Chloroplast and cytoplasmic enzymes

Summary Chloroplastic and cytoplasmic forms of pea (Pisum sativum L.) leaf carbonic anhydrase were separated by isoelectric focusing. The two forms have identical pH optima, 7.0 for the hydration reaction and 7.5 for the dehydration reaction, and identical Michaelis constants for CO₂, 0.03 M. Neither isozyme is affected by any of several compounds involved in carbon metabolism in the green plant.IV=Anderson and Pacold (1972).     

Chloroplast division

Chloroplasts are descendants of cyanobacteria and divide by binary fission. Several components of the division apparatus have been identified in the past several years and we are beginning to appreciate the plastid division process at a mechanistic level. In this review, we attempt to summarize the most recent developments in the field and assemble these observations into a working model of plastid division in plants.     

Chloroplast movement

Abstract. Chloroplasts redistribute and/or reorientate in the cell as a response to the light direction, resulting in patterns typical for light of low or high fluence rate, respectively. Usually, the main photoreceptor pigment is a blue-UV-absorbing pigment ('cryptochrome'), but in a few exceptional cases, the reversible red/far-red system phytochrome is involved. Detection of light direction is based on light refraction and/or on dichroic orientation of photoreceptor molecules. Membrane effects, intracellular calcium redistribution and calcium-calmodulin interaction are discussed as likely steps in signal transduction . In the response mechanism the actin-myosin system is involved. However, several details of perception, transduction and response are still unsolved and open for discussion. Particularly interesting are the cases of multiple photoreceptor systems , i.e. those where separate transduction chains are started which coact or interact with each other. This raises the question as to the evolution of multiple photoreceptor systems under the assumption that light-oriented chloroplast movements serve to optimize photosynthesis.     

Chloroplast Proteases

The chloroplast within the plant cell has a dynamic environment where proteases play an important role in processing of precursor proteins, degradation of incomplete proteins lacking cofactors, stress-induced degradation and removal of damaged proteins. A number of proteases in the chloroplast are well characterized and found to be localized within different compartments such as stroma, thylakoids and lumen. In recent years, an increasing number of proteases in chloroplasts have been discovered and identified as bacterial protease homologues. These include the stromal Clp, thylakoidal FtsH and lumenal DegP. The current focus is to understand their role in chloroplast regulation both at the enzyme-substrate and genetic levels.     

Chloroplast dielectrophoresis

Dielectrophoretic coefficients of chloroplasts, untreated and treated with cationized ferritin, have been measured in axisymmetric ac electric fields at different frequencies. The treated chloroplasts have surface charge density 2.4 times smaller than the untreated ones.The dielectrophoretic coefficients are in the range 10^-25 F · m² to 7x10^-25 F · m² for frequencies from 6,000 Hz to 1 MHz. Dielectrophoretic effects have not been observed for frequencies from 1 to 6,000 Hz and from 1 MHz and 10 MHz. The surface charge decrease leads to an increase of 2–3 times in the dielectrophoretic coefficients and slight shift of the dielectrophoretic mobility of lower frequencies.These results may be qualitatively explained on the basis of the existing theories for cell and vesicle dielectrophoresis.Abbreviations DCMU 3-(3,4-Dichlorophenyl)-1,1-Dymethylurea - EDTA Ethylenediaminetetraacetic acid - HEPES (N-2 Hydroxyethylpiperozine-N-2-ethanesulphonic acid) - MES (2[N-morpholino]ethane sulfonic acid) - TRICINE (N-tris[Hydroxymethyl]methyl glycine) - DMPA (N,N-dimethyl-1,3-propanediamine)     

Behavior of Chloroplast Nucleus during Chloroplast Development and Degeneration in Chlamydomonas reinhardii

Changes in morphology of chloroplast nuclei (cp-nuclei), totalcp-DNA content, number of cp-nuclei, oxygen-evolution activityand chlorophyll (a and b) content were examined during the degenerationand development of chloroplasts, using Chlamydomonas reinhardiicells which had been incubated on solid medium for various periods. Under 4'-6-diamidino-2-phenylindole (DAPI) epifluorescence microscopy,each cell that had been incubated for 7 days had one cell nucleus,one cup-shaped chloroplast and about 10 small, dispersed cp-nucleiin the chloroplast. One day after incubation of these cellson fresh medium, the cell volume and cp-nuclei increased insize 2-3 fold, but rapidly decreased in size after cell division.After about 7 days of incubation, cells ceased to divide andcp-nuclei began to associate with each other. At about 20 daysthey formed a ring-shaped structure surrounding the pyrenoid,followed by condensation into one cp-nuclear particle near thepyrenoid. When 41-day-old cells, having only one cp-nucleus,were reinoculated on fresh solid medium, the cp-nucleus increasedin size 2–3 fold, divided into several cp-nuclear particlesand then dispersed into the chloroplast, forming a bead-likestructure, before cell division. From microscopic fluorometry,a 4-fold increase in total cp-DNA content per chloroplast, withoutan increase in the number of cp-nuclear particles per chloroplast,occurred one day after the start of the experiment and one dayafter reinoculation of 41-day-old cells onto fresh medium. Theprocess of condensation of dispersed cp-nuclear particles intoone cp-nucleus during degeneration of the chloroplast was notaccompanied by any change in total cp-DNA content per chloroplast.A large peak of oxygen-evolution (0.6–0.9 pmoles/cell/hour)was seen one day after inoculation and reinoculation of thecells. The chlorophyll content (a+b) was high (1.2–2.2pg/cell) during the first week of incubation, after which itgradually decreased. (Received December 18, 1985; Accepted April 2, 1986)     

Complete Chloroplast Genome of Sedum sarmentosum and Chloroplast Genome Evolution in Saxifragales

Comparative chloroplast genome analyses are mostly carried out at lower taxonomic levels, such as the family and genus levels. At higher taxonomic levels, chloroplast genomes are generally used to reconstruct phylogenies. However, little attention has been paid to chloroplast genome evolution within orders. Here, we present the chloroplast genome of Sedum sarmentosum and take advantage of several available (or elucidated) chloroplast genomes to examine the evolution of chloroplast genomes in Saxifragales. The chloroplast genome of S. sarmentosum is 150,448 bp long and includes 82,212 bp of a large single-copy (LSC) region, 16.670 bp of a small single-copy (SSC) region, and a pair of 25,783 bp sequences of inverted repeats (IRs).The genome contains 131 unique genes, 18 of which are duplicated within the IRs. Based on a comparative analysis of chloroplast genomes from four representative Saxifragales families, we observed two gene losses and two pseudogenes in Paeonia obovata, and the loss of an intron was detected in the rps16 gene of Penthorum chinense. Comparisons among the 72 common protein-coding genes confirmed that the chloroplast genomes of S. sarmentosum and Paeonia obovata exhibit accelerated sequence evolution. Furthermore, a strong correlation was observed between the rates of genome evolution and genome size. The detected genome size variations are predominantly caused by the length of intergenic spacers, rather than losses of genes and introns, gene pseudogenization or IR expansion or contraction. The genome sizes of these species are negatively correlated with nucleotide substitution rates. Species with shorter duration of the life cycle tend to exhibit shorter chloroplast genomes than those with longer life cycles.     

Chloroplast glutathione reductase

Glutathione reductase (EC 1.6.4.2) activity is present in spinach (Spinacia oleracea L.) chloroplasts. The pH dependence and substrate concentration for half-maximal rate are reported and a possible role in chloroplasts is proposed.     

Chloroplast translation regulation

Chloroplast gene expression is primarily controlled during the translation of plastid mRNAs. Translation is regulated in response to a variety of biotic and abiotic factors, and requires a coordinate expression with the nuclear genome. The translational apparatus of chloroplasts is related to that of bacteria, but has adopted novel mechanisms in order to execute the specific roles that this organelle performs within a eukaryotic cell. Accordingly, plastid ribosomes contain a number of chloroplast-unique proteins and domains that may function in translational regulation. Chloroplast translation regulation involves cis-acting RNA elements (located in the mRNA 5′ UTR) as well as a set of corresponding trans-acting protein factors. While regulation of chloroplast translation is primarily controlled at the initiation steps through these RNA-protein interactions, elongation steps are also targets for modulating chloroplast gene expression. Translation of chloroplast mRNAs is regulated in response to light, and the molecular mechanisms underlying this response involve changes in the redox state of key elements related to the photosynthetic electron chain, fluctuations of the ADP/ATP ratio and the generation of a proton gradient. Photosynthetic complexes also experience assembly-related autoinhibition of translation to coordinate the expression of different subunits of the same complex. Finally, the localization of all these molecular events among the different chloroplast subcompartments appear to be a crucial component of the regulatory mechanisms of chloroplast gene expression.     

Chloroplast Deg proteases

For some chloroplast proteases ATP binding and hydrolysis is not necessary for their catalytic activity, most probably because even strongly unfolded substrates may penetrate their catalytic chamber. Deg1, 2, 5 and 8 are the best known of Arabidopsis thaliana ATP- independent chloroplast proteases, encoded by orthologues of genes coding for DegP, DegQ and DegS proteases of Escherichia coli. Current awareness in the area of structure and functions of chloroplast Degs is much more limited vs the one about their bacterial counterparts. Deg5 and Deg8 form a catalytic heterododecamer which is loosely attached to luminal side of thylakoid membrane. The complex catalyses--supported by Deg1 and one of FtsH proteases--the degradation of PsbA damaged due to plant exposition to elevated irradiance and thus these protease are of key importance for the plants' sensitivity to photoinhibition. Deg2 role in the disposal of damaged PsbA has not been elucidated. Recombinant Deg1 may degrade PsbO and plastocyanin in vitro but it is not clear whether this reaction is performed in vivo as well.     

Chloroplast inorganic pyrophosphatase

An alkaline, Mg²⁺-dependent inorganic pyrophosphatase has been isolated from previously isolated spinach chloroplast. The activity of the enzyme was increased 100-fold, with a 42% yield, upon purification from the total soluble chloroplast enzymes. The pH optimum for the enzyme shifts from 9.0 at 5 mM Mg²⁺ to 7.0 at 40 mM Mg²⁺. The substrate for the reaction appears to be magnesium pyrophosphate, and anionic pyrophosphate is an effective inhibitor. There seems to be also an activating effect of Mg²⁺ on the enzyme at pH 7. No other cation substitutes for Mg²⁺ in activating the hydrolysis of pyrophosphate. Among anions tested, only F⁻ caused severe inhibition. The enzyme is inactive towards fructose 1,6-diphosphate, thiamine pyrophosphate, ATP, and ADP. The possibility that this enzyme is subject to metabolic regulation is discussed in relation to an indicated role of pyrophosphate in the regulation of photosynthetic carbon reduction.     

Chloroplast manganese and superoxide.

Particles prepared from spinach chloroplast membranes with Triton X-100 inhibited the superoxide-mediated reduction of nitro-blue tetrazolium by riboflavin. This superoxide dismutase-like activity was of two kinds, one inactivated by heating and inhibited by H₂O₂ and the other insensitive to both of these treatments; both activities were destroyed by washing with concentrated Tris buffer or with EDTA. Attempts at reconstitution with transition metal ions suggested that two different forms of bound manganese may be responsible and it is proposed that the inhibition by H₂O₂ is indicative of three different oxidation states of particle-bound manganese. The possibility that the photosynthetic water-splitting system and superoxide dismutase have evolved from a single precursor is discussed.     

Chloroplast pigments and algal systematics

The chloroplast pigments of one typical representative (Pleurochloris magna) and two potential members (clone BSG Sticho and an isolate called Tunis) of the new class Eustigmatophyceae have been examined by modern methods including mass spectrometry. The three cultures all exhibited the same chloroplast pigments: Chlorophyll a, but no b or c, δ-carotene (I), canthaxanthin (II), violaxanthin (IIIa), and esterified vaucheriaxanthin (IVb) plus some free vaucheriaxanthin (IVa). Furanoid rearrangement of the epoxidic carotenoids complicated the analysis. The unique pigment complement hereby indicated for Eustigmatophyceae is clearly different from the pigment distribution patterns reported for Chlorophyceae and Xanthophyceae.     

Effect of High Temperature on Chloroplast Ribosomes and Biosynthesis of Chloroplast Proteins in Wheat

The chloroplasts of wheat have chanced greatly at high temperature condition(34℃). When wheat grown at 34℃ for 10 days, its chlorophyll content was 6 times less than that under the normal condition(22℃). The ribosomes were isolated from the leaves by sucrose density gradient centrifugation. It is found that only 80 S ribosomes existed in wheat leaves grown at the high temperature and the formation of 70 S ribosomes is specifically prevented. Since the absence of 70 S ribosomes in chloroplast, proteins synthesis can no longer proceed. Analysis of SDS-polyacrylamide gel electrophoresis indicates that the bands of chloroplast proteins from the leaves of wheat at the high temperature are less than those under normal condition. One of the poly- peptides the large subunit(MW=57000 daltons) of ribulose bisphosphate carboxylase, which is coded for by chloroplast genome and synthesized on 70 S ribosome in chloroplast, was lost. The photosynthetic intensity is decreased due to the blocking synthesis in chloroplast of some polypeptides which play the important role in photosynthesis.