Arahidopsis CHLOROPHYLLASE 1 protects young leaves from long-term photodamage by facilitating FtsH-mediated D1 degradation in photosystemⅡrepair

The proteolytic degradation of the photodamaged D1 core subunit during the photosystemⅡ(PSⅡ)repair cycle is well understood,but chlorophyll turnover during D1 degradation remains unclear.Here,we report that Arabidopsis thaliana CHLOROPHYLLASE 1(CLH1)plays important roles in the PSII repair process.The abundance of CLH1 and CLH2 peaks in young leaves and is induced by high-light exposure.Seedlings of clh1 single and clh1-1/2-2 double mutants display increased photoinhibition after long-term high-light exposure,whereas seedlings overexpressing CLH1 have enhanced light tolerance compared with the wild type.CLH1 is localized in the developing chloroplasts of young leaves and associates with the PSⅡ-dismantling complexes RCC1 and RC47,with a preference for the latter upon exposure to high light.Furthermore,degradation of damaged D1 protein is retarded in young clh1-1/2-2 leaves after 18-h highlight exposure but is rescued by the addition of recombinant CLH1 in vitro.Moreover,overexpression of CLH1 in a variegated mutant(var2~2)that lacks thylakoid protease FtsH2,with which CLH1 interacts,suppresses the variegation and restores D1 degradation.A var2-2 clh1-1/2-2triple mutant shows more severe variegation and seedling death.Taken together,these results establish CLH1 as a long-sought chlorophyll dephytylation enzyme that is involved in PSⅡrepair and functions in long-term adaptation of young leaves to high-light exposure by facilitating FtsH-mediated D1 degradation.

AtCLH2, a Typical but Possibly Distinctive Chlorophyllase Gene in Arabidopsis

Chlorophyllase （EC 3.1.1.14） is involved in the first step of chlorophyll degradation. Isolation of chlorophyllase genes greatly facilitates characterization of chlorophyllase properties and elucidation of molecular regulation of their in vivo activities. There are two chlorophyllase genes, AtCLH1 and AtCLH2, in Arabidopsis thaliana. The in vivo roles of AtCLH1 have been reported previously. However, few studies have been carried out on AtCLH2. Here, we show that purified recombinant Chlase2, encoded by AtCLH2, exhibits in vitro chlorophyllase activity. Interestingly, ＂activation＂ of in vitro activity of the recombinant Chlase2 required higher concentrations of a detergent or a polar solvent. To determine its activity in vivo, the expression of AtCLH2 was inhibited by RNA interference. RNAi plants showed decreased contents of chlorophyllide without a substantial change in the total amount of the extractable chlorophyll and consequently presented lower chlorophyllide to chlorophyll ratios in their leaves. In addition, the two AtCLHs exhibited differential expression patterns. Our results suggest that AtCLH2 might play a distinctive role in chlorophyll catabolism in vivo.

Research on the Construction and Function of a Chlorophyll Degradation Recombinant Engineering Strain

In order to effectively reduce the chlorophyll content in flue-cured tobacco, improve the overall quality of tobacco leaves, chlorophyllase gene was cloned from Arabidopsis thaliana. After the expression of the expression vector in E. coil, the recombinant engineering strain was obtained. Afterwards, IPTG （isopropy-β-D-thiogalactopyranoside）was used to induce the goal protein, and the chlorophyllase activity of the recombinant engineering strain was measured, so as to investigate its degradation effect on the chlorophyll in the extracts of tobacco leaves. The results were as follows： （1） the amplified chlorophyllase gene At- CLH1 constructed the expression vector pET28a-AtCLH1 successfully, obtaining the recombinant engineering strain; （2） induced under 30 ℃ for 22 h, the strain could well express the recombinant protein AtCLH1 with 0.5 mmol/L IPTG, and the molecular weight was about 35 kDa; （3） the strain showed good chlorophyllase producing capability, and the activity of the produced chlorophyllase could reach up to 24.9 U/mL, which could degrade the chlorophyll in tobacco extract and had a good application prospect in improving the quality of low quality tobacco; （4） based on the results of orthogonal test, the enzyme extract from the strain was added to the tobacco leaf surface, which could make the degradation rate of chlorophyll in the tobacco leaf reach 17.06% under the temperature of 37 ℃ at the humidity of 75% for 48 h; （5） after treated by the enzyme liquid, the test tobacco showed increase in the content of aromatic substances, enhancement of tobacco fragrance quality and amount, significant decrease of offensive odor and irritation, significant improvement of agreeable aftertaste, making the overall sensory quality of the tobacco leaf significantly improved.