ISSN 1021-4437, Russian Journal of Plant Physiology, 2007, Vol. 54, No. 4, pp. 450–455. © Pleiades Publishing, Ltd., 2007.Original Russian Text © A.N. Deryabin, M.S. Sin’kevich, S.V. Klimov, N.V. Astakhova, T.I. Trunova, 2007, published in Fiziologiya Rastenii, 2007, Vol. 54, No. 4, pp. 511–516.

450

INTRODUCTION

Plant chilling tolerance largely depends on carbohy-drate metabolism and source–sink relations (SSR) gov-erning the level of sugars accumulated by the cells ofdifferent organs. A multifunctional role of sugars inchilling tolerance, most thoroughly investigated infrost-resistant plants, is first of all associated with theircryoprotective influence on the membrane system ofthe cell and depends on their participation in metabolicprocesses as a source of energy and precursors to othersubstances of protective nature. There are data concern-ing a modifying effect of sugars on the plasma mem-brane, which promotes its homeoviscous adaptation tohypothermia [1]. It was shown that the accumulation ofsugars and their distribution in the leaves and crowns ofwinter cereals during adaptation to cold depend on cru-cial changes in their translocation and modification in

ëé

2

exchange. For instance, low hardening tempera-tures suppressed stronger respiration than photosynthe-sis [2]. Preservation of a high rate of photosynthesis at

low positive temperatures is an important factor of coldhardening and subsequent survival of plants duringoverwintering [3]. In chilling-sensitive plant species,photosynthesis is the first to be suppressed. At thebeginning, this suppression is reversible and becomesirreversible after longer exposures [4]. For instance, incucumber plants grown at

22°C

, a prolonged exposureto low temperature of

10°C

(for 5 days) in the light irre-versibly inhibited photosynthesis, whereas in morechilling-resistant tomato, the suppression of photosyn-thesis caused by the same exposure was reversible.

In contrast to frost-resistant plants, the role of carbo-hydrate metabolism and SSR in the formation of toler-ance in chilling-resistant plants is much less investi-gated. It is quite expedient to study this issue in a typi-cal chilling-resistant potato plant. The potato plantexpressing a gene for yeast invertase (

inv

) under thecontrol of tuber-specific patatin class I

B33

promoter(for apoplastic enzyme) is an organism with modifiedSSR [5]. Source ability therein is reduced because of anelevated activity of acid insoluble invertase cleaving thetransport form of sugars (sucrose) to monosaccharides(glucose and fructose); as a result, the efflux of assimi-

CO

2

Exchange and Structural Organization of Chloroplasts under Hypothermia in Potato Plants Transformed

with a Gene for Yeast Invertase

A. N. Deryabin, M. S. Sin’kevich, S. V. Klimov, N. V. Astakhova, and T. I. Trunova

Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya ul. 35, Moscow, 127276 Russia;fax: 7 (495) 977-8018; e-mail: trunova@ippras.ru

Received September 21, 2006

Abstract

—Growth,

CO

2

exchange, and the ultrastructure of chloroplasts were investigated in the leaves ofpotato plants (

Solanum

tuberosum

L., cv. Désirée) of wild type and transformed with a gene for yeast invertaseunder the control of patatin class I

B33

promoter (for apoplastic enzyme) grown in vitro on the Murashige andSkoog medium supplemented with 2% sucrose. At a temperature of

22°C

optimal for growth, the transformedplants differed from the plants of wild type in retarded growth and a lower rate of photosynthesis as calculatedper plant. On a leaf dry weight basis, photosynthesis of transformed plants was higher than in control plants.Under hypothermia (

5°C

), dark respiration and especially photosynthesis of transformed plants turned out tobe more intense than in control material. After a prolonged exposure to low temperature (6 days at

5°C

), in theplants of both genotypes, the ultrastructure of chloroplasts changed. Absolute areas of sections of chloroplastsand starch grains rose, and the area of plastoglobules decreased; in transformed plants, these changes were morepronounced. By some ultrastructural characteristics: a reduction in the cold of relative total area of sections ofstarch grains and plastoglobules (in percents of the chloroplast section area) and in the number of granal thyla-koids (per a chloroplast section area), transformed plants turned out to be more cold resistant than wild-typeplants. The obtained results are discussed in connection with changes in source–sink relations in transformedpotato plants. These changes modify the balance between photosynthesis and retarded efflux of assimilates,causing an increase in the intracellular level of sugars and a rise in the tolerance to chilling.

DOI:

10.1134/S1021443707040036

Key words: Solanum tuberosum - yeast invertase gene - patatin B33 promoter - source–sink relations - photo-synthesis - respiration - chloroplast ultrastructure - growth - chilling stress

Abbreviations

:

inv

—yeast invertase gene; MS—Murashige andSkoog nutrient medium; SSR—source–sink relations.

RESEARCH PAPERS

RUSSIAN JOURNAL OF PLANT PHYSIOLOGY

Vol. 54

No. 4

2007

CO

2

EXCHANGE AND STRUCTURAL ORGANIZATION OF CHLOROPLASTS 451

lates from photosynthesizing tissues is suppressed, andthey accumulate in the leaves [6, 7]. These transformedplants have a lower threshold of sucrose concentration(1–2%) necessary for the initiation of tuber formation[8] and display a higher tolerance to oxidative stressinduced by hypothermia [7, 9]. Since the chilling toler-ance of plants depends on the tolerance of their photo-synthetic machinery [10], it was important to study theeffect of expression in potato plants of a foreign genethat caused changes in SSR on the structure of chloro-plasts and on the rate of photosynthesis and dark respi-ration.

It is known that long-term potato plant micropropa-gation in vitro accelerates the processes of autoselec-tion aiming at the selection of plants with better growthcharacteristics under the given conditions, in particularat heterotrophic nutrition. As a result, the selection byheterotrophy leads to a decline in the photosyntheticactivity of these plants [11]. The measurements takenby these researchers showed that the amount of carbondioxide diffusing from the ambient air through the cot-ton wool plug ensured only 5–6% of photosyntheticdemands of the test-tube plants, and due to nutrientmedia rich in organic compounds, their growth waspredominantly heterotrophic. This suggests that photo-synthesis of potato plants in vitro is largely restricted bythe shortage of carbon dioxide. At the same time, thecontent of pigments and the level of light saturation ofphotosynthesis indicated that the plants possessed aproperly formed photosynthetic machinery. Theresearchers believe that, taking into consideration thelow level of gas exchange between the environment andthe medium within the test-tube, the photosyntheticfunction of the plants comes to the assimilation of

ëé

2

released as a result of heterotrophic growth, and oxygenemanated by the plants contributes to heterotrophicgrowth [12]. In the palisade cells of potato leavesexposed to low temperatures (

5°C

for 10 days followedby a gradual fall of temperature to 0 and

–2°C

), Bal-agurova et al. [13] detected some ultrastructuralchanges: the density of cellular contents rose; the mem-brane surface in the cells increased, which appeared asa rise in the number of elements of the endoplasmicreticulum and mitochondrial cristae; numerous invagi-nations of the tonoplast filled with membrane structureswere formed; thylakoids became sinuous, and starchgrains disappeared from the chloroplasts. At the sametime, these researchers revealed a greater tolerance ofpotato leaves to hypothermia as compared with plantsthat were not subjected to chilling.

In relation to the foregoing, the aim of this work wasto elucidate a possible relationship between the greaterchilling resistance of transformed potato plants and thestructure and functions of their photosynthetic machin-ery, in particular, with chloroplast ultrastructure and therates of photosynthesis and dark respiration. To thisend, the transformed plants grown in vitro were studiedat normal growth temperature and after chilling.

MATERIALS AND METHODS

The experiments were conducted with potato(

Solanum tuberosum

L., cv. Désirée) plants expressinga gene for yeast invertase (

inv

) under the control oftuber-specific patatin class I

Ç33

promoter and contain-ing a sequence for the leader peptide of the proteinaseinhibitor II for apoplastic enzyme localization (

B33-

inv

plants). The transformed plant also carried a marker

nptII

gene for tolerance to kanamycin. Common potatoplants of the same cultivar (wild type) served as controlmaterial. The plants were taken from the collection ofclones produced as a result of cooperation between theresearchers of Max Planck Institute of Molecular PlantPhysiology (Golm, Germany) and Chailakhyan Labo-ratory of Growth and Development, Timiryazev Insti-tute of Plant Physiology, Russian Academy of Sciences(Moscow, Russia).

The plants were micropropagated in vitro and grownin a controlled-climate chamber at the Institute of PlantPhysiology, Russian Academy of Sciences, at

22°C

and16-h illumination from the luminescent lamps produc-ing white light (illuminance of 4 klx) for 5 weeks intest-tube culture on MS agar medium containing 2%sucrose and the vitamins: 0.5 mg/l thiamine, 0.5 mg/lpyridoxine, and 60.0 mg/l meso-inositol. The plantswere subjected to chilling for 6 days at

5°C, 16

-h pho-toperiod, and an illuminance of 4 klx. Judging by theresults obtained by other researchers, who worked withthis potato cultivar [14], the temperature regime and theduration of chilling, we employed, were optimal for theinvestigations of this kind.

Relative growth rate (RGR) of the plants was calcu-lated according to the equation:

where

W

1

and

W

2

are the dry weights of plant or thelengths of shoot at the moments of time

T

1

and

T

2

.

ëé

2

exchange (photosynthesis and dark respira-tion) was measured using an open type unit equippedwith a URAS 2T infrared gas analyzer (Hartmann undBraun, Germany) and attendant devices produced bythe same manufacturer according to the proceduredescribed earlier [15]. The content of

ëé

2

in the airblown through the leaf chamber was 0.0419% by vol-ume, and a sensitivity of the gas analyzer was 0.005%by volume over the whole scale. A difference betweenthe content of

ëé

2

at the inlet and outlet of the leafchamber did not exceed 1–2% of the content of

ëé

2

inthe air. In order to measure gas exchange, by 4 testtubes, each with one plant, were accommodated in theexposure chamber placed within the working space of aGrönland climate cabinet (ILKA, Germany). The rateof a temperature fall within the climate chamber was

1°

C/min, and the accuracy of its maintenance was

±

0.5°C

. Within the leaf chamber, temperature waschecked using a fixed mercury thermometer. As a

RGRW2 W1–

T2 T1–( )W1-----------------------------,=

452

RUSSIAN JOURNAL OF PLANT PHYSIOLOGY

Vol. 54

No. 4

2007

DERYABIN et al.

source of light, we used a Proton slide projector(Diaproektor, Russia) with a 300 W incandescent lampplaced outside the climate cabinet. The plants in theleaf chamber were illuminated through a special win-dow closed as needed with a lightproof gate. Because ofa heat-insulating effect of double glass in the windowof the climate cabinet, the temperature in the leaf cham-ber both in the light and in the dark did not deviate fromthe desired value. The light intensity on the plant levelof 1000

µ

mol/(m

2

s) was saturating for photosynthesisof the investigated objects. Gas exchange was recordedright after attaining a desired temperature in the leafchamber. In order to reduce the experimental errorrelated to diurnal dynamics of photosynthesis, gasexchange in the plants of both types was determinedwithin the same time interval (from 9:30 to 14:00).Duration of a single measurement (or one replicate)was 15–20 min.

Gas exchange was evaluated from the rate of true

ëé

2

assimilation determined during the light–darktransition and the rate of dark respiration measured 10–15 min after turning off the light. True assimilation of

ëé

2

was a result of apparent assimilation and light res-piration evaluated from the release of

ëé

2

during thefirst 3–5 min after turning off the light. Table 1 showsthe means of three measurements and their standarderrors. Each measurement was taken using 4 plantswith 3 replicates.

In order to investigate the ultrastructure of the mes-ophyll cells, the leaves from the middle part of 5-week-old plants were detached and fixed for 4 h with 2.5%glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. Afterfour washings in the same buffer, the material was fixedwith 1% solution of

OsO

4

and embedded in the Epon812 resin. Ultrathin sections of palisade parenchymawere prepared using an LKB 3 ultra microtome (LKB,Sweden) and contrasted with a saturated solution ofuranyl acetate at

37°C

for 30 min followed by lead cit-rate at

20–22°C

for 15 min. The sections were exam-ined with a TEMSCAN 100CX2 electron microscope

(Jeol, Japan). Morphometric investigation of the cellsand chloroplasts was made using a MOP-VIDEO-PLAN apparatus (Reichert, Austria). For electron-microscopic examinations, the samples were takenfrom 5 leaves each of 4 plants of every type of treat-ment. Morphometric measurements were based on theexamination of 70 chloroplasts. Differences reliable ata 95% level of significance are discussed.

RESULTS AND DISCUSSION

Genetic transformation of potato plants associatedwith the insertion of a gene for yeast invertase causedretardation of their linear growth (Table 1). As com-pared with the plants of wild type, relative growth rateof

B33-

inv

plants decreased by 21%, and relative rate ofdry weight accumulation declined more than twice.

The obtained results concerning the rate of photo-synthetic gas exchange in potato plants of investigatedgenotypes are shown in Table 1. It is apparent that therate of photosynthesis upon light saturation calculatedon a leaf dry weight basis was higher in

B33-

inv

plantsboth at normal (

22°C

) and low (

5°C

) temperature. Webelieve that a greater rate of photosynthesis observed inthe transformed plants depends on a more pronouncedsuppression of leaf dry weight accumulation, on whichthe calculation of photosynthesis was based.

The photosynthetic machinery of

B33-

inv

plantswas more cold-resistant. Whereas in the plants of wildtype, the fall of temperature from 22 to

5°C

caused afull cessation of photosynthesis, in

B33-

inv

plants, pho-tosynthesis declined by only 86%. The rate of dark res-piration was also higher in transformed plants at bothtemperatures. The same as photosynthesis, dark respi-ration of

B33-

inv

plants was more resistant to the low-ering of temperature and declined by 64% as comparedwith 70% in control plants. However, these differencesare not great and fall within the experimental error.

At

22°C

, the rates of photosynthesis and dark respi-ration the same as the ratio of photosynthesis to respi-

Table 1.

The rate of growth and CO

2

exchange in control potato plants of wild type (C) and B33-

inv

plants at normal tem-perature (22

°

C) and after the exposure to cold (5

°

C)

Characteristic22

°

C 5

°

C 5

°

/22

°

C

C B33-

inv

C B33-

inv

C B33-

inv

Relative growth rate:

mm/(cm day) 0.23 0.19 – – – –

mg/(g day) 43 20 – – – –

True photosynthesis, mg CO

2

/(g dry wt of the leaf h) 7.2

±

0.6* 10.9

±

0.8* 0 1.5

±

0.9 0 14

Dark respiration, mg CO

2

/(g dry wt of the leaf h) 0.80

±

0.03 1.40

±

0.25 0.20

±

0.02 0.50

±

0.06 30 36

True photosynthesis (P), mg CO

2

/(plant h) 868

±

79* 576

±

45* 0 79

±

46 0 14

Dark respiration (R), mg CO

2

/(plant h) 300

±

13 284

±

50 79

± 9 99 ± 11 26 35

Ratio P/R 2.9 2.0 0 0.8 0 40

* Differences between control and B33-inv plants are reliable at 95% level of significance.

RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 54 No. 4 2007

CO2 EXCHANGE AND STRUCTURAL ORGANIZATION OF CHLOROPLASTS 453

ration calculated on a whole plant basis in B33-invplants were lower than in control material. However, atlow temperature (5°C), these characteristics in trans-formed plants were greater than in control material.Upon a fall of temperature from 22 to 5°C, the ratio ofphotosynthesis to respiration in control plantsapproached zero; at the same time, in B33-inv plants, itdecreased by 60%. Because the value of this ratio at lowtemperatures is one of the important indicators of coldand frost tolerance in plants [15], these results corrobo-rate a higher cold tolerance of B33-inv plants as com-pared with the plants of wild type we revealed earlier[16].

The greater cold tolerance of some plant species isassociated with their ability to suppress the formationof free radicals by means of coupling the electron trans-port in chloroplasts to the reduction of ëé2 in thecourse of dark reactions [17, 18]. In our experiments[7], this was confirmed by a lower rate of peroxidationof membrane lipids and lower activity of superoxidedismutase in B33-inv plants under hypothermia ascompared with the plants of wild type.

Insertion of the gene for yeast invertase broughtabout a change in the structure of chloroplasts in B33-inv plants (Table 2). The area of chloroplast sections inB33-inv plants turned out to be by 34% greater than incontrol material, which agrees with a higher rate ofphotosynthesis in transformed plants (calculated on aleaf dry weight basis). By the section area of the starchgrains, potato plants of both genotypes were identical;however, starch grains were more numerous in trans-

formed plants. As a result of expression of the gene foryeast invertase, the number of grana and thylakoids inB33-inv plants increased by 1.5 times. The area of plas-toglobules was also greater in B33-inv plants as com-pared with the plants of wild type. However, we did notfind differences in the number of plastoglobules perchloroplast and the number of thylakoids per granumbetween the plants of investigated genotypes.

Long (6 days) exposure of plants to low tempera-tures (5°C) caused differences between the genotypesin the rates of growth and photosynthesis and inducedmorphometric changes in the chloroplasts and theirstructural elements (Table 2). In the plants of investi-gated genotypes, the area of chloroplast sectionincreased; in B33-inv plants, it was by 55% greater thanin control material. It should be noted that swelling ofchloroplasts in the cold is one of the typical responsesof the cellular ultrastructure observed not only in plantssensitive to cold [15, 18] but also in frost-resistantplants in the course of hardening [19]. In plants of bothgenotypes, long exposure to low temperatures caused adecrease in the area of plastoglobules and their numberin the chloroplasts; in B33-inv plants, this reductionwas more pronounced. Moreover, in contrast to controlplants, hypothermia resulted in a decrease in the num-ber of thylakoids in the chloroplasts.

In order to verify differences in chloroplast ultra-structure in potato plants of investigated genotypes, weemployed another method of calculation when the sec-tion area of the elements of the cellular ultrastructurewas expressed in percent of the area of chloroplast sec-

Table 2. Structural organization of chloroplasts in control potato plants of wild type (C) and B33-inv plants at normal tem-perature (22°C) and after 6 days of chilling at 5°C

Characteristic22°C 5°C 5°/22°C

C B33-inv C B33-inv C B33-inv

Section area, µm2

Chloroplast 10.1 ± 0.4 13.5 ± 0.3 11.7 ± 0.6 18.1 ± 0.8 1.16 1.34

Starch grain 1.5 ± 0.1 1.4 ± 0.1 1.8 ± 0.1 1.6 ± 0.1 1.20 1.14

Plastoglobule 0.053 ± 0.03 0.062 ± 0.003 0.045 ± 0.002 0.032 ± 0.002 0.85 0.52

No. per chloroplast section

Grana 13.4 ± 0.6 20.1 ± 0.5 16.1 ± 0.7 18.3 ± 0.8 1.20 0.91

Thylakoids 99 153 116 136 1.17 0.88

Thylakoids per granum 7.4 ± 0.1 7.7 ± 0.2 7.2 ± 0.2 7.4 ± 0.2 0.97 0.96

Starch grains 1.7 ± 0.1 2.4 ± 0.1 1.8 ± 0.1 2.3 ± 0.1 1.06 0.96

Plastoglobules 3.4 ± 0.2 3.5 ± 0.3 2.0 ± 0.3 1.8 ± 0.3 0.59 0.51

Normalized characteristics

Starch grains, % of the chloroplastsection area

26 25 28 20 1.08 0.80

Plastoglobules, % of the chloroplastsection area

1.8 1.6 0.8 0.3 0.44 0.19

No. granal thylakoids per µm2

of chloroplast area9.9 11.4 9.9 7.5 1.00 0.66

454

RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 54 No. 4 2007

DERYABIN et al.

tion. It turned out that at 22°C B33-inv plants did notdiffer from control plants in such characteristics as theaverage area of starch grains and plastoglobules; signif-icant differences were observed in such a normalizedparameter as the number of granal thylakoids per areaunit of the chloroplast section. When B33-inv plantswere kept for 6 days at 5°C, the proportion of chloro-plast section occupied by starch grains decreased 1.3times because their number declined. In contrast, incontrol plants this characteristic even somewhatincreased.

Hydrolysis of starch grains in the chloroplasts is oneof the most important indicators of plant adaptationoccurring on the cellular level [18]. It was shown earlierthat, when the temperature went down, the proportionof chloroplast area occupied by starch grains decreasedboth in frost-resistant cereals (winter rye and wheat)and frost-sensitive but chilling-resistant tomato; how-ever, it remained almost the same in chilling-sensitivecucumber [20, 21]. By this parameter, the transformedpotato plants were more chilling-resistant than the con-trol plants of wild type. Probably, this is corroboratedby a more pronounced decrease in the proportion ofchloroplast section area occupied by plastoglobules andobserved after the transfer of B33-inv plants to the cold(respective decrease in this parameter turned out to betwo times more pronounced than in control material).

One of the symptoms of adaptation to cold on thelevel of chloroplast organization is a reduction in thenumber of granal thylakoids per chloroplast area unit[22]. By this parameter, the transformed plants alsoturned out to be more cold resistant than control mate-rial. Whereas in the plants of wild type, the number ofgranal thylakoids remained the same, in B33-inv plants,it decreased 1.5 times.

Thus, the transformed plant of potato with insertedgene for yeast invertase is a plant with modified SSR[5]. Elevated activity of acid-insoluble invertase inB33-inv plants, we have discovered earlier, suppressedthe efflux of assimilates from the photosynthesizingcells and tissues [6]. Our investigations have shown thata disturbance of the balance between photosynthesisand assimilate efflux manifests itself in a decrease inthe relative growth rate of the transformed plants. At thesame time, they preserve the higher rate of photosyn-thesis in the leaf tissues under hypothermia as com-pared with control plants (Table 1). In accordance withthe mechanism of plant adaptation realized through thechanges in SSR [23], the transformed plants with sucha type of SSR must have a greater content of sugars andhigher tolerance to hypothermia, which was shown inour previous investigations [6, 9, 16].

ACKNOWLEDGMENTS

This work was supported by the Russian Foundationfor Basic Research, project no. 04-04-48476.

REFERENCES

1. Uemura, M. and Steponkus, P.L., Artificial Manipulationof the Intracellular Sucrose Content Alters the Incidenceof Freeze-Induced Membrane Lesions of Isolated Proto-plasts of Arabidopsis thaliana, Cryobiology, 1997,vol. 35, p. 336.

2. Klimov, S.V., Specific Features of Autumnal CO2-Exchange in the Overwintering Plants, Agric. Sci. Art.,2004, vol. 2, pp. 97–102.

3. Klimov, S.V., Bioenergetic Aspects of Adaptation andFrost-Tolerance of Wintering Cereals, Usp. Sovrem.Biol., 1987, vol. 104, pp. 251–267.

4. Klimov, S.V., Astakhova, N.V., and Trunova, T.I., ColdTolerance of Tomato and Cucumber Plants and Low-Temperature Photosynthetic Activity, Dokl. Akad. Nauk,1999, vol. 365, pp. 279–282.

5. Stitt, M., von Schaewen, A., and Willmitzer, L., “Sink”Regulation of Photosynthetic Metabolism in TransgenicTobacco Plants Expressing Yeast Invertase in Their CellWall Involves a Decrease of the Calvin-Cycle Enzymesand an Increase of Glycolytic Enzymes, Planta, 1990,vol. 183, pp. 40–50.

6. Deryabin, A.N., Trunova, T.I., Dubinina, I.M., Burakha-nova, E.A., Sabel’nikova, E.P., Krylova, E.M., andRomanov, G.A., Chilling Tolerance of Potato PlantsTransformed with a Yeast-Derived Invertase Gene underthe Control of the B33 Patatin Promoter, Russ. J. PlantPhysiol., 2003, vol. 50, pp. 449–454.

7. Deryabin, A.N., Sin’kevich, M.S., Dubinina, I.M., Bura-khanova, E.A., and Trunova, T.I., Effect of Sugars on theDevelopment of Oxidative Stress Induced by Hypother-mia in Potato Plants Expressing a Gene for Yeast Inver-tase, Russ. J. Plant Physiol., 2007, vol. 54, pp. 32–38.

8. Aksenova, N.R., Konstantinova, T.N., Golyanov-skaya, S.A., Kossmann, J., Willmitzer, L., andRomanov, G.A., Transformed Potato Plants as a Modelfor Studying the Hormonal and Carbohydrate Regula-tion of Tuberization, Russ. J. Plant Physiol., 2000,vol. 47, pp. 370–379.

9. Deryabin, A.N., Dubinina, I.M., Burakhanova, E.A.,Astakhova, N.V., Sabelnikova, E.P., and Trunova, T.I.,Influence of Yeast-Derived Invertase Gene Expression inPotato Plants on Membrane Lipid Peroxidation at LowTemperature, J. Therm. Biol., 2005, vol. 30, pp. 73–77.

10. Levitt, J., Responses of Plants to EnvironmentalStresses, vol. 1, Chilling, Freezing and High Tempera-ture Stresses, New York: Academic, 1980.

11. Tsoglin, L.N. and Gabel’, B.V., Autoselection duringPlant Micropropagation: Ppotato Phototrophic Micro-propagation In Vitro, Russ. J. Plant Physiol., 1994,vol. 41, pp. 384–388.

12. Tsoglin, L.N., Melik-Sarkisov, O.S., Andreenko, T.I.,and Rozanov, V.V., Gas Exchange and Photosynthesis ofPotato Plants In Vitro, Dokl. Akad. Nauk, 1991, vol. 316,pp. 1020–1024.

13. Balagurova, N.I., Drozdov, S.N., Tikhova, M.A., andSulimova, G.M., Effects of Low Positive Temperatureson the Cell Ultrastructure in Potato Leaves, Bot. Zh.(Leningrad), 1980, vol. 65, pp. 1156–1161.

14. Svensson, A.S., Johansson, F.I., Mollerb, I.M., and Ras-musson, A.G., Cold Stress Decreases the Capacity for

RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 54 No. 4 2007

CO2 EXCHANGE AND STRUCTURAL ORGANIZATION OF CHLOROPLASTS 455

Respiratory NADH Oxidation in Potato Leaves, FEBSLett., 2002, vol. 517, pp. 79–82.

15. Klimov, S.V., Astakhova, N.V., and Trunova, T.I.,Changes in Photosynthesis, Dark Respiration Rates, andPhotosynthetic Carbon Partitioning in Winter Rye andWheat Seedlings during Cold Hardening, J. Plant Phys-iol., 1999, vol. 155, pp. 734–739.

16. Deryabin, A., Dubinina, I., Burakhanova, E., Astakhova, A.,Sabel’nikova, E., Sinkevich, M., and Trunova, T., Study-ing of Cold Tolerance of Potato Plants Transformed withYeast Invertase Gene, Agric. Sci. Art., 2004, vol. 2,pp. 22–29.

17. Walker, M.A., McKersie, B.D., and Pauls, K.P., Effectsof Chilling on the Biochemical and Functional Proper-ties of Thylakoid Membranes, Plant Physiol., 1991,vol. 97, pp. 663–669.

18. Kratsch, H.A. and Wise, R.R., The Ultrastructure ofChilling Stress, Plant, Cell Environ., 2000, vol. 23,pp. 337–350.

19. Stefanowska, M., Kuras, M., and Kacperska, A., LowTemperature-Induced Modifications in Cell Ultrastruc-ture and Localization of Phenolics in Winter OilseedRape (Brassica napus L. var. oleifera) Leaves, Ann. Bot.,2002, vol. 90, pp. 637–645.

20. Klimov, S.V., Astakhova, N.V., and Trunova, T.I., Rela-tionship between Plant Cold Tolerance, Photosynthesisand Ultrastructural Modifications of Cells and Chloro-plasts, Russ. J. Plant Physiol., 1997, vol. 44, pp. 759–765.

21. Trunova, T.I. and Astakhova, N.V., Effect of Low Tem-perature on Protein Content and Chloroplast Ultrastruc-ture in Cucumber and Tomato Plants, Proc. Int. Conf.Frost Hardiness of Plants, Poznan, 1999, pp. 58–61.

22. Klimov, S.V., Pathways of Plant Adaptation to Low Tem-peratures, Usp. Sovrem. Biol., 2001, vol. 211, pp. 3–22.

23. Klimov, S.V., Trunova, T.I., and Mokronosov, A.T.,Mechanism of Plant Adaptation to Unfavorable Environ-ments via Changes in the Source–Sink Relations, Sov.Plant Physiol., 1990, vol. 37, pp. 1024–1035.