How the Climatic Change Affect Animal Production, and the Impact of Dairy Animals on Environment

765 Pages·2016·24.11 MB·English

Updated on 25.11.2016
27th International Scientific-Expert Congress of Agriculture and Food Industry, 26-28 September 2016, Bursa, Turkey
How the Climatic Change Affect Animal Production, and the Impact of Dairy Animals
on Environment

Ahmed A.K. SALAMA1, Ekin SUCU2 and Gerardo CAJA1
1Grup de Recerca en Remugants (G2R), Departament de Ciència Animal i dels Aliments, Universitat Autònoma de Barcelona, 08193
Bellaterra, SPAIN
2Department of Animal Science, Faculty of Agriculture, Uludag University, 16059 Bursa, TURKEY
* Corresponding author: [email protected]

ABSTRACT
This paper describes the contribution of ruminants on greenhouse gasses and how the potential of greenhouse gasses’ from
ruminants on global warming is mispresented. In addition, this paper hopes to review the responses of dairy ruminants to thermal
stress (heat and cold) and some nutritional modifications to reduce the adverse effects of thermal stress.

Keywords: Emission of greenhouse gases, Livestock, Thermal stress, Reduction of thermal stress

INTRODUCTION

The observed earth’s surface temperature increased by up to 2.5°C from 1901 to 2012 according to the
assessment report of the Intergovernmental Panel on Climate Change (IPCC, 2013). Additionally, the Northern
Hemisphere had the warmest 30-year period from 1938 to 2012 of the last 1400 years. Continued emissions of
greenhouse gases will cause further warming. By the end of the 21st century, the models of IPCC projected a 3–
10°C warming in annual temperature over the global land area, with greater warming in the Northern
Hemisphere.
The theoretical low critical temperature for ruminants (-35 to -20 ºC) led to the idea that cold stress is
less important compared to heat stress. However, performance data clearly showed a reduction in production at
ambient temperatures well above this theoretical low critical temperature. In the future and regardless the
production system, it is likely that livestock animals will suffer heat and cold waves with a higher frequency,
intensity and duration, which will affect their health, welfare, and performance.
The objective of the current short review is to shed some light on the emission of greenhouse gases
from livestock, responses of dairy ruminants to thermal stress (heat and cold), and some nutritional strategies to
reduce the impact of thermal stress.

The Overestimation of Warming Potential of Greenhouse Gases Emitted from Ruminants
The emission of methane (CH ), nitrous oxide (N O) and carbon dioxide (CO ) gases from livestock is a concern
4 2 2
in scientific and media articles. This concern is based on the fact that CH has global warming potential (GWP)
4
21 times more than CO . However, comparing the GWP of one greenhouse gas with another should be based on
2
the behavior of the gases at the molecular level. In other words, the comparison should deal with equal numbers
of molecules of the two gases. This would not be possible without the correction for the molecular mass of each
gas (Flood, 2011). Keeping in mind the masses of atoms of C (12), H (1) and O (16), then the mass of gas
molecules are 16 and 44 for CH and CO , respectively. So, a CH molecule does not have a warming ability that
4 2 4
is 21 times that of a CO molecule since the GWP is based on comparing equal masses. In 1 kg of methane there
2
are 2.75 (44/16) times the number of molecules that there are in 1 kg of CO . Molecule for molecule, the GWP
2
of CH compared to CO is only 7.6 (21/2.75) times.
4 2
Furthermore, a study was conducted to compare the environmental impact of modern (2007) dairy
production with historical production practices as exemplified by dairy system in 1944 (Capper et al., 2009).
Although there is a perception that pasture based, low-input dairy systems characteristic of the 1940s were more
environmental friendly than modern milk production systems, Capper et al. (2009) found that the carbon
footprint per billion kilograms of milk produced from high genetic merit cows in 2007 reduced by 63%
compared to equivalent milk production in 1944. The use of nutritionally precise diets and improving genetic
merit not only increase profit, but also improve environmental sustainability. From the previous presented
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information, it is clear that there are some “misconceptions” that resulted in the exaggeration of the role of dairy
animals in greenhouse gas emissions. In fact, animals are dramatically affected by the climate change rather than
being inducers of it.

Reponses of Dairy Ruminants to Thermal Stress
As shown by Salama et al. (2016), the 5 welfare freedoms (freedom from thirst, hunger and malnutrition;
freedom from discomfort; freedom from pain, injury and disease; freedom to express normal behavior; and
freedom from fear and distress) are all negatively affected by the thermal stress. When ambient temperatures are
high, animals reduce their feed intake to decrease heat production. Additionally, in regions where animals graze,
besides the high ambient temperature in summer there is a clear reduction in the forage quality (high fiber and
lower protein contents), which negatively affects the animal welfare. Heat-stressed animals are uncomfortable
and move a lot seeking cooler zones. In addition, below the lower critical temperature, the acute response of
animals is shivering to increase heat production.
Besides the negative effects on welfare, both heat and cold stress reduced milk yield and growth in
cattle, sheep, and goats (Baumgard and Rhoads, 2013; Salama et al., 2016). Additionally, feed intake reduced by
heat stress resulted in decreasing the absorbed propionate arriving to liver for the gluconeogenesis (Figure 1),
which would decrease glucose produced via propionate. However, we detected no difference in blood glucose
between heat-stressed and thermo-neutral goats (Hamzaoui et al., 2013). Alternatively, there was some degree of
muscle degradation by heat stress as indicated by greater blood creatinine. It is possible that some amino acids
resulting from muscle degradation are used for liver gluconeogenesis (Figure 1). This could partially compensate
for the reduction in glucose production via propionate and helps in keeping blood glucose levels.
Not surprisingly, acute heat and cold stress induce the expression of several genes, especially heat
shock transcription factor 1 and heat shock proteins. Those proteins exert cytoprotective effects to assist folding
of newly synthesized proteins and repairing and refolding damaged proteins under stress conditions. When the
stress persisted, changes in gene expression led to an altered physiological state referred to as “acclimation”, a
process largely controlled by altered gene expression in response to endocrine signals. There were dramatic
changes in the transcriptomic profile of blood and milk cells by heat stress. These changes included modification
in lipid metabolism and consequently functions of immunity cells. It was also clear that heat stress exerts its
negative effect on milk production by inhibiting mammary synthetic capacity and increasing the apoptosis of
mammary cells.

Feed intake
Glucose
Propionate
Amino acids Glucose
Creatinine (muscle degradation)

Figure 1. Metabolic changes in dairy animals exposed to heat stress.
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Some Nutritional Strategies to Alleviate Thermal Stress Effects
Cattle under high temperatures selectively decrease the intake of forage to reduce heat production. Increasing the
proportion of concentrate in the diet had positive effects on DM intake and on milk energy produced per unit of
feed energy. Furthermore, high concentrate rations fed to cattle under low temperatures can help mitigate cold
stress because the extra metabolic energy derived from starch is more advantageous than the extra heat
increment derived from fiber. However, supplementation with concentrates under heat stress conditions should
be considered with care as heat-stressed animals are highly prone to ruminal acidosis. The addition of fat to the
diet of ruminants also is a common practice to increase dietary energy density. Feeding fat is associated with
reduced metabolic heat production per unit of energy fed and, compared with starch and fiber, fat has a much
lower heat increment in the rumen. Heat-stressed dairy goats supplemented with 4% soybean oil produced milk
with greater milk fat without effects on milk protein content or milk yield. Supplementation with yeast
(Saccharomyces cerevisiae) during summer increased milk and milk solids yield by 5% and tended to increase
blood glucose and niacin levels (Salvati et al., 2015). Furthermore, cows supplemented with yeast had lower
respiratory rate, but had similar rectal temperature compared with non-supplemented animals (Salvati et al.,
2015). We recently hypothesized that L-carnitine would improve lipid metabolism and decrease the use of amino
acids as energy source under heat stress conditions. However, when we supplemented heat-stressed dairy goats
with rumen-protected L-carnitine, there was no effect on milk yield, milk fat or milk protein.

ACKNOWLEDGMENTS

Part of information presented herein comes from a research project funded by the Spanish Ministry of Economy
and Competitiveness (Plan Nacional I+D+I; Project AGL-2013-44061R).

CONCLUSIONS

Some misconceptions resulted in the exaggeration of the role of livestock animals as greenhouse gas emitters.
Instead, animals suffer the climate changes at various levels including welfare, metabolism, gene expression, and
production. Some nutritional modifications and supplements could be helpful to mitigate the effects of thermal
stress.

REFERENCES

Baumgard, L.H. and R.P. Rhoads. 2013. Effects of heat stress on post absorptive metabolism and energetics. Ann. Rev. Anim. Biosci. 1:311–
337.
Capper, J.L., R.A. Cady, and D.E. Bauman. 2009. The environmental impact of dairy production 1944 compared with 2007. J. Anim. Sci.
87:2160–2167.
Flood, W. 2011. The methane misconceptions. Energy Environment, 22:233–239.
Hamzaoui, S. A.A.K. Salama, E. Albanell, X. Such, and G. Caja. 2013. Physiological responses and lactational performances of late lactating
dairy goats under heat stress conditions. J. Dairy Sci. 96:6355–6365.
IPCC. (Intergovernmental Panel on Climate Change). 2013. Summary for Policymakers. In: Climate Change 2013: The Physical Science
Basis. Contribution of Working Group I to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change [Stocker,
T.F., D. Qin, G.-K. Plattner, M. Tignor, S. K. Allen, J. Boschung, A. Nauels, Y. Xia, V. Bex and P.M. Midgley (eds.)]. Cambridge
University Press, Cambridge, United Kingdom and New York, NY, USA.
Salama, A.A.K., G. Caja, S. Hamzaoui, B. Badaoui, A. Castro-Costa, D.E. Façanha, M.M. Guilhermino, and R. Bozzi. 2014. Different levels
of response to heat stress in dairy goats. Small Ruminant Research 121:73–79.
Salama, A.A.K., B. Badaoui, S. Hamzaoui, and G. Caja. 2015. RNA-sequencing analysis of milk somatic cells in heat-stressed dairy goats. J.
Dairy Sci. 98(E-Suppl. 1): 62.
Salama, A.A.K., G. Caja, S. Hamzaoui, X. Such, E. Albanell, B. Badaoui, and J.J. Loor. 2016. Thermal stress in ruminants: Responses and
strategies for alleviation. Pages 11 to 36 (Chapter 2) in Animal Welfare in Extensive Production Systems. J. J. Villalba, ed. 5M
publishing, Sheffield, United Kingdom (ISBN: 9781910455548).
Salvati, G.G.S., N.N. Morais Júnior, A.C.S. Melo, R.R. Vilela, F.F. Cardoso, M. Aronovich, R.A.N. Pereira, and M.N. Pereira. 2015.
Response of lactating cows to live yeast supplementation during summer. J. Dairy Sci. 98:4062–4073.

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Changes in Peroxidase Isoenzyme and Protein Patterns in Oat Cultivars under Cold
Stress

İmren KUTLU1* and Ece TURHAN2
1 Eskişehir Osmangazi University, Faculty of Agriculture, Department of Biosystem Engineering, 26160 Eskişehir, TURKEY
2 Eskişehir Osmangazi University, Faculty of Agriculture, Department of Agricultural Biotechnology, 26160 Eskişehir, TURKEY
* Corresponding author: [email protected]

ABSTRACT
The possible role of peroxidase isoenzymes and protein patterns induced by cold stress were investigated in 'Faikbey' (relatively
sensitive) and 'Yeniçeri' (relatively tolerant) oat (Avena sativa L.) cultivars. The seeds of the oat cultivars were sown in
perlite:torf:sand (1:1:1) media and germinated at 25/15 °C (day/night) in a controlled greenhouse. Plants were harvested after 21
days and half of the plants for 12 hours while the other half was acclimated for 24 hours at 4 °C to low temperatures. After that, the
leaves were exposed to controlled freezing test at 0, -6, -12 and -18 ºC for 12 hours in manually-controlled low temperature freezer.
Samples were then removed from the freezer at each temperature and placed at 4 °C overnight for slow thawing. Peroxidase (POX)
profiles and protein patterns of the leaf tissues were performed by native polyacrylamide gel electrophoresis (PAGE) and SDS-
PAGE, respectively. In native PAGE, different acidic and basic isoperoxidase bands (between Rf=0.04 and Rf=0.88) were observed
commonly with different band intensities in both cultivars with respect to the cold treatments and duration of acclimation. In
addition, according to the SDS-PAGE analysis different protein bands, in mass range 9-94 kDa, were detected in the oat cultivars.
The intensities of some proteins generally decreased and/or disappeared and a few new proteins appeared in response to cold stress
and duration of acclimation. In conclusion, we supposed that isoperoxidases and proteins may responsible to cold tolerance in oat
cultivars.

Keywords: Avena sativa L., Cold acclimation, Low temperature stress, Peroxidase, Soluble protein

INTRODUCTION

Low temperatures (cold and frost) represent a severe threat to plant survival and economic crop productivity in
many regions. The process whereby certain plants increase in freezing tolerance in response to low non-freezing
temperatures are known as cold-acclimation (or cold-hardiness) (Thomashow 2010). In temperate latitudes, cold-
acclimation is established in the autumn, when the temperatures are low but positive and the photoperiod
decreases, and it reaches a maximum in winter (Reulland et al. 2009). The ability of plants to acclimate to low
temperatures is crucial for their survival.
Plant responses to low temperature stress have been associated with activated forms of oxygen,
including superoxide (O .-), hydrogen peroxide (H O ), hydroxyl (-OH) radicals and singlet oxygen (O 1) (Foyer
2 2 2 2
and Noctor 2005, Suzuki and Mittler 2006). Cold stress induces or enhances the active oxygen species-
scavenging enzymes like, catalase (CAT: EC 1.11.1.6), peroxidase (POX:EC 1.11.1.7), ascorbate peroxidase
(APX: EC 1.11.1.11), and glutathione reductase (GR: EC 1.6.4.2) (Turhan et al. 2012a, 2012b). Peroxidases are
a large group of isoenzymes with an extreme range of isoelectric points, serving a multitude of functions
(Huystee 1987). Peroxidases a class of enzymes in plant, catalyze oxido reduction between H O and various
2 2
reductans (Hiraga et al. 2001). Therefore, peroxidases are known one of the important parts of the enzymatic
defence system of plant cells under stress conditions (Gaspar et al. 1982).
Biochemical changes that have been associated with cold-acclimation include increased sugar and
soluble protein content, expression of specific proteins and the appearance of new isozymes (Eris et al. 2007,
Gulen et al. 2008, Turhan 2012, Liu et al. 2013). Protein folding is influenced by temperature changes.
Temperature-induced changes in the physical state of membranes and proteins are expected to change the
metabolic reactions and thus the metabolite concentrations. Therefore, plant cells can sense cold stress through
membrane rigidification, protein/nucleic acid conformation, and/or metabolite concentration (Chinnusamy et al.
2010).
The commercial value of oat is derived both from its high-energy grain and from superior break-crop
benefits. Today oat is mainly used as animal feed, but it is one of the most promising future cereals in the
functional food area (Bräutigam et al. 2005). Oats are grown in Turkey where occasional frost may occur. In
Turkey, yields are limited by the fact that oat cannot be used as a winter crop. In order to develop cold hardy oat
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cultivars, more knowledge about mechanisms of cold tolerance in oat is required (Bräutigam et al. 2005).
Livingston et al. (2005) reported that under freezing temperatures, most of the aerial plant tissue in oat is
destroyed and recovery from freezing is mainly dependent on surviving cells from the crown. In addition, the
close involvement of soluble carbohydrates in cold tolerance is well established (Livingston and Henson 1998)
and cold tolerance has been found to be correlated to the activity of antioxidative enzymes in oat plant (Liu et al.
2013, Kutlu and Turhan 2015). However, more detailed data are needed on isozymes and proteins to understand
and explain the mechanisms of cold-hardiness in oat. Changes in the qualitative activities of POX and proteins at
two durations of acclimation (12 and 24 hours) and non-stressed plants (control) in two oat cultivars were under
cold stress assessed in the present study. Changes associated with cold stress were analysed for a better
understanding of the general cultivar differences and for providing preliminary information for later, more
detailed, studies on oat cultivars.

MATERIALS AND METHODS

The seeds of the oat (Avena sativa L.) cultivars ('Faikbey'; relatively cold sensitive and 'Yeniçeri; relatively cold
tolerant') (Kutlu and Turhan 2015) were sown in perlite:torf:sand (1:1:1) media and germinated at 25/15 °C in a
controlled greenhouse. Plants were harvested after 21 days and half of the plants for 12 hours while the other
half was acclimated for 24 hours at 4 °C to low temperatures. Samples were exposed to low temperatures
according to the method of Arora et al. (1992) with some modifications. Briefly, leaves from each cultivar were
wrapped (10 leaves per temperature in each replication) in aluminium foil along with moistened paper and
placed in a manually controlled low-temperature freezer. The plant tissue temperature was monitored with a
copper-constant thermocouple (Testo 925, Omni Inst., Scotland, UK) inserted in the foil pouch. The temperature
was decreased stepwise at approximately 1.5ºC/h to 0ºC and 6ºC/h thereafter to a final value of -18ºC. The
samples were exposed to low temperatures of 0, -6, -12 and -18 ºC for 12 h. The samples were then removed
from the freezer at each temperature and placed at 4ºC overnight for slow thawing. The leaf material was
collected from each plant group at each temperature application step. Triplicate samples of leaf tissues were
frozen immediately put in liquid N and stored at −80 °C until used for POX isozymes and protein analysis. In
2
addition, plants that are unstressed used as control plants. POX was extracted from leaf tissues using the
extraction methods described by Gulen et al. (2002), with modifications as follows. Ground leaf tissues (0.1 g)
were homogenized at 4 °C in 0.6 mL extraction buffer (0.1M potassium phosphate pH 7.5, 30mM boric acid,
50mM L-ascorbic acid, 17mM sodium metabisulfite, 16mM diethyldithiocarbamic Acid (DIECA), 1mM
ethylenediaminetetraacetic acid (EDTA), and 2% (w/v) polyvinylpolypyrrolidone (PVPP); 4% (w/v) PVP-40
was omitted and the final pH was readjusted to 7.5 with sodium hydroxide). Homogenates were centrifuged at
21000g for 20 min and the supernatant was used for electrophoresis. Discontinuous polyacrylamide gel
electrophoresis (PAGE) was performed with a PROTEAN tetra vertical electrophoresis unit (Bio-Rad, Hercules,
CA) for acidic and basic POX, respectively, according to Davis (1964) and Reisfeld et al. (1962). Five percent
stacking gels and 10% separating gels were prepared for both systems. Equal volumes of the protein (30 µg)
were loaded for each sample. Electrophoresis was performed at 20 mA for 30 min, followed by 40 mA for 3 h.
Gels were stained for POX using the method of Wendel and Weeden (1989). The relative distance (Rf-value) of
the bands on the gel was calculated as described by Manganaris and Alston (1992) using Rf =1.0, distance to the
fastest band and Rf = 0.0, the starting point. Scanned isozymes profiles gels were analyzed by using the Public
Domain NIH Image program (available on the internet at http://rsb.info.NIH.gov/nih-image/ (verified 28 July
2008) ImageJ Software. Total soluble proteins (TPS) were extracted in the light of Shen et al. (2003) method
with some modifications. Exactly 0.25 g of each sample was homogenized in 1 mL homogenate buffer
containing 25 mM Tris-base (pH 7.8), 275 mM sucrose, 2 mM EDTA, 10 mM Dithiothreitol (DTT), 0.5 mM
Phenylmethylsulfonyl fluoride (PMSF), and 1% PVPP. The homogenate was transferred into an eppendorf tube
and then centrifuged at 10000 rpm for 10 min at 4°C. Protein content was measured using the Bradford assay
method (Bradford 1976). Discontinuous sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE) was performed with a PROTEAN tetra vertical electrophoresis unit (Bio-Rad, Hercules, CA, USA) using
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0.04 stacking gel and 0.125 separating gel. An equal amount of total protein (10 mg) was loaded for each sample
and gels were stained with Coomassie Brilliant Blue G-250.

RESULTS

Native PAGE analysis of the samples was repeated at least three times with similar results and data from a
single, representative analysis are presented herein. The results showed that there were changes in intensities and
number of isozyme bands of leaf tissues subjected to low temperatures for two durations of acclimation.
Analysis of acidic POX zymograms patterns, showed varied expression of isoforms in two oat cultivars studied,
the number of these isozymes varies from 1 to 4 depending on each cultivar studied. These isoforms, named
POX1-POX4 were expressed in both tolerant and sensitive cultivars but variations in the intensity of the bands
were observed among the cold treatments in comparison with the control (Figure 1 to Figure 2). We noticed, in
cv.'Faikbey', three isozymes of POX (Rf 0.14, POX1; Rf 0.27, POX2; Rf 0.88, POX4) in cv.'Yeniçeri' four
isozymes of POX (Rf 0.14, POX1; Rf 0.27, POX2; Rf 0.83, POX3; Rf 0.88, POX4) in both durations of
acclimation (Figure 1A, B).

Figure 1. Native PAGE of acidic POX activities of the leaf tissues of oat cultivars under low temperature treatments at two
durations of acclimation (12h: Panel A and 24 h: Panel B) and unstressed (Control:C) plants and their band intensities based
on the quantitative measurements. Equal volumes of the crude extracts, 20 µL, were loaded in each lane. POX1 (Rf 0.14),
POX2 (Rf 0.27), POX3 (Rf 0.83), POX4 (Rf 0.88).

In general POX activities were the highest level in control plants, whereas low temperatures
(particularly 4ºC) inhibited the POX activity in cv.'Faikbey' at both durations of acclimation. However the band
intensity generally increased with low temperature treatments in cv.'Yeniçeri' especially at 12h duration of
acclimation. Indeed POX band intensities of cv.'Yeniçeri' were higher than cv. 'Faikbey' in both durations of
acclimation (Fig.1A, B). Moreover, only relatively cold tolerant cultivar cv.'Yeniçeri' has a capacity to activate
POX3 under cold stress.

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Figure 2. Native PAGE of basic POX activities of the leaf tissues of oat cultivars under low temperature treatments at two
durations of acclimation (12h: Panel A and 24 h: Panel B) and unstressed (Control:C) plants and their band intensities based
on the quantitative measurements. Equal volumes of the crude extracts, 20 µL, were loaded in each lane. POX1 (Rf 0.04),
POX2 (Rf 0.36), POX3 (Rf 0.48), POX4 (Rf 0.63).

Four basic POX isozymes were detected at two durations of acclimation in both cultivars during cold
stress at (Figure 2A, B). The intensity of basic POX isoenzymes in cv.‘Faikbey’ generally increased, while the
intensity of four basic isozymes, POX1 (Rf 0.04), POX2 (Rf 0.36), POX3 (Rf 0.48) and POX4 (Rf 0.63)
decreased at 12h duration of acclimation in cv.'Yeniçeri' and was stronger in cv.‘Faikbey’ than in cv.‘Yeniçeri’.
The bands were observed commonly with different band intensities in all over the low temperature treatments at
two durations of acclimation. The intensities of the bands generally significantly increased especially at 0°C and
-6 °C treatments at 12h acclimation and at 0 °C at 24 h acclimation in both cultivars. Band intensities of basic
isoperoxidases of both cultivars showed some fluctuations (up and down) at two durations of acclimation under
cold stress (Figure 2A, B).
SDS-PAGE analyses of samples of leaf proteins were repeated at least three-times with similar results.
Data from a single, representative SDS-PAGE profile are presented (Figure 3A, B). According to the SDS-
PAGE profiles, at 12 h acclimation stage, four protein bands estimated at 9, 15, 18 and 26 kDa were considered
in cv. 'Faikbey'. 13, 15, 34, 72 and 88 kDa size of bands have been observed in all cold treatments in
cv.'Yeniçeri' (Figure 3A). While, the intensities of 9, 15 and 26 kDa size of bands decreased at -18 °C, 18 kDa
protein band disappeared at 0 C° in cv.'Faikbey' at 12 h acclimation stage. The 94 kDa protein band was
observed from the control to -18 ºC in cv.'Faikbey at 24 h acclimation stage'. However, the 94 kDa protein band
disappeared after 4 °C treatment in cv.'Yeniçeri'. New protein bands with 13, 15, 18, 20, 26, 28, 36, 40, 94 kDa
appeared and with 9 kDa band disappear in cv. 'Faikbey' at 24 h acclimated stage. However generally all protein
bands disappeared except 13 kDa detected at 12 h acclimated stage and new protein bands appeared with 84 and
94 kDa protein bands in cv.'Yeniçeri' (Figure 3B).

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Figure 3. Total soluble protein (TSP) profiles (SDS-PAGE) of the leaf tissues of oat cultivars under low temperature
treatment at 12h (Panel A) and 24 h (Panel B) durations of acclimation and unstressed (control) plants. 10 µg of protein was
loaded on each lane. Molecular weight markers (M) and their molecular masses (kDa) are shown on the left side of each
Panel.

DISCUSSION

Plant peroxidases are important in diverse cellular functions such as lignin biosynthesis, hormone generation,
and detoxification of hydrogen peroxide (Conroy et al. 1982). Plant peroxidases are usually classified as acidic,
neutral, or basic, according to their isoelectric points (Yoshida et al. 2003). Each group is thought to have a
different function in the cell. Acidic peroxidases are the isoenzymes most likely involved in lignin formation and
wall-associated, whereas function of basic isoenzymes has been suggested that they might provide H O for
2 2
other peroxidases (Walter 1992). In the present study, data from native PAGE indicated three and four asidic
isoperoxidases with different intensities in cv. 'Faikbey' and in cv.'Yeniçeri', respectively at both durations of
acclimation under cold stress (Figure 1, 2). The increased POX activities under low temperature in oat were
linked with expression of isoform POX3 only in relatively cold tolerant cultivar. It seems that the cold tolerant
cultivar display a better overall POX activity. In addition, four basic isoperoxidases stated at two durations of
acclimation in both cultivars with different intensities. Since data indicated a linear relationship between band
intensities and the low temperature treatment, it is correlated to cold-acclimation of oat leaf tissues. On the other
hand, the data indicated that expression of isozymes in response to cold stress depends on the temperatures and is
higher in tolerant cultivar than in sensible one. Similar results also obtained in Medicago under cold stress
(Nourredine et al. 2015). In addition Liu et al. (2013) reported that POX activities in naked oats (Avena nuda L.)
were higher under low temperature than normal temperature. Kuk et al. (2003) showed, in rice plants, that
isozyme profile and activity of peroxidase were significantly expressed under cold stress and deduced that
peroxidases were most important for cold acclimation and chilling tolerance. Cansev et al. (2005) reported
expression of acidic peroxidase bands with different band intensities which are responsible for tolerance to cold
stress of 15 olive cultivars. Gulen et al. (2008) also pointed out that one acidic (Rf=0.23) and one basic
(Rf=0.17) POX bands were observed commonly in cold subjected strawberry plants with different band
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intensities, and, they suggested that it may be associated with cold-acclimation in strawberry plants under low
temperature stress.
The effects of low temperature stress on protein profile on both cultivars were different. Protein banding
patterns of cv.'Faikbey' were between 9 kDa and 26 kDa at 12 h duration of acclimation and between 15 kDa and
94 kDa at 24 h duration of acclimation with reference to the marker (Figure 3). As shown in Figure 3, protein
banding patterns of cv. 'Yeniçeri' were between13 kDa and 88 kDa at 12 h duration of acclimation and between
13 kDa and 94 kDa at 24 h duration of acclimation with reference to the marker under low temperature stress.
Different band positions between cv.'Faikbey' and cv.'Yeniçeri' indicated their specific response towards the
given low temperature stress and also it reflects their different genetic variation. The banding intensity of
different polypeptide bands (15kDa) of cv.'Yeniçeri' was higher than cv.'Faikbey' under low temperature
treatment. It was founded that protein banding patterns of rice plant between 30 kDa and 15 kDa with reference
to the marker under low temperature stress (Perveen et al. 2013).
In conclusion, our data indicated that acidic POX3 isoform (Rf 0.83) can be associated with cold-
acclimation in oat plants under cold stress. In addition we supposed TSP may responsible to cold tolerance in oat
cultivars. Differences in the TSP contents of leaf tissues occurred between the duration of acclimation. The 84
kDa leaf proteins appeared that to be related to cold-acclimation in oat. Further research is in progress to
characterise these proteins and to immunolocalise them.

REFERENCES

Arora R, Wisniewski ME, Scorza R (1992). Cold acclimation in genetically related (sibling) deciduous and evergreen peach (Prunus persica
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