Downregulation of chloroplast protease AtDeg5 leads to changes in chronological progression of ontogenetic stages, leaf morphology and chloroplast ultrastructure in Arabidopsis

The chloroplast protein AtDeg5 is a serine-type protease peripherally attached to thylakoid membrane at its lumenal side. Since reliable data regarding the role of AtDeg5 in controlling the course of growth and developmental processes are extremely limited, two independent T-DNA insertional lines with different extent of AtDeg5 reduction were prepared and ontogenesis stage-based analysis performed. Both mutant lines displayed a compensatory overaccumulation of AtDeg8. The repression of AtDeg5 protease altered a range of phenotypic features in at least one of the mutants, with the most prominent being changes in chronological progression of development and growth of individual rosette leaves, flower production and silique ripening as well as in the area of fully expanded leaves and chloroplast ultrastructure. By analyzing the results of parallel-mutant screening we conclude that AtDeg8 overdose may rescue 23% of AtDeg5 deficiency with regard to some AtDeg5-controlled traits; alternatively AtDeg5 may have catalytic sites in excess so that these traits might remain unaltered when AtDeg5 pool is reduced by 23%. For some other AtDeg5-dependent traits the absence of excessive amount of AtDeg5 catalytic sites, lack of AtDeg5 dosage effect and inability of AtDeg8 to compensate deficiency or absence of AtDeg5 occurred

Physiological functions of chloroplast protease AtDeg5

Wydział Biologii: Instytut Biologii EksperymentalnejChloroplastowe białko AtDeg5 jest proteazą typu serynowego peryferycznie zasocjowaną z błoną od strony światła tylakoidu. Ze względu na słabo poznaną rolę, jaką odgrywa AtDeg5 w kontroli przebiegu procesów ontogenezy przebadano dwie niezależne linie mutantów insercyjnych o różnym stopniu wyciszenia AtDEG5 i w oparciu o stadia ontogenezy przeanalizowano szereg cech. Obie linie mutantów wykazywały kompensacyjną hiperakumulację AtDeg8. Represja proteazy AtDeg5 spowodowała zmianę wielu cech fenotypowych przynajmniej u jednego z mutantów, przy czym najbardziej znaczące zmiany dotyczyły chronologicznej progresji wzrostu i rozwoju indywidualnych liści rozetkowych, produkcji kwiatów, dojrzewania łuszczynek oraz morfologii w pełni rozwiniętych liści, ultrastruktury chloroplastów, akumulacji apobiałka Lhcb6 w chloroplastach, organizacji strukturalnej cząsteczek PSII, tworzenia rozgałęzień pędów kwiatostanowych oraz długości i szerokości nasion. W odniesieniu do cech fenotypowych regulowanych przez AtDeg5 można wnioskować, że nadmiar AtDeg8 może efektywnie kompensować deficyt 23% puli AtDeg5 lub chloroplast dysponuje nadmiarem centrów katalitycznych AtDeg5 w stosunku do potrzeb, a efekt dawki AtDeg5 odgrywa bądź nie odgrywa istotnej roli. Alternatywnie nadmiar AtDeg8 nie kompensuje deficytu 23%puli AtDeg5, a chloroplast nie dysponuje nadmiarem centrów katalitycznych AtDeg5 w stosunku do potrzeb.The chloroplast protein AtDeg5 is a serine-type protease peripherally attached to thylakoid membrane at its lumenal side. Since reliable data regarding the role of AtDeg5 in controlling the course of growth and developmental processes are extremely limited, two independent T-DNA insertional lines with different extent of AtDeg5 reduction were prepared and ontogenesis stage-based analysis performed. Both mutant lines displayed a compensatory overaccumulation of AtDeg8. The repression of AtDeg5 protease altered a range of phenotypic features in at least one of the mutants, with the most prominent being changes in chronological progression of development and growth of individual rosette leaves, flower production, silique ripening and in the morphology of fully expanded leaves, chloroplast ultrastructure, starch metabolism in chloroplasts, accumulation of Lhcb6 apoprotein in chloroplasts and structural organization of PSII molecules as well as in formation of inflorescence debranching and in length and width of seeds. By analyzing the results of parallel-mutant screening we conclude that AtDeg8 overdose may rescue 23% of AtDeg5 deficiency with regard to some AtDeg5-controlled traits; alternatively AtDeg5 may have catalytic sites in excess so that these traits might remain unaltered when AtDeg5 pool is reduced by 23%. For some other AtDeg5-dependent traits the absence of excessive amount of AtDeg5 catalytic sites, lack of AtDeg5 dosage effect and inability of AtDeg8 to compensate deficiency or absence of AtDeg5 occurred

Selected small molecules as inducers of pluripotency

The general idea of regenerative medicine is to fix or replace tissues or organs with live and patient-specific implants. Pluripotent stem cells are capable of indefinite self-renewal and differentiation into all cell types of the body. An easily accessible source of induced pluripotent stem cells (iPSCs) may allow obtaining and culturing tissues in vitro. Many approaches in the methods leading to obtain iPSCs have been tested in order to limit immunogenicity and tumorigenesis, and to increase efficiency. One of the approaches causing pluripotency is usage of small molecule compounds. It would be of great importance to assess their specific properties and reveal their new capacity to induce pluripotent stem cells and to improve reprogramming efficiency. Identification of the epigenetic changes during cellular reprogramming will extend our understanding of stem cell biology and many therapeutic applications. In this paper we discuss mainly the nucleotide derivatives, already proven or for now only putative inducers of the cells' pluripotency, that modulate the epigenetic status of the cell

<i>Rindera graeca</i> (A. DC.) Boiss. & Heldr. (Boraginaceae) In Vitro Cultures Targeting Lithospermic Acid B and Rosmarinic Acid Production

The in vitro cultures of Rindera graeca, a rare endemic plant, were developed as a sustainable source of phenolic acids. Various shoot and root cultures were established and scaled up in a sprinkle bioreactor. A multiplication rate of 7.2 shoots per explant was achieved. HPLC–PDA–ESI–HRMS analysis revealed the presence of rosmarinic acid (RA) and lithospermic acid B (LAB) as the main secondary metabolites in both the shoot and root cultures. The maximum RA (30.0 ± 3.2 mg/g DW) and LAB (49.3 ± 15.5 mg/g DW) yields were determined in root-regenerated shoots. The strongest free radical scavenging activity (87.4 ± 1.1%), according to 2,2-diphenyl-1-picrylhydrazyl-hydrate assay, was noted for roots cultivated in a DCR medium. The highest reducing power (2.3 µM ± 0.4 TE/g DW), determined by the ferric-reducing antioxidant power assay, was noted for shoots cultivated on an SH medium containing 0.5 mg/L 6-benzylaminopurine. A genetic analysis performed using random amplified polymorphic DNA and start codon targeted markers revealed genetic variation of 62.8% to 96.5% among the investigated shoots and roots. This variability reflects the capacity of cultivated shoots and roots to produce phenolic compounds

Polyurethane Foam Rafts Supported In Vitro Cultures of Rindera graeca Roots for Enhanced Production of Rinderol, Potent Proapoptotic Naphthoquinone Compound

Unique phytochemical profile of plants belonging to Boraginaceae family provides a prolific resource of lipophilic pigments from the group of naphthoquinone derivatives. To overcome low compound content, the major obstacle of plant-based production, immobilization of Rindera graeca roots in in vitro cultures was implemented for efficient production of rinderol, novel furanonaphthoquinone derivative with anticancer properties. Chromatographic procedures revealed rinderol presence in extracts of all investigated root lines, derived both from root biomass and post-culture medium. Unexpectedly, in the second stage of the experiment, rinderol production was ceased in control, unmodified culture systems. On the contrary, roots immobilized on PUF rafts uniformly and stably produced rinderol, and its highest amount was noted for transformed root lines after 42 days of cultivation (222.98 &plusmn; 10.47 &micro;g/flask). PUF occurred to be the main place of compound accumulation. Moreover, investigation of rinderol biological activity revealed its fast-acting cell death induction in HeLa cervical cancer cells at relatively low concentrations. Presented results revealed successful application of R. graeca roots immobilization on PUF rafts for production and in situ product removal of rinderol, novel lipophilic furanonaphthoquinone with suggested proapoptotic activity