ISSN 10683674, Russian Agricultural Sciences, 2014, Vol. 40, No. 1, pp. 14–17. © Allerton Press, Inc., 2014. Original Russian Text © Ye.N. Baranova, E.N. Akanov, A.A. Gulevich, L.V. Kurenina, S.A. Danilova, M.R. Khaliluev, 2013, published in Doklady Rossiiskoi Akademii Sel’skokhozyaistvennykh Nauk, 2013, No. 6, pp. 13–16.
PLANT CULTIVATION
Dark Respiration Rate of Transgenic Tomato Plants Expressing FeSOD1 Gene under Chloride and Sulfate Salinity Ye. N. Baranovaa, E. N. Akanovb, A. A. Gulevicha, L. V. Kureninaa, S. A. Danilovac, and M. R. Khalilueva, d aAllRussia
Research Institute of Agricultural Biotechnology, Moscow, 127550 Russia email:
[email protected] bPryanishnikov AllRussia Research Institute of Agrochemistry, Moscow, 127550 Russia c Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Moscow, 127276 Russia dRussian State Agrarian University–Timiryazev Moscow Agricultural Academy, Moscow, 127550 Russia Received May 13, 2013
Abstract—Dark respiration of transgenic tomato regenerants expressing Fedependent superoxide dismutase under conditions of sulfate and chloride salinity has been studied. The method and system of measuring the dark respiration rate by means an infrared gas analyzer have been modified, making possible the use of indi vidual regenerants in vitro in a small volume test tube. It is shown that transgenic lines expressing the FeSOD1 gene from Arabidopsis thaliana L. demonstrates a differently directed response to the effect of NaCl and Na2SO4. Keywords: Solanum lycopersicum, transgenic plants, Fedependent superoxide dismutase, dark respiration rate DOI: 10.3103/S1068367414010029
Salinity is one of the principal stress factors limiting cultivation of most agricultural crops in many agrocli matic zones [1]. Areas of salinized agricultural lands are increasing every year. This is caused to a greater degree by socalled secondary salinization related not only to the presence in soil of salt ions that can’t be removed by chemical amelioration methods but also the need to use irrigation with saline water [2].
where they participate in the formation of a hetero complex with chloroplast DNA, thereby protecting the genome from injuries causes by ROS [6]. It has been shown [7] that the ultrastructural organization of chloroplasts of photosynthetic tissues, nuclear com partment, and mitochondria changes considerably in transgenic tomato and tobacco plants expressing the FeSOD1 gene from A. thaliana. Data on the ultrastruc tural organization of mitochondria allow judging the passage of the most important process of plant cell metabolism—respiration.
As is known, an important factor injuring plant cells under salinity conditions is oxidative stress related to the formation of reactive oxygen species (ROS) such as hydrogen peroxide, superoxide anion radical, hydroxyl radical, and others [3]. Of great importance among antioxidant enzymes actively involved in the first stage of oxidative stress are super oxide dismutases (SOD; EC 1.15.1.1) catalyzing the conversion of superoxide anion radicals to molecular oxygen and hydrogen peroxide. Furthermore, the hydrogen peroxide forming serves as a signal molecule triggering a cascade of protective reactions under stress conditions of a biotic and abiotic nature [4].
Dark respiration, which is customarily condition ally divided into growth respiration and maintenance respiration, is important for supporting vital functions of plants [8]. Respiration rate is a comprehensive index whose value varies strongly depending on the object, development stage, physiological state, and effect of environmental factors [9, 10]. An analysis of the literature [11] indicates that the rate of respiratory processes can both increase and decrease under salin ity conditions. The purpose of the present investigation was to develop a system of quantitative measurement of dark respiration of plants under in vitro conditions and to assess the effect of expression of the A. thaliana FeSOD1 gene on dark respiration rate of transgenic tomato regenerants without salinity as well as under chloride and sulfate salinity.
Plant cells contain several isoforms of SOD differ ing in the metal (Fe2+, Mn2+, and Cu2+) in the active site of the enzyme as well as their localization in sub cellular compartments [5]. Thus, in Arabidopsis thaliana L., one of the isoforms of ironcontaining SOD (FeSOD1) is localized in the cytosol, whereas two others (FeSOD2 and FeSOD3) are in plastids, 14
DARK RESPIRATION RATE OF TRANSGENIC TOMATO PLANTS EXPRESSING
METHOD The plant material for the experiments was tomato regenerants of generations T0 (lines No. 4, No. 6, and No. 8) and T1 (line No. 19) expressing the FeSOD1 gene from A. thaliana with a leader sequence providing localization of the product in chloroplasts. The pri mary transgenic tomato regenerants were cultured on a Murashige–Skoog (MS) medium with the addition of 2% sucrose, 0.2 mg/L indole3butyric acid, 50 mg/L kanamycin, and 0.7% agar for rhizogenesis. Multiplication of transgenic regenerates was accom plished by cuttinggrafting a shoot onto segments con taining one or two nodes and subsequent rooting on a medium of the same composition. The seeds of the T1 generation of tomato line No. 19 were introduced into the in vitro culture by means of surface sterilization for 5 min in a 1.5% sodium hypochlorite solution with the addition of 0.01% Tri ton X100. The disinfected seeds were placed in cul ture vessels with agarized MS nutrient medium sup plemented with kanamycin in a concentration of 75 mg/L. Leaves of kanamycinresistant tomato seed lings were used as explants for obtaining regenerants. Shoot organogenesis processes were induced on MS medium supplemented with 5 mg/L 6benzylami nopurine, 0.2 mg/L indole3acetic acid, and kana mycin in a concentration of 30 mg/L. Regenerated kanamycinresistant shoots were separated from the callus tissue and transferred to a medium for rhizogen esis with the addition of a selective antibiotic in a con centration of 50 mg/L. Expression of the FeSOD1 gene in generation T1 transgenic tomato plants was determined by RTPCR analysis with specific primers FeS1 and FeS2 (Sintol, Russia) [7]. The dark respiration rate (DRR) of T0 and T1 gen eration transgenic regenerants was determined under normal conditions as well as under oxidative stress. For this purpose, the rooted regenerants were trans ferred to test tubes with agarized MS nutrient medium with the addition of sodium chloride or sulfate in a concentration of respectively 96.2 and 76.5 mM, which corresponds to an increase of osmotic pressure of the medium by 400 kPa. As the control we used regenerants of wildtype tomato (variety Belyi Naliv) obtained from leaves with their culturing on a nutrient medium for shoots organogenesis with the aforemen tioned composition of growth regulator. Each variant of the experiment was carried out in threefold replica tion with ten regenerants in each. Regenerants of rel atively the same size were used in the experiment. We determined DRR after culturing the regener ants for 7 days. Because it was necessary to determine CO2 being released by an individual regenerant being cultivated in a small volume (50 cm3) test tube, we modified the gas analyzer system and recording method: the dark respiration rate was estimated by means of a closed system (Fig. 1); 2 h before measure RUSSIAN AGRICULTURAL SCIENCES
Vol. 40
No. 1
15
IR gas analyzer CO2 0–500 ppm
4 1
2
3
5
Fig. 1. Diagram of closed system for determining DRR of plants in vitro: (1) pump; (2) air dryer; (3) rotameter; (4) infrared gas analyzer; (5) monitor.
ments the test tubes with the regenerants were unsealed and kept open; 2 h before the measurements the test tubes were placed in the dark; a constant tem perature (22–23°C) was maintained immediately dur ing recording of CO2 being released by the regener ants. Measurements were taken by a GOA4 infrared gas analyzer (Khimavtomatika, Russia) with a 0–0.05% CO2 scale. We determined DRR per 1 mg dry weight of the regenerant, for which after determining the amount of released CO2, the regenerants were removed from the test tubes and incubated at 70°C for 72 h and then their weight was determined. We deter mined DRR (μg CO2/h) by the formula: DRR = [ ΣVΔC/100M × 0.2 ]K, where ΣV is the total volume of the closed system, including the volume of the test tube (50 cm3) and measuring system (50 cm3); ΔC is the change in the concentration of CO2 in the total volume during expo sure, %; M is the dry weight of the regenerant, mg; 0.2 is exposure time, h; K is the conversion factor of the amount of CO2 from volume units (cm3) to weight units (μg) reduced to normal conditions (temperature 0°C and pressure 1 atm). RESULTS AND DISCUSSION Nine independent transgenic lines containing the FeSOD1 gene were obtained earlier as a result of a series of experiments on Agrobacteriummediated transformation of tomato variety Belyi Naliv with the use of the binary vector pB1121 FeSOD. The presence of the target gene was confirmed by PCR analysis [13]. From the results of RTPCR, expression of gene FeSOD1 was found in seven of nine independent trans genic tomato lines [9]. Transgenic lines with con firmed expression of the FeSOD1 gene were clonally multiplied to obtain a sufficient amount of plant mate rial. It was established during multiplication that dif 2014
16
BARANOVA et al. 1
2
3
4
5
6
645 bp
Fig. 2. Expression of FeSOD1 gene in T1 transgenic tomato lines by means of RTPCR analysis: (1) molecular marker; (2) positive control (cDNA of T0 transgenic tomato line); (3–6) cDNA of T1 transgenic tomato lines.
ferent shoot segments differ in the ability to grow and rooting on the selective medium. Most of them actively formed a root system, grew vigorously on the nutrient medium, and didn’t show signs of a negative effect of the selective agent, whereas the formation of short, thickened primary roots occurred in some. Rhizogenesis was absent in a number of cases. In that case, stem segments kept a characteristic green color for a long time. Rooted transgenic regenerants adapted success fully to soil conditions and grew under conditions of sheltered ground. During growth, the transgenic lines were analyzed for the presence of somatic deviations from the wildtype plants. Lines differing from the control by a greater height and number of internodes as well as thickened leaf blades with a changed shape were observed. Fruits didn’t form for a long time in three transgenic lines expressing gene FeSOD1. Nev ertheless, after long culturing we succeeded in produc ing fruit in them, which, however, didn’t contain vig orous seeds. Instead of them, only small undeveloped ovules were found in the locules of the ovaries, which indicated fruit development as a result of partheno carpy. Thus, seed progeny was obtained only in four of the seven independent transgenic tomato lines expressing the FeSOD1 gene. The T1 transgenic lines were analyzed for the pres ence of expression of the target gene. From the results of RTPCR with the use of specific primers for the sequence of gene FeSOD1, its expression was estab
lished in all analyzed lines (Fig. 2), which attests to the absence of the effect of transgene silencing in the seed generation. Regenerants of generation T0 and T1 transgenic lines were selected for assessing the effect of FeSOD1 gene expression on DRR. In connection with the need to determine DRR of each individual regenerant cul tured in vitro in a small volume test tube, we modified the measurement method and system. Initially, we measured the volume of the closed system consisting of connecting hoses, measuring cuvette of the gas ana lyzer, micropump, and other elements (Fig. 1). The total volume of the closed system, 50 cm3, was estab lished. Since culturing the regenerants in test tubes was done over a comparatively long time, the CO2 concen tration in the test tube was substantially higher than in atmospheric air, which can introduce an additional error into the measurement accuracy. To obtain cor rect results, before the start of measurements the test tubes with regenerants were held open for 24 h. After this, the test tubes were sealed with a rubber plug with two holes (entry and exit) connected with flexible hoses. The flexible hoses, successively passing through a pump, air dryer, rotameter, and infrared gas analyzer, were connected to the entry hole, and in this way a closed system was formed with a valve for access of atmospheric air for equalizing the CO2 content (the valve is opened before starting the measurement). The gas analyzer is equipped with a monitor with an auto matic recorder. It is known that the DRR of plants depends substantially on temperature [8, 9]. In con nection with this, a constant temperature was main tained during the measurements. Statistically significant differences with respect to the DRR index were determined between wildtype plants and transgenic lines (table). Thus, this value for the tested transgenic lines was 1.3–1.9 times lower than for the control lines, and significantly higher for line No. 19 (T1) than for the other tested lines. The results obtained indicate an effect of FeSOD1 gene expression on respiratory processes. Under sulfate salinity conditions, DRR of the con trol plants as well as transgenic line No. 19 (T1) decreased substantially, whereas that of lines No. 4 and
Dark Respiration Rate of in vitro cultured tomato regenerants as a result of FeSOD1 gene expression Line Control No. 4 (T0) No. 6 (T0) No. 8 (T0) No. 19 (T1)
Regenerant weight, mg
Rate of accumulation of respiratory CO2 in closed system, µg CO2/h
fresh
dry
0.640 ± 0.097 0.508 ± 0.096 0.414 ± 0.059 0.675 ± 0.025 0.720 ± 0.123
421.4 ± 36.3 669.33 ± 53.7 406.6 ± 137.9 826.5 ± 62.5 572.3 ± 46.8
38.3 ± 4.3 60.0 ± 19.0 39.4 ± 11.9 79.5 ± 15.5 57.7 ± 5.8
RUSSIAN AGRICULTURAL SCIENCES
DRR, µg CO2/h 0.0167 ± 0.0014 0.0085 ± 0.0010 0.0105 ± 0.0013 0.0085 ± 0.0014 0.0125 ± 0.0015 Vol. 40
No. 1
2014
DARK RESPIRATION RATE OF TRANSGENIC TOMATO PLANTS EXPRESSING
17
µg CO2/h
µg CO2/h 200
(b)
200
(a)
175
175
150
150
125
125
100
100
75
75 50
50 C
№4
№6
C
№ 19 (T1)
№6
№ 19 (T1)
Fig. 3. DRR (µg CO2/h) of regenerants of wildtype tomato (Belyi Naliv) and transgenic lines expressing the FeSOD1 gene with out salinity (left column) and under conditions of sulfate (a) and chloride (b) salinity (right column).
No. 6 increased (Fig. 3). In this case, statistically sig nificant differences weren’t found in No. 4 and No. 6; consequently, there is no negative effect of Na2SO4 on respiratory processes. A tendency toward an increase of DRR in control and transgenic plants of line No. 19 (T1) under chloride salinity conditions was noted. However, expression of the FeSOD1 gene in transgenic line No. 6 reduced the value of this index considerably. Thus, an efficient closed system was developed for determining DRR by an infrared gas analyzer in plants cultured in vitro in small vessels. It is shown that expression of gene FeSOD1 from A. thaliana in trans genic tomato plants substantially affects DRR. A differ ently directed response of respiratory metabolism of transgenic lines to the effect of Na2SO4 was established. ACKNOWLEDGMENTS The study was supported by the Russian Founda tion for Basic Research (project no. 130801323a). REFERENCES 1. Flowers, T.J., Improving crop salt tolerance, J. Exp. Bot., 2004, vol. 55, pp. 307–319. 2. Yeo, A.R., Predicting the interaction between the effects of salinity and climate change on crop plants, Sci. Hortic., 1999, vol. 78, pp. 159–174. 3. Baranova, E.N. and Gulevich, A.A., Problems and per spectives of genetic engineering approach to the improvement of the plant resistance to salinity, Skh. Biol., 2006, no. 1, pp. 39–52. 4. Kreslavski, V.D., Allakhverdiev, S.I., Los, D.A., and Kuznetsov, V.V., Signaling role of reactive oxygen spe cies in plants under stress, Russ. J. Plant Physiol., 2012, vol. 59, no. 2, pp. 141–154. 5. Baranenko, V.V., Superoxide dismutase in plant cells, Tsitologiya, 2006, vol. 48, no. 6, pp. 465–474. RUSSIAN AGRICULTURAL SCIENCES
Vol. 40
No. 1
6. Myouga, F., Hosoda, Ch., Umezawa, T., Lizumi, H., Kuromori, T., Motohashi, R., Shono, Y., Nagata, N., Ikeuchi, M., and Shinozaki, K., A heterocomplex of iron superoxide dismutases defends chloroplast nucle oids against oxidative stress and is essential for chloro plast development, Plant Cell, 2008, vol. 20, pp. 3148– 3162. 7. Serenko, E.K., Kurenina, L.V., Gulevich, A.A., May surian, A.N., Baranova, E.N., Balakhnina, T.I., Koso bruhov, A.A., and Polyakov, V.Yu., Structural organiza tion of chloroplast of tomato plants Solanum lycopersi cum transformed by Fecontaining superoxide dismutase, Biochem. (Moscow) Suppl. Ser. A: Membr. Cell Biol., 2011, vol. 5, no. 2, pp. 177–184. 8. Amthor, J.S., The McCreede WitPenning de Vries Thornley respiration paradigms: 30 years later, Ann. Bot., 2000, vol. 86, pp. 1–20. 9. Poorter, H., Gifford, R.M., Kriedemann, P.E., and Wong, S.C., A quantitative analysis of dark respiration and carbon content as factors in the growth response of plants to elevated CO2, Aust. J. Bot., 1992, vol. 40, pp. 501–513. 10. Lambers, H., Respiration in intact plants and tissues: its regulation and dependence on environmental fac tors, metabolism, and invaded organisms, in Higher Plant Cell Respiration (Encyclopedia of Plant Physiology, New Series, Berlin: SpringerVerlag, 1985, vol. 18, pp. 418–473. 11. Klimachev, D.A., Kuznetsova, S.A., and Starikova, V.T., Alternation of plant respiration intensity under salt stress, Vestn. Mosk. Gos. Univ.: Ser. Estestv. Nauki, 2011, no. 1, pp. 30–33. 12. Murashige, T. and Skoog, F., A revised medium for rapid growth and bioassays with tobacco tissue culture, Physiol. Plant., 1962, vol. 15, pp. 473–497. 13. Serenko, E.K., Ovchinnikova, V.N., Kurenina, L.V., Baranova, E.N., Gulevich, A.A., Maisuryan, A.N., and Kharchenko, P.N., Production of transgenic tomato plants with the Fedependent superoxide dismutase gene, Russ. Agricult. Sci., 2009, vol. 35, no. 4, pp. 223–226.
Translated by J. Slep 2014
