Genetic And Cell Biological Analysis Of Plant Ca²⁺ Signaling In Response To Low Temperature Stress

Low temperature is an important environmental factor influencing plant growth and development,and it often extensively affects crop quality and productivity.During the past few years,many intermediates in the cold signal transduction pathway have been identified in Arabidopsis and considerable progress in genetic research has been made in dissecting their functional significance.It has been demonstrated that the earliest reported event in plant responses to low temperature is a transient increase in the concentration of cytosolic free calcium.Despite the fact that many genes are being identified at a rapid pace,the relationships between those genes and the Ca²⁺ are not clear.To address the relationship between those genes and Ca²⁺,we transformed the atfry1,atlos1, atlos5,atice1,atcbl1,athos1,atcipk15,atcbl9-Imutants and CBF1,CBF2,CBF3 overexpression lines with the plasmid of pMAQ2 and then measured cold-triggered calcium responses.The results demonstrated that cold-triggered calcium responses in athos1,atcbl9-1 mutants and CBF1 overexpression lines were higher than those in control,but were similar in atfry1,atlos1,atlos5, atcbl1,ateipk15,atcbl1,CBF2,CBF3 mutants.These findings could help us define the intermediates of Ca²⁺-mediated signal transduction into nucleus and their spatial and temporal relationships in cold signaling.However,at present,little is known about the function of AtCBL9 in cold stress signaling in plants.Here,we report the function of AtCBL9 response to low temperature.Under normal growth conditions,atebl9-1 plants grow like wild-type plants,but chilling injury became apparent after 4℃treatment.The atcbl9-1 leaves showed little increase in electrolyte leakage than the wild type during chilling treatment.The atcbl9-1 mutant plants have shown substantially more tolerant to freezing than wild-type plants under both cold-acclimating and nonacclimating conditions.These results from these studies indicated that atcbl9-1 is more tolerance to cold.It has been identified that an increase in intracellular calcium can activate the expression of transcription factors that control the subsequent transcription of many genes which must be switched on to provide cold tolerance in the plant.We generated transgenic Arabidopsis plants stably transformed with the plasmids of pMAQ2 and HVA1-2.2.Then,we measured cold-triggered calcium responses in atcbl9-1 and wild-type plants.The atcbl9-1 mutant plants exhibited a higher mean peak[Ca²⁺]_c than the wild-type plants,but the calcium signature adjacent to the vacuolar membrane rose to similar levels.When Arabidopsis seedlings were pretreated with EGTA or LaCl₃, the mean peak values of wild-type and mutant plants descend to similar levels.LiCl which prevents calcium release from intracellular pools has little effect on the decreased extent.These results may indicate the main involvement of extracellular calcium in this response.The N-terminal sequence of AtCBL9 harbours a conserved myristoylation motif suggesting a membrane localization of the proteins.The expression pattern at the subcellular level using GFP-AtCBL9 transgenic plants shows the localization of AtCBL9 on the plasma membrane.We speculated that AtCBL9 sensor cold signal in the membrane and regulate the expression of genes whose products are required for stress tolerance.In the atcbl9-1 mutants,transcript levels of CBF2, RD29A,KIN1 and COR15A were substantially higher than in the wild-type.Taken together,as a Ca²⁺ sensor in the membrane AtCBL9 participates in the cold signal transduction and acts to negatively control the expression of the COR genes.