Ion homeostasis in the Chloroplast

peer reviewedThe chloroplast is an organelle of high demand for macro- and micro-nutrient ions, which are required for the maintenance of the photosynthetic process. To avoid deficiency while preventing excess, homeostasis mechanisms must be tightly regulated. Here, we describe the needs for nutrient ions in the chloroplast and briefly highlight their functions in the chloroplastidial metabolism. We further discuss the impact of nutrient deficiency on chloroplasts and the acclimation mechanisms that evolved to preserve the photosynthetic apparatus. We finally present what is known about import and export mechanisms for these ions. Whenever possible, a comparison between cyanobacteria, algae and plants is provided to add an evolutionary perspective to the description of ion homeostasis mechanisms in photosynthesis

A simple method for quantification of protochlorophyllide in etiolated <i>Arabidopsis </i>seedlings

Etiolated seedlings accumulate the chlorophyll biosynthesis intermediate protochlorophyllide (Pchlide) and measuring Pchlide can be important for characterizing photomorphogenic mutants that may be affected in chloroplast development. In this chapter we outline a simple and sensitive method for quantifying Pchlide in extracts of Arabidopsis seedlings using fluorescence spectroscopy. This method can be easily adapted to study chloroplast development in a wide range of plant species.</p

Refolding and enzyme kinetic studies on the ferrochelatase of the cyanobacterium synechocystis sp. PCC 6803

Heme is a cofactor for proteins participating in many important cellular processes, including respiration, oxygen metabolism and oxygen binding. The key enzyme in the heme biosynthesis pathway is ferrochelatase (protohaem ferrolyase, EC 4.99.1.1), which catalyzes the insertion of ferrous iron into protoporphyrin IX. In higher plants, the ferrochelatase enzyme is localized not only in mitochondria, but also in chloroplasts. The plastidic type II ferrochelatase contains a C-terminal chlorophyll a/b (CAB) motif, a conserved hydrophobic stretch homologous to the CAB domain of plant light harvesting proteins and light-harvesting like proteins. This type II ferrochelatase, found in all photosynthetic organisms, is presumed to have evolved from the cyanobacterial ferrochelatase. Here we describe a detailed enzymological study on recombinant, refolded and functionally active type II ferrochelatase (FeCh) from the cyanobacterium Synechocystis sp. PCC 6803. A protocol was developed for the functional refolding and purification of the recombinant enzyme from inclusion bodies, without truncation products or soluble aggregates. The refolded FeCh is active in its monomeric form, however, addition of an N-terminal His6-tag has significant effects on its enzyme kinetics. Strikingly, removal of the C-terminal CAB-domain led to a greatly increased turnover number, kcat, compared to the full length protein. While pigments isolated from photosynthetic membranes decrease the activity of FeCh, direct pigment binding to the CAB domain of FeCh was not evident.The authors are thankful to the Royal Swedish Academy (to C.F.) and the Kempe foundation (to P.S.) for granting their positions. The work was supported by the Swedish Energy Agency and Umeå University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. </p