Eduardo Zabaleta

Carbonic Anhydrase Subunits Form a Matrix-exposed Domain Attached to the Membrane Arm of Mitochondrial Complex I in Plants

Complex I of Arabidopsis includes five structurally related subunits representing γ-type carbonic anhydrases termed CA1, CA2, CA3, CAL1, and CAL2. The position of these subunits within complex I was investigated. Direct analysis of isolated subcomplexes of complex I by liquid chromatography linked to tandem mass spectrometry allowed the assignment of the CA subunits to the membrane arm of complex I. Carbonate extraction experiments revealed that CA2 is an integral membrane protein that is protected upon protease treatment of isolated mitoplasts, indicating a location on the matrix-exposed side of the complex. A structural characterization by single particle electron microscopy of complex I from the green alga Polytomella and a previous analysis from Arabidopsis indicate a plant-specific spherical extra-domain of about 60 Å in diameter, which is attached to the central part of the membrane arm of complex I on its matrix face. This spherical domain is proposed to contain a heterotrimer of three CA subunits, which are anchored with their C termini to the hydrophobic arm of complex I. Functional implications of the complex I-integrated CA subunits are discussed.

MADS-box genes expressed during tomato seed and fruit development

MADS-box genes in plants are putative transcription factors involved in regulating numerous developmental processes, such as meristem and organ identity in inflorescences and in flowers. Recent reports indicate that they are involved in other processes than flower development such as the establishment of developing embryos, seed coat and ultimately in root and fruit development. We have identified seven tomato MADS-box genes that are highly expressed during the first steps of tomato fruit development. According to comparisons of their deduced amino acid sequences, they were classified into two groups: (1) already identified tomato MADS-box genes previously defined as flower identity genes (TAG1, TDR4 and TDR6) and (2) new tomato MADS-box genes (TAGL1, TAGL2, TAGL11 and TAGL12). With the exception of TAGL12, which is expressed near uniformly in every tissue, the other genes show an induction during the tomato fruit development phase I (anthesis) and phase II, when active cell division occurs. In situ hybridization analyses show a specific expression pattern for each gene within the fruit and embryo sac tissues suggesting an important role in the establishment of tissue identity. Yeast two-hybrid analyses indicate that some of these proteins could potentially form dimers suggesting they could act together to accomplish their proposed role.

Gamma carbonic anhydrases in plant mitochondria

Three genes from Arabidopsis thaliana with high sequence similarity to gamma carbonic anhydrase (γCA), a Zn containing enzyme from Methanosarcina thermophila(CAM), were identified and characterized. Evolutionary and structural analyses predict that these genes code for active forms of γCA. Phylogenetic analyses reveal that these Arabidopsis gene products cluster together with CAM and related sequences from α and γ proteobacteria, organisms proposed as the mitochondrial endosymbiont ancestor. Indeed, in vitro and in vivo experiments indicate that these gene products are transported into the mitochondria as occurs with several mitochondrial protein genes transferred, during evolution, from the endosymbiotic bacteria to the host genome. Moreover, putative CAM orthologous genes are detected in other plants and green algae and were predicted to be imported to mitochondria. Structural modeling and sequence analysis performed in more than a hundred homologous sequences show a high conservation of functionally important active site residues. Thus, the three histidine residues involved in Zn coordination (His 81, 117 and 122), Arg 59, Asp 61, Gin 75, and Asp 76 of CAM are conserved and properly arranged in the active site cavity of the models. Two other functionally important residues (Glu 62 and Glu 84 of CAM) are lacking, but alternative amino acids that might serve to their roles are postulated. Accordingly, we propose that photosynthetic eukaryotic organisms (green algae and plants) contain γCAs and that these enzymes codified by nuclear genes are imported into mitochondria to accomplish their biological function.

Physiological, biochemical and molecular aspects of mitochondrial complex I in plants

Respiratory complex I of plant mitochondria has to date been investigated with respect to physiological function, biochemical properties and molecular structure. In the respiratory chain complex I is the major entry gate for low potential electrons from matrix NADH, reducing ubiquinone and utilizing the released energy to pump protons across the inner membrane. Plant complex I is active against a background of several other NAD(P)H dehydrogenases, which do not contribute in proton pumping, but permit and establish several different routes of shuttling electrons from NAD(P)H to ubiquinone. Identification of the corresponding molecular structures, that is the proteins and genes of the different NADH dehydrogenases, will allow more detailed studies of this interactive regulatory network in plant mitochondria. Present knowledge of the structure of complex I and the respective mitochondrial and nuclear genes encoding various subunits of this complex in plants is summarized here. Copyright 1998 Elsevier Science B.V.

oiwa, a Female Gametophytic Mutant Impaired in a Mitochondrial Manganese-Superoxide Dismutase, Reveals Crucial Roles for Reactive Oxygen Species during Embryo Sac Development and Fertilization in Arabidopsis

Traditionally considered to be deleterious molecules, reactive oxygen species (ROS) are emerging as essential signaling molecules in numerous organisms. Here, we show how ROS play a crucial role during female gametogenesis and fertilization in plants. Mitochondrial Mn-superoxide dismutase appears critical for maintaining ROS localization and important for embryo sac patterning. Reactive oxygen species (ROS) can function as signaling molecules, regulating key aspects of plant development, or as toxic compounds leading to oxidative damage. In this article, we show that the regulation of ROS production during megagametogenesis is largely dependent on MSD1, a mitochondrial Mn-superoxide dismutase. Wild-type mature embryo sacs show ROS exclusively in the central cell, which appears to be the main source of ROS before pollination. Accordingly, MSD1 shows a complementary expression pattern. MSD1 expression is elevated in the egg apparatus at maturity but is downregulated in the central cell. The oiwa mutants are characterized by high levels of ROS detectable in both the central cell and the micropylar cells. Remarkably, egg apparatus cells in oiwa show central cell features, indicating that high levels of ROS result in the expression of central cell characteristic genes. Notably, ROS are detected in synergid cells after pollination. This ROS burst depends on stigma pollination but precedes fertilization, suggesting that embryo sacs sense the imminent arrival of pollen tubes and respond by generating an oxidative environment. Altogether, we show that ROS play a crucial role during female gametogenesis and fertilization. MSD1 activity seems critical for maintaining ROS localization and important for embryo sac patterning.

A basal carbon concentrating mechanism in plants

Many photosynthetic organisms have developed inorganic carbon (Ci) concentrating mechanisms (CCMs) that increase the CO₂ concentration within the vicinity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). Several CCMs, such as four carbon (C4) and crassulacean acid metabolism (CAM), bicarbonate accumulation systems and capsular structures around RubisCO have been described in great detail. These systems are believed to have evolved several times as mechanisms that acclimate organisms to unfavourable growth conditions. Based on recent experimental evidence we propose the occurrence of another more general CCM system present in all plants. This basal CCM (bCCM) is supposed to be composed of mitochondrial carbonic anhydrases (a β-type carbonic anhydrase and the γ-type carbonic anhydrase domain of the mitochondrial NADH dehydrogenase complex) and probably further unknown components. The bCCM is proposed to reduce leakage of CO₂ from plant cells and allow efficient recycling of mitochondrial CO₂ for carbon fixation in chloroplasts.

Gamma carbonic anhydrase like complex interact with plant mitochondrial complex I

We report the identification by two hybrid screens of two novel similar proteins, called Arabidopsis thaliana gamma carbonic anhydrase like1 and 2 (AtγCAL1 and AtγCAL2), that interact specifically with putative Arabidopsis thaliana gamma Carbonic Anhydrase (AtγCA) proteins in plant mitochondria. The interaction region that was located in the N-terminal 150 amino acids of mature AtγCA and AtγCA like proteins represents a new interaction domain. In vitro experiments indicate that these proteins are imported into mitochondria and are associated with mitochondrial complex I as AtγCAs. All plant species analyzed contain both AtγCA and AtγCAL sequences indicating that these genes were conserved throughout plant evolution. Structural modeling of AtγCAL sequences show a deviation of functionally important active site residues with respect to γCAs but could form active interfaces in the interaction with AtγCAs. We postulate a CA complex tightly associated to plant mitochondrial complex.

Nitric oxide, nitrosyl iron complexes, ferritin and frataxin: A well equipped team to preserve plant iron homeostasis

Iron is a key element in plant nutrition. Iron deficiency as well as iron overload results in serious metabolic disorders that affect photosynthesis, respiration and general plant fitness with direct consequences on crop production. More than 25% of the cultivable land possesses low iron availability due to high pH (calcareous soils). Plant biologists are challenged by this concern and aimed to find new avenues to ameliorate plant responses and keep iron homeostasis under control even at wide range of iron availability in various soils. For this purpose, detailed knowledge of iron uptake, transport, storage and interactions with cellular compounds will help to construct a more complete picture of its role as essential nutrient. In this review, we summarize and describe the recent findings involving four central players involved in keeping cellular iron homeostasis in plants: nitric oxide, ferritin, frataxin and nitrosyl iron complexes. We attempt to highlight the interactions among these actors in different scenarios occurring under iron deficiency or iron overload, and discuss their counteracting and/or coordinating actions leading to the control of iron homeostasis.

Recombinant plant gamma carbonic anhydrase homotrimers bind inorganic carbon

MINT‐7266036: gamma CA2 (uniprotkb:Q9C6B3) and gamma CA2 (uniprotkb:Q9C6B3) physically interact (MI:0914) by molecular sieving (MI:0071)

Ectopic expression of mitochondrial gamma carbonic anhydrase 2 causes male sterility by anther indehiscence

Plant mitochondria include gamma-type carbonic anhydrases (γCAs) of unknown function. In Arabidopsis, the γCAs form a gene family of five members which all are attached to the NADH dehydrogenase complex (complex I) of the respiratory chain. Here we report a functional analysis of gamma carbonic anhydrase 2 (CA2). The gene encoding CA2 is constitutively expressed in all plant organs investigated but it is ten fold induced in flowers, particularly in tapetal tissue. Ectopic expression of CA2 in Arabidopsis causes male sterility in transgenic plants. In normal anther development, secondary thickenings of the endothecial cell wall cause anthers to open upon dehydration. Histological analyses revealed that abnormal secondary thickening prevents anther opening in 35S::CA2 transgenic plants. CA2 abundance in transgenic plants is increased 2–3 fold compared to wild-type plants as revealed by Western blotting analyses. Moreover, abundance of other members of the CA family, termed CA3 and CAL2, is increased in transgenic plants. Oxygen uptake measurements revealed that respiration in transgenic plants is mainly based on NADH reduction by the alternative NADH dehydrogenases present in plant mitochondria. Furthermore, the formation of reactive oxygen species (ROS) is very low in transgenic plants. We propose that reduction in ROS inhibits H2O2 dependent lignin polymerization in CA2 over-expressing plants, thereby causing male sterility.