Molekularna struktura kloroplastnih nukleoida špinata (Spinacea oleracea)

The ultrastructure and molecular composition of spinach chloroplast nucleoids were studied. Isolated nucleoids retained their three-dimensionally folded structure observed in situ. Fluorescence microscopy and staining with DAP1 reaffirmed the mostly DNA-containing nature of these complexes. The presence of chloroplast DNA was confirmed by restriction fragment analysis. Electron microscopy revealed a highly condensed, chromatin-like, beaded substructure. Naked DNA supercoils or loops were not observed. Integral nucleoid proteins were isolated and analysed on SDS-polyacrylamide gels. A distinct pattern of the nucleoid proteins, covering the entire range of molecular masses from 5 kDa to >100 kDa revealed that the nucleoid fraction was highly depleted of major thylakoid and stromal constituents.Istraživali smo ultrastrukturu i molekularnu građu kloroplastnih nukleoida špinata (Spinacea oleráceo). Izolirani nukleoidi zadržavaju svoju trodimenzionalnu građu vidljivu in situ. Fluorescencijskom mikroskopijom i bojenjem s fluorokromom DAPI potvrdili smo visok sadržaj DNA u ovim kompleksima. Prisustvo kloroplastne DNA dokazali smo restrikcijskom analizom. Pod elektronskim mikroskopom vidljiva je kondenzirana (gusta), kromatinu slična, kugličasta građa. Gole DNA petlje ili superzavojnice nisu primjećene. Proteini nukleoida pokrivaju čitav raspon molekulskih masa, od 5 do više od 100 kDa. Glavni proteini tilakoidnih membrana i strome kloroplasta nisu prisutni u nukleoidnoj frakciji

Dinamičke promjene plastidnih nukleoida u divljeg-tipa i aurea-tipa listova kaline (Ligustrum ovalifolium Hassk. var. aureurn)

The changes in number, size, distribution and DNA content of plastid nucleoids in wild- and mutant-type leaves of an aurea variety of privet (Ligustrum ovalifolium. Hassk. var. aureum) were studied. Wild-type leaves did not show changes (in nucleoid number, size and distribution)! induced by different light intensities. Mutant leaves responded to changes in light conditions by changes in colour. During yellowing of regreened aurea leaves and bleaching of yellow aurea leaves the number of plastid nucleoids decreased with concomitant increase of their volume in such a fashion that the total volume of nucleoids and the DNA content per plastid remained constant. No degradation of chlo- roplast DNA before and during yellowing and bleaching of the aurea leaves was observed.Istraživali smo promjene broja, veličine, razmještaja i sadržaja DNA, plastidnih nukleoida u divljih i mutiranih listova kaline (Ligustrum ovalifolium Hassk. var. aureum). Listovi divljeg tipa ne pokazuju promjene (broja, veličine i razmještaja nukleoida) uzrokovane različitim intenzitetima svjetlosti. Mutirani listovi mijenjaju boju s obzirom na intenzitet osvijetljenosti. Tijekom žućenja ozelenjelih listova i izbljeđivanja žutih listova aurea, broj nukleoida se smanjuje s istovremenim povećanjem njihova volumena. Stoga, ukupni volumen nukleoida, a time i količina DNA, ostaju konstantni u svim fazama. Razgradnja kloroplastne DNA prije i tijekom žućenja i izbljeđivanja nije opažena

Effects of TROL Presequence Mutagenesis on Its Import and Dual Localization in Chloroplasts

Thylakoid rhodanase-like protein (TROL) is involved in the final step of photosynthetic electron transport from ferredoxin to ferredoxin: NADP+ oxidoreductase (FNR). TROL is located in two distinct chloroplast compartments—in the inner envelope of chloroplasts, in its precursor form ; and in the thylakoid membranes, in its fully processed form. Its role in the inner envelope, as well as the determinants for its differential localization, have not been resolved yet. In this work we created six N-terminal amino acid substitutions surrounding the predicted processing site in the presequence of TROL in order to obtain a construct whose import is affected or localization limited to a single intrachloroplastic site. By using in vitro transcription and translation and subsequent protein import methods, we found that a single amino acid exchange in the presequence, Ala67 to Ile67 interferes with processing in the stroma and directs the whole pool of in vitro translated TROL to the inner envelope of chloroplasts. This result opens up the possibility of studying the role of TROL in the chloroplast inner envelope as well as possible consequence/s of its absence from the thylakoids

Balancing chloroplast redox status – regulation of FNR binding and release

Working in synchrony, photosynthetic charge separation, electron transfer, and redox reactions generate proton motive force necessary for the synthesis of ATP and funneling of electrons toward stromal reducing equivalent NADPH. The last step of electron transfer from ferredoxin to NADP+ is catalyzed by ferredoxin-NADP+ oxidoreductase (FNR). Two proteins, TROL (thylakoid rhodanese-like) and Tic62 (62 kDa component of the translocon at the inner envelope of chloroplasts), have been characterized and shown to form dynamic complexes with FNR. Inactivation of TROL leads to changes in efficiency of electron transfer and induction of non-photochemical quenching. TROL-deficient plants have changed nuclear gene expression with up-regulation of NADPH-dependent malic enzyme, which can form NADPH in an alternative pathway. Thus, NADPH synthesis, mediated by FNR-TROL interaction, may be the source element in metabolic retrograde signal-transduction pathway linking light reactions with nuclear gene expression

Beginners guide to sample preparation techniques for transmission electron microscopy

Background purpose: The revolution in microscopy came in 1930 with the invention of electron microscope. Since then, we can study specimens on ultrastructural and even atomic level. Besides transmission electron microscopy (TEM), for which specimen preparation techniques will be described in this article, there are also other types of electron microscopes that are not discussed in this review. Materials and methods: Here, we have described basic procedures for TEM sample preparation, which include tissue sample preparation, chemical fixation of tissue with fixatives, cryo-fixation performed by quick freezing, dehydration with ethanol, infiltration with transitional solvents, resin embedding and polymerization, processing of embedded specimens, sectioning of samples with ultramicrotome, positive and negative contrasting of samples, immunolabeling, and imaging. Conclusion: Such collection of methods can be useful for novices in transmission electron microscopy