Identification of A Novel bZIP Transcription Factor in Camellia sinensis as A Negative Regulator of Freezing Tolerance in Transgenic Arabidopsis

Basic region/leucine zipper (bZIP) transcription factors play vital roles in the abiotic stress response of plants. However, little is known about the function of bZIP genes in Camellia sinensis. Here, we show that CsbZIP6 is induced during cold acclimation in tea plant. Constitutive overexpression of CsbZIP6 in Arabidopsis lowered the plants’ tolerance to freezing stress and ABA exposure during seedling growth. Compared to wildtype (WT) plants, CsbZIP6 overexpression (OE) lines exhibited increased levels of electrolyte leakage (EL) and malondialdehyde (MDA) contents and reduced levels of total soluble sugars (TSS) under cold stress conditions. Microarray analysis of transgenic Arabidopsis revealed that many differentially expressed genes (DEGs) between OE lines and WT plants could be mapped to ‘response to cold’ and ‘response to water deprivation’ terms based on GO analysis. Interestingly, CsbZIP6 overexpression repressed most of the cold- and drought-responsive genes as well as the starch metabolism under cold stress conditions. Taken together, our data suggests that CsbZIP6 functions as a negative regulator of the cold stress response in Arabidopsis thaliana, potentially by down-regulating cold-responsive genes. To obtain insights into the molecular mechanisms by which CsbZIP6 mediates senstivity to cold stress in Arabidopsis plants, gene expression profiles in leaves of two CsbZIP6 OE lines and WT plants under normal (22ºC) and cold (4ºC) conditions were compared. The Agilent Arabidopsis Gene Expression (4×44K, Design ID: 021169) was used in this experiment. Arabidopsis seedlings were germinated, grown on 1/2 MS for 2 weeks and then transferred to soil. Thirty-day-old plants (WT, OE-1, OE-2) were grown at 4 oC (‘cold’) or normal conditions (‘normal’) for 4 days before the leaves were sampled, snap frozen in liquid nitrogen and stored at -80ºC until RNA isolation. Each replicate was collected from one seedling. Three biological replicates were used for the analysis except for the ‘normal’ OE-2 samples which lack a replicate because of substandard RNA quality. In total, 17 arrays were used in the study.