Role of magnesium chelatase activity in the early steps of the tetrapyrrole biosynthetic pathway

Abstract

Magnesium-protoporphyrin IX chelatase (Mg-chelatase) is located at the branchpoint of tetrapyrrole biosynthesis, at which point protoporphyrin IX is distributed for the synthesis of chlorophyll and heme. We investigated the regulatory contribution of Mg-chelatase to the flow of metabolites. In plants, the enzyme complex consists of three subunits, designated CHL D, CHL I, and CHL H. Transgenic tobacco (Nicotiana tabacum) plants expressing antisense RNA for the Mg-chelatase subunit CHL H were analyzed to elucidate further the role of Mg-chelatase in the distribution of protoporphyrin IX into the branched tetrapyrrolic pathway. The transgenic plants displayed a reduced growth rate and chlorophyll deficiency. Both phenotypical properties were correlated with lower Mg-chelatase activity. Unexpectedly, less protoporphyrin IX and heme accumulated, and a decrease in 5-aminolevulinate (ALA)-synthesizing capacity and ALA dehydratase activity paralleled the progressive reduction in Mg-chelatase activity in the transformants compared with control plants. The reduced activities of the early enzymatic steps corresponded with lower levels of transcripts encoding glutamyl-tRNA reductase and ALA-dehydratase. The decreased expression and activities of early enzymes in the pathway could be explained by a feedback-controlled mechanism in response to lower Mg-chelatase activity. We discuss intercompartmental signaling that synchronizes the activities of the first steps in tetrapyrrolic metabolism with the late steps for the synthesis of end products.

Figure 1

Primary tobacco transformants expressing Chl H antisense RNA and a wild-type plant (SNN) grown in the greenhouse at 300 μmol m⁻² s⁻¹ for 5 weeks. Top panel, Transformants with a progressive loss of chlorophyll. Left to right, Wild-type plant SNN, Pa 1/14, Pa 1/60, Pa 1/54, and Pa 1/56. Bottom panel, Sixth leaves of the same transformants (except of wild-type-like Pa 1/60) shown for comparison. Transgenic plants remain slightly green along the vascular bundles.

Figure 2

Northern-blot analysis of the expression of Mg-chelatase subunits. Plants were grown for 5 weeks in the greenhouse at 300 μmol m⁻² s⁻¹. For the determination of steady-state RNA levels of the three Mg-chelatase subunits in selected Chl H antisense plants, total RNA was extracted from leaves 4, 6, and 8 of wild-type plants and transformants. Fifteen micrograms of isolated RNA was subjected to northern-blot hybridization and subsequently hybridized with the Chl H, Chl D, and Chl I cDNA. The Chl H cDNA probe recognized both the endogenous transcript and the antisense RNA.

Figure 3

Analysis of Mg-chelatase activity in chloroplasts of selected Chl H antisense transformants and control plants. Mg-chelatase activity was determined in chloroplasts isolated from 5-week-old plants. Values represent means ± sd from three separate preparations.

Figure 4

Analysis of porphyrins and Mg porphyrins in selected transgenic and wild-type plants. A through C, Contents of protoporphyrin IX, Mg-protoporphyrin (Mg-Proto) IX, and Mg-protoporphyrin IX monomethylester (Mg-Proto IX-MME) of leaf extracts from leaf 4 (white column) and 6 (striped column) were prepared and subjected to HPLC analysis with fluorescence detection as described in “Materials and Methods.” D and E, Leaf discs of leaf 4 (white columns) and 6 (striped columns) were incubated in 5 mm ALA in the dark for 4 h and analyzed for protoporphyrin IX and Mg-protoporphyrin IX accumulation. Values represent means ± sd from three separate extractions.

Figure 5

Analysis of ALA-synthesizing capacity (top) and ALA- dehydratase activity (bottom) of control plants and selected transgenic plants expressing Chl H antisense RNA. Top panel, Leaf discs of the fourth leaf were incubated in the light for 6 h in levulinic acid, an inhibitor of ALA-dehydratase. Amounts of accumulated ALA were determined spectrophotometrically after reaction with Ehrlichs reagent. Values represent means ± sd from three separate experiments. Bottom panel, ALA-dehydratase activity was measured from total extracts of the fourth leaf. Values represent means ± sd from three separate preparations.

Figure 6

Steady-state levels of RNA that encodes proteins of the early steps of the tetrapyrrole biosynthetic pathway and the antenna complex of photosystem II in antisense plants for CHL H (lines Pa 1/14, Pa 1/54, and Pa 1/56) and wild-type plants. Total RNA was isolated from leaves 3, 5, and 7 (counted from the top of each plant). Fifteen micrograms of RNA was loaded per lane, separated on a 1% (w/v) formaldehyde-agarose gel, and probed against cDNA encoding glutamyl-tRNA reductase (Hem A), Glu 1-semialdehyde aminotransferase (gsa), ALA-dehydratase (Ala), and the light-harvesting chlorophyll-binding proteins of photosystem II (Lhc). A cDNA probe for actin was subsequently hybridized to the RNA on the same filter to prove equal loading (data not shown). Numbers below each northern blot represent the relative levels of each transcript of the transgenic lines compared with those in the corresponding leaves of control plants.