MCU proteins dominate in vivo mitochondrial Ca2+ uptake in Arabidopsis roots

Ca²⁺ signaling is central to plant development and acclimation. While Ca²⁺-responsive proteins have been investigated intensely in plants, only a few Ca²⁺-permeable channels have been identified, and our understanding of how intracellular Ca²⁺ fluxes is facilitated remains limited. Arabidopsis thaliana homologs of the mammalian channel-forming mitochondrial calcium uniporter (MCU) protein showed Ca²⁺ transport activity in vitro. Yet, the evolutionary complexity of MCU proteins, as well as reports about alternative systems and unperturbed mitochondrial Ca²⁺ uptake in knockout lines of MCU genes, leave critical questions about the in vivo functions of the MCU protein family in plants unanswered. Here, we demonstrate that MCU proteins mediate mitochondrial Ca²⁺ transport in planta and that this mechanism is the major route for fast Ca²⁺ uptake. Guided by the subcellular localization, expression, and conservation of MCU proteins, we generated an mcu triple knockout line. Using Ca²⁺ imaging in living root tips and the stimulation of Ca²⁺ transients of different amplitudes, we demonstrated that mitochondrial Ca²⁺ uptake became limiting in the triple mutant. The drastic cell physiological phenotype of impaired subcellular Ca²⁺ transport coincided with deregulated jasmonic acid-related signaling and thigmomorphogenesis. Our findings establish MCUs as a major mitochondrial Ca²⁺ entry route in planta and link mitochondrial Ca²⁺ transport with phytohormone signaling.

Monitoring of subcellular Ca²⁺ dynamics in living Arabidopsis roots reveals that MCU proteins provide the dominant mitochondrial Ca²⁺ uptake mechanism in vivo.     

The CD95/CD95 ligand system is not the major effector in anticancer drug-mediated apoptosis

Many anticancer drugs are able to induce apoptosis in tumor cells but the mechanisms underlying this phenomenon are poorly understood. Some authors reported that the p53 tumor suppressor gene may be responsible for drug-induced apoptosis; however, chemotherapy-induced apoptosis can also be observed in p53 negative cells. Recently, doxorubicin (DXR) was reported to induce CD95L expression to mediate apoptosis through the CD95/CD95L system. Thus, an impairment of such a system may be involved in drug resistance. We evaluated the in vitro antitumor activity of several cytotoxic drugs on two human p53-negative T-cell lymphoma cell lines, the HUT78-B1 CD95L-resistant cell line and the HUT78 parental CD95L-sensitive cell line. We demostrated by Western blotting assay that DXR and etoposide (VP-16) were able to induce CD95L expression after 4 h of treatment. In contrast, they were unable to induce the expression of p53. DXR, at concentrations ranging from 0.001 - 1 microg/ml, and VP16, at concentrations ranging from 0.05 - 1 microg/ml, were equally cytotoxic and induced apoptosis in both cell lines as assessed by fluorescence microscopy and flow cytometry analyses. Although we observed a slightly reduced percentage of apoptotic cells in HUT78B1 when compared with the parental HUT78 cells after few hours of drug exposure, this difference was no longer evident at 48 or 72 h. Similarly, the exposure of HUT78 cells to a CD95-blocking antibody partially reduced early apoptosis (24 h) without affecting the long-term effects of the drugs including cytotoxicity. Furthermore, as observed with DXR and VP-16, both the CD95L-sensitive and the CD95L-resistant cell lines resulted equally sensitive to the cytotoxic effects of a number of different cytotoxic drugs (vincristine, camptothecin, 5-fluorouracil and methotrexate). The treatment with the Caspase-3 tetrapeptide aldehyde inhibitor, Ac-DEVD-CHO, did not affect the DXR-induced apoptosis whereas it only modestly inhibited apoptosis and cytotoxicity of VP-16, while Z-VAD.FMK, a Caspase inhibitor that prevents the processing of Caspase-3 to its active form, was able to block DXR-induced apoptosis at 24 h but not at 48 h. Thus, our results do not confirm a crucial role for the CD95/CD95L system in drug-induced apoptosis and suggest the involvement of alternative p53-independent pathways at least in this experimental model system.     

Ribosomal RNA genes ofPhaseolus coccineus V. Relationship between rDNA phenotype and somatic differentiation

Summary Size variations in the intergenic spacer of ribosomal DNA were detected between individual plants of openly pollinatedPhaseolus coccineus. Eleven days after sowing, two plant samples were examined: slowly developing plants with a length less than 40 cm; and fast developing plants with a length greater than 70 cm. The two samples were characterized by different plant weight and, at maturity, by highly distinctive seed yield. They also exhibited distinct patterns of protein expression as analyzed by 2-D electrophoresis. In particular a 38 kDa protein, related to malate dehydrogenase on the basis of its N-terminal sequence, was present at higher concentration and higher activity levels in fast developing plants. Intergenic spacer length variants were detected in both samples at approximately 180 bp intervals. More than one spacer length variant was present in each individual plant. At least 13 different intergenic spacer hybridization patterns were in fact detected: some patterns occurred equally in both slowly and fast developing samples while the majority of patterns was significantly different between the two samples.Abbreviations FDP fast developing plants - IGS intergenic spacer - MDH malate dehydrogenase - rDNA SLV spacer length variant of ribosomal DNA - SDP slowly developing plants     

The Athb-1 and -2 HD-Zip domains homodimerize forming complexes of different DNA binding specificities.

The Arabidopsis Athb-1 and -2 proteins are characterized by the presence of a homeodomain (HD) with a closely linked leucine zipper motif (Zip). We have suggested that the HD-Zip motif could, via dimerization of the leucine zippers, recognize dyad-symmetric DNA sequences. Here we report an analysis of the DNA binding properties of the Athb-1 homeodomain-leucine zipper (HD-Zip-1) domain in vitro. DNA binding analysis performed using random-sequence DNA templates showed that the HD-Zip-1 domain, but not the Athb-1 HD alone, binds to DNA. The HD-Zip-1 domain recognizes a 9 bp dyad-symmetric sequence [CAAT(A/T)ATTG], as determined by selecting high-affinity binding sites from random-sequence DNA. Gel retardation assays demonstrated that the HD-Zip-1 domain binds to DNA as a dimer. Moreover, the analysis of the DNA binding activity of Athb-1 derivatives indicated that a correct spatial relationship between the HD and the Zip is essential for DNA binding. Finally, we determined that the Athb-2 HD-Zip domain recognizes a distinct 9 bp dyad-symmetric sequence [CAAT(G/C)ATTG]. A model of DNA binding by the HD-Zip proteins is proposed.     

Chromatin assembly in Xenopus oocytes: In vitro studies

We describe and characterize a complex reaction that catalyzes DNA supercoiling and chromatin assembly in vitro. A Xenopus oocyte extract supplemented with ATP and Mg⁺⁺ converts DNA circles into minichromosomes that display a native, 200 bp periodicity. When supercoiled DNA is added to this extract it undergoes a time-dependent series of topological changes, which precisely mimic those found when the DNA is microinjected into oocytes. As judged by the conformation of the subsequently deproteinized DNA, the supercoiled DNA is first relaxed, in a reaction that takes 4 min, and then it is resupercoiled in a slower process that takes 4 hr. The relaxation is partially inhibited by EDTA, to an extent that suggests that that it is catalyzed by a type I DNA topoisomerase. The resupercoiling, on the other hand, requires ATP and Mg⁺⁺, is completely inhibited by EDTA, and is inhibited by novobiocin in a manner that suggests it is catalyzed by a type II DNA topoisomerase. These findings, and the ones reported in the preceding paper (Ryoji and Worcel, 1984), lead us to propose that chromatin assembly is an active, ATP-driven process.