Chlorophyll degradation: the tocopherol biosynthesis-related phytol hydrolase in Arabidopsis seeds is still missing

Abstract

Phytyl diphosphate (PDP) is the prenyl precursor for tocopherol biosynthesis. Based on recent genetic evidence, PDP is supplied to the tocopherol biosynthetic pathway primarily by chlorophyll degradation and sequential phytol phosphorylation. Three enzymes of Arabidopsis (Arabidopsis thaliana) are known to be capable of removing the phytol chain from chlorophyll in vitro: chlorophyllase1 (CLH1), CLH2, and pheophytin pheophorbide hydrolase (PPH), which specifically hydrolyzes pheophytin. While PPH, but not chlorophyllases, is required for in vivo chlorophyll breakdown during Arabidopsis leaf senescence, little is known about the involvement of these phytol-releasing enzymes in tocopherol biosynthesis. To explore the origin of PDP for tocopherol synthesis, seed tocopherol concentrations were determined in Arabidopsis lines engineered for seed-specific overexpression of PPH and in single and multiple mutants in the three genes encoding known dephytylating enzymes. Except for modestly increasing tocopherol content observed in the PPH overexpressor, none of the remaining lines exhibited significantly reduced tocopherol concentrations, suggesting that the known chlorophyll-derived phytol-releasing enzymes do not play major roles in tocopherol biosynthesis. Tocopherol content of seeds from double mutants in NONYELLOWING1 (NYE1) and NYE2, regulators of chlorophyll degradation, had modest reduction compared with wild-type seeds, although mature seeds of the double mutant retained significantly higher chlorophyll levels. These findings suggest that NYEs may play limited roles in regulating an unknown tocopherol biosynthesis-related phytol hydrolase. Meanwhile, seeds of wild-type over-expressing NYE1 had lower tocopherol levels, suggesting that phytol derived from NYE1-dependent chlorophyll degradation probably doesn't enter tocopherol biosynthesis. Potential routes of chlorophyll degradation are discussed in relation to tocopherol biosynthesis.

Figure 1.

The substrate PDP directing toward tocopherol biosynthesis is primarily derived from chlorophyll degradation. Two phytol-releasing activities are known, i.e. CLH catalyzing release from chlorophyll and PPH dephytylating pheophytin. Phytol is then converted to PDP by sequential kinase reactions catalyzed by VTE5 and a second, unknown kinase. Condensation of PDP and homogentisate by HPT marks the initial reaction of tocopherol biosynthesis. phy, Phytyl. [See online article for color version of this figure.]

Figure 2.

Deficiency of PPH does not result in a decrease in total tocopherol in mature Arabidopsis seeds. Total tocopherol concentrations of mature seeds from the wild type (WT) and two independent T-DNA insertion lines (pph-2 and pph-3) are shown. Data represent mean and se of at least three biological replicates.

Figure 3.

Impact of seed-specific overexpression of PPH on seed morphology and contents in total tocopherol and free phytol. A, Siliques of the wild type (WT) and one wild-type line overexpressing PPH seed, specifically (PPH-2) at 12 DAP. Note that compared with the wild type, PPH-2 seeds contain less chlorophyll. B, Mature seeds from four independent PPH-overexpressing lines (PPH-1–PPH-4) have slightly increased total tocopherol concentrations. C, Free phytol content of seeds from the wild type and PPH-2 at 12 DAP. Data represent mean and se of at least three biological measurements. **P < 0.01, *P < 0.05; Student’s t test.

Figure 4.

Total tocopherol content increases rapidly in developing seeds of PPH-overexpressing lines between 6 and 9 DAP. Tocopherol content was determined in the wild type and four independent PPH-overexpressing lines (PPH-1–PPH-4) at 6, 9, 12, and 15 DAP. Data represent mean and se of at least three measurements. For the statistical difference between the wild type and each of four independent PPH-overexpressing lines (PPH-1–PPH-4) at the same developmental stage, double asterisks indicate P < 0.01 and a single asterisk indicates P < 0.05, and for the statistical difference between stages for each line, A, B, and C indicates P < 0.01 and a, b, and c indicates P < 0.05 (Student’s t test).

Figure 5.

Analysis of expression of some genes involved in tocopherol biosynthesis in seeds of one biological replicate. The expression of the indicated genes was determined by real-time PCR in seeds of the wild type (WT) and a PPH-overexpressing line (PPH-1) at 6, 9, 12, and 15 DAP. A, HPPD. B, VTE5. C, PES2. D, HPT. Data represent mean and se of three technical measurements. Gene expression was normalized to At5G09810. **P < 0.01, *P < 0.05; Student’s t test.

Figure 6.

CLH1 and CLH2 deficiency does not result in a decrease in total tocopherol concentration in mature Arabidopsis seeds. Total tocopherol concentration of mature seeds from the wild type (WT) and clh1-1 and clh2-1 single and clh1-1,2-1 double mutants was assayed. Data represent mean and se of at least three biological replicates. *P < 0.05; Student’s t test.

Figure 7.

Simultaneous absence of CLHs and PPH does not alter tocopherol content in mature seeds. A, Phenotype of mature seeds from the wild type (WT) and different mutants. B, Total tocopherol in seeds of the wild type and different mutants at the onset of maturation (12 DAP). Data in B represent mean and se of at least three biological replicates.

Figure 8.

Simultaneous absence of CLHs and PPH does not alter tocopherol and chlorophyll contents in developing seeds on the onset of maturation. The chlorophyll content (A) and total tocopherol content (B) in seeds of the wild type (WT) and different mutants at the onset of maturation (12 DAP). Data represent mean and se of at least three biological replicates.

Figure 9.

Deficiency of NYEs has only a small impact on seed tocopherol. A, Phenotype of mature seeds from the wild type and different mutants. Note that seeds of nye1-1,nye2-1 retain chlorophyll and thus exhibit a stay-green phenotype. B, Total tocopherol content in mature seeds from the wild type and different nye mutants is shown. Data represent mean and se of at least three biological replicates. **P < 0.01; Student’s t test. WT, Wild type.

Figure 10.

Impact of seed-specific overexpression of NYE1 on seed morphology. A, Siliques of the wild type and one line overexpressing NYE1 seed specifically (NYE1-2) at 12 DAP. Note that, compared to the wild type, NYE1-2 seeds contain less chlorophyll. B, Chlorophyll quantification in seeds of the wild type and lines overexpressing NYE1 or PPH at 12 DAP. Note that chlorophyll content in seeds of overexpressing NYE1 or PPH is less than that of wild-type seeds at the same developmental stage. WT, Wild type.

Figure 11.

Impact of seed-specific overexpression of NYE1 on total tocopherol and free phytol contents. A, Levels of total tocopherol content in mature seeds of the wild type (WT) and four independent NYE1-overexpressing lines (NYE1-1–NYE1-4). B, Free phytol content in developing seeds from the wild type and NYE1-2 at 12 DAP. Data represent mean and se of at least three biological replicates. **P < 0.01; Student’s t test.

Figure 12.

Tentative model of the relation of chlorophyll breakdown to tocopherol biosynthesis in seeds of Arabidopsis. Destabilization of LHCII by NYEs is essential for chlorophyll degradation. Furthermore, two activities, i.e. CLH (supposed to be localized in cytosol) and PPH (localized in plastid), have been shown to be able to release phytol. The data presented here indicate that neither CLH nor PPH provide phytol for tocopherol biosynthesis. Instead, we postulate a novel, NYE-independent dephytylase that is active in seeds and that produces free phytol, which, after two-step phorphorylation involving VTE5, is fed into tocopherol biosynthesis. The curved line is representative of NYE/PPH-derived phytol seemingly does not contribute to the pool of PDP used for tocopherol synthesis. Chl, Chlorophyll.