Influences Of AZI1 On Cold Resistance And Lignin Synthesis Of Arabidopsis Thaliana

Alignment of the deduced amino acid sequences and the nucleotide sequences by Clustal W algorithm showed that AZI1 (AZELAIC ACID INDUCED1) possessed high similarity with other genes of EARLI1 subfamily. AZI1 contains conservative PRD (proline-rich domain) and 8CM (eight-cysteine-motif).Promoter analysis in the PLACE database showed the cis-acting element CCGAC involved in response to low temperature existed in upstream regulation sequences of AZI1, indicating AZI1 might be related to cold responsiveness of Arabidopsis thaliana. The results of RNA gel blot analysis demonstrated that AZI1 could be activated by cold environment indeed, but more than 6h treatment at 4℃was required to detect an increase in mRNA abundance. Along with the elongation of treatment time, mRNA of AZI1 increased slowly and peaked after 24h. However, the high expression state could not be maintained stably and would decline to basal level when the plants were transferred to room temperature.In order to clarify the function of AZI1 in resistance to low temperature, overexpressing, RNA interference and T-DNA knockout lines of this gene were used in electrolyte leakage assays. Overexpression of AZ11 resulted in reduced electrolyte leakage during freezing damage. In contrast, AZI1 knockdown and knockout lines showed increased tendencies in cellular damage after freezing treatment. To further validate the potential resistance of AZI1 to low-temperature stress, Saccharomyces cerevisiae cells were transformed with pESC-AZI1 in which AZI1 was under the control of GAL1 promoter. Compared to yeast cells containing empty pESC-URA, the survival rate of yeast cells harboring AZI1 increased obviously after freezing treatment. All these results suggested that AZI1 was related to cold tolerance of Arabidopsis.Phenotype observation exhibited that AZI1 overexpressing plants flowered later than the wild-type plants, while the T-DNA knockout mutants flowered one week earlier in comparison with the wild-type control. Statistical analysis after 40 days of growth revealed the bolting rate of FLAG 327H06-3 homozygous AZI1 mutant was more than twice compared to wild-type Ws and the bolting rate of SALK 017709-1 homozygous AZI1 mutant was 1.5 times of wild-type Col-0. Especially, when the bolting rate of RNAi lines reached 100%, the wild-type late flowering Col-FRI-Sf2 plants was still lingered in vegetative growth stage. Besides, the amount of the total lignin in AZI1 mutants decreased compared to the wild-type control. It suggested that AZI1 may also affect the flowering time and the lingin accumulation directly or indirectly except its tolerance to low temperature.