The Arabidopsis-accelerated cell death Gene ACD1 is Involved in Oxygenation of Pheophorbide a: Inhibition of the Pheophorbide a Oxygenase Activity does not Lead to the “Stay-Green” Phenotype in Arabidopsis

Abstract

Oxygenation of pheophorbide a is a key step in chlorophyll breakdown. Several biochemical studies have implicated that this step was catalyzed by an iron-containing and ferredoxin-dependent monooxygenase, pheophorbide a oxygenase (PaO). It has been proposed that inhibition of its activity arrests the chlorophyll breakdown and leads to the "stay-green" phenotype. We searched the Arabidopsis genome for a possible PaO-encoding gene and hypothesized that it has homology to known iron-containing Rieske-type monooxygenase sequences. We identified three such open reading frames, Tic55, ACD1 and ACD1-like. We produced transgenic Arabidopsis plants which expressed antisense RNA as a method to inhibit the expression of these genes. The appearance of these antisense plants were indistinguishable from that of the wild type under illumination. However, after they were kept under darkness for 5 d and again illuminated, the leaves of the antisense ACD1 plants (AsACD1) were bleached. Leaves of AsACD1 accumulated 387 nmol (g FW)(-1) pheophorbide a which corresponded to 60% of chlorophyll a degraded. The rate of decrease in chlorophyll a was not influenced in senesced AsACD1 leaves. These results demonstrated that ACD1 is involved in PaO activity, and its inhibition led to photooxidative destruction of the cell instead of the "stay-green" phenotype.

The Arabidopsis Spontaneous Cell Death1 gene, encoding a ζ-carotene desaturase essential for carotenoid biosynthesis, is involved in chloroplast development, photoprotection and retrograde signalling

“…It is interesting to notice that spc1-1 has a lower level of chlorophylls a and b, which appears to be correlated to the reduced expression of PORB and CAO. The impaired chlorophyll metabolism and abnormal chloroplast development have been attributed as an important mechanism to trigger spontaneous cell death in a number of mutants, including Arabidopsis-accelerated cell death2 (acd2) [63,64], acd1 [65][66][67] and maize lethal leaf spot-1 (lls1) [68,69]. ACD1 and LLS1 are believed to be ortholog genes in Arabidopsis and maize [65,66,69].…”

“…The impaired chlorophyll metabolism and abnormal chloroplast development have been attributed as an important mechanism to trigger spontaneous cell death in a number of mutants, including Arabidopsis-accelerated cell death2 (acd2) [63,64], acd1 [65][66][67] and maize lethal leaf spot-1 (lls1) [68,69]. ACD1 and LLS1 are believed to be ortholog genes in Arabidopsis and maize [65,66,69]. These three genes, all encoding key enzymes for chlorophyll degradation, presumably function to prevent the formation of ROS or free radicals generated from photooxidation, thereby negatively regulating cell death [63,64,69].…”

Update on the biochemistry of chlorophyll breakdown

“…This is explained, at least in part, by the substrate specificity of PAO for pheophorbide (Pheide) a (Hörtensteiner et al 1995) and suggests that conversion of Chl b to Chl a is a prerequisite for further degradation via PAO. However, mutants that are devoid of PAO specifically accumulate Pheide a (Tanaka et al 2003;Pružinská et al 2003), indicating that conversion of Chl b to Chl a occurs upstream of Pheide formation.…”

“…As mentioned above, PAO exhibits an intriguing specificity for Pheide a, with Pheide b being a competitive inhibitor. In 2003, two groups succeeded in identifying Arabidopsis PAO at the molecular level (Pružinská et al 2003;Tanaka et al 2003). PAO is identical to ACCELERATED CELL DEATH (ACD) 1 (Greenberg and Ausubel 1993) and the ortholog of LETHAL LEAF SPOT 1 in maize (Gray et al 1997).…”

Carbohydrate, phenolic and antioxidant level in relation to chlorophyll a content in oilseed winter rape (Brassica napus L.) inoculated with Leptosphaeria maculans

“…Mur et al (2010) showed that reactive oxygen species caused chlorophyll degradation in the course of Pseudomonas syringae infection and that light-excited products of chlorophyll degradation, e.g., pheophorbide a (Pheide) may be an additional source of ROS. It was suggested in several other studies that pheophorbide a can act as a photosensitizer that generates ROS in response to light (Tanaka et al 2003;Hörtensteiner 2004;Pruzinska et al 2005;Tanaka and Tanaka 2006). Pheophorbide a content was not analyzed in this study.…”