Consumer acceptance of food crops developed by genome editing

One of the major problems regarding consumer acceptance of genetically modified organisms (GMOs) is the possibility that their transgenes could have adverse effects on the environment and/or human health. Genome editing, represented by the CRISPR/Cas9 system, can efficiently achieve transgene-free gene modifications and is anticipated to generate a wide spectrum of plants. However, the public attitude against GMOs suggests that people will initially be unlikely to accept these plants. We herein explored the bottlenecks of consumer acceptance of transgene-free food crops developed by genome editing and made some recommendations. People should not pursue a zero-risk bias regarding such crops. Developers are encouraged to produce cultivars with a trait that would satisfy consumer needs. Moreover, they should carefully investigate off-target mutations in resultant plants and initially refrain from agricultural use of multiplex genome editing for better risk–benefit communication. The government must consider their regulatory status and establish appropriate regulations if necessary. The government also should foster communication between the public and developers. If people are informed of the benefits of genome editing-mediated plant breeding and trust in the relevant regulations, and if careful risk–benefit communication and sincere considerations for the right to know approach are guaranteed, then such transgene-free crops could gradually be integrated into society.     

Evaluation of the catalytic specificity,biochemical properties,and milk clotting abilities of an aspartic peptidase from <Emphasis Type="Italic">Rhizomucor miehei</Emphasis>

In this study, we detail the specificity of an aspartic peptidase from Rhizomucor miehei and evaluate the effects of this peptidase on clotting milk using the peptide sequence of k-casein (Abz-LSFMAIQ-EDDnp) and milk powder. Molecular mass of the peptidase was estimated at 37 kDa, and optimum activity was achieved at pH 5.5 and 55 °C. The peptidase was stable at pH values ranging from 3 to 5 and temperatures of up 45 °C for 60 min. Dramatic reductions in proteolytic activity were observed with exposure to sodium dodecyl sulfate, and aluminum and copper (II) chloride. Peptidase was inhibited by pepstatin A, and mass spectrometry analysis identified four peptide fragments (TWSISYGDGSSASGILAK, ASNGGGGEYIFGGYDSTK, GSLTTVPIDNSR, and GWWGITVDRA), similar to rhizopuspepsin. The analysis of catalytic specificity showed that the coagulant activity of the peptidase was higher than the proteolytic activity and that there was a preference for aromatic, basic, and nonpolar amino acids, particularly methionine, with specific cleavage of the peptide bond between phenylalanine and methionine. Thus, this peptidase may function as an important alternative enzyme in milk clotting during the preparation of cheese.     

HmuS and HmuQ of <Emphasis Type="Italic">Ensifer</Emphasis>/<Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> degrade heme in vitro and participate in heme metabolism in vivo

Ensifer meliloti is a nitrogen-fixing symbiont of the alfalfa legume able to use heme as an iron source. The transport mechanism involved in heme acquisition in E. meliloti has been identified and characterized, but the fate of heme once inside the cell is not known. In silico analysis of E. meliloti 1021 genome revealed no canonical heme oxygenases although two genes encoding putative heme degrading enzymes, smc01518 and hmuS, were identified. SMc01518 is similar to HmuQ of Bradyrhizobium japonicum, which is weakly homologous to the Staphylococcus aureus IsdG heme-degrading monooxygenase, whereas HmuS is homolog to Pseudomonas aeruginosa PhuS, a protein reported as a heme chaperone and as a heme degrading enzyme. Recombinant HmuQ and HmuS were able to bind hemin with a 1:1 stoichiometry and displayed a K_d value of 5 and 4 µM, respectively. HmuS degrades heme in vitro to the biliverdin isomers IX-β and IX-δ in an equimolar ratio. The HmuQ recombinant protein degrades heme to biliverdin IX-δ only. Additionally, in this work we demonstrate that humS and hmuQ gene expression is regulated by iron and heme in a RirA dependent manner and that both proteins are involved in heme metabolism in E. meliloti in vivo.     

No need to breed for enhanced colonization by arbuscular mycorrhizal fungi to improve low-P adaptation of West African sorghums

Background and aims

Herbaspirillum seropedicae (Hs) Z67 a diazotrophic endophyte was genetically engineered for secretion of 2-keto-D-gluconic acid by heterologous expression of genes for pqq synthesis and gluconate dehydrogenase to study its beneficial effect on plants.

Methods

Two plasmids, pJNK5, containing a 5.1 Kb pqq gene cluster of Acinetobacter calcoaceticus and pJNK6, carrying in addition the Pseudomonas putida KT2440 gluconate dehydrogenase (gad) operon were constructed in pUCPM18Gm^r under Plac promoter. H. seropedicae Z67 transformants were monitored for P and K solubilization, cadmium (Cd) tolerance and rice growth promotion.

Results

Hs (pJNK5) secreted 23.5 mM gluconic acid and Hs (pJNK6) secreted 3.79 mM gluconic acid and 15.8 mM 2-ketogluconic acid respectively. Under aerobic conditions, Hs (pJNK5) and Hs (pJNK6) solubilized 239.7 μM and 457.7 μM P on HEPES rock phosphate and, 76.7 μM and 222.7 μM K on HRPF (feldspar), respectively, in minimal medium containing 50 mM glucose. Under N free minimal medium, similar effects of P and K solubilization were obtained. Hs (pJNK5) and Hs (pJNK6) inoculation increased the biomass, N, P, K content of rice plants (Gujarat – 17). These plants also accumulated 0.73 ng/g PQQ, and had improved growth and tolerance to CdCl₂.

Conclusions

Incorporation of pqq and gad gene clusters in H. seropedicae Z67 imparted additional plant growth promoting traits of P and K solubilization and ability to alleviate Cd toxicity to the host plant.

     

Isolation of a <Emphasis Type="Italic">Latimeria menadoensis</Emphasis><Emphasis Type="Italic">heat shock protein 70</Emphasis> (<Emphasis Type="Italic">Lmhsp70</Emphasis>) that has all the features of an inducible gene and encodes a functional molecular chaperone

Molecular chaperones facilitate the correct folding of other proteins, and heat shock proteins form one of the major classes of molecular chaperones. Heat shock protein 70 (Hsp70) has been extensively studied, and shown to be critically important for cellular protein homeostasis in almost all prokaryotic and eukaryotic systems studied to date. Since there have been very limited studies conducted on coelacanth chaperones, the main objective of this study was to genetically and biochemically characterize a coelacanth Hsp70. We have successfully isolated an Indonesian coelacanth (L. menadoensis) hsp70 gene, Lmhsp70, and found that it contained an intronless coding region and a potential upstream regulatory region. Lmhsp70 encoded a typical Hsp70 based on conserved structural and functional features, and the predicted upstream regulatory region was found to contain six potential promoter elements, and three potential heat shock elements (HSEs). The intronless nature of the coding region and the presence of HSEs suggested that Lmhsp70 was stress-inducible. Phylogenetic analyses provided further evidence that Lmhsp70 was probably inducible, and that it branched as a clade intermediate between bony fish and tetrapods. Recombinant LmHsp70 was successfully overproduced, purified and found to be functional using ATPase activity assays. Taken together, these data provide evidence for the first time that the coelacanth encodes a functional molecular chaperone system. K. W. Modisakeng and M. Jiwaji contributed equally to this study.     

Somatic Embryogenesis and in vitro Flowering in <Emphasis Type="Italic">Saposhnikovia divaricata</Emphasis>

Efficient somatic embryogenesis (SE) and in vitro flowering and fruiting were achieved in Saposhnikovia divaricata (Turcz.) Schischk. Friable embryogenic callus developed from the root, internode, and leaf explants on Murashige and Skoog medium (MS) with 2.26 μM 2,4-dichlorophenoxyacetic acid (2,4-D), and subsequently developed into somatic embryos on MS medium containing 4–5% sucrose, 1.74 μM naphthaleneacetic acid (NAA), 4.44 μM 6-benzylaminopurine (BA), and 1.90 μM abscisic acid (ABA). Then the mature embryos were separated and transferred onto MS with 3% sucrose and 0.6% agar for further development and conversion to plantlets. In vitro flowering and fruiting were obtained when the subcultures were carried out for over 15 months. Paclobutrazol (PP333) or ethephon (ETH) at low levels promoted flowering significantly. Also, abnormal rootless somatic embryos of S. divaricata could form flowers and fruits in vitro.     

Ontogenetic changes in foraging tactics of the piscivorous cornetfish <Emphasis Type="Italic">Fistularia commersonii</Emphasis>

Foraging behaviors of the piscivorous cornetfish Fistularia commersonii were observed at shallow reefs in Kuchierabu-jima Island, southern Japan. This fish foraged on two types of prey fishes: one was reef fish that typically dwell on or near substrata (e.g., Tripterygiidae and Labridae), and the other was pelagic fish that shoal in the water column (e.g., Clupeidae and Carangidae). The prey sizes, prey types and foraging behaviors changed as the predator size increased. Prey sizes were largely limited by gape size of the cornetfish, and small predators consumed small prey. The small cornetfish (10–30 cm in total length) fed only on reef fish captured after stalking (where the fish slowly approaches the prey and then suddenly attacks). The stalking was done either solitarily or in foraging association with conspecifics. Large fish (30–120 cm) fed on both types of fishes by stalking and/or chasing (where the fish chases the prey using its high mobility and attacks), either solitarily or in foraging association with con- or heterospecifics. Thus, chasing was only performed by the large cornetfish against pelagic prey fish in associative foraging with other con- and heterospecific predators. As their body sizes increased, F. commersonii began to show a diversification of foraging behaviors, which was strongly related not only to the habitat types and anti-predatory behaviors of the prey fishes but also to associative foraging with con- or heterospecifics, which improves their foraging success.     

Plant meristems: <Emphasis Type="Italic">CLAVATA3/ESR</Emphasis>-related signaling in the shoot apical meristem and the root apical meristem

The plant meristems, shoot apical meristem (SAM) and root apical meristem (RAM), are unique structures made up of a self-renewing population of undifferentiated pluripotent stem cells. The SAM produces all aerial parts of postembryonic organs, and the RAM promotes the continuous growth of roots. Even though the structures of the SAM and RAM differ, the signaling components required for stem cell maintenance seem to be relatively conserved. Both meristems utilize cell-to-cell communication to maintain proper meristematic activities and meristem organization and to coordinate new organ formation. In SAM, an essential regulatory mechanism for meristem organization is a regulatory loop between WUSCHEL (WUS) and CLAVATA (CLV), which functions in a non-cell-autonomous manner. This intercellular signaling network coordinates the development of the organization center, organ boundaries and distant organs. The CLAVATA3/ESR (CLE)-related genes produce signal peptides, which act non-cell-autonomously in the meristem regulation in SAM. In RAM, it has been suggested that a similar mechanism can regulate meristem maintenance, but these functions are largely unknown. Here, we overview the WUS–CLV signaling network for stem cell maintenance in SAM and a related mechanism in RAM maintenance. We also discuss conservation of the regulatory system for stem cells in various plant species. S. Sawa is the recipient of the BSJ Award for Young Scientist, 2007.     

Oxidative Stress in Cerebrospinal Fluid of Patients with Aseptic and Bacterial Meningitis

This study aimed to determine whether patients with aseptic and bacterial meningitis presented alterations in oxidative stress parameters of cerebrospinal fluid (CSF). A total of 30 patients were used in the research. The CSF oxidative stress status has been evaluated through many parameters, such as lipid peroxidation through thiobarbituric acid reactive substances (TBARS) and antioxidant defense systems such as superoxide dismutase (SOD), glutathione S-transferase (GST), reduced glutathione (GSH) and ascorbic acid. TBARS levels, SOD and GST activity increase in aseptic meningitis and in bacterial meningitis. The ascorbic acid concentration increased significantly in patients with both meningitis types. The reduced glutathione levels were reduced in CSF of patients with aseptic and bacterial meningitis. In present study we may conclude that oxidative stress contributes at least in part to the severe neurological dysfunction found in meningitis.