Yuichi Uno

A novel yeast two‐hybrid approach to identify CDPK substrates: Characterization of the interaction between AtCPK11 and AtDi19, a nuclear zinc finger protein1

Calcium‐dependent protein kinases (CDPKs) are sensor‐transducer proteins capable of decoding calcium signals in diverse phosphorylation‐dependent calcium signaling networks in plants and some protists. Using a novel yeast two‐hybrid (YTH) approach with constitutively active and/or catalytically inactive forms of AtCPK11 as bait, we identified AtDi19 as an AtCPK11‐interacting protein. AtDi19 is a member of a small family of stress‐induced genes. The interaction was confirmed using pull‐down assays with in vitro translated AtCPK11 and GST–AtDi19 and localization studies in Arabidopsis protoplasts cotransfected with AtCPK11:GFP and AtDi19:DsRed2 protein fusions. We further showed that the interaction of AtDi19 is specific to both AtCPK4 and AtCPK11, whereas other closely related CPKs from Arabidopsis interacted weakly (e.g., AtCPK12) or did not interact (e.g., AtCPK26, AtCPK5 and AtCPK1) with AtDi19. Deletion analyses showed that a region containing two predicted nuclear localization signals (NLS) and a nuclear export signal (NES) of AtDi19 is essential for interaction with AtCPK11. We further demonstrated that AtDi19 is phosphorylated by AtCPK11 in a Ca2+‐dependent manner at Thr105 and Ser107 within the AtDi19 bipartite NLS using in vitro kinase assays. Our data suggest that disruption of the autoinhibitor domain leading to the formation of a constitutively active CDPK may stabilize kinase–substrate interactions without affecting specificity.

Molecular Responses to Water Stress in Arabidopsis thaliana

Plants respond and adapt to environmental changes including drought, high salinity and low temperature. abscisic acid (ABA) plays important roles in these stress responses. A number of plant genes are induced by water stress, such as drought, high salinity and low temperature, and are thought to function in the stress tolerance and responses of the plant. At least four signal transduction pathways control these genes inArabidopsis thaliana: two are ABA-dependent, and two are ABA-independent. Acis-acting element named DRE (Dehydration Responsive Element) is involved in one of the ABA-independent signal transduction pathways, and its DNA binding proteins have been characterized. Drought- and ABA-inducible MYC and MYB homologues are involved in ABA-responsive gene expression inarabidopsis. Roles of thesecis andtrans-acting factors in water stress responses are discussed. In addition, a number of genes for protein kinases, enzymes involved in phosphatidyl inositol metabolism (PI turnover) and transcription factors are also induced by water stress, and thought to be involved in the stress signal transduction cascades. Possible signaling processes in water stress response are discussed.

Functional characterization and expression profiling of a DREB2-type gene from lettuce (Lactuca sativa L.)

Dehydration responsive element binding factors (DREBs) play important roles in plant responses to abiotic stress. In this study, a DREB2-type gene, LsDREB2A, was isolated from lettuce (Lactuca sativa L.) and characterized. LsDREB2A encoded a protein of 322 amino acid residues with the conserved apetala 2 domain and was classified into the A2 subgroup of DREBs. LsDREB2A has no sequence rich in proline, glutamic acid, serine, and threonine to trigger protein degradation. Southern blot analysis indicated that there could be more than ten DREB homologs in lettuce. A gel shift assay indicated that LsDREB2A could specifically recognize the dehydration responsive element (DRE) sequence in vitro. In a yeast one-hybrid assay, LsDREB2A bound specifically to the DRE sequence and activated the expression of both His3 and LacZ reporter genes. A transient expression experiment in lettuce protoplasts showed that LsDREB2A was localized to the nucleus. These results suggest that LsDREB2A might function as a transcription factor. Quantitative real-time polymerase chain reaction revealed that the expression of LsDREB2A was significantly increased by hyperosmotic and high salinity treatment, but not cold, heat, nor abscisic acid (ABA) treatment. Overexpression of LsDREB2A increased the tolerance of salt stress in transgenic plants. These results suggested that LsDREB2A plays an important role during high salinity stress in an ABA-independent pathway.

Relationship between the expression of Rab family GTPases and neuropeptide hormones in the brain of Bombyx mori.

Rab proteins are small GTPases that play essential roles in vesicle transport. In this study, we examined the expression of Rab proteins and neuropeptide hormones in the brain of the silkworm, Bombyx mori. We produced antibodies against B. mori Rab1 and Rab14 in rabbits. Immunoblotting of samples of brain tissue from B. mori revealed a single band for each antibody. Rab1 and Rab14 immunohistochemical labeling in the brain of B. mori was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum. Rab1, Rab7 and Rab14 co-localized with bombyxin. Rab1 and Rab7 co-localized with eclosion hormone. Rab1 co-localized with prothoracicotropic hormone. These results suggest that Rab1, Rab7 and Rab14 may be involved in neuropeptide transport in the brain of B. mori. This is the first report on the specificity of Rab proteins for the secretion of different neuropeptides in insects.

Bioconversion of small molecules by cytochrome P450 species expressed in Escherichia coli

P450 (cytochrome P450) enzymes catalyse the mono‐oxygenation of a wide range of compounds such as steroids, fatty acids, vitamins and drugs. In the present paper we demonstrate a system for bioconverting diverse compounds [flavanone, DHEA (dehydroepiandrosterone) and 7‐ethoxycoumarin] using P450 species expressed in Escherichia coli. First, we expressed four P450 species: rabbit CYP2B (P450 family 2, subfamily B), fruitfly (Drosophila) CYP317A, rat CYP3A23 and mouse CYP2J5. Next, we added substrates directly to the incubation medium. The resulting metabolites were extracted and analysed by HPLC and spectrofluorimetry. The first substrate, 7‐ethoxycoumarin, was de‐ethylated by CYP2B; CYP2J5 and CYP3A23 showed weak activity, and CYP317A had no activity for 7‐ethoxycoumarin. We next used flavanone, a flavonoid, as a substrate for these four P450 species and other P450 species expressed previously. As a result, CYP2B, CYP2C43 and CYP2C29 catalysed flavanone 2‐hydroxylation. CYP2A5 catalysed 2‐ and 4‐hydroxylations. Finally, to produce diverse modified compounds, variants of CYP2A5 with point mutations were incubated with a steroid (DHEA) and an antioxidant (flavanone) in vivo. HPLC analysis indicated that two P450 species produced a 7‐β‐hydroxy‐DHEA and two P450 species produced a 2‐α‐hydroxy‐DHEA. Four P450 species catalysed flavanone 2‐ and 4‐hydroxylations. These results indicate that bioconversion by P450 is a useful technique to modify small molecules (steroids, coumarin and flavanone) and produce new, diverse hydroxylated compounds, which could be used for high‐throughput screening for drug discovery.

Rab14 from Bombyx mori (Lepidoptera: Bombycidae) shows ATPase activity

Rab GTPases are essential for vesicular transport, whereas adenosine triphosphate (ATP) is the most important and versatile of the activated carriers in the cell. But there are little reports to clarify the connection between ATP and Rab GTPases. A cDNA clone (Rab14) from Bombyx mori was expressed in Escherichia coli as a glutathione S-transferase fusion protein and purified. The protein bound to [3H]-GDP and [35S]-GTPγS. Binding of [35S]-GTPγS was inhibited by guanosine diphosphate (GDP), guanosine triphosphate (GTP) and ATP. Rab14 showed GTP- and ATP-hydrolysis activity. The Km value of Rab14 for ATP was lower than that for GTP. Human Rab14 also showed an ATPase activity. Furthermore, bound [3H]-GDP was exchanged efficiently with GTP and ATP. These results suggest that Rab14 is an ATPase as well as GTPase and gives Rab14 an exciting integrative function between cell metabolic status and membrane trafficking.

In vitro evaluation of osmotic stress tolerance using a novel root recovery assay

We established a novel in vitro method, termed the root recovery assay, to evaluate the survival under osmotic stress of lettuce (Lactuca sativa L.) seedlings. Under salinity and drought stress, combination of the root-bending assay and root recovery assay showed the same trends in dry weight and survival rate as a hydroponic culture. Both in vitro assays and hydroponics ranked the three lettuce cultivars in the same order of drought tolerance. The root-bending assay evaluated the plant’s growth and the root recovery assay indicated the plant’s survival. In addition, the combined assay required less space and approximately half the time period compared with the hydroponic culture. These results suggested that application of the root-bending and root recovery assay should be a rapid and space-saving method with which to evaluate the osmotic stress tolerance of lettuce from both growth and survival standpoints.

Small GTPases of the Rab family in the brain of Bombyx mori

Small GTPases of the Rab family are key regulators of membrane trafficking. We produced antibodies against the Rab7 protein of Bombyx mori (BRab7) in rabbits, and against the Rab11 protein of B. mori (BRab11) in mice. The antibodies recognized BRab7 and BRab11 proteins, but did not recognize other Rab proteins. Immunoblotting of samples from brain tissue of B. mori revealed a single band for each antibody. Rab11 was expressed in most tissues, whereas Rab7 was expressed in the brain, ovary, and testis. Immunohistochemical reactivity of Rab7 and Rab11 in the brain of B. mori was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum. Double-labeling experiments demonstrated that immunohistochemical reactivity of Rab7 co-localized with that of Rab11 and partially with that of Rab8. Immunohistochemical reactivity of Rab11 and Rab8 co-localized with that of PERIOD, one of the proteins associated with circadian rhythm. These findings suggest that Rab7, Rab8, and Rab11 are involved in protein transport in the neurons of the brain of B. mori and might play a role in the control of circadian rhythm.

Characterization of Rab-interacting lysosomal protein in the brain of Bombyx mori

AbstractRab guanosine triphosphatases in eukaryotic cells are key regulators of membrane-trafficking events, such as exocytosis and endocytosis. Rab7 regulates traffic from early to late endosomes and from late endosomes to vacuoles/lysosomes. The Rab7-interacting lysosomal protein (RILP) was extracted from the silkworm, Bombyx mori (B. mori), and expressed in Escherichia coli (E. coli), followed by its purification. The glutathione sulfotransferase pull-down assay revealed that Rab7 of B. mori interacted with RILP of B. mori. We then produced antibodies against RILP of B. mori in rabbits for their use in Western immunoblotting and immunohistochemistry. Western immunoblotting of brain tissue for RILP revealed a single band, at approximately 50 kD. RILP-like immunohistochemical reactivity (RILP-ir) was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum. Furthermore, RILP-ir was colocalized with the eclosion hormone-ir and bombyxin-ir. However, RILP-ir was not colocalized with prothoracicotropic hormone-ir. These results were similar to those of Rab7 from our previous study. These findings suggest that RILP and Rab7 are involved in the neurosecretion in a restricted subtype of neurons in B. mori. Thus, our study is the first to report of a possible relationship between an insect Rab effector and neurosecretion.

Development of Method for Non-Destructive Measurement of Nitrate Concentration in Vegetable Leaves by Near-Infrared Spectroscopy

Abstract The purpose of this research is to develop a method for non-destructive measurement of nitrate concentration in vegetable leaves. Near-infrared (NIR) spectroscopy was adopted for the measurement. Spinach and komatsuna leaves were examined. Absorption spectra at small areas in the leaves were measured by a NIR spectrometer. The measured areas were cut away from the leaves and their nitrate concentrations were measured by a liquid chromatography analyzer. Mathematical models to estimate the nitrate concentration from the spectra were developed by PCR or PLS methods with a wavelength selection algorithm. As a result, the maximum coefficient of determination between the measured and estimated concentrations became more than 0.8.