Regulation of 5-Aminolevulinic Acid Synthesis in Developing Chloroplasts

Abstract

Intact chloroplasts isolated from greening cucumber (Cucumis sativus L. var Beit Alpha) cotyledons regenerated protochlorophyllide (Pchlide) in the dark with added cofactors from either exogenous glutamate or endogenous substrates. No other intermediates of the chlorophyll biosynthetic pathway accumulated. When inhibitors of 5-aminolevulinic acid (ALA) dehydratase were added, the Pchlide that failed to form was replaced by an excessive amount of ALA. When greening seedlings were retumed to the dark, ALA-synthesizing activity in the isolated chloroplasts decreased dramatically and recovered if the dark-treated seedlings were again exposed to continuous white light prior to chloroplast isolation. Both the decline and the recovery of ALAsynthesizing activity were complete in approximately 50 minutes. Changes in chloroplast structure during in vivo light to dark and dark to light transitions (as evidenced by electron microscopy) were much slower. Exposing isolated chloroplasts from darktreated seedlings to short white flashes before incubation transformed nearly all the endogenous Pchlide, but hardly stimulated ALA synthesis, suggesting that Pchlide does not act as a feedback inhibitor on ALA synthesis. Chloroplasts isolated from darktreated tissue did not form Pchlide from glutamate when incubated in the dark with added cofactors; moreover, the endogenous Pchlide did not tum over in organello. However, these chloroplasts did synthesize Pchlide from added ALA at the normal rate and synthesized ALA from glutamate at a reduced, but still significant, rate. Mg chelation was not affected by in vivo dark treatment.

Characterization of a Family of Chlorophyll-Deficient Wheat (Triticum) and Barley (Hordeum vulgare) Mutants with Defects in the Magnesium-Insertion Step of Chlorophyll Biosynthesis

“…During the dark cycle the MV Pchlide levels initially increased at the same rate for a11 three plants. However, the wild-type MV Pchlide pool leveled off sooner, at about 1700 pmol g-' fresh weight, a value previously reported in the literature for cucumbers (Huang and Castelfranco, 1989), whereas the mutant levels continued to increase. When the time-course experiment was extended for an additional 8 h (shaded area) to determine whether the Pchlide level in the mutants might also level off, it was found that the mutant MV Pchlide content still continued to increase.…”

“…Altematively, although the pathway is clearly blocked during both day and night, the block could be more extensive during the daytime. Thus, the mutations could be affecting the regulation of the enzyme's activity, or possibly the mutations could indirectly perturb the phytochrome-dependent mechanism (Huang and Castelfranco, 1989) that shuts off ALA synthesis at night, which involves heme and the products of the tigrina loci (in barley) (reviewed by Somerville, 1986). Clearly, these results will be more easily addressed in the future when both the Mg-chelatase and the nature of these mutants are defined at the molecular level.…”

Regulation of 5-Aminolevulinic Acid (ALA) Synthesis in Developing Chloroplasts

“…The decline and recovery were complete in approximately 50 min (12). In the present study, the light-mediated in vivo restoration of the ALA-synthesizing capacity has been further examined.…”

“…We have previously observed (12) that ALA-synthesizing capacity in isolated chloroplasts greatly declines when etiolated cucumber seedlings that have been exposed to white light (60 ME.-m2 s') for 20 h are returned to the dark, and largely recovers when the dark-treated seedlings are again exposed to continuous white light (12). The decline and recovery were complete in approximately 50 min (12).…”

“…Until recently, it has been generally thought that the phytochrome regulation is limited to the early stages of greening and that feedback inhibition of ALA synthesis by pchlide controls ALA synthesis later on (14). In the companion paper (12), we have examined the regulation of ALA synthesis in chloroplasts isolated from partially greened cucumber cotyledons and have found that feedback inhibition of ALA synthesis by Pchlide is not significant. However, we have observed that approximately two-thirds ofthe ALA-synthesizing capacity exhibited in organello is modulated by light/dark in vivo treatments, prior to chloroplast isolation.…”