No abstract

Chlorophyll synthesis in higher plants involves the light-induced formation of ALA2 (4) by an enzyme system which, in all probability, is not identical with ALA synthetase (1, 10). In previous papers we presented evidence that light induces the synthesis, rather than the activation of this ALA-producing enzyme(s) with a half-life of about 80 min (3,8). It was also shown that the cessation of ALA accumulation after a light-dark transition in leaves treated with LA, a specific inhibitor of the ALA dehydratase, is in agreement with the proposed half-life of the enzyme (3). The rapid cessation of PChl synthesis in the dark after a light period in leaves not treated with LA could not be explained by the properties of the enzyme system alone and suggested a feedback inhibition of the activity of the ALAproducing enzyme, which could be removed by an additional illumination. It in batches of 1 to 2 g, and then placed in a single row with their bases in transport cuvettes, 0.6 cm wide and 2.5 cm high, containing either distilled H20 or a 50 mm LA solution, brought to pH 5.8 to 6.0 with KOH. After a 2-hr preincubation period in the dark, the leaves were exposed for 10 min to light from a Xenon source, filtered through water-cooled interference filters (Intraflex B40, Balzers, Liechtenstein). Light intensity was measured with an YSI Kettering model 65 radiometer, and is expressed as erg* cm-2 sec-'. After the illumination, the leaves were returned to the dark for 4 hr and, when required, exposed again to white fluorescent light of 1.2 x 103 erg* cm-2 -sec-' (80 ft-c) for 3 hr. Concomitantly, etiolated leaves treated with or without LA were kept in the dark for 0 hr, 2 hr, 4 hr, and 7 hr, or exposed for 3 hr to white light without preillumination. Some of the samples were then extracted with 80% acetone and PChl and Chl determined spectrophotometrically, while from others trichloroacetic acid extracts were prepared for ALA determinations, as described earlier (8, 10 PChl and Chl contents are expressed as nmol ALA/g fresh weight of the leaves (8 mol ALA = 1 mol Chl or PChl). Per cent PChl-Chl transformation is defined as Chlp x 100/PChl.. Chl and ALA content were also determined at the end of the 3-hr period of continuous illumination.The pyrrolle formed with acetylacetone was identified as ALA-pyrrolle on thin layer chromatograms (5). RESULTSEffect of Short ilmination on PChl-Chl Conversion in Etiolated Leaves and on ALA Accumulation during a Subsequent Dark Period. Detached maize leaves treated with LA were exposed for 10 min to various intensities of light filtered through interference filters and returned for 4 hr to the dark. Figure 1 shows the dose response curves for the PChl-Chl conversion which occurred during illumination with light filtered through filters with maximal transmissions at 650 nm (R..) and 659 nm (RIta). Figure 2 gives the dose response curve for total ALA accumulation (PChl + free ALA) during the subsequent 4-hr dark period, which was induced by Rcw and R<,,. From these and similar dose response cu...

Levulinic acid inhibited the greening of etiolated maize (Zea mays) and bean (Phaseolus vulgaris) leaves and caused accumulation of 5-aminolevulinic acid (ALA). ALA accumulation in maize was equivalent to the decrease in chlorophyll, over a wide range of experimental conditions. It was saturated at low light intensities and was not limited by the supply of substrates during the early hours of greening. During 20 hours in light, levulinic acid had little effect on the structural development of thylakoids in bundle sheath chloroplasts but significantly reduced the number and size of thylakoids in grana of mesophyll chloroplasts. Recrystallization of prolamellar bodies and their reformation was inhibited. Mitochondria appeared not to be affected.The accumulation of ALA in bean leaves differed from that in maize in regard to its time course and the effect of levulinic acid concentration and light intensity. The amount of ALA accumulated exceeded that expected from the degree of inhibition of chlorophyll synthesis. Levulinic acid caused abnormalities in the structural development of bean chloroplasts and marked swelling of mitochondria.Chloramphenicol and cycloheximide inhibited ALA accumulation, whlile inhibitors of RNA synthesis had no effect. The extent of inhibition depended on the time the inhibitor was applied during the greening process. The use of ALA accumulation as a tool for studying the control of chlorophyll synthesis is discussed.

Intact plastids from greening maize (Zea mays L.) leaves converted I'4Ciglutamate and 114C12-ketoglutarate (KG) to 114C15-aminolevulinic acid (ALA). Glutamate appeared to be the immediate precursor of ALA, while KG was first converted to glutamate, as shown by the effect of various inhibitors of amino acid metabolism. Plastids from greening leaves contained markedly higher activity as compared with etioplasts or chloroplasts. The synthesis of ALA by intact plastids was light dependent. The enzyme system resides in the stroma of plastids or may be lightly bound to membranes. The solubilized system showed maximal activity around pH 7.9 and required Mg2+, ATP, and NADPH although dependence on the latter was not clear-cut. A relatively high level of activity could be extracted from etioplasts. Maximal activty was obtained from plastids of leaves which had been ilhuminated for 90 minutes, after which activity declined sharply. The enzyme system solubilized from plastids also catalyzed the conversion of putative glutamate 1-semialdehyde to ALA in a reaction which was not dependent on the addition of an amino donor.The system in maize greatly resembled the one which had been reported from barley. It is suggested that this system is the one responsible for the biosynthesis of ALA destined for chlorophyll formation.

Polyphenol oxidases (PPOs) are nuclear-encoded chloroplast proteins that are targeted to the thylakoid lumen by a bipartite presequence. The N-terminal part of this sequence is removed by a stromal processing peptidase (SPP), and the resulting intermediate is translocated across the thylakoid and processed to the mature protein. A 4800-fold-purified SPP processed a PPO precursor (pPPO) at a site identical to that occurring in organelle. The in vitro product of SPP action on pPPO was further processed and translocated by thylakoids. This SPP processed other precursors but was inactive toward those of light-harvesting chlorophyll binding proteins. The enzyme appeared to be a metalloendopeptidase, like previously reported SPPs. However, it differed in substrate specificity, apparent size, and, most significantly, cleavage site of pPPO. Whereas the processing sites of lumen proteins determined so far were relatively distant from the hydrophobic core of the thylakoid targeting domain, pPPO was cleaved immediately before this domain. Cleavage removed the twin arginine motif characteristic of thylakoid targeting domains of lumen proteins, which are translocated by the ⌬pH-dependent pathway. The possible significance of these observations to PPO translocation mechanism is discussed. It is suggested that several SPPs may exist in chloroplasts with preferences for different subsets of precursors.Nuclear-encoded plastid proteins are synthesized in the cytosol. Targeting is conferred by a positively charged transit peptide rich in hydroxyamino acids (1, 2). The presequence is removed by a stromal processing peptidase (SPP) 1 upon import of the precursor (3). Some imported proteins are subsequently directed to the thylakoid membrane or lumen. Transit peptides of lumen proteins contain, in addition to the stromal-targeting domain, a thylakoid-targeting domain that is removed by TPP while crossing the thylakoid or immediately thereafter (2, 4). A single peptidase is believed to be responsible for the stromal processing of all plastid proteins (1, 2). A partially purified SPP acted on several precursors of plastid proteins (3). Two polypeptides that processed the precursors of LHCPII and other proteins were purified (5), and a gene coding for an antigenically related metalloprotease was cloned (6). The deduced protein (CPE) contained a zinc binding motif, and its relation to subunit ␤ of the mitochondrial processing peptidase was pointed out. Escherichia coli-expressed CPE was reported to process a wide range of protein precursors, including some lumen proteins (7). Two serine-protease SPPs from Chlamydomonas processed pSSU (8, 9). One catalyzed the formation of an intermediate form of SSU, and the other formed the mature protein. A consensus cleavage site, (Ile/Val)-X-(Ala/Cys)-2-Ala, was derived from comparing precursors of chloroplast proteins (10). However, this motif was absent in many cases. In general, SPP cleavage sites lack significant primary sequence similarity or a conserved secondary structure motif (1, 2, 4, 11). ...