QQT proteins colocalize with microtubules and are essential for early embryo development in Arabidopsis

During Arabidopsis embryogenesis, the control of division between daughter cells is critical for pattern formation. Two embryo-defective (emb) mutant lines named quatre-quart (qqt) were characterized by forward and reverse genetics. The terminal arrest of qqt1 and qqt2 embryos was at the octant stage, just prior to the round of periclinal divisions that establishes the dermatogen stage . Homozygous embryos of a weaker allele of qqt1 were able to divide further, resulting in aberrant periclinal divisions. These phenotypic analyses support an essential role of the QQT proteins in the correct formation of the tangential divisions. That an important proportion of qqt1 embryos were arrested prior to the octant stage indicated a more general role in cell division. The analysis of QQT1 and QQT2 genes revealed that they belong to a small subgroup of the large family encoding ATP/GTP binding proteins, and are widely conserved among plants, vertebrates and Archaea. We showed that QQT1 and QQT2 proteins interact with each other in a yeast two-hybrid system, and that QQT1 and QQT2 tagged by distinct fluorescent probes colocalize with microtubules during mitosis, in agreement with their potential role in cell division and their mutant phenotype. We propose that QQT1 and QQT2 proteins participate in the organization of microtubules during cell division, and that this function is essential for the correct development of the early embryo.     

Fluorescence decay curves under pulsed excitation: distinction between free and bound NAD(P)H on isolated living cells

Under pulsed excitation the decay curve of the intrinsic cellular fluorescence has been recorded using an original equipment. Cell by cell experiments are feasible so that the NAD(P)H bound/free ratio and the relaxation time characteristic of bound NAD(P)H can be used in order to build up histograms characterizing the cell population.     

Cloning,Sequencing, Expression and Structural Investigation of Mnemiopsin from <Emphasis Type="Italic">Mnemiopsis leidyi</Emphasis>: An Attempt Toward Understanding Ca<Superscript>2+</Superscript>-Regulated Photoproteins

A comparison of the two most famous groups of calcium-regulated photoproteins, cnidarians and ctenophores, showed unexpectedly high degree of structural similarity regardless of their low sequence identity. It was suggested these photoproteins can play an important role in understanding the structural basis of bioluminescence activity. Based on this postulate, in this study the cDNA of mnemiopsin from luminous ctenophore Mnemiopsis leidyi was cloned, expressed, purified and sequenced. The purified cDNA, with 621 base pairs, coded a 206 residues protein. Sequence of mnemiopsin showed 93.5 and 51% similarity to other ctenophore proteins and cnidarians, respectively. The cDNA encoding apo-mnemiopsin of M. leidyi was expressed in Escherichia coli. The purified apo-protein showed a single band on SDS-PAGE (molecular weight ~27 kDa). A semi-synthetic mnemiopsin was prepared using coelenterazine and EDTA and its luminescence activity was measured in the presence of CaCl₂. The results showed an optimum pH of 9.0 and lower calcium sensitivity compared to aequorin. Comparison of amino acid residues in substrate binding site indicated that binding pocket of ctenophores contains less aromatic residues than cnidarians. This can lead to a decline in the number of stacking interactions between substrate and protein which can affect the stability of coelenterazine in binding cavity. Structural comparison of photoproteins with low sequence identity and high 3D structural similarity, can present a new insight into the mechanism of light emission in photoproteins.     

Molecular characterization of Iranian Trichogrammatids (Hymenoptera: Trichogrammatidae) and their Wolbachia endosymbiont

During 2009–2010, a field survey of native Trichogramma species was carried out in six provinces of Iran, including Khorasan Razavi, Tehran, Mazandaran, Guilan, Golestan, and Qom. In this study, a molecular method for identifying Trichogramma and for determining the prevalence of Wolbachia in those species was used. Based on ITS2 (internal transcribed spacer 2) sequence, 14 populations were identified as the species T. embryophagum, T. evanescens, or T. brassicae. Wolbachia infection in these Trichogrammatids was detected using wsp gene sequencing. The highest infection rates in Trichogramma were found in Mazandaran and Golestan provinces. There was no evidence of infection in Trichogramma species in Guilan and Qom provinces. Of the three infected populations, two populations of T. evanescens were infected with only one Wolbachia strain from sib subgroup and one population was superinfected. Here, we report the first data on molecular characterization of Iranian Trichogrammatids and their Wolbachia-endosymbionts.     

RNA‐directed DNA methylation requires an AGO4‐interacting member of the SPT5 elongation factor family

Recent studies have identified a conserved WG/GW‐containing motif, known as the Argonaute (AGO) hook, which is involved in the recruitment of AGOs to distinct components of the eukaryotic RNA silencing pathways. By using this motif as a model to detect new components in plant RNA silencing pathways, we identified SPT5‐like, a plant‐specific AGO4‐interacting member of the nuclear SPT5 (Suppressor of Ty insertion 5) RNA polymerase (RNAP) elongation factor family that is characterized by the presence of a carboxy‐terminal extension with more than 40 WG/GW motifs. Knockout SPT5‐like mutants show a decrease in the accumulation of several 24‐nt RNAs and hypomethylation at different loci revealing an implication in RNA‐directed DNA methylation (RdDM). Here, we propose that SPT5‐like emerged in plants as a facultative RNAP elongation factor. Its plant‐specific origin and role in RdDM might reflect functional interactions with plant‐specific RNA Pols required for RdDM.     

DOMINO1, a member of a small plant-specific gene family, encodes a protein essential for nuclear and nucleolar functions

Arabidopsis embryos carrying the domino1 mutation grow slowly in comparison with wild type embryos and as a consequence reach only the globular stage at desiccation. The primary defect of the mutation at the cellular level is the large size of the nucleolus that can be observed soon after fertilization in the nuclei of both the embryo and the endosperm. The ultrastructure of mutant nucleoli is drastically different from wild type and points to a fault in ribosome biogenesis. DOMINO1 encodes a protein, which belongs to a plant-specific gene family sharing a common motif of unknown function, present in the tomato DEFECTIVE CHLOROPLASTS AND LEAVES (LeDCL) protein. Using a GFP protein fusion, we show that DOMINO1 is targeted to the nucleus. We propose that inactivation of DOMINO1 has a negative effect on ribosome biogenesis and on the rate of cell division.     

A chloroplastic RNA-binding protein is a new member of the PPR family

P67, a new protein binding to a specific RNA probe, was purified from radish seedlings [Echeverria, M. and Lahmy, S. (1995) Nucleic Acids Res. 23, 4963–4970]. Amino acid sequence information obtained from P67 microsequencing allowed the isolation of genes encoding P67 in radish and Arabidopsis thaliana. Immunolocalisation experiments in transfected protoplasts demonstrated that this protein is addressed to the chloroplast. The RNA-binding activity of recombinant P67 was found to be similar to that of the native protein. A significant similarity with the maize protein CRP1 [Fisk, D.G., Walker, M.B. and Barkan, A. (1999) EMBO J. 18, 2621–2630] suggests that P67 belongs to the PPR family and could be involved in chloroplast RNA processing.     

Host-induced gene silencing of Pectobacterium carotovorum quorum sensing gene enhances soft rot disease resistance in potato plants

Quorum sensing is a regulatory mechanism of gene expression in bacteria which regulated many physiological processes such as production of virulence factors in Pectobacterium carotovorum. In this research, the plant expression vector, pBI121, harbouring the anti-quorum sensing gene, aiiA, was transformed into Potato (Solanum tuberosum cv. Agria) explants by Agrobacterium mediated transformation procedure and putative transformants containing pBI121/aiiA were obtained. The results of bioassay test revealed that the expression of aiiA gene in potato plant induces the resistance to the early stage of bacterium pathogenesis. The soft rot symptoms such as wilting, yellowing and tissue maceration were not observed until 48?hr after inoculation of transgenic potatoes with P. carotovorum. Although in non-transgenic plants, the disease symptoms were appeared after 24?hr of plants infection. Hence, this study proves that the heterologous expression of aiiA gene enhances the resistance against potato soft rot disease.