Photocontrol of phenylalanine ammonia-lyase in barley seedlings treated with pyridazinone inhibitors of chloroplast development

Seedlings imbibed for 48 hr in aqueous solutions of the pre-emergent herbicide Sandoz 6706, or its presumably active conversion product Norflurazon, grow into albino plants in white light. Neither herbicide has any effect on PAL in dark grown barley shoots. In white light, however, pretreatment with 100 μM herbicide causes an increase in barley shoot PAL of about 50% over that found in untreated plants. Barley root PAL is stimulated by 0.1 μM Sandoz 6706 but inhibited by higher concentrations. Mung bean primary leaves show dose responses similar to barley roots. The herbicides have no effect in continuous red light, yet blue light is as effective as white light in eliciting PAL responses. The results are discussed in relation to the subcellular distribution of PAL.     

Isolation and properties of a natural inhibitor of the chloroplast adenosine triphosphatase

The complete sequence of protein L17 which is a component of the large subunit of the E. coli ribosome has been determined. Peptides deriving from enzymatic hydrolysis with trypsin, thermolysin, chymotrypsin and S. aureus and A. mellea protease were isolated and sequenced by the DABITC/PITC double coupling method. Some overlapping peptides were obtained after mild acid cleavage of the protein. According to the amino acid sequence protein L17 contains 127 residues and has a molecular mass of 14 365. The primary structure of protein L17 agrees well with the amino acid analysis of the intact protein and its N-terminal sequence as derived from automatic sequencing in an improved Beckman sequencer. Secondary predictions and a search for homologous sequence stretches to other ribosomal proteins were made.     

Interactions of chlorophyll a and terpenoid alcohols with chloroplast acyl lipids in monomolecular films

Surface pressure-area measurements of purified chlorophyll a and monogalactosyl diacylglycerol in mixed monolayers were performed at 20°C with an automatic recording surface film balance at a constant compression rate. In addition structural parts of the chlorophyll and the monogalactosyl diacylglycerol as phytol and geranylgeraniol on one hand and different fatty acids on the other were studied pure and in mixed films. All components studied formed stable monomolecular films. Chlorophyll a and monogalactosyl diacylglycerol showed miscibility. Phytol was immiscible with synthetized monogalactosyl diacylglycerol containing only stearic acid. Mixed monolayers of phytol and monogalactosyl diacylglycerol isolated from barley containing 83 mol % a-linolenic acid showed a strong interaction. An increased miscibility and association were found between phytol and fatty acids with increasing unsaturation. The results are discussed as a model for the localization of part of chlorophyll a in the thylakoid membrane.     

Convertibility of a saponifiable lipoidal derivative of 18-hydroxycorticosterone

Metabolic properties and subcellular localization of the biosynthesis of SM, a saponifiable 18-OH-B (18-Hydroxycorticosterone) derivative, were investigated. Homogenates biosynthesized SM at a nearly constant rate of 463 pmol/50 mg tissue during 30 min. This biosynthesis was more efficient at pH 7.4 than at pH 4.8. Not only 18-OH-B but also its less polar anhydride 18-DAL (18-Deoxyaldosterone) were good precursors. SM was reverted to these precursors both enzymatically and spontaneously, 4.8 being a more suitable pH for this reversion than 7.4. Trapping experiments demonstrated a sequence comprising, in this order, the following echelons: SM, 18-OH-B, 18-DAL, Aldosterone. The first two steps are reversible and the last two ones depend on proton concentrations. It is postulated that SM could be on a dead-end to which 18-OH-B could be deviated if Aldosterone biosynthesis became temporarily unnecessary. Also, that 18-OH-B may convert to either 18-DAL or SM for selective membrane transports, according to homeostatic requirements.     

Lidocaine and ATPase inhibitor interaction with the chloroplast envelope

Photosynthetic capacity of isolated intact chloroplasts is known to be sensitive to K⁺ fluxes across the chloroplast envelope. However, little is known about the system of chloroplast envelope proteins that regulate this K⁺ movement. The research described in this report focused on characterizing some of the components of this transport system by examining inhibitor effects on chloroplast metabolism. Digitoxin, an inhibitor of membrane-bound Na⁺/K⁺ ATPases, was found to reduce stromal K⁺ at a range of external K⁺ and inhibit photosynthesis. Scatchard plot analysis revealed a specific protein receptor site with a K_m for digitoxin binding of 13 nanomolar. Studies suggested that the receptor site was on the interior of the envelope. The effect of a class of amine anesthetics that are known to be K⁺ channel blockers on chloroplast metabolism was also studied. Under conditions that facilitate low stromal pH and concomitant photosynthetic inhibition, the anesthetic, lidocaine, was found to stimulate photosynthesis. This stimulation was associated with the maintenance of higher stromal K⁺. Comparison of the effects on photosynthesis of lidocaine analogs which varied in lipophilicity suggested a lipophilic pathway for anesthetic action. The results of experiments with lidocaine and digitoxin were consistent with the hypothesis that a K⁺ channel and a K⁺-pumping envelope ATPase contribute to overall K⁺ flux across the chloroplast envelope. Under appropriate assay conditions, photosynthetic capacity of isolated chloroplasts was shown to be much affected by the activity of these putative envelope proteins.     

Melittin: An inhibitor of chloroplast photochemical reactions

Melittin, a polypeptide component of bee venom, is an inhibitor of photochemical reactions in chloroplasts isolated from higher plants. At concentrations around 5 μM, melittin acts as an uncoupler of photophosphorylation and abolishes the 518 nm light induced absorbance changes. At higher concentrations (30–50 μM), melittin abolishes both the light-induced photooxidation of cytochrome $\text{f}$, and partially inhibits other reactions of photosynthetic electron transfer, without causing lysis of the membrane. The observed inhibitions appear to be due to changes in the properties of the membrane lipid bilayer, caused by penetration of melittin molecules.     

Cellular content of chloroplast DNA and chloroplast ribosomal RNA genes in Euglena gracilis during chloroplast development.

The cellular content of chloroplast DNA in Euglena gracilis has been quantitatively determined. DNA was extracted from Euglena cells at various stages of chloroplast development and renatured in the presence of trace amounts of 3H-labeled chloroplast DNA. From the kinetics of renaturation of the 3H-labeled chloroplast DNA, compared with the kinetics of renaturation of excess nonradioactive chloroplast DNA, the fraction of cellular DNA represented by chloroplast DNA was calculated. The content of chloroplast DNA was found to increase from 4.9 to 14.6% of cellular DNA during light-induced chloroplast development. Correcting for the change in DNA mass per cell, the number of copies of chloroplast DNA is found to vary from 1400 to 2900 per cell. During this developmental transition, the cellular content of the chloroplast ribosomal RNA genes varies from 1900 to 5200 copies per cell. The ratio of the number of copies of rRNA genes to chloroplast genomes per cell remains in the range of 1-2 throughout chloroplast development, ruling out selective amplification of chloroplast rRNA genes as a means of regulation of rRNA gene expression. Direct measurement of the number of rRNA cistrons per 9.2 X 10(7) dalton genome yields a value of 1 or 2.     

Protoplasts as a means of studying chloroplast development in vitro

Protoplasts obtained enzymically from etiolated primary leaves of oat were illuminated in vitro, and the process of etioplast chloroplast transformation followed. Chloroplast development proceeded up to 6 hours of incubation in the light (20 C). During this period, complete photosynthetic light and dark reactions were constituted, in addition to prolamellar body-degrading protease activity.     

Disruption of chloroplast biogenesis and plant development upon down-regulation of a chloroplast processing enzyme involved in the import pathway

Typically, nuclear-encoded chloroplast proteins are synthesized as precursors and require proteolytic processing upon import before their assembly into functional complexes within the organelle. A cDNA encoding a chloroplast processing enzyme (CPE), which was originally identified as a protease that cleaves the precursor for the major light-harvesting chlorophyll binding protein (preLHCP), was introduced into the tobacco genome in an antisense orientation to investigate the role of the enzyme in vivo . The presence of the antisense-CPE gene resulted in chlorotic leaves, and retarded shoot and root growth. The introduction of the antisense-CPE gene disrupted the normal pattern of plastid division. Chloroplast numbers in cotyledon and first leaf cells were reduced 25% compared to the control plants. Chloroplasts contained fewer thylakoids and large starch grains, the latter an indication of a change in carbon flux. CPE levels and activity were significantly lower in stromal extracts in the transgenic plants. Interestingly, in vitro import of precursor proteins was defective. Most of the preLHCP remained on the exterior of the organelle, and only a small fraction of preRBCA was imported, suggesting that a change in CPE levels can influence translocation across the envelope. Our in vivo results support the conclusion that CPE plays a critical role during chloroplast biogenesis, and that the pleiotropic effects of CPE down-regulation reflect its function as a general stromal processing peptidase as part of the import machinery. Furthermore, these findings indicate the importance of regulating the expression of components of the import machinery for normal plant development.     

Inhibition of chloroplast development by tentoxin

Light-dependent chloroplast development in detached pea shoots was measured in terms of chlorophyll synthesis and the synthesis of Fraction 1 protein. Both synthetic processes were inhibited more than 90% by the fungal metabolite, tentoxin (1 or 10 μg/ml). These results place Pisum sativum in the class of tentoxin-sensitive higher plants. Tentoxin, actinomycin D, lincomycin, D-threo-chloramphenicol and carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) were compared in their ability to inhibit RNA and protein synthesis by isolated pea chloroplasts. Energy for the synthetic reactions was supplied either by light or by added ATP. Only CCCP gave the same pattern of inhibition as tentoxin, i.e. inhibition of both RNA and protein synthesis in the light-driven system but no inhibition in the ATP-driven system. It is concluded that chloroplast developmental processes are inhibited by tentoxin through the inhibition of photophosphorylation.     

Young Seedling Stripe1 encodes a chloroplast nucleoid-associated protein required for chloroplast development in rice seedlings

Planta - Young Seedling Stripe1 (YSS1) was characterized as an important regulator of plastid-encoded plastid RNA polymerase (PEP) activity essential for chloroplast development at rice seedling...     

Tentoxin An uncompetitive inhibitor of lettuce chloroplast coupling factor 1

The interaction of tentoxin $\text{[cyclo}\text{(-}\text{l}\text{-leucyl}\text{-N-}\text{methyl-(Z)-dehydrophenyl-analyl-glycyl}\text{-N-}\text{methyl-}\text{l}\text{-alanyl-)}\text{]}$ with solubilized lettuce chloroplast coupling factor 1 was characterized by direct binding studies, measurement of the time course of ATPase inhibition, and steady-state enzyme kinetics. Neither substrates, products or Ca²⁺ competed with the tentoxin binding site, nor did they induce any large change in tentoxin affinity. The inhibition of lettuce chloroplast coupling factor 1 ATPase was found to be the time dependent, and at equilibrium the affinities estimated by equilibrium ultrafiltration and enzyme inhibition were similar (1.8 · 10⁸M^?1). The steady-state kinetics best fit an uncompetitive pattern suggesting that the inhibited steps follow an irreversible step occurring after ATP binding.     

POR - import and membrane association of a key element in chloroplast development

The development of proplastids or etioplasts to chloroplast is visualized by the accumulation of chlorophyll in leaves of higher plants. The biosynthesis of chlorophyll includes a light-dependent reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide). This light-dependent step is catalysed by the nucleus-encoded NADPH:Pchlide oxidoreductase (POR, EC 1.6.99.1). POR is active within plastids and therefore has to be translocated over the plastid envelope membranes. The import of chloroplast proteins seems to follow a general import pathway using translocons at the outer and inner envelope membrane. POR cross-linking to Toc75, one of the major translocon components at the outer envelope membrane, indicates its use of the general import pathway. However, since variations exist within the so-called general import pathway one has to consider previous data suggesting a novel totally Pchlide-dependent import pathway of one POR isoform, PORA. The suggested Pchlide dependency of POR import is discussed since recent observations contradict this idea. In the stroma the POR transit peptide is cleaved off and the mature POR protein is targeted to the plastid inner membranes. The correct and stable association of POR to the membrane requires the cofactor NADPH. Functional activity of POR calls for formation of an NADPH–Pchlide–POR complex, a formation that probably takes place after the membrane association and is dependent on a phosphorylation reaction.     

叶绿体增殖调控机制研究进展

叶绿体为内共生起源的细胞器。利用电镜观察发现叶绿体分裂时具有中央缢缩现象,并且缢缩过程中存在环状结构。在大肠杆菌中,FtsZ蛋白最早在分裂位点组成一个环状结构（Z-环,FtsZ protein ring）,其他分裂相关蛋白再与之结合,共同组成一个复杂的分裂装置,最终导致原核细胞分裂的完成。其分裂位点的选择受到min操纵子（包括MinC,MinD。MinE基因）的精细调控。叶绿体分裂的分子调控机制与原核细胞类似。原核起源与真核起源的分裂相关蛋白组成分裂复合体,确保叶绿体的正常分裂。