The Effects of Phytohormones and 5-Azacytidine on Apoptosis in Etiolated Wheat Seedlings

The development of etiolated wheat (Triticum aestivum L.) seedlings is necessarily accompanied by apoptosis in their coleoptiles and first leaves. Internucleosome DNA fragmentation, which is characteristic of apoptosis, was detected in the coleoptile as soon as six days after germination. After eight days of germination, DNA fragmentation was clearly expressed in the coleoptile and was noticeable in the apical part of the first-leaf blade. Growing of intact seedlings or incubation of their shoots in the presence of such phytohormones as benzyladenine, gibberellin A₃, fusicoccin C, and 2,4-D at the concentration of 10^–5 M did not essentially affect DNA fragmentation in the coleoptile. As distinct from antioxidants, none of the phytohormones used prevented apoptosis in wheat seedlings. In contrast, ABA (10^–5 M) and an ethylene producer, ethrel (2-chloroethylphosphonic acid, 10^–2–10^–3 M), stimulated sharply DNA fragmentation in the coleoptile. An inhibitor of DNA methylation, 5-azacytidine, was very efficient in the stimulation of DNA fragmentation in the coleoptiles of eight-day-old seedlings at its concentration of 100 g/ml. Thus, some phytohormones can regulate apoptosis, and DNA methylation is involved in this process. Our results indicate that apoptosis activation by some phytohormones may be mediated by their regulation of DNA methylation/demethylation, which is responsible for the induction of genes encoding apoptogenic proteins and/or the repression of antiapoptotic genes.     

Exogenous Spermidine Promotes Somatic Embryogenesis of <i>Cunninghamia lanceolata</i> by Altering the Endogenous Phytohormone Content

In order to study how exogenous hormones in C. lanceolata (gymnosperm) regulate somatic embryogenesis, we measured the endogenous phytohormones of two genotypes with different somatic embryogenesis efficiency and found that an increase in endogenous concentrations of IAA and ABA may be correlated to more efficient somatic embryogenesis. By applying exogenous spermidine, we found that exogenous hormones may affect somatic embryogenesis efficiency through affecting the endogenous phytohormone content. Based on these results, further studies can be conducted whereby the concentration of exogenous hormones or the levels of endogenous phytohormones by molecular methods are regulated to promote somatic embryogenesis. Our research may benefit the long-term economic output of the forestry industry and lays the foundation to studying the molecular mechanism that controls somatic embryogenesis efficiency.     

Plant Parasitic Nematodes: Habitats, Hormones, and Horizontally-Acquired Genes

Abstract Plant parasitic nematodes are ubiquitous and cosmopolitan pathogens of vascular plants and exploit all parts of the roots and shoots, causing substantial crop damage. Nematodes deploy a broad spectrum of feeding strategies, ranging from simple grazing to the establishment of complex cellular structures (including galls) in host tissues. Various models of feeding site formation have been proposed, and a role for phytohormones has long been speculated, although whether they perform a primary or secondary function is unclear. On the basis of recent molecular evidence, we present several scenarios involving phytohormones in the induction of giant cells by root-knot nematode. The origin of parasitism by nematodes, including the acquisition of genes to synthesize or modulate phytohormones also is discussed, and models for horizontal gene transfer are presented.     

Physical characterization of plasmids from Streptomyces kasugaensis MB273

Plasmid DNA of molecular weight 6.8 × 10⁶ was isolated from Streptomyces kasugaensis MB273. The plasmid DNA showed a single CsCl-ethidium bromide density gradient centrifugation, in neutral sucrose gradient centrifugation, and in agarose gel electrophoresis. When this DNA was digested with BamHI or SalI endonucleases, an unexpected number of fragments were found on agarose gel electrophoresis. Molecular weight summation of fragments obtained from double restriction enzyme digestions suggested that the plasmid DNA was a mixture of two different plasmids. This was confirmed by constructing recombinant plasmids between S. kasugaensis plasmid DNA and pBR322, and then by isolating two plasmids after SalI endonuclease treatment followed by sucrose gradient centrifugation. One of the plasmids (pSK1) had a single recognition site for BamHI, EcoRI, and SalI, and three sites for BglII. The other plasmid (pSK2) had a single recognition site for EcoRI and BglII, two recognition sites for BamHI, and no cleavage site for SalI. The cleavage maps of these plasmids were constructed using several restriction endonucleases.     

植物激素调控丛枝菌根发育的作用机制研究进展

丛枝菌根（arbuscular mycorrhiza,AM）是土壤中AM真菌和绝大多数维管植物根系长期进化过程中相互识别、相互作用形成的互利共生体。AM的发育与功能效应依赖AM真菌-寄主植物之间精准的“分子对话”,同时受到环境条件特别是土壤养分水平、干旱和盐渍化的制约。植物激素作为低浓度的小分子有机物,是参与调控AM共生过程的重要信号分子。其中,主要有9种植物激素参与AM发育过程且分工各有不同：独脚金内酯（strigolactones,SLs）参与AM真菌-寄主植物之间最初的共生识别,脱落酸（abscisic acid,ABA）和油菜素内酯（brassinosteroid,BR）促进前期的菌丝入侵,但水杨酸（salicylic acid,SA）和乙烯（ethylene,ET）抑制前期的菌丝入侵,生长素（auxin,Aux）、ABA和BR促进随后的丛枝形成而ET和赤霉素（gibberellin,GA）的作用则相反,茉莉酸（jasmonic acid,JA）对菌丝入侵与丛枝形成均可能存在正调控或负调控作用。目前细胞分裂素（cytokinin,CTK）在AM发育中的作用尚不明确。更为复杂的是,通常植物激素信号之间的交叉互作决定AM的发育进程。本文针对AM发育过程总结了不同植物激素的调控作用特点和不同植物激素信号之间的互作（协同或拮抗）,以及胁迫条件下不同植物激素信号的可能调控机制。深入研究和系统阐明植物激素调控AM真菌-寄主植物共生的生理/分子机制,将有助于促进生物共生学理论研究及菌根技术的应用。     

Possible involvement of DNA-repair events in the transdifferentiation of mesophyll cells ofZinnia elegans into tracheary elements

In cultures of isolated mesophyll cells ofZinnia elegans, transdifferentiation into tracheary elements is induced by a combination of auxin and cytokinin and is blocked by inhibitors of DNA synthesis and poly (ADP-ribose) synthesis. During transdifferentiation, a very low level of synthesis of nuclear DNA was found in some cultured cells by microautoradiography after pulse-labeling with [³H]thymidine. Density profiles of nuclear DNA that had been double-labeledin vivo with bromodeoxyuridine (BrdU) and [³H]thymidine indicated that this DNA synthesis was repair-type synthesis. The sedimentation velocity of nucleoids increased during the culture of isolated mesophyll cells and the increase was dependent on phytohormones. This phenomenon may reflect the rejoining of DNA strand breaks after repair-type DNA synthesis during transdifferentiation. Treatment of cells with inhibitors of DNA synthesis or of poly(ADP-ribose) synthesis prevented the increase in the sedimentation velocity of nucleoids. The data suggest the involvement of DNA-repair events in the transdifferentiation of mesophyll cells into tracheary elements.     

DNA–DNA sequence homology recognition: physical mechanisms and open questions

First, we summarize recent experimental facts on homologous DNA pairing in vitro and discuss possible mechanisms of DNA–DNA sequence recognition. Then, we overview the mechanism of DNA–DNA recognition based on complementarity of DNA charge patterns. The theory predicts the recognition energy up to 10 k_BT for close parallel homologous DNA fragments of gene‐relevant lengths. We argue why this estimate cannot be directly applied to pairing of homologous DNA loci in experiments on yeast chromosomes. Namely, DNA–DNA distances assessed from experiments are much larger than those typically used in theory. Finally, we suggest some experiments that could help to judge whether short‐range electrostatic forces indeed govern DNA pairing. This viewpoint paper introduces recently developed theoretical concepts to molecular biologists, with a hope to generate a junction between theory and future experiments on DNA recognition. Copyright © 2010 John Wiley & Sons, Ltd.     

A Mycobacterium leprae-specific gene encoding an immunologically recognized 45 kDa protein

By screening a Mycobacterium leprae lambda gt11 expression library with a serum from an Ethiopian lepromatous leprosy (LL) patient a clone was isolated (LL4) belonging to hybridization group III of a panel of previously isolated M. leprae clones. Members of this hybridization group encode a serologically recognized 45 kDa protein. The complete DNA sequences of the partially overlapping clones LL4 and L1 (hybridization group III) are presented and these revealed the presence of an open reading frame (ORF) predicting a protein with a molecular size of 42 448 Da. Southern hybridizations on total genomic DNA of M. Ieprae, Mycobacterium tuberculosis and eight atypical mycobacteria showed that the LL4 DNA fragment is specific for M. Ieprae DNA even under low-stringency conditions. The M. Ieprae specificity of LL4 DNA was further confirmed by the polymerase chain reaction using four different sets of primers. Western blotting analyses showed that the M. Ieprae 45 kDa protein is frequently recognized by antibodies from leprosy patients and that this recognition is specific since no antibodies could be detected in sera of tuberculosis patients. T-cell proliferation assays also demonstrated T-cell recognition by leprosy patients and healthy contacts of the M. Ieprae 45 kDa protein. The specificity of the LL4 DNA region and the 45 kDa antigen that is encoded by hybridization group III could provide unique tools for the development of M. Ieprae-specific immunological and DNA reagents.     

A Single-Amino-Acid Substitution in the C Terminus of PhoP Determines DNA-Binding Specificity of the Virulence-Associated Response Regulator from Mycobacterium tuberculosis

The Mycobacterium tuberculosis PhoP-PhoR two-component system is essential for virulence in animal models of tuberculosis. Genetic and biochemical studies indicate that PhoP regulates the expression of more than 110 genes in M. tuberculosis. The C-terminal effector domain of PhoP exhibits a winged helix-turn-helix motif with the molecular surfaces around the recognition helix (α8) displaying strong positive electrostatic potential, suggesting its role in DNA binding and nucleotide sequence recognition. Here, the relative importance of interfacial α8-DNA contacts has been tested through rational mutagenesis coupled with in vitro binding-affinity studies. Most PhoP mutants, each with a potential DNA contacting residue replaced with Ala, had significantly reduced DNA binding affinity. However, substitution of nonconserved Glu215 had a major effect on the specificity of recognition. Although lack of specificity does not necessarily correlate with gross change in the overall DNA binding properties of PhoP, structural superposition of the PhoP C-domain on the Escherichia coli PhoB C-domain-DNA complex suggests a base-specific interaction between Glu215 of PhoP and the ninth base of the DR1 repeat motif. Biochemical experiments corroborate these results, showing that DNA recognition specificity can be altered by as little as a single residue change of the protein or a single base change of the DNA. The results have implications for the mechanism of sequence-specific DNA binding by PhoP.     

Homology modeling and molecular dynamics simulations of HgiDII methyltransferase in complex with DNA and S-adenosyl-methionine: Catalytic mechanism and interactions with DNA

M.HgiDII is a methyltransferase (MTase) from Herpetosiphon giganteus that recognizes the sequence GTCGAC. This enzyme belongs to a group of MTases that share a high degree of amino acid similarity, albeit none of them has been thoroughly characterized. To study the catalytic mechanism of M.HgiDII and its interactions with DNA, we performed molecular dynamics simulations with a homology model of M.HgiDII complexed with DNA and S-adenosyl-methionine. Our results indicate that M.HgiDII may not rely only on Glu119 to activate the cytosine ring, which is an early step in the catalysis of cytosine methylation; apparently, Arg160 and Arg162 may also participate in the activation by interacting with cytosine O2. Another residue from the catalytic site, Val118, also played a relevant role in the catalysis of M.HgiDII. Val118 interacted with the target cytosine and kept water molecules from accessing the region of the catalytic pocket where Cys79 interacts with cytosine, thus preventing water-mediated disruption of interactions in the catalytic site. Specific recognition of DNA was mediated mainly by amino acids of the target recognition domain, although some amino acids (loop 80–88) of the catalytic domain may also contribute to DNA recognition. These interactions involved direct contacts between M.HgiDII and DNA, as well as indirect contacts through water bridges. Additionally, analysis of sequence alignments with closely related MTases helped us to identify a motif in the TRD of M.HgiDII that may be relevant to specific DNA recognition.     

Understanding and optimizing the addition of phytohormones in the culture of microalgae for lipid production

Some studies have described the use of phytohormones in microalgal culture for the production of biodiesel or selected fatty acids. However, no study has determined the amount of phytohormones that maximizes lipid yield. We determined the optimal concentration of auxins and gibberellins (which is between 40 and 60 μM) in two strains (Scenedemus abundans and Chlorella ellipsoidea) suitable for biodiesel production. More than 3‐fold increment was reached with S. abundans and near 7‐fold increment with C. ellipsoidea. Furthermore, this work suggests that the improved growth of the microalgae in the presence of the phytohormones was due to a reduction in the level of reactive oxygen species (ROS) in the cells. An economic analysis showed that, due to its low cost, auxin offers a positive cost‐benefit balance and therefore could be used at large scale. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1203–1211, 2016     

cGAS is activated by DNA in a length‐dependent manner

Cytosolic DNA stimulates innate immune responses, including type I interferons (IFN), which have antiviral and immunomodulatory activities. Cyclic GMP‐AMP synthase (cGAS) recognizes cytoplasmic DNA and signals via STING to induce IFN production. Despite the importance of DNA in innate immunity, the nature of the DNA that stimulates IFN production is not well described. Using low DNA concentrations, we show that dsDNA induces IFN in a length‐dependent manner. This is observed over a wide length‐span of DNA, ranging from the minimal stimulatory length to several kilobases, and is fully dependent on cGAS irrespective of DNA length. Importantly, in vitro studies reveal that long DNA activates recombinant human cGAS more efficiently than short DNA, showing that length‐dependent DNA recognition is an intrinsic property of cGAS independent of accessory proteins. Collectively, this work identifies long DNA as the molecular entity stimulating the cGAS pathway upon cytosolic DNA challenge such as viral infections.     

The binding process of a nonspecific enzyme with DNA

Protein-DNA recognition of a nonspecific complex is modeled to understand the nature of the transient encounter states. We consider the structural and energetic features and the role of water in the DNA grooves in the process of protein-DNA recognition. Here we have used the nuclease domain of colicin E7 (N-ColE7) from Escherichia coli in complex with a 12-bp DNA duplex as the model system to consider how a protein approaches, encounters, and associates with DNA. Multiscale simulation studies using Brownian dynamics and molecular-dynamics simulations were performed to provide the binding process on multiple length- and timescales. We define the encounter states and identified the spatial and orientational aspects. For the molecular length-scales, we used molecular-dynamics simulations. Several intermediate binding states were found, which have different positions and orientations of protein around DNA including major and minor groove orientations. The results show that the contact number and the hydrated interfacial area are measures that facilitate better understanding of sequence-independent protein-DNA binding landscapes and pathways.     

Searching for a Common Centromeric Structural Motif: Drosophila Centromeric Satellite DNAs Show Propensity to form Telomeric-Like Unusual DNA Structures

The molecular basis of centromere formation in a particular chromosomal region is not yet understood. In higher eukaryotes, no specific DNA sequence is required for the assembly of the kinetochore, but similar centromeric chromatins are formed on different centromere DNA sequences. Although epigenesis has been proposed as the main mechanism for centromere specification, DNA recognition must also play a role. Through the analysis of Drosophilacentromeric DNA sequences, we found that dodeca satellite and 18HT satellite are able to form unusual DNA structures similar to those formed by telomeric sequences. These findings suggest the existence of a common centromeric structural DNA motif which we feel merits further investigation. This revised version was published online in July 2006 with corrections to the Cover Date.     

EVALUATION OF PLANT GROWTH REGULATORS TO INCREASE NICKEL PHYTOEXTRACTION BY ALYSSUM SPECIES

Recent studies have shown that application of phytohormones to shoots of Alyssum murale increased biomass production but did not increase Ni shoot concentration. Increased biomass and Ni phytoextraction efficiency is useful to achieve economically viable phytomining. The objective of this study was to evaluate the effect of two types of phytohormones on the Ni phytoextraction capacity of four Alyssum species. Two different commercially available phytohormones (Cytokin® and Promalin®) based on cytokinins and/or gibberellins were applied on shoot biomass of four Ni hyperaccumulating Alyssum species (A. corsicum, A. malacitanum, A. murale, and A. pintodasilvae). Cytokin was applied in two concentrations and promalin in one concentration. The application of phytohormones had no clear positive effect on biomass production, Ni accumulation and Ni phytoextraction efficiency in the studied Alyssum species. A. malacitanum was the only species in which a significantly negative effect of these treatments was observed (in Ni uptake). A slightly positive response to promalin treatment was observed in the biomass production and Ni phytoextraction efficiency of A. corsicum. Although this effect was not significant it does indicate a potential application of these approaches to improve phytoextraction ability. Further studies will be needed to identify the most adequate phytohormone treatment as well as the appropriate concentrations and application times.