УДК 581.1



© 2014 B. Borawska-Jarmułowicz*, G. Mastalerczuk*, H. M. Kalaji**, R. Carpentier***, 

S. Pietkiewicz**, S. I. Allakhverdiev****

* Department of Agronomy, Warsaw University of Life Sciences SGGW, Warsaw, Poland

** Department of Plant Physiology, Warsaw University of Life Sciences SGGW, Warsaw, Poland

*** Groupe de Recherche en Biologie Végétale (GRBV), Université du Québec à Trois-Rivières, Québec, Canada

**** K.A. Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Moscow, Russia

Received March 26, 2013

Resistance to low temperature is crucial for overwintering crops. In this work we compared the resistance to low temperature treatment of some varieties of two forage grass species Dactylis glomerata L. and Lolium perenne L. in order to elucidate the reason for the better resistance found in some species. The variety Amila of D. glomerata and Diament of L. perenne were more tolerant to low temperature stress during the emergence and tillering phases as compared to the varieties Amera and Gagat. The improved tolerance and ability for recovery after stress were associated with better recovery of photosynthetic efficiency of these varieties and better survival of their shoots after low temperature stress.


 This text was submitted by the authors in English.


Abbreviation: PSII  photosystem II.

Corresponding authors: Suleyman I. Allakhverdiev and Hazem M. Kalaji. S.I.A., K.A. Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya ul. 35, Moscow, 127276 Russia; fax: +7 (499) 977-8018; e-mail: suleyman.allakhverdiev@gmail.com; H.M. Kalaji, Department of Plant Physiology, Warsaw University of Life Sciences SGGW, Nowoursynowska 159, 02-776 Warsaw, Poland; e-mail: hazem@kalaji.pl

Keywords: Dactylis glomerata  Lolium perenne  chlorophyll fluorescence  low temperature  photosynthesis  photosystem II



For sustainable agricultural grassland in northern countries, there is always a need to develop varieties with better winter survival. For example, Dactylis glomerata L. and Lolium perenne L. are very popular and widely grown forage grasses in Poland. The latter species is already well known to tolerate below-zero temperatures [1, 2]. However, D. glomerata exhibits tolerance to unfavorable environmental conditions, such as drought [3], but its ability to tolerate low temperature, especially during early spring time, seems to be crucial for survival [4]. 

Nowadays, various physiological parameters are being used as reliable indicators of plant stress tolerance. Photosynthetic efficiency of plants, based on chlorophyll a fluorescence measurements, is one of them [5]. It is a fast, non-destructive, and informative tool for the comparative study of photosynthetic efficiency of control and stressed plants [6]. It helps to get better understanding of the plant responses to environmental condition fluctuations [7, 8]. The development of freezing tolerance is crucial for the survival and productivity of overwintering crops. Hence, it is very important to understand the basis of their tolerance to low temperature conditions [9, 10]. The present investigation aims at elucidating the reasons standing behind the tolerance of some D. glomerata and L. perenne varieties to low temperature stress using their photosynthetic efficiency as the main indicator.



The experiments were conducted under controlled conditions in plant growth chambers (Phytotron) of Warsaw University of Life Sciences. Fifty seeds of each of Polish forage varieties of Dactylis glomerata L.  Amera and Amila and Lolium perenne L.  Diament and Gagat were sown in pots filled with 3 kg of mineral brown soil of 70% capillary water capacity (optimum moisture content) in five replications. The concentrations of available soil phosphorus (6.6 mg/100 g), potassium (13.3 mg/100 g), and magnesium (6.4 mg/100 g) were medium and the soil pHKCl was 5.1. Fertilization was applied once before sowing (g per pot): N  1.039, P  0.268, and K  0.162. At emergence and tillering phases (22 and 43 days after sowing, respectively), plants were exposed for 24 h to either 5°C or 10°C. 

Leaf greenness index (relative chlorophyll content) was measured with the SPAD-502 Chlorophyll Meter (“Minolta”, Japan) and chlorophyll a fluorescence with the Fluorescence Imaging System  FluorCam800MF (“Photon Systems Instruments”, Czech Republic). Measurements were carried out once before (control) and twice after each low temperature treatment (directly and after 48 h). Leaf greenness was measured only during tillering phase, while chlorophyll a fluorescence was measured during both emergence and tillering phases. The chlorophyll fluorescence measurements were done after 30 min of plant dark adaption using light saturation pulse intensity of 3750 µmol photons/(m2 s) for 800 ms. Subsequently, the minimum fluorescence signal (when all PSII centers are in the open state) from dark-adapted material (F0) and the maximum fluorescence signal (when all PSII centers are in the closed state) from dark-adapted material (Fm) were measured, and photosystem II (PSII) maximum efficiency within dark-adapted material (Fv/Fm) was calculated. The amount of survived shoots, dry matter of above-ground mass and roots (g per leaf or shoot depending on the growth phase) after one week from cold stress were also recorded. 

All measurements were done in 15 replications on fully developed leaves of randomly selected plants. The experimental data were analyzed by multifactor analysis of variance. The significance of differences between means was determined using the Tukey’s test at the significance level P < 0.05 and P < 0.01.



Low temperature causes damage to the plant photosynthetic apparatus [5, 11, 12]. This is mainly due to its influence on both chlorophyll content [1315] and photosynthetic efficiency [10, 16]. The activity of the enzymes involved in the photosynthetic processes is negatively affected under low temperature stress, thus decreasing the rate of photosynthesis [17]. The chlorophyll content decrease is more expressed due to the fact that membrane-bound chlorophyll is destroyed by the free radicals of oxygen despite the protective action of carotenoids [18, 19]. This drop in chlorophyll content has also been associated to metabolic blocks in the porphyrin pathway that leads to the chlorophyll synthesis [20].

The lower leaf chlorophyll contents expressed as leaf greenness (SPAD units) was indeed observed here (fig. 1) during the tillering phase of D. glomerata and L. perenne treated at both 5°C or 10°C. However, highly significant differences in the relative chlorophyll content were observed between the investigated species and varieties of grasses. These differences depended on the low temperature applied, varieties, species, and the time of measurement (table 1).

Low temperatures (5°C and 10°C) caused significant decreases in the chlorophyll contents of all investigated varieties soon after its application. However, varieties of L. perenne showed the lower relative chlorophyll content values as compared to D. glomerata. The chlorophyll content in both varieties of D. glomerata treated with the lower temperature (10°C) was reduced stronger (mean 25.1%) then at 5°C treatment (17.0% for Amila and 24.1% for Amera). After 48 h following the end of the freezing period at 5°C, the chlorophyll content significantly increased in Amera and Amila, indicating a recovery from stress. However, these values remained lower than those of the plants grown without temperature stress (control). The plants of L. perenne exhibited a significant decrease in the chlorophyll content immediately after freezing compare to the control treatment. This decrease was more expressed in plants treated at 10°C independently of varieties (25.728.5%). No recovery was observed in 48 h after the end of the freezing treatment, in place chlorophyll content even dropped further especially after 10°C.

The change in chlorophyll a fluorescence parameters is one the first responses of the plant photosynthetic apparatus to any fluctuations in plant growth environment/stress. It thus expresses the actual physiological state of the plant much ahead of any other parameter (e.g., enzymatic, morphological, or anatomical features) [10].

One of the main targets in the photosynthetic apparatus to be affected by a variety of environmental stresses is photosystem II (PSII). The activity of PSII is frequently restricted by strong light [2126] and low temperature [24, 2729], a major stresses limiting the crop productivity. We found here highly significant differences in Fv/Fm values between tested species and varieties of grasses, temperatures, and the time of measuring (table 2). Statistical analysis showed also highly significant relationships among all investigated factors. 

The measurement of chlorophyll a fluorescence showed that, during both growth stages (emergence and tillering phases), the maximum quantum efficiency of PSII (Fv/Fm) of D. glomerata and L. perenne varieties was distinctly lower after stress application in comparison with the control conditions (fig. 2). This result is in agreement with that of previous studies by Harrison et al. [2]. During the emergence phase, after 48 h following the end of the application of freezing temperature of 5°C, plants of all varieties showed the recovery of their photosynthetic efficiency (based on Fv/Fm values) (fig. 3). Amila variety of D. glomerata exhibited the higher values of Fv/Fm (0.683) than Amera (0.575) while Diament variety of L. perenne showed the higher value (0.766) than Gagat (0.698). No significant recovery was observed after 10°C application except for the variety Diament of L. perenne. A significant reduction in Fv/Fm value was denoted for all plants of tested varieties treated with both low temperatures during the tillering phase. It was also found that after freezing at 5°C, plants of all varieties of L. perenne with similar values of Fv/Fm (0.7460.792) and Amila of D. glomerata (0.562) showed recovery. These results also did not show the recovery of plants from thermal stress at 10°C (fig. 2). 

Minimal (F0) and maximal (Fm) fluorescence values were negatively affected by low temperature treatment. The values of these parameters were evidently affected by the lowest applied temperature, and recovery was observed only after treatment at 5°C (table 3). Temperature of 10°C caused significant damage to the photosynthetic apparatus. Low Fm values indicated that the level of reaction center reduction and reduced plastoquinone pool were almost equal to zero in most cases. The above changes could result from changes in membrane properties, including membrane fluidity that are known to occur when temperature deviates from optimal. These changes are commonly accompanied by alterations in the membrane composition, including fatty acid saturation, which in turn influences many biochemical events occurring in the thylakoid membrane, in particular photosynthetic electron transport [10]. Low temperature stress has also negative effects on the photosynthetic apparatus due to the inhibition of sucrose synthesis, which leads to the restriction of phosphate recycling and photophosphorylation [30]. This sequentially lowers the ADP/ATP ratio, causing the decrease of photosynthetic electron transport [28], which also increases the risk of photoinhibition occurrence [28, 30]. 

The survival of shoots estimated one week after thermal stress in all investigated varieties was observed only in plants exposed to 5°C (fig. 4). All plants treated with stress temperature of 10°C did not survive (there were no recovered plants). The lack of survival of L. perenne plants after freezing for a few hours at a similar temperature (8°C) was also reported by Lorenzetti et al. [1]. In the present study, it was also found that, at the tillering phase, the survival was clearly better than at the emergence phase. Especially, L. perenne varieties showed better survival of plants compared to D. glomerata. It was noticed that both investigated varieties of L. perenne showed similarly high survival (81% in Diament and 78% in Gagat), while variety Amila of D. glomerata showed better survival as compared to Amera (38.5 and 16.6%, respectively). 

Significant differences in shoot dry weight and all plant biomass among species, varieties, and development phases were also found. It was noticed that dry weight of roots highly depended on the growth phase (table 4). The dry weight of aboveground organs (measured 10 days after the end of temperature stress) during the emergence phase was slightly higher than dry weight of roots independent on species and variety, while in the tillering phase it was significantly less (fig. 5).



Our results indicate that the exposure of both D. glomerata and L. perenne varieties to low temperatures causes a significant decrease in the relative chlorophyll content. The greater reduction in the maximum quantum efficiency of PSII (Fv/Fm) than in chlorophyll content was also observed. As a response to freezing stress (5°C and 10°C) a significant reduction in Fv/Fm values in comparison with the control (before stress) of two others measurements (directly after stress and 48 h after stress) was observed in plants during both emergence and tillering growth phases. The photosynthetic apparatus of all varieties was more sensitive to 10°C at the emergence growth phase. One week after thermal stress, the survival of shoots was observed only in plants treated with 5°C and it was clearly better in tillering phase. 

It was found, that low temperatures significantly influenced only the dry weight of aboveground plant organs of investigated grass species and varieties in both growing phases. The variety Amila of D. glomerata and Diament of L. perenne were more tolerant to low temperature stress during the emergence and tillering phases as compared to the variety Amera and Gagat, respectively. That could be due to their better recovery of photosynthetic efficiency from stress after withholding of low temperature application and better survival of their shoots as compared to the varieties Amera and Gagat. Varieties of L. perenne are more tolerant than those of D. glomerata. The reason behind that seems to be that the former can efficiently run its photosynthetic machinery by reducing the chlorophyll content, minimizing the loss of energy as heat dissipation and increasing the trapping level of absorbed energy. We highly recommend the use of chlorophyll fluorescence measurements to study the tolerance of different varieties and species to low temperature stress.

The authors express their thanks to Ms. Marlena Dublińska for her help in laboratory work. 

S.I.A. was supported by grants from the Russian Foundation for Basic Research (nos. 11-04-01389, 12-04-92101, 13-04-91372, and 13-04-92711) and by the Presidium of RAS (program “Molecular and Cell Biology”).



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Fig. 1. Leaf greenness index (SPAD units) of D. glomerata (Amila and Amera) and L. perenne (Diament and Gagat) varieties at the tillering phase before (1, control) and after low temperatures (5°C and 10°C) application (2, immediately; 3, after 48 h).


Fig. 2. Maximum quantum efficiency of PSII (Fv/Fm) of D. glomerata (Amila and Amera) and L. perenne (Diament and Gagat) varieties at emergence and tillering phases before (1, control) and after low temperatures (5°C and 10°C) application (2, immediately; 3, after 48 h).


Fig. 3. Maximum quantum efficiency of PSII (Fv/Fm) of D. glomerata (Amila and Amera) and L. perenne (Diament and Gagat) varieties at emergence phase before (1, control) and after 48 h of freezing temperature at 5°C (2).


Fig. 4. Survival of shoots of D. glomerata (Amila and Amera) and L. perenne (Diament and Gagat) varieties in emergence (1) and tillering (2) phases one week after 5°C temperature treatment (%).


Fig. 5. Dry weight of aboveground plant organs and roots (g per leaf or shoot depending on the growth phase) of D. glomerata and L. perenne varieties.