Effects Of Abscisic Acid Treatment On Cold Resistance Of Kiwifruit And Functional Identification Of Transcription Factor AchnABF1

Low temperature storage(LTS)is one of the most commonly used methods to prolong the shelf life of postharvest products.However,chilling injury(CI)caused by low temperature limited the healthy development of postharvest industry.’Hongyang’(Actinidia chinensis cv.Hongyang)is a typical cold-sensitive kiwifruit cultivar,which is prone to CI during LTS.In this study,postharvest ’Hongyang’ fruit was treated with abscisic acid(ABA)at different concentrations and then transferred to cold storage.On the one hand,the effects of exogenous ABA treatment at different concentrations on CI of ’Hongyang’ was elucidated.On the other hand,key cold-responsive transcription factor family genes were identified through transcriptome sequencing.The main research contents and results are described as follows:(1)The dynamic CI evaluation was conducted during storage,including CI phenotype observation,lignified cell microstructure analysis,lignin content determination,lignin synthase enzymes activity determination as well as gene expression quantification.The results showed that there is dose effect on controlling lignification.For example,50 mg/L ABA treatment increased phenylalanine ammonialyase(PAL),cinnamyl alcohol dehydrogenase(CAD)and peroxidase(POD)activity,which are crucial for lignin synthesis,and sharply upregulated expression of the Ac PAL,Ac CAD and Ac POD genes.However,20 mg/L ABA alleviated black patch symptoms,increased lignin content in the peel,and did not negatively affect fruit quality.Furthermore,microscope observations showed that 20 mg/L ABA decreased the number of lignin cells with a diameter of 40-80 μm,thus reducing the chance of lignification in the pulp.(2)In this study,we identified 81 bZIP family proteins in kiwifruit and classified them into 11 groups,which correspond to the A-I,K and S subgroups of Arabidopsis.Further transcriptome analysis revealed that the expression of members of the AREB/ABF family was strongly induced by low temperature and ABA.The phylogenetic analysis revealed that the six AchnABFs were conserved in protein sequence and motif,had consistent expression characteristics induced by low temperatures.Their expression profile response to ABA was detected by qRT-PCR.It is speculated that the sustained high expression of AchnABF1 response to ABA during storage may have positive significance in regulating the chilling tolerance of ’Hongyang’ fruit.(3)Over-expression of AchnABF1 gene enhanced the resistance of transgenic Arabidopsis thaliana plants to abiotic stress at germination stage,post-germination stage and seedling development stage.The transgenic lines(OE)showed more radicle elongation,higher survival rate,less ion leakage and malondialdehyde(MDA)content than the wild type(WT)plants under osmotic stress,such as Na Cl,mannitol,cold acclimation cold and non-cold acclimation stress conditions.Some of the stress response gene(RD29A,COR15 A,COR47,KIN1,LTI29,CBF1 and CBF2)expressions were induced.Specifically,the germination rate of OE lines showed higher ABA sensitivity than that of the WT.The multiple stresses tolerance assay showed that the OE lines may improve the abiotic stress resistance,resulting in less ROS accumulation and membrane lipid peroxide damage under osmotic stress.(4)Over-expressing AchnABF1 gene in Micro Tom tomato(Lycopersicon esculentum Mill.)increased chilling tolerance of tomato fruit.The evidence from microscopic and ultrastructure observation proved that the symptoms of CI in transgenic tomato were light.The OE lines inhibited the damages of cell structure,plasma membrane and the chloroplast;However,the CI symptoms of WT fruit were serious.The cell structure and plasma membrane were damaged,the organelle was scattered.The chloroplast became round and expanded.The capsule was broken,and the starch grains inside the chloroplast were scattered and swelled with water absorption.Meanwhile,starch content and soluble sugar content analysis proved that AchnABF1 gene promoted the starch hydrolysis,increased soluble sugar content in cells,maintained the cell osmotic potential,enhanced water retention,thereby enhanced the cold resistance of transgenic tomato fruit.