Low-fluence blue light-induced phosphorylation of Zmphot1 mediates the first positive phototropism.

Phototropin1 (phot1) perceives low- to high-fluence blue light stimuli and mediates both the first and second positive phototropisms. High-fluence blue light is known to induce autophosphorylation of phot1, leading to the second positive phototropism. However, the phosphorylation status of phot1 by low-fluence blue light that induces the first positive phototropism had not been observed. Here, we conducted a phosphoproteomic analysis of maize coleoptiles to investigate the fluence-dependent phosphorylation status of Zmphot1. High-fluence blue light induced phosphorylation of Zmphot1 at several sites. Notably, low-fluence blue light significantly increased the phosphorylation level of Ser291 in Zmphot1. Furthermore, Ser291-phosphorylated and Ser369Ser376-diphosphorylated peptides were found to be more abundant in the low-fluence blue light-irradiated sides than in the shaded sides of coleoptiles. The roles of these phosphorylation events in phototropism were explored by heterologous expression of ZmPHOT1 in the Arabidopsis thaliana phot1phot2 mutant. The first positive phototropism was restored in wild-type ZmPHOT1-expressing plants; however, plants expressing S291A-ZmPHOT1 or S369AS376A-ZmPHOT1 showed significantly reduced complementation rates. All transgenic plants tested in this study exhibited a normal second positive phototropism. These findings provide the first indication that low-fluence blue light induces phosphorylation of Zmphot1 and that this induced phosphorylation is crucial for the first positive phototropism.

[Recognition of developmental disorders and the information sources among the Japanese adult population].

Objectives　This study aimed to examine methods of providing information to improve the recognition of people with developmental disorders by comparing recognition of developmental disorders among education and health care professionals within the general public.Methods　A cross-sectional internet-based survey was conducted in January 2016. The subjects were Japanese adults aged between 20 and 69 years. Of all the respondents, 418 were males and 424 were females. The questionnaire was used to assess recognition of the names of developmental disorders and to determine the corresponding methods through which the information was obtained. We analyzed cognitive states by classifying the respondents by occupation (education and health care professionals) and by whether the subject had a developmental disorder or was a family member or friend of a person with a developmental disorder separately.Results　The percentage of respondents that reported having heard about developmental disorders was 91.5%. However, only 26.5% reported having provided some form of support to people with developmental disabilities. The percentages of education and health care professionals who were aware of developmental disorders were close to 100%; however, only 63.9% and 42.9%, respectively, could cite how they had become aware of developmental disorders. With regard to the sources of information on developmental disorders, the most frequent answers were television and radio programs (67.1%), followed by the internet. Of all the respondents, 11.3% had heard about developmental disorders in a school setting and 9.9% had heard about the disorders in the workplace.Conclusion　To increase understanding of developmental disorders among education and health care professionals, basic and in-service education must be improved. Furthermore, it is important to provide information on developmental disorders through the media.

Efficient and cost effective production of active-form human PKB using silkworm larvae.

Protein kinase B (PKB) also known as Akt is involved in many signal transduction pathways. As alterations of the PKB pathway are found in a number of human malignancies, PKB is considered an important drug target for cancer therapy. However, production of sufficient amounts of active PKB for biochemical and structural studies is very costly because of the necessity of using a higher organism expression system to obtain phosphorylated PKB. Here, we report efficient production of active PKBα using the BmNPV bacmid expression system with silkworm larvae. Following direct injection of bacmid DNA, recombinant PKBα protein was highly expressed in the fat bodies of larvae, and could be purified using a GST-tag and then cleaved. A final yield of approximately 1 mg PKBα/20 larvae was recorded. Kinase assays showed that the recombinant PKBα possessed high phosphorylation activity. We further confirmed phosphorylation on the activation loop by mass spectrometric analysis. Our results indicate that the silkworm expression system is of value for preparation of active-form PKBα with phosphorylation on the activation loop. This efficient production of the active protein will facilitate further biochemical and structural studies and stimulate subsequent drug development.

Identification of proteins enriched in rice egg or sperm cells by single-cell proteomics.

In angiosperms, female gamete differentiation, fertilization, and subsequent zygotic development occur in embryo sacs deeply embedded in the ovaries. Despite their importance in plant reproduction and development, how the egg cell is specialized, fuses with the sperm cell, and converts into an active zygote for early embryogenesis remains unclear. This lack of knowledge is partly attributable to the difficulty of direct analyses of gametes in angiosperms. In the present study, proteins from egg and sperm cells obtained from rice flowers were separated by one-dimensional polyacrylamide gel electrophoresis and globally identified by highly sensitive liquid chromatography coupled with tandem mass spectroscopy. Proteome analyses were also conducted for seedlings, callus, and pollen grains to compare their protein expression profiles to those of gametes. The proteomics data have been deposited to the ProteomeXchange with identifier PXD000265. A total of 2,138 and 2,179 expressed proteins were detected in egg and sperm cells, respectively, and 102 and 77 proteins were identified as preferentially expressed in egg and sperm cells, respectively. Moreover, several rice or Arabidopsis lines with mutations in genes encoding the putative gamete-enriched proteins showed clear phenotypic defects in seed set or seed development. These results suggested that the proteomic data presented in this study are foundational information toward understanding the mechanisms of reproduction and early development in angiosperms.