بررسی روند تغییرات عوامل فلورسانس کلروفیل در طی تنش یخ‌زدگی در دو توده باقلا (Vicia faba L.)

Introduction
Freezing tolerance is an important factor determining natural geographic distribution of plant species as well as growth and yield of many crop plants. Many plants from temperate and cold climates, including several important crop species, are able to increase their freezing tolerance in response to low, nonfreezing temperatures in a process termed cold acclimation. It is generally accepted that cellular membranes are the primary targets of freezing damage in plants. So, besides whole plant survival, determination of electrolyte leakage from plant tissues after freezing and thawing, using conductivity measurements has been the most frequently used method reported in the literatures. This method mainly monitors the ability of the plasma membrane to function as a semi-permeable barrier towards intracellular ions, but the intactness of the vacuole, as the major storage compartment for inorganic ions, may also impact the measurements. Under stress conditions, the possibility of over excitation of photosystem II (PSII) increases, and this reduces the photosynthetic rate and lead to an increase in the dissipation of absorbed energy through nonradiative processes. The measurement of chlorophyll fluorescence has been used for many years as a sensitive, reliable, and rapid method to determine the effect of environmental stresses, like drought, temperature, excessive light and air pollution on green plants. Decrease of the maximal rate of the fast rise of fluorescence after exposure of leaves to chilling temperatures, correlated well with the visual symptoms of chilling injury in several species
 
Materials & Methods
 This study was carried out to measure chlorophyll fluorescence and determine the survival of two Vicia faba ecotypes (Borujerd and Neyshabur) in the fall and winter of 2015 at Research Center for Plant Sciences, Ferdowsi University of Mashhad. Treatments were arranged as factorial based on completely randomized block design with four replications. Plants were grown in pots under natural conditions until four to six leaf stage for acclimation, and then were subjected to freezing temperatures (0, -4, -8, -12, -16, -20, -24 °C) in a thermo gradient freezer. Freezer temperature was 5 °C at the beginning and reached to -24 °C, decreasing two degrees centigrade per hour. Ice nucleation active bacteria was sprayed to nucleation formation in plants at -3°C. Plants were kept for an hour in each temperature and then were transported to a cold chamber (5±2 °C) and kept for 24 hours to avoid rapid melting. Chlorophyll fluorescence was recorded with a pulse amplitude modulation fluorometer (PAM-2000, Walz, Effeltrich, Germany) before and 3, 6, 12, 24, 48 and 72 hour after freezing stress. The efficiency of excitation energy capture by open PSII reaction centers (F´v/F´m) and the quantum yield of electron transport at photosystem II (PSII), were determined. Survival percentage was determined after three weeks recovery in greenhouse condition. Data were analyzed using a two-way ANOVA model, followed by Duncanʼs test for mean comparison at 95 % confidence level by Minitab 16 program.
 
Results & Discussion
Result indicated that maximum quantum yield of PSII photochemistry (F'v/F'm) was four percent higher in Borujerd ecotype compared to Neyshabur ecotype. No significant change was found in chlorophyll fluorescence parameters with decreasing temperature from zero to -12°C but more temperature reduction caused chlorophyll fluorescence parameters to decrease in a way that the lowest mean was observed at -24°C. Rapid reductions were found in chlorophyll fluorescence parameters such as F'v/F'm until 24 hours after freezing stress which was followed by gently slope increasing trend, but the photochemical efficiency of photosystem II, 72 hours after stress, did not return to the level before freezing stress. Survival rate was decreased as freezing temperature decreased and reached to 83% at -12°C. More temperature reduction to -16°C led to sever decrease in survival rate in a way that no plant survival and only five percent was observed in Borujerd and Neyshabur ecotypes, respectively. High regression coefficients were found between F'v/F'm and survival rate in both ecotypes (R2=0.99 and R2=0.98 for Borujerd and Neyshabur ecotypes, respectively)
 
Conclusion
Generally, evaluation of chlorophyll fluorescence parameters of both Vicia faba ecotypes showed that chlorophyll fluorescence has a direct relationship with survival three weeks after freezing stress and can be used as an index for assessment of freezing tolerance