Controlled Partitioning of Glutamyl-tRNA Reductase in Stroma- and Membrane-Associated Fractions Affects the Synthesis of 5-Aminolevulinic Acid.

The synthesis of 5-aminolevulinic acid (ALA) determines adequate amounts of metabolites for the tetrapyrrole biosynthetic pathway. Glutamyl-tRNA reductase (GluTR) catalyzes the rate-limiting step of ALA synthesis and was previously considered to be exclusively localized in the chloroplast stroma of light-exposed plants. To assess the intraplastidic localization of GluTR, we developed a fast separation protocol of soluble and membrane-bound proteins and reassessed the subplastidal allocation of GluTR in stroma and membrane fractions of Arabidopsis plants grown under different light regimes as well as during de-etiolation and dark incubations. Under the examined conditions, the amount of stroma-localized GluTR correlated with the ALA synthesis rate. The transfer to dark repression of ALA synthesis resulted in a loss of soluble GluTR. Arabidopsis mutants lacking one of the GluTR-interacting factors FLUORESCENT (FLU), the GluTR-binding protein (GBP) or ClpC, a chaperone of the Clp protease system, were applied to examine the amount of GluTR and its distribution to the stroma or membrane in darkness and light. Taking into consideration the different compartmental allocation of GluTR, its stability and ALA synthesis rates, the post-translational impact of these regulatory factors on GluTR activity and plastidic sublocalization is discussed.

Posttranslational Control of ALA Synthesis Includes GluTR Degradation by Clp Protease and Stabilization by GluTR-Binding Protein.

5-Aminolevulinic acid (ALA) is the first committed substrate of tetrapyrrole biosynthesis and is formed from glutamyl-tRNA by two enzymatic steps. Glutamyl-tRNA reductase (GluTR) as the first enzyme of ALA synthesis is encoded by HEMA genes and tightly regulated at the transcriptional and posttranslational levels. Here, we show that the caseinolytic protease (Clp) substrate adaptor ClpS1 and the ClpC1 chaperone as well as the GluTR-binding protein (GBP) interact with the N terminus of GluTR Loss-of function mutants of ClpR2 and ClpC1 proteins show increased GluTR stability, whereas absence of GBP results in decreased GluTR stability. Thus, the Clp protease system and GBP contribute to GluTR accumulation levels, and thereby the rate-limiting ALA synthesis. These findings are supported with Arabidopsis (Arabidopsis thaliana) hema1 mutants expressing a truncated GluTR lacking the 29 N-terminal amino acid residues of the mature protein. Accumulation of this truncated GluTR is higher in dark periods, resulting in increased protochlorophyllide content. It is proposed that the proteolytic activity of Clp protease counteracts GBP binding to assure the appropriate content of GluTR and the adequate ALA synthesis for chlorophyll and heme in higher plants.

GluTR2 complements a hema1 mutant lacking glutamyl-tRNA reductase 1, but is differently regulated at the post-translational level.

Arabidopsis HEMA1 and HEMA2 encode glutamyl-tRNA reductase (GluTR) 1 and 2, the two isoforms of the initial enzyme of tetrapyrrole biosynthesis. HEMA1 is dominantly expressed in photosynthetic tissue, while HEMA2 shows low constitutive expression and is induced upon stress treatments. We introduce a new HEMA1 knockout mutant which grows only heterotrophically on MS (Murashige and Skoog) medium at low light, indicating that the remaining GluTR2 does not sufficiently compensate for the extensive needs of metabolic precursors for Chl. While hema1 accumulates low amounts of Chl, it contains more than half of the wild-type heme content. The functional diversity of the two GluTR isoforms was analyzed by means of complementation studies of the hema1 mutant by expression of pHEMA1::HEMA2 and p35S::HEMA1, respectively. Expression of both transgenes complements hema1, indicating that GluTR2 can likewise be involved in the synthesis of Chl and is not exclusively assigned to heme synthesis. In comparison with p35S::HEMA1-complemented hema1 and the wild type, GluTR2 expression under control of the HEMA1 promoter (pHEMA1) in pHEMA1::HEMA2-complemented hema1 mutants causes elevated protochlorophyllide levels under extended dark periods as well as in short-day-grown adult plants, resulting in the formation of necrotic leaf tissue. Although both GluTR isoforms have similar activity and contribute to 5-aminolevulinic acid synthesis for adequate accumulation of Chl and heme, it is proposed that the two proteins experience a different post-translational control in darkness and light. While GluTR2 continues 5-aminolevulinic acid synthesis in darkness, GluTR1 is efficiently inactivated by the interaction with the FLU (FLUORESCENT) protein, thereby preventing an accumulation of protochlorophyllide.

Overexpression of HEMA1 encoding glutamyl-tRNA reductase.

5-Aminolevulinic acid (ALA) synthesis has been shown to be the rate limiting step of tetrapyrrole biosynthesis. Glutamyl-tRNA reductase (GluTR) is the first committed enzyme of plant ALA synthesis and is controlled by interacting regulators, such as heme and the FLU protein. Induced inactivation of the HEMA1 gene encoding GluTR by RNAi expression in tobacco resulted in a reduced activity of Mg chelatase and Fe chelatase indicating a feed-forward regulatory mechanism that links ALA synthesis posttranslationally with late enzymes of tetrapyrrole biosynthesis (Hedtke et al., 2007). Here, the regulatory impact of GluTR was investigated by overexpression of AtHEMA1 in Arabidopsis and tobacco plants. Light-dependent ALA synthesis cannot benefit from an up to 7-fold induced expression of GluTR in Arabidopsis. While constitutive AtHEMA1 overexpression in tobacco stimulates ALA synthesis by 50-90% during light-exposed growth of seedlings, no increase in heme and chlorophyll contents is observed. HEMA1 overexpression in etiolated and dark-grown Arabidopsis and tobacco seedlings leads to additional accumulation of protochlorophyllide. As excessive accumulation of GluTR does not correlate with increased ALA formation, it is hypothesized that ALA synthesis is additionally limited by other effectors that balance the allocation of ALA with the activity of enzymes of chlorophyll and heme biosynthesis.