The N-Terminal Domain of Chlorophyllide a Oxygenase Confers Protein Instability in Response to Chlorophyll b Accumulation in Arabidopsis

Abstract

Plants acclimate to variations in light intensity by changing the antenna size of photosystems. This acclimation allows them to undergo efficient photosynthesis and creates a protective strategy to minimize photodamage. Chlorophyll b synthesis by chlorophyllide a oxygenase (CAO) is a key regulatory step in the control of antenna size. Recently, we found that higher plant CAOs consist of three domains (A, B, and C domains) and confirmed that the C domain possesses catalytic function. To investigate the function of the A domain, we fused various combinations of these three domains with green fluorescent protein (GFP) and introduced them into Arabidopsis thaliana. When a full-length CAO-GFP fusion protein was introduced into a chlorophyll b-less chlorina1-1 mutant, chlorophyll b accumulated to almost the same levels as in the chlorophyll b-containing Columbia wild type, but the CAO-GFP could not be detected by immunoblotting. By contrast, when a GFP-C domain fusion was introduced into chlorina1-1 or Columbia wild type, a large amount of GFP-C domain protein accumulated and the chlorophyll a/b ratio decreased drastically from 3.6 to 2.2 in Columbia wild type. When an A domain-GFP was introduced into Columbia wild type, A domain-GFP levels were very low. Conversely, a large amount of the protein accumulated when it was introduced into the chlorina1-1 mutant. These results indicate that the A domain may sense the presence of chlorophyll b and regulate the accumulation of CAO protein in the chloroplasts.

Chlorophyll b degradation by chlorophyll b reductase under high-light conditions

“…Feedback regulation of chlorophyll b synthesis by CAO has also been reported (Yamasato et al 2005). CAO is degraded in the presence of its product, chlorophyll b.…”

Functional analysis of N-terminal domains of Arabidopsis chlorophyllide a oxygenase

“…These reports suggest that the A domain is involved in the regulation of the CAO protein levels. Interestingly, this regulatory mechanism did not operate when the chlorophyll-b synthesizing activity was deficient [37]. This observation indicates that the level of CAO protein is regulated according to the amount of chlorophyll b.…”

“…In order to overexpress CAO-GFP fusion proteins, we modified the plasmid vector that was constructed in our previous study [37]. The vector contained the CaMV promoters, the tobacco mosaic virus ω sequence, the GFP (S65T) sequence (kindly provided by Dr. Niwa, University of Shizuoka) and the nopaline terminator in the backbone of pGreenII-0029 (kindly provided from R.P.…”

“…Effects of B domain on the accumulation of the full-length CAO protein: Comparison GFP-ABC and GFP-AC overexpressors Since we focused on the analysis of the function of A domain in our previous study [37], we examined all possible combinations of CAO domains ( Figure 2) with an emphasis on the function of the B domain in this study. At first, in order to examine the effects of B domain on the accumulation of the full-length CAO protein, we compared the phenotypes of GFP-ABC and GFP-AC overexpressors.…”

“…It has been demonstrated that C domain alone is sufficient to catalyze the conversion of chlorophyll a to b [24]. The deletion of the A domain drastically increased the CAO protein level and decreased the chlorophyll a/b ratio in transgenic Arabidopsis plants [37]. Similarly, when a PhCAO sequence which only contains C domain was overexpressed in Arabidopsis thaliana, the chlorophyll a/b ratio drastically reduced [16].…”