A New Plant Breeding Technique Using ALSV Vectors to Shorten the Breeding Periods of Fruit Trees

Fruit trees have a long juvenile phase. For example, the juvenile phase of apple lasts for 6–12 years and is a serious constraint for creating new varieties by breeding based on crossing and selection. In this chapter, we report a novel technology using the apple latent spherical virus (ALSV) vector to accelerate flowering time and life cycle in apple and pear seedlings. Inoculation of apple and pear cotyledons immediately after germination with ALSV-AtFT/MdTFL1 concurrently expressing Arabidopsis FLOWERING LOCUS T (AtFT) gene and suppressing apple TERMINAL FLOWER 1-1 (MdTFL1-1) gene can shorten the period from seeding to flowering to 1.5–3 months after germination and generation times in order to obtain next-generation seeds in 1 year or less. Most next-generation seedlings obtained from ALSV vector–infected plants were free of the virus. We also developed a method for eliminating ALSV vectors from infected apple and pear plants by only high-temperature treatment. A method combining the promotion of flowering in apple and pear by ALSV vector with an ALSV elimination technique is expected to see future application as a new plant breeding technique that can significantly shorten the breeding periods of apple and pear

Efficient virus-induced gene silencing in apple, pear and Japanese pear using Apple latent spherical virus vectors

<p>Abstract</p> <p>Background</p> <p>Virus-induced gene silencing (VIGS) is an effective technology for the analysis of gene functions in plants. Though there are many reports on virus vectors for VIGS in plants, no VIGS vectors available for <it>Rosaceae </it>fruit trees were reported so far. We present an effective VIGS system in apple, pear, and Japanese pear using <it>Apple latent spherical virus </it>(ALSV) vectors.</p> <p>Results</p> <p>Inoculation of ALSV vectors carrying a partial sequence of endogenous genes from apple [ribulose-1, 5-bisphosphate carboxylase small subunit (<it>rbcS</it>), alpha subunit of chloroplast chaperonin (<it>CPN60a</it>), elongation factor 1 alpha (<it>EF-1a</it>), or actin] to the cotyledons of seeds by a particle bombardment induced highly uniform knock-down phenotypes of each gene on the true leaves of seedlings from 2~3 weeks after inoculation. These silencing phenotypes continued for several months. Northern blot and RT-PCR analyses of leaves infected with ALSV containing a fragment of <it>rbcS </it>gene showed that the levels of <it>rbcS</it>-mRNA drastically decreased in the infected apple and pear leaves, and, in reverse, <it>rbcS-</it>siRNAs were generated in the infected leaves. In addition, some of apple seedlings inoculated with ALSV vector carrying a partial sequence of a <it>TERMINAL FLOWER 1 </it>gene of apple (<it>MdTFL1</it>) showed precocious flowering which is expected as a knock-down phenotype of the silencing of <it>MdTFL1 </it>gene.</p> <p>Conclusions</p> <p>The ALSV-based VIGS system developed have provides a valuable new addition to the tool box for functional genomics in apple, pear, and Japanese pear.</p

Hsp105 but not Hsp70 family proteins suppress the aggregation of heat-denatured protein in the presence of ADP

AbstractHsp105α and Hsp105β are mammalian members of the Hsp105/110 family, a diverged subgroup of the Hsp70 family. Here, we show that Hsp105α and Hsp105β bind non-native protein through the β-sheet domain and suppress the aggregation of heat-denatured protein in the presence of ADP rather than ATP. In contrast, Hsc70/Hsp40 suppressed the aggregation of heat-denatured protein in the presence of ATP rather than ADP. Furthermore, the overexpression of Hsp105α but not Hsp70 in COS-7 cells rescued the inactivation of luciferase caused by ATP depletion. Thus, Hsp105/110 family proteins are suggested to function as a substitute for Hsp70 family proteins to suppress the aggregation of denatured proteins in cells under severe stress, in which the cellular ATP level decreases markedly

ALSV Vector Substantially Shortens Generation Time of Horticultural Plants

Flowering of plants is tightly regulated by both plant maturity and seasons in the year. Now that the Flowering LocusT (FT) gene has been revealed to encode the flowering hormone florigen, researchers are seeking to regulate and modify flowering behaviours by using florigen as a genetic tool. In place of transgenic approaches, Apple latent spherical virus (ALSV) vector was successful in promoting flowering of both model plants (Arabidopsis and tobacco), and fruit trees (e.g. apple, pear, and loquat), vegetables (e.g. tomato and cucumber), legumes (e.g. soybean), and ornamental flowers (e.g. petunia, Japanese gentian and Eustoma). In so doing, FT was expressed and/or TFL1 was suppressed by the ALSV vector. ALSV is a latent (non-pathogenic) virus isolated from an apple tree. After induction of flowering and seed production in crops, ALSV is not transferred to most of the next-generation seedlings, or it can be artificially removed from the infected plant by incubation at high temperature. Thus, the generation times of horticultural plants are approximately halved, and the generation time of apple plants is substantially shortened to within one year. Hence, ALSV technology is expected to be useful as a part of New Plant Breeding Techniques (NPBT) for agricultural application

Investigating accuracy of pitch-accent annotations in neural network-based speech synthesis and denoising effects

We investigated the impact of noisy linguistic features on the performance of a Japanese speech synthesis system based on neural network that uses WaveNet vocoder. We compared an ideal system that uses manually corrected linguistic features including phoneme and prosodic information in training and test sets against a few other systems that use corrupted linguistic features. Both subjective and objective results demonstrate that corrupted linguistic features, especially those in the test set, affected the ideal system's performance significantly in a statistical sense due to a mismatched condition between the training and test sets. Interestingly, while an utterance-level Turing test showed that listeners had a difficult time differentiating synthetic speech from natural speech, it further indicated that adding noise to the linguistic features in the training set can partially reduce the effect of the mismatch, regularize the model, and help the system perform better when linguistic features of the test set are noisy.Comment: Accepted for Interspeech 201

Training Multi-Speaker Neural Text-to-Speech Systems using Speaker-Imbalanced Speech Corpora

When the available data of a target speaker is insufficient to train a high quality speaker-dependent neural text-to-speech (TTS) system, we can combine data from multiple speakers and train a multi-speaker TTS model instead. Many studies have shown that neural multi-speaker TTS model trained with a small amount data from multiple speakers combined can generate synthetic speech with better quality and stability than a speaker-dependent one. However when the amount of data from each speaker is highly unbalanced, the best approach to make use of the excessive data remains unknown. Our experiments showed that simply combining all available data from every speaker to train a multi-speaker model produces better than or at least similar performance to its speaker-dependent counterpart. Moreover by using an ensemble multi-speaker model, in which each subsystem is trained on a subset of available data, we can further improve the quality of the synthetic speech especially for underrepresented speakers whose training data is limited.Comment: Submitted to Interspeech 2019, Graz, Austri

Crucial role of nitric oxide synthases system in endothelium-dependent hyperpolarization in mice

The endothelium plays an important role in maintaining vascular homeostasis by synthesizing and releasing several relaxing factors, such as prostacyclin, nitric oxide (NO), and endothelium-derived hyperpolarizing factor (EDHF). We have previously demonstrated in animals and humans that endothelium-derived hydrogen peroxide (H2O2) is an EDHF that is produced in part by endothelial NO synthase (eNOS). In this study, we show that genetic disruption of all three NOS isoforms (neuronal [nNOS], inducible [iNOS], and endothelial [eNOS]) abolishes EDHF responses in mice. The contribution of the NOS system to EDHF-mediated responses was examined in eNOS−/−, n/eNOS−/−, and n/i/eNOS−/− mice. EDHF-mediated relaxation and hyperpolarization in response to acetylcholine of mesenteric arteries were progressively reduced as the number of disrupted NOS genes increased, whereas vascular smooth muscle function was preserved. Loss of eNOS expression alone was compensated for by other NOS genes, and endothelial cell production of H2O2 and EDHF-mediated responses were completely absent in n/i/eNOS−/− mice, even after antihypertensive treatment with hydralazine. NOS uncoupling was not involved, as modulation of tetrahydrobiopterin (BH4) synthesis had no effect on EDHF-mediated relaxation, and the BH4/dihydrobiopterin (BH2) ratio was comparable in mesenteric arteries and the aorta. These results provide the first evidence that EDHF-mediated responses are dependent on the NOSs system in mouse mesenteric arteries

Serum uric acid distribution according to SLC22A12 W258X genotype in a cross-sectional study of a general Japanese population

<p>Abstract</p> <p>Background</p> <p>Although <it>SLC22A12 258X </it>allele was found among those with hypouricemia, it was unknown that serum uric acid distribution among those with <it>SLC22A12 258X </it>allele. This study examined serum uric acid (SUA) distribution according to <it>SLC22A12 </it>W258X genotype in a general Japanese population.</p> <p>Methods</p> <p>Subjects were 5,023 health checkup examinees (3,413 males and 1,610 females) aged 35 to 69 years with creatinine < 2.0 mg/dL, who were participants of a cohort study belonging to the Japan Multi-Institutional Collaborative Cohort Study (J-MICC Study). <it>SLC22A12 </it>W258X was genotyped with a polymerase chain reaction with confronting two-pair primers.</p> <p>Results</p> <p>The genotype frequency was 4,793 for <it>WW</it>, 225 for <it>WX</it>, and 5 for <it>XX</it>, which was in Hardy-Weinberg equilibrium (p = 0.164) with <it>X </it>allele 0.023 (95% confidence interval [0.021-0.027]). Mean (range) SUA was 6.2 (2.1-11.4) mg/dL for <it>WW</it>, 3.9 (0.8-7.8) mg/dL for <it>WX</it>, and 0.8 (0.7-0.9) mg/dL for <it>XX </it>among males, and 4.5 (1.9-8.9) mg/dL, 3.3 (2.0-6.5) mg/dL, and 0.60 (0.5-0.7) mg/dL among females, respectively. Six individuals with SUA less than 1.0 mg/dL included two males with <it>XX </it>genotype, one male with <it>WX </it>genotype, and three females with <it>XX </it>genotype. Subjects with <it>WX </it>genotype were 14 (77.8%) of 18 males with a SUA of 1.0-2.9 mg/dL, and 28 (34.6%) of 81 females with the same range of SUA. The corresponding values were 131 (25.1%) of 522 males and 37 (3.5%) of 1,073 females for SUA 3.0-4.9 mg/dL, and 8 (0.4%) of 2,069 males and 5 (1.1%) of 429 females for SUA 5.0-6.9 mg/dL. The <it>X </it>allele effect for SUA less than 3 mg/dL was significantly (p < 0.001) higher in males (OR = 102.5, [33.9-309.8]) than in females (OR = 25.6 [14.4-45.3]).</p> <p>Conclusions</p> <p>Although <it>SLC22A12 </it>W258X was a determining genetic factor on SUA, SUA of those with <it>WX </it>genotype distributed widely from 0.8 mg/dL to 7.8 mg/dL. It indicated that other genetic traits and/or lifestyle affected SUA of those with <it>WX </it>genotype, as well as those with <it>WW </it>genotype.</p