Mechanism Of Arabidopsis ADF5 In Response To Cold Stress

Low temperature is a major adverse environment factor that limits the normal plant growth.Extreme low temperature often leads to a large area of crop yield reduction.Therefore,it has important theoretical significance and practical application value to study the molecular cytological basis of plant adaptation to low temperature environment.Previous studies had observed that low temperature can cause the rearrangement of plant actin cytoskeleton,suggesting that actin cytoskeleton may play an important role in plant response to cold stress.However,the regulatory mechanism of plant actin cytoskeleton and its dynamic regulatory by actin-binding proteins(ABPs)in cold stress is still unclear.In addition,cold acclimation can make plants obtain better freezing resistance.C-repeat binding factors(CBFs)play a key role in this process,but whether they participate in the regulation of actin cytoskeletal remodeling has not been reported.Actin-depolymerizing factor(ADF)is a class of conserved actin cytoskeleton severing/depolymerizing proteins in eukaryotes.Our previous study found that ADF5 and ADF9,members of the ADF subfamily III in Arabidopsis thaliana,have evolved new functions of F-actin bundling function.However,the biological significance of their new functions is still unclear.This study explored the physiological function and molecular regulation mechanism of ADF5 in Arabidopsis thaliana in response to cold stress.The mainly results are listed as followed.1.ADF5 improves basic and acquired freezing resistance in Arabidopsis.The results showed that the expression of ADF5 was up-regulated by low temperature,and the loss of ADF5 function of Arabidopsis led to the sensitivity to freezing in normal growth state and cold acclimation conditions,which indicates that ADF5 improves the cold adaptation ability of Arabidopsis.2.ADF5 is the downstream target gene of CBFs.Through q RT-PCR analysis of cold induced expression of ADF5 in cbf3,cbf1 cbf3 and cbf1 cbf2 cbf3(cbfs)mutants,it was found that the induced expression of ADF5 was inhibited in the mutants,and the expression of ADF5 was greatly inhibited in the cbfs.The results showed that CBFs could bind to ADF5 promoter relying on CBFs recognition sites(CCGAC).It was also found that CBF3 could activate ADF5 promoter activity through CBFs recognition sites and bind ADF5 promoter in vivo,and this binding was regulated by low temperature.3.ADF5 positively regulates low-temperature adaptation of Arabidopsis by partially relying on the CBF pathway.Under non-cold acclimation conditions(NA),the adf5 cbfs quadruple mutant mainly showed the freezing sensitivity of adf5 and the ion leakage of adf5 cbfs was higher than that of adf5 or cbfs.Under cold acclimation conditions(CA),the adf5 cbfs mainly showed the freezing sensitivity of cbfs,the ion leakage of adf5 cbfs mainly showed the characteristics of cbfs.4.Low temperature disturbs the actin cytoskeleton in adf5 and cbfs.By observing actin cytoskeleton in vivo,the results showed that the actin cytoskeleton of WT remained stable during cold acclimation,but the actin cytoskeleton of all mutants was disturbed to varying degrees.The adf5 cbfs quadruple mutant was the most severe.It suggested that non-CBF signaling pathway may regulate ADF5 in this process,which is consistent with q RT-PCR and physiological data.5.The endocytosis rate of adf5 and cbfs mutants decreases significantly during cold acclimation compared with WT.The results showed that low temperature reduced the rate of endocytosis in plant cells.In normal growth,the endocytosis rate of adf5-1,cbfs-1 and adf5 cbfs was faster than WT,but in the cold acclimation,the decreased endocytosis rate of adf5-1,cbfs-1 and adf5 cbfs was greater than WT.These results suggested that the endocytosis rate of the mutants was inhibited during cold acclimation compared with WT,which may damage the cold acclimation effect of the mutants.6.ADF5 may be a negative regulator of CBF signaling pathway.In this study,we found that ADF5 interacted with FVE,a component of histone deacetylase complex.The results showed that the expression levels of CBF1,CBF2 and CBF3 in adf5-1 were increased,and CORs(COR47 and KIN1)as CBFs downstream genes were up-regulated in varying degrees compared with WT.The expression patterns of these genes in adf5-1 were similar to those in acg1 as a mutant of fve,suggesting that ADF5 and FVE may regulate the expression of these genes together for negative feedback regulation in CBF signaling pathway.7.The ADF subfamily I negatively regulate the freezing resistance of Arabidopsis.The results showed that the expression of ADF4 was inhibited by low temperature and the freezing resistance of adf4 and ADF1-4 Ri increased compared with WT under normal growth and cold acclimation conditions.In addition,cold acclimation increased markedly the freezing resistance of ADF1-4 Ri compared with adf4.In conclusion,this study revealed that the newly functionalized ADF5 is downstream of CBF signaling pathway,and participates in plant response and resistance to cold stress by regulating the dynamics of actin cytoskeleton.This study will provide a new theoretical basis for elucidating the regulation mechanism of plant actin cytoskeleton and the molecular mechanism of plant resistance to abiotic stress.