The pattern of CBF and P5CS genes expression under freezing stress and effects of cold acclimation on free proline changes in chickpea (Cicer arietinum)

IntroductionCold and freezing stress is one of the most important environmental stresses that affect the growth and yield of crops. One of the important processes in plant resistance to cold stress is known to acclimatize. In this process, the internal mechanisms of plants causes higher resistance to freezing stress after exposure to temperatures close to zero. Low temperature stress induce or inhibit the expression of certain genes. P5CS protein and transcription factor DREB (CBF) play an important role in adaptation and cold tolerance in plants. P5CS activates the enzymes in proline synthesis pathway and cause accumulation of this substance in plant that protects membranes and proteins against different effects of high concentrations of inorganic ions and high and low temperatures. Also DREB proteins with a conserved sequence bind to specific DNA sequences upstream of responsible genes in resistance to cold stress and leads to their expression. In this study the expression of P5CS and (DREB1)CBF genes that have an important role in the face of many plants to freezing and cold stress were examined in chickpea in both resistant and susceptible genotypes and used of Real time PCR method to evaluate the expression of these genes under stress and non-stress.
Materials & MethodsIn the first experiment, the expression pattern of P5CS and CBF (DREB1) genes were evaluated at normal (23°C), acclimation (10°C) and freezing (-10°C)temperatures in two resistant (MCC426) and susceptible (MCC505) genotypes of chickpea, in two organs (leaf and stem). Briefly pots containing plants of both genotypes stored at 23°C for two weeks. After this period, plants are divided into two parts, half remained in the normal conditions and the other half were transferred to 10°C for cold acclimation. After 10 days, seedlings of normal and acclimation conditions transferred to a thermogradient freezer and after 15 minutes at -10°C, sampling was carried out from the leaves and stems. The simultaneous sampling of the control plants (normal conditions) were also done. After RNA extracting from samples and determine the concentration and quality by nanodrop and treatment with DNase I enzyme, cDNA synthesis was performed. After designing appropriate primers, the PCR process was optimized and Real time PCR was performed under optimized process conditions and gene expression levels were calculated. In the Second experiment free proline changes of seedlings in the acclimation phase were evaluated. Briefly after transferring of plants in the acclimation conditions, amount of proline at them were measured at appropriate intervals after exposure in acclimation conditions.
Results & DiscussionResults of analysis showed that the expression of these genes were significantly higher in resistant genotype than the susceptible one. Because of these genes are the important factors in plant resistance to stress especially cold and freezing stress, this can confirm that MCC426 genotype is resistant and MCC505 genotype is sensitive to cold. Also the interaction of genotype and organ sample in the control condition showed significant difference only for expression of P5CS gene. Since this gene is an important gene in freezing and cold stress, increased expression of this gene in MCC426 genotype that is resistant to cold not unexpected. But expression of CBF gene occurred only after cold stress induction and in the absence of stress is poor expression. In addition, it was revealed that the interaction of genotype and acclimation treatment on CBF genes in both genotypes had significant differences, but in P5CS gene only in MCC426, this trend was observed. However, changes of free proline as one of the mechanisms of cell protection in acclimation period in both genotypes showed that proline contents of MCC426 in response to acclimation treatment was gradually increased, but this increase in MCC505 genotype occurred during the early stages of acclimation, and proline has decreased over time.
ConclusionAccording to the results, it generally seems, one of the main reason for tolerance to chilling is the high expression levels of the mentioned genes during chilling and freezing stress and therefore it can be used for inducing freezing tolerance into sensitive plants.