Supraoptimal activity of CHLOROPHYLL DEPHYTYLASE1 results in an increase in tocopherol level in mature arabidopsis seeds

ABSTRACT

Tocopherols are synthesized in photosynthetic organisms, playing a role in plant stress tolerance. Recent studies showed that the phytol moiety of tocopherols comes from the salvaged phytol chain during chlorophyll degradation. However, the enzyme(s) responsible for chlorophyll dephytylation remains unclear. Recently, we reported the identification and characterization of CHLOROPHYLL DEPHYTYLASE1 (CLD1) of Arabidopsis, suggesting its role in chlorophyll turnover at steady state. In this addendum to the report, we presented and discussed the results related to the function of CLD1 in tocopherol biosynthesis. The tocopherol levels in the mature seeds were not altered in the transgenic lines with reduced CLD1 expression but were moderately increased in the plants with supraoptimal CLD1 activity compared to wild type. These results suggest that manipulating CLD1 activity could affect tocopherol biosynthesis to a certain extent and that other dephytylating enzymes are sharing redundant function in contributing the phytol pool in plant cells.

Tocopherols are lipophilic antioxidants produced in oxygenic photosynthetic organisms, i.e., plants, alga, and cyanobacteria. The compounds are widely recognized for their important function in the human diet, better known as vitamin E. They also play an important role for the well-being of plants. Under stress conditions, such as high light intensity, drought, toxic heavy metals, and unfavorable temperatures, the photosynthetic organisms accumulate elevated levels of tocopherols (see the review article ¹ and the references therein). Mutants unable to synthesize tocopherols showed increased sensitivity to stresses.^2-4 Seeds of Arabidopsis mutants devoid of tocopherols have reduced longevity.⁵ These reports point to the pivotal role of tocopherols in plant stress tolerance.

Four tocopherol isoforms (α, β, γ, and δ) can be detected in plants and the relative ratios vary among different species. In Arabidopsis, α-tocopherol is the primary one presented in the leaf, and the γ-isoform is predominant in mature seeds.^6,7 These compounds are produced in a pathway started from the condensation of homogentisate and phytyl diphosphate (Fig. 1). While homogentisate is derived from the shikimate pathway, phytyl diphosphate (PDP) could come from two sources, reduction of geranylgeranyl pyrophosphate by geranylgeranyl reductase⁸ and consecutive phosphorylation of the free phytol chain recycled during chlorophyll degradation (Fig. 1).^9,10 Recent genetic studies indicate that chlorophyll-derived phytol is the primary source for tocopherol biosynthesis.^9,11,12

Figure 1.

A simplified scheme of tocopherol biosynthesis coupled with the production of free phytol. The phytol moiety of tocopherols could be derived from chlorophyll and pheophytin dephytylation during chlorophyll turnover and breakdown, respectively. The biosynthesis of tocopherols commences by the condensation of homogentisate (HGA) and phytyl diphosphate (phytyl-PP) catalyzed by HGA phytyltransferase (HPT). Phytyl-PP is formed by the consecutive phosphorylation of phytol catalyzed by phytol kinase (VTE5) and phytyl-phosphate kinase (VTE6). In addition to tocopherol biosynthesis, phytyl-PP can be used by chlorophyll synthase (CHLG) to form chlorophyll. The enzymes that produce free phytol are denoted in red ovals, and other enzymes are in black ovals. CLD1, chlorophyll dephytylase; PPH, pheophytinase; SGR, STAY-GREEN (chlorophyll a Mg-dechelatase); PAO, pheophorbide a oxygenase; Phein a, pheophytin a; Pheide a, pheophorbide a; Chlide a, chlorophyllide a; NCCs, nonfluorescent chlorophyll catabolites.

Free phytol chain is produced during chlorophyll turnover in green tissues and chlorophyll breakdown in the senescent leaves, ripening fruits, and maturing seeds.^13-16 For chlorophyll breakdown, chlorophyll b is first converted to chlorophyll a, dechelated to form pheophytin a, then dephytylated by pheophytinase (PPH) to yield pheophorbide a, which eventually enters the PHEOPHORBIDE A OXYGENASE (PAO) pathway for a complete breakdown (Fig. 1).^17,18 The phytol chain released from pheophytin a during chlorophyll breakdown was thought to be the main source for tocopherol biosynthesis.¹⁶ However, the tocopherol levels were not significantly affected in the Arabidopsis mature seeds of a PPH null mutant,¹⁶ suggesting that chlorophyll breakdown may not be the sole source of free phytol. On the contrary, overexpression of PPH moderately increased tocopherol levels in transgenic Arabidopsis plants.¹⁶ These results imply that in addition to PPH there is another phytol-releasing enzyme(s) involved in tocopherol biosynthesis.

Free phytol can also be produced by hydrolysis of chlorophylls released from damaged photosystems that are subjected to repair at steady state when chlorophyll levels are relatively stable. Dephytylation of chlorophyll during its turnover at steady state was attributed to the activity of chlorophyllase. However, it was shown that Arabidopsis mutant devoid of chlorophyllase accumulated wild-type level of tocopherols,¹⁶ indicating that the enzyme is not responsible for providing free phytol for tocopherol biosynthesis. Recently, the studies on Arabidopsis heat sensitive mutants in our laboratory revealed a chlorophyll salvage pathway, in which chlorophylls are dephytylated and rephytylated by CHLOROPHYLL DEPHYTYLASE1 (CLD1) and CHLOROPHYLL SYNTHASE (CHLG), respectively (Fig. 1).^14,19 A missense mutation of CLD1, cld1-1, results in a mutant enzyme with a specific activity 15-fold higher than that of the wild-type enzyme. To see whether CLD1 is involved in tocopherol biosynthesis, the tocopherol levels in wild type, cld1-1, and transgenic lines with reduced or overexpressed CLD1 activity were analyzed and compared. Our data showed that γ-tocopherol content in the seeds was increased by 15% in cld1-1 and 30% in WT(cld1-1) transgenic lines that overexpressed the mutant enzymes in wild type background, but the tocopherol level was not affected in CLD1-silenced lines (Fig. 2). This phenomenon is similar to the case of the transgenic lines with PPH overexpression, suggesting the functional redundancy of the dephytylases in tocopherol biosynthesis. Given the existence of two more CLD1 and PPH homologs in Arabidopsis genome,¹⁴ more studies are needed to elucidate their exact roles in tocopherol biosynthesis in the future.

Figure 2.

The effect of modification of CLD1 activity on tocopherol content in Arabidopsis seeds. Mature seeds were ground in liquid nitrogen and extracted with 100% acetone pre-cooled at -20^oC (1 mL per 100 mg fresh weight). Tocopherols in the extract were separated using an ultra-performance liquid chromatography system (UPLC; Waters, http://www.waters.com/) and a BEH C18 reverse-phase column with a mobile phase described in the previous study.²⁰ The transgenic lines are labeled as WT(cld1-1), the wild type transformed with ectopic cld1-1 genomic DNA; amiR-CLD1, transgenic lines expressing artificial microRNA targeting CLD1. Numbers after the symbol “#” indicate the line designations derived from independent transformation events. Data are means ± SD of three biological replicates; * indicates the significant difference from WT in Student's t-test with p < 0.05.