High-quality sugar production by osgcs1 rice.

Carbohydrates (sugars) are an essential energy-source for all life forms. They take a significant share of our daily consumption and are used for biofuel production as well. However, sugarcane and sugar beet are the only two crop plants which are used to produce sugar in significant amounts. Here, we have discovered and fine-tuned a phenomenon in rice which leads them to produce sugary-grain. We knocked-out GCS1 genes in rice by using CRISPR technology, which led to fertilization failure and pollen tube-dependent ovule enlargement morphology (POEM) phenomenon. Apparently, the POEMed-like rice ovule ('endosperm-focused') can grow near-normal seed-size unlike earlier observations in Arabidopsis in which gcs1 ovules ('embryo-focused') were aborted quite early. The POEMed-like rice ovules contained 10-20% sugar, with extremely high sucrose content (98%). Trancriptomic analysis revealed that the osgcs1 ovules had downregulation of starch biosynthetic genes, which would otherwise have converted sucrose to starch. Overall, this study shows that pollen tube content release is sufficient to trigger sucrose unloading at rice ovules. However, successful fertilization is indispensable to trigger sucrose-starch conversion. These findings are expected to pave the way for developing novel sugar producing crops suited for diverse climatic regions.

Anesthetics, Anesthesia, and Plants.

General anesthesia, its nature, and how exactly it works are still poorly understood. Plants can also be anesthetized and lose their responses to external stimuli. Interestingly, plants are known to produce endogenous anesthetic compounds to deal with stress. Plants offer an excellent model object for studies on anesthetics and anesthesia.

MES Buffer Affects Arabidopsis Root Apex Zonation and Root Growth by Suppressing Superoxide Generation in Root Apex.

In plants, growth of roots and root hairs is regulated by the fine cellular control of pH and reactive oxygen species (ROS). MES, 2-(N-morpholino)ethanesulfonic acid as one of the Good's buffers has broadly been used for buffering medium, and it is thought to suit for plant growth with the concentration at 0.1% (w/v) because the buffer capacity of MES ranging pH 5.5-7.0 (for Arabidopsis, pH 5.8). However, many reports have shown that, in nature, roots require different pH values on the surface of specific root apex zones, namely meristem, transition zone, and elongation zone. Despite the fact that roots always grow on a media containing buffer molecule, little is known about impact of MES on root growth. Here, we have checked the effects of different concentrations of MES buffer using growing roots of Arabidopsis thaliana. Our results show that 1% of MES significantly inhibited root growth, the number of root hairs and length of meristem, whereas 0.1% promoted root growth and root apex area (region spanning from the root tip up to the transition zone). Furthermore, superoxide generation in root apex disappeared at 1% of MES. These results suggest that MES disturbs normal root morphogenesis by changing the ROS homeostasis in root apex.

Production and removal of superoxide anion radical by artificial metalloenzymes and redox-active metals.

Generation of reactive oxygen species is useful for various medical, engineering and agricultural purposes. These include clinical modulation of immunological mechanism, enhanced degradation of organic compounds released to the environments, removal of microorganisms for the hygienic purpose, and agricultural pest control; both directly acting against pathogenic microorganisms and indirectly via stimulation of plant defense mechanism represented by systemic acquired resistance and hypersensitive response. By aiming to develop a novel classes of artificial redox-active biocatalysts involved in production and/or removal of superoxide anion radicals, recent attempts for understanding and modification of natural catalytic proteins and functional DNA sequences of mammalian and plant origins are covered in this review article.

UV-B Induced Generation of Reactive Oxygen Species Promotes Formation of BFA-Induced Compartments in Cells of Arabidopsis Root Apices.

UV-B radiation is an important part of the electromagnetic spectrum emitted by the sun. For much of the period of biological evolution organisms have been exposed to UV radiation, and have developed diverse mechanisms to cope with this potential stress factor. Roots are usually shielded from exposure to UV by the surrounding soil, but may nevertheless be exposed to high energy radiation on the soil surface. Due to their high sensitivity to UV-B radiation, plant roots need to respond rapidly in order to minimize exposure on the surface. In addition to root gravitropism, effective light perception by roots has recently been discovered to be essential for triggering negative root phototropism in Arabidopsis. However, it is not fully understood how UV-B affects root growth and phototropism. Here, we report that UV-B induces rapid generation of reactive oxygen species which in turn promotes the formation of BFA-induced compartments in the Arabidopsis root apex. During unilateral UV-B irradiation of roots changes in auxin concentration on the illuminated side have been recorded. In conclusion, UV-B-induced and ROS-mediated stimulation of vesicle recycling promotes root growth and induces negative phototropism.

Light as stress factor to plant roots - case of root halotropism.

Despite growing underground, largely in darkness, roots emerge to be very sensitive to light. Recently, several important papers have been published which reveal that plant roots not only express all known light receptors but also that their growth, physiology and adaptive stress responses are light-sensitive. In Arabidopsis, illumination of roots speeds-up root growth via reactive oxygen species-mediated and F-actin dependent process. On the other hand, keeping Arabidopsis roots in darkness alters F-actin distribution, polar localization of PIN proteins as well as polar transport of auxin. Several signaling components activated by phytohormones are overlapping with light-related signaling cascade. We demonstrated that the sensitivity of roots to salinity is altered in the light-grown Arabidopsis roots. Particularly, light-exposed roots are less effective in their salt-avoidance behavior known as root halotropism. Here we discuss these new aspects of light-mediated root behavior from cellular, physiological and evolutionary perspectives.

Root photomorphogenesis in laboratory-maintained Arabidopsis seedlings.

In nature, root systems of most terrestrial plants are underground in darkness. Nevertheless, several photoreceptors have been found in roots and light-responsive mechanisms allowing roots to escape from strong light conditions have been discovered. In transparent Petri dishes, regular light exposure affects root morphology and behavior. We advocate the use of darkened Petri dishes to allow roots to be kept in darkness, thus mimicking more closely the conditions in nature.

Illumination of Arabidopsis roots induces immediate burst of ROS production.

Arabidopsis roots are routinely exposed to light both during their cultivation within transparent Petri dishes and during their confocal microscopy analysis. Here we report that illumination of roots which naturally grow in darkness, even for a few seconds, induces an immediate and strong burst of reactive oxygen species (ROS). Plant scientists studying roots should pay great attention to the environment of living roots, and keep them in darkness as long as possible. Results obtained using illuminated roots during in vivo microscopic analysis should also be interpreted with great caution.

Prevention of oxidative DNA degradation by copper-binding peptides.

Free divalent ions of copper (Cu) are capable of generating radical species such as hydroxyl radicals in the presence of hydrogen peroxide or ascorbic acid through Harbor-Weiss-like reactions under physiological conditions. It has been reported that radical-mediated damage to DNA molecules in animal cells leads to programmed cell death. Hence it is important to seek for methods to prevent Cu-mediated DNA damage. In this study we identified on effect of Cu binding of short peptides (chiefly Gly-Gly-His tripeptide) in the prevention of DNA degradation caused by Cu-mediated reactions in the presence of hydrogen peroxide and of ascorbic acid.

Copper-binding peptides from human prion protein and newly designed peroxidative biocatalysts.

A previous work suggested that peptides from the histidine-containing copper-binding motifs in human prion protein (PrP) function as peroxidase-like biocatalysts catalyzing the generation of superoxide anion radicals in the presence of neurotransmitters (aromatic monoamines) and phenolics such as tyrosine and tyrosyl residues on proteins. In this study, using various phenolic substrates, the phenol-dependent superoxide-generating activities of PrP-derived peptide sequences were compared. Among the peptides tested, the GGGTH pentapeptide was shown to be the most active catalyst for phenol-dependent reactions. Based on these results, we designed a series of oligoglycyl-histidines as novel peroxidative biocatalysts, and their catalytic performances including kinetics, heat tolerance, and freezing tolerance were analysed.

Superoxide generation catalyzed by the ozone-inducible plant peptides analogous to prion octarepeat motif.

Ozone-inducible (OI) peptides found in plants contain repeated sequences consisting of a hexa-repeat unit (YGH GGG) repeated 7-9 times in tandem, and each unit tightly binds copper. To date, the biochemical roles for OI peptides are not fully understood. Here, we demonstrated that the hexa-repeat unit from OI peptides behaves as metal-binding motif catalytically active in the O2•--generation. Lastly, possible mechanisms of the reaction and biological consequence of the reactions are discussed by analogy to the action of human prion octarepeat peptides.

Prevention of copper-induced calcium influx and cell death by prion-derived peptide in suspension-cultured tobacco cells.

Impact of copper on the oxidative and calcium signal transductions leading to cell death in plant cells and the effects of the copper-binding peptide derived from the human prion protein (PrP) as a novel plant-protecting agent were assessed using a cell suspension culture of transgenic tobacco (Nicotiana tabacum L., cell line BY-2) expressing the aequorin gene. Copper induces a series of biological and chemical reactions in plant cells including the oxidative burst reflecting the production of reactive oxygen species (ROS), such as hydroxyl radicals, and stimulation of calcium channel opening, allowing a transient increase in cytosolic calcium concentrations. The former was proven by the action of specific ROS scavengers blocking the calcium responses and the latter was proven by an increase in aequorin luminescence and its inhibition by specific channel blockers. Following these early events completed within 10 min, the development of copper-induced cell death was observed during additional 1 h in a dose-dependent manner. Addition of a synthetic peptide (KTNMKHMA) corresponding to the neurotoxic sequence in human PrP, prior to the addition of copper, effectively blocked both calcium influx and cell death induced by copper. Lastly, a possible mechanism of peptide action and future applications of this peptide in the protection of plant roots from metal toxicity or in favour of phytoremediation processes are discussed.

Thermo-stable nature of aromatic monoamine-dependent superoxide-generating activity of human prion-derived Cu-binding peptides.

Human prion protein has four distinct Cu-binding motifs that catalyze the generation of superoxide coupled to oxidation of phenols and amines. Here, the thermostability of the superoxide-generating prion-derived peptides was tested. Among the peptides tested, two maintained high catalytic activity even after heating and repeated freezing/thawing cycles. The biological roles for these thermostable catalysts are discussed.

Free tyrosine and tyrosine-rich peptide-dependent superoxide generation catalyzed by a copper-binding, threonine-rich neurotoxic peptide derived from prion protein.

Previously, generation of superoxide anion (O(2)(*-)) catalyzed by Cu-binding peptides derived from human prion protein (model sequence for helical Cu-binding motif VNITKQHTVTTTT was most active) in the presence of catecholamines and related aromatic monoamines such as phenylethylamine and tyramine, has been reported [Kawano, T., Int J Biol Sci 2007; 3: 57-63]. The peptide sequence (corresponding to helix 2) tested here is known as threonine-rich neurotoxic peptide. In the present article, the redox behaviors of aromatic monoamines, 20 amino acids and prion-derived tyrosine-rich peptide sequences were compared as putative targets of the oxidative reactions mediated with the threonine-rich prion-peptide. For detection of O(2)(*-), an O(2)(*-)-specific chemiluminescence probe, Cypridina luciferin analog was used. We found that an aromatic amino acid, tyrosine (structurally similar to tyramine) behaves as one of the best substrates for the O(2)(*-) generating reaction (conversion from hydrogen peroxide) catalyzed by Cu-bound prion helical peptide. Data suggested that phenolic moiety is required to be an active substrate while the presence of neither carboxyl group nor amino group was necessarily required. In addition to the action of free tyrosine, effect of two tyrosine-rich peptide sequences YYR and DYEDRYYRENMHR found in human prion corresponding to the tyrosine-rich region was tested as putative substrates for the threonine-rich neurotoxic peptide. YYR motif (found twice in the Y-rich region) showed 2- to 3-fold higher activity compared to free tyrosine. Comparison of Y-rich sequence consisted of 13 amino acids and its Y-to-F substitution mutant sequence revealed that the tyrosine-residues on Y-rich peptide derived from prion may contribute to the higher production of O(2)(*-). These data suggest that the tyrosine residues on prion molecules could be additional targets of the prion-mediated reactions through intra- or inter-molecular interactions. Lastly, possible mechanism of O(2)(*-) generation and the impacts of such self-redox events on the conformational changes in prion are discussed.