending on the line (Fig. 4a). The sizes of leaf epidermal cells of the transgenic plants of lines 3 and 12 were reduced by 26 and 14% respectively. It is possible that the reduc
tion of the leaf sizes of these plants was caused prima
rily by the reduction of the cell sizes; however, it seems to be most likely that the reduction of the cell number also plays some role in this case. It is of interest that, in plants of lines 9, 14, and 17, for which organ size was not decreased, some cells were larger than control cells (Fig. 4a). For example, the cell sizes of plant line 9 were increased by 16%, those of the line 14 were increased by 51%, and those of line 17 were increased by 41%. The cell areas of flower epidermis of plant lines 3 and 12 were reduced a little (by 11 and 5% respectively), whereas those of lines 9, 14, and 17 were increased (by 5, 18 and 10% respectively) as compared to control (Fig. 4b). Approximately the same picture was observed when plants transgenic in fragment of the NtЕХРА4 gene were studied. The plant lines, which possessed decreased organ size, were characterized as well by a reduction of the cell sizes of the leaf epidermis (Fig. 4a). The cell sizes of plant line 15 were decreased by 25%, those of plant line 19 were decreased by 16%, and those of line 29 were decreased by 21% as compared to control. The lines of transgenic plants for which organ size remained within norm, on the contrary, were characterized by an increase in the cell sizes of the lower leaf epidermis (Fig. 4a). For example, the cell sizes of line 30 were increased by 37%. The cell sizes of the external flower epidermis of all transgenic plants expressing the NtEXPA4 gene were characterized by an insignificant (by 12% on average) reduction as com
pared to control (Fig. 4b). Thus these plants differed from transgenic plants expressing the fragment of the АRGOS gene in antisense orientation, part of which possessed an increased cell size of the flower epidermis. Earlier we have shown that the overexpression of the АRGOS LIKE gene promotes an increase in the level of the tobacco expansin expression [8]. Based on the homology of the АRGOS and АRGOS LIKE genes, it is possible to assume that in transgenic plants expressing the fragment of the АRGOS gene in anti
sense orientation the level of the gene expression of
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(e) 2.2 1.8 1.4 1.0 1301
ARG3 ARG12 ARG17 EXP15 EXP27 EXP30 SR1 ARG9 ARG14 EXP5 EXP19 EXP29
Fig. 2. Morphological parameters of transgenic and control plants; (a) the length of leaves in flowering, cm; (b) the area of leaves in flowering, cm2; (c) the stem height in flowering, cm; (d) the length of flowers, cm; (e) the length of boxes, cm; 1301 is control transgenic tobacco plants that contain the insertion of TDNA of pCambia 1301 binary vector without of the target gene. SR1 is the control tobacco plants of wild type; ARG3–ARG17 are the lines of experimental tobacco plants that express the fragment of the ARGOS gene in antisense orientation; EXP5–EXP30 are the lines of experimental tobacco plants that express the fragment of the NtEXPA4 gene in antisense orientation. RUSSIAN JOURNAL OF GENETICS
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(a)
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Fig. 3. Flowers of the control and transgenic plants that express the fragment of the NtEXPA4 gene in antisense ori entation; (a) the flower of the control plants of wild type; (b) the flower of transgenic tobacco plant of the line no. 15.
some expansins may be lowered. To verify this assump
tion, the semiquantitative RT PCR of the NtЕХРА1 and NtЕХРА4 genes in young growing leaves of tobacco plant was performed [6]. The level of the expression of the NtЕХРА1 and NtЕХРА4 genes was high enough in the tops of sprouts, young flowers, and leaves (the data are not presented) to support the par
ticipation of the expansins in organ growth (Fig. 5, 1 and 2). At the same time, an appreciable reduction of the level of the expression of the NtЕХРА1 and NtЕХРА4 genes in young leaves of the transgenic plants of lines 3 and 12 was observed (Fig. 5, 3 and 5). It should be noted that the reduction of organ size was shown for these plants. And in the leaves of transgenic plants of line 9, the level of the expression of the (a)
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Fig. 4. Comparative analysis of the epidermal cell sizes of the leaves and flowers of control and experimental plants; (a) the area of the cells of the lower epidermis of tobacco leaves, µm2; (b) the cell area of the external epidermis of tobacco flowers, µm2; 1301 and SR1 are control plants; ARG3–ARG17and EXP5–EXP30 are the lines of experimental plants. RUSSIAN JOURNAL OF GENETICS
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NtЕХРА1 and NtЕХРА4 genes remained within the norm (Fig. 5, 4).
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NtEXPA1
DISCUSSION Predictably, the part of transgenic tobacco plants that express the fragments of the АRGOS and the NtЕХРА4 genes in antisense orientation are really characterized by a reduction of organ sizes. At the same time, the morphological signs of the majority lines of transgenic plants were the same as the signs of control plants. Because the plants for which organ size did not change possessed an increase in the sizes of some cells, it is possible to assume the existence of a compensatory mechanism that promotes mainte
nance of organ size within norm. For example, when the level of АRGOS gene expression is decreased, the number of cells per organ should be decreased; how
ever, because of the stimulation of cell expansion, organ size remains within the norm (in the lines 9, 14, and 17). However, the cell sizes of plant lines 3 and 12 were not larger, but smaller than those of control plants. It is exactly for this reason that the organ size of these plants were smaller than in control. It is very dif
ficult to answer the question of why the compensatory increase in cell sizes was not observed exactly in these lines of plants. It is possible to assume that in these plants not only the level of the expression of the АRGOS gene, which regulates cell division, was decreased [1], but also the expression of the АRGOS
LIKE gene, which is homologous to the АRGOS gene and regulates cell expansion [10], was reduced. According to our data, the expression of the АRGOS
LIКЕ gene promotes an increase in the level of expansin gene transcription [8]. For this reason we analyzed the level of expression of the NtЕХРА1 and the NtЕХРА4 genes in the investigated plants. This experiment revealed that the reduction of organ size and cells in the lines of transgenic plants 3 and 12 may be connected with a decrease in the level of expression of some expansins. As in our previous work [8], it was shown that the sizes of the flowers of transgenic tobacco plants were changed to a lesser degree than the sizes of the leaves and stem. The sizes of flowers were decreased in four plant lines containing the fragment of the АRGOS gene, but the sizes of the flower cells were decreased in lines 3 and 12 only, testifying to the partial blocking of compensatory mechanisms in these plants. In this case, it is possible that a decrease in the level of expres
sion of some expansins, which are connected with the АRGOS and the АRGOS LIKE genes in a unified gene network, was also observed. The decrease in the sizes of organs due to a decrease in the sizes of some cells was revealed in three lines of transgenic tobacco plants expressing the part of the NtЕХРА4 gene in antisense orientation. Accord
ing to the literature data, these peculiar properties of the morphology of transgenic plants may be really RUSSIAN JOURNAL OF GENETICS
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NtEXPA4
α Tubuline
Fig. 5. Results of RTPCR in the young leaves of the trans genic plants expressing the fragment of the ARGOS gene in antisense orientation; (1) control transgenic tobacco plants, which contain the TDNA of the pCambia 1301 binary vector without a target gene; (2) the control tobacco plants of wild type; (3–5) the experimental tobacco plants of the lines 3, 9, and 12 respectively.
caused by a decrease in the level of the expression of the NtЕХРА4 gene, because the protein product of this gene takes part in cell expansion [5]. It is interesting to notice that unlike the АRGOS gene, the NtEXPA4 gene can influence stem length (Fig. 2c). Apparently, the organs of tobacco plants transgenic in the part of the NtЕХРА4 gene were decreased not only because of a reduction of the cell sizes, but also because of a reduc
tion of the number of cells per organ. For this reason, the lines of tested plants for which organ size did not change were characterized by an increase in the sizes of some cells. In this case, the compensatory mecha
nism, which influences cell expansion, can be caused by the functioning of others expansins in tobacco leaves [6]. It should be noted that the reduction of cell sizes was observed in the flowers of all tested plants expressing the fragment of the NtЕХРA4 gene (Fig. 4b). It means that the flower sizes of the lines 27 and 30, for example, reached normal sizes because of the stimulation of cell division, in which the АRGOS gene takes part also. Unfortunately, the АRGOS and the АRGOS LIKE genes of the tobacco plant are not sequenced, and it is impossible meanwhile to check the level of the expres
sion of these genes in transgenic plants. Unlike leaves, the compensatory increase in flower cell size was observed in none of the plant lines, which could indi
cate the great importance of the NtЕХРА4 expansin in the regulation of cell expansion in flowers. In leaves, possibly, others expansins function also, accomplish
ing the function of NtЕХРА4. It should be noted that the expression of the frag
ments of the АRGOS and the NtЕХРА4 genes in anti
sense orientation did not led to considerable reduction (for example, by 2–3 times as compared to control) of the organ size of transgenic plants. It is possible that other approaches used for the inhibition of expression 2014
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in plants are necessary for this purpose. At the same time, the gene engineering constructions created by us can be applied to obtain transgenic plants with reduced organ size. However, to achieve the more notable result, a careful selection of the corresponding lines and the application of methods of selection are required. ACKNOWLEDGMENTS
5.
6.
7.
This work was supported by the Russian Founda tion for Basic Research, project no. 120431292. 8.
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Translated by E. Ladyzhenskaya
RUSSIAN JOURNAL OF GENETICS
Vol. 50
No. 1
2014
