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The CRISPR–Cas9 genome-editing tool and the availability of whole-genome sequences from plant species have revolutionized our ability to introduce targeted mutations into important crop plants, both to explore genetic changes and to introduce new functionalities. Here, we describe protocols adapting the CRISPR–Cas9 system to apple and grapevine plants, using both plasmid-mediated genome editing and the direct delivery of CRISPR–Cas9 ribonucleoproteins (RNPs) to achieve efficient DNA-free targeted mutations in apple and grapevine protoplasts. We provide a stepwise protocol for the design and transfer of CRISPR–Cas9 components to apple and grapevine protoplasts, followed by verification of highly efficient targeted mutagenesis, and regeneration of plants following the plasmid-mediated delivery of components. Our plasmid-mediated procedure and the direct delivery of CRISPR–Cas9 RNPs can both be utilized to modulate traits of interest with high accuracy and efficiency in apple and grapevine, and could be extended to other crop species. The complete protocol employing the direct delivery of CRISPR–Cas9 RNPs takes as little as 2–3 weeks, whereas the plasmid-mediated procedure takes >3 months to regenerate plants and study the mutations.

Two orthologues of FLORICAULA/LEAFY, AFL1 and AFL2 (apple FLO/LFY), were isolated from the floral buds of apple trees. Their expression was detected in various tissues and during differentiation of the floral buds. Furthermore, the flowering effectiveness of each gene was assessed with transgenic Arabidopsis. Both AFL1 and AFL2 showed high homology to each other (90%) and a high degree of similarity to PTLF and PEAFLO (70%), which are homologues of FLO/LFY from poplar and pea, respectively. RNA blot analysis showed that AFL1 was expressed only in the floral bud during the transition from vegetative to reproductive growth, whereas AFL2 was expressed in vegetative shoot apex, floral buds, floral organs and root. Genomic Southern analysis showed that apple had other homologues in addition to AFL1 and AFL2. The transgenic Arabidopsis with over-expressed AFL2 showed accelerated flowering and gave rise to several solitary flowers from rosette axils directly. AFL1 had similar effects, but the phenotypes of the transgenic Arabidopsis with AFL1 were weaker than those with AFL2. These results suggest that both genes are involved in flower differentiation in apple.

Electrically controlled fiber variable-optical-attenuator arrays with polymer-network liquid crystals are shown to be compact and to have a large attenuation range (30–40 dB) and low residual loss (0.55 dB) at wavelengths from 1.3 to 1.6 µm. Their estimated power consumption is very low (<30 nW/channel), and arrays with more than ten channels can be made. The manufacturing process is simple: Trenches 30–100 µm wide are cut across parallel conductive-layer-coated optical fibers and are filled with a polymer-network liquid crystal. The attenuation properties depend on UV-curing conditions and on trench width.

We cloned novel Na+-ATPase (HANA) cDNA from marine alga Heterosigma akashiwo. The full-length HANA cDNA was 4467 bp long and coded for a 1330 amino acid protein with a molecular weight of 146 306. The deduced product exhibited around 40% identity in amino acids with Na+/K+-ATPase α-subunits. A hydrophilic sequence of 285 amino acid residues that showed no homology with any sequence listed in databases existed in the M7–M8 junction of HANA. This is the first report on the primary structure of putative Na+-transporting ATPase from plant cells.

Two cDNA clones (HAA13 and HAA1) which include conserved regions of genes of P-type ATPases were isolated from the marine alga Heterosigma akashiwo by a method that included the polymerase chain reaction. The longer cDNA (3286 bp), HAA13, consisted of an open reading frame that encoded a 106 kDa polypeptide of 977 amino acids with several possible transmembrane domains and conserved regions of eukaryotic P-type ATPases. One transmembrane domain had a leucine zipper structure. HAA1 was not a full-length gene (2054 bp) and lacked the 5' region, but it also included the conserved regions and putative transmembrane domains. Antibodies against the polypeptides encoded by HAA13 and HAA1 that have been expressed in Escherichia coli reacted with 100 kDa and 95 kDa polypeptides, respectively, on intracellular membranes of H. akashiwo cells. Immunostaining of H. akashiwo cells revealed that the HAA13 antigen was distributed on membranes around chloroplasts and the HAA1 antigen was located on small vesicles.

An 18 kDa extracellular insoluble protein (EIP18) was found previously in amorphous particles suspended in the culture medium and in the interspaces of cell clusters of carrot (Daucus carota L.) callus, as well as in the extracellular spaces of carrot seeds, being located both in the embryo and at the inner edge of the endosperm. We purified EIP18 by washing the amorphous particles with the mixture of Triton X-100, NaCl and ethylenediaminetetraacetic acid (EDTA). We determined several partial amino acid sequences, and then we cloned and sequenced a cDNA for EIP18. EIP18 was found to consist of 133 amino acid residues that included a signal sequence, but it did not contain cysteine, sites for N-linked glycosylation or hydrophobic regions. Since its sequence was found to be homologous to that of inhibitors of cysteine proteinases, namely cystatins, EIP18 was renamed EICC (extracellular insoluble cystatin of carrot). EICC expressed in yeast was also found in an insoluble form in yeast cell walls. EICC prepared from the culture medium of carrot cells inhibited commercial cysteine proteinases and a proteinase extracted from germinating carrot seeds. The expression of the gene for EICC was detected in developing seeds, and the level of its transcript was markedly enhanced upon treatment of somatic embryos with abscisic acid.

ABSTRACTIntact particles of rice dwarf phytoreovirus adsorbed to and entered monolayer-cultured cells of the insect vector Nephotettix cincticepsand multiplied within the cells. Particles that lacked the P2 protein neither attached to nor infected such cells. Furthermore, P2-free particles obtained from a transmission-competent isolate of the virus were unable to infect insect vectors that had been allowed to feed on these virus particles through a membrane. However, when such virus particles were injected into insects via a glass capillary tube they successfully infected the insects, which became able to transmit the virus. These results support the hypothesis that, while P2-free particles can neither interact with nor infect cells in the intestinal tract of the insect vector, they do retain the ability to infect such cells when physically introduced into the hemolymph by injection.

A new type of 18-crown-6 derivative having two carboxylic acids on the side arms transported calcium and potassium ions in opposite directions across a dichloromethane membrane by pH control. Calcium and potassium ions were concentrated in the acidic and the basic phases, respectively. The presence of picrate ion in the acidic phase plays an important role in the transport of potassium ion from the acidic to the basic phase.

Immunological homology was investigated between Heterosigma akashiwo(a marine alga) Na +-activated ATPase and animal Na +,K +-ATPase. The former polypeptide [(1989) Plant Cell Physiol. 30, 923-928] reacted with anti-serum raised against the amino-terminal half of the pig kidney Na +,K +-ATPase α subunit. It is suggested that the Na +,K +-ATPase epitope within the amino-terminal region is conserved in the plant Na +-activated ATPase, and the region containing the epitope may be important for Na ion transport.

Immunological homology was investigated between Heterosigma akashiwo(a marine alga) Na+‐activated ATPase and animal Na+,K+‐ATPase. The former polypeptide [(1989) Plant Cell Physiol. 30, 923‐928] reacted with anti‐serum raised against the amino‐terminal half of the pig kidney Na+,K+‐ATPase α subunit. It is suggested that the Na+,K+‐ATPase epitope within the amino‐terminal region is conserved in the plant Na+‐activated ATPase, and the region containing the epitope may be important for Na ion transport.

We demonstrate a mode-locked Er:fiber laser with a compact fiber-pigtailed optical bench containing a free-space electro-optic modulator for high-speed feedback control. The achieved servo bandwidth of the beat frequency between a given comb component and a reference laser is approximately 850 kHz. The realization of a narrow-linewidth and highly stable frequency comb is confirmed by measuring both in-loop and out-of-loop beat frequencies. We also demonstrate the frequency measurement of two iodine-stabilized Nd:YAG lasers through the narrow-linewidth comb without stability degradation. The developed optical frequency comb can work continuously for weeks without maintenance.

The marine raphidophycean biflagellate, Heterosigma akashiwo, clearly showed diurnal vertical migration under a 12 h light-12 h dark photoperiod appearing at the surface of the culture medium during the light period and at the bottom during the dark period. The upward migration commenced a few hours before the light was turned on and the downward migration a few hours before it was turned off. The diuranal vertical migration behavior was closely correlated with diurnal changes in the specific gravity of the cells, those near the surface of the culture medium had a smaller specific gravity than those at the bottom. The migration behavior was also correlated with the direction of cell swimming. More cells had flagella furrow facing upward than downward in the light phase, and vice versa in the dark phase. Phototaxis was not the main factor inducing the vertical migration, though the cells did show a tactic respose to light. Chemotactic responses to O2, N2 or CO2 gas did not occur.

The effect of growth temperature on the compositions of phospholipids and fatty acids of the psychrotrophic bacterium Pseudomonas sp. strain E-3 was studied. The composition of phospholipids (phosphatidylethanolamine, phosphatidylglycerol and cardiolipin) did not differ significantly in cells grown at 5�, 15�, and 30�C. Phosphatidylethanolamine (PE) was the most abundant, amounting to over 70% of total phospholipids. When the growth temperature was lowered, palmitoleic [16:1(Δ9)] and cis-vaccenic [18:1(Δ11)] acids increased at the expense of palmitic (16:0) acid, especially in PE. In vivo experiments using [l-14C]16:0 showed that incorporation of radioactivity into 16:1(Δ9) was oxygen-dependent and cerulenin-insensitive, and incorporation into 18:1(Δ11) was cerulenin-sensitive. These results suggest that 16:0 was aerobically desaturated to 16:1(Δ9) and subsequently elongated to 18:1(Δ11). Desaturation activity of 16:0 in the cells grown at 25�C was enhanced by lowering the assay temperature.

Highly purified plasma membranes were isolated from Heterosigma akashiwo cells, a marine raphidophycean unicellular biflagellate, by the silica microbead method, and the ATPase activity of the membranes was characterized. The ionic requirements and spectrum of effective inhibitors enable us to identify a novel Na+-activated ATPase in the plasma membrane of this organism. Furthermore, we detected two phosphorylated intermediate forms of ATPases, with molecular weights of 150 kDa and 95 kDa as judged by acid SDS-polyacrylamide gel electrophoresis of extracts of isolated plasma membrane. The 150 kDa intermediate was phosphorylated in the presence of both Mg2+ and Na+, while the 95 kDa intermediate was phosphorylated in the presence of Mg2+ alone. Both were dephosphorylated in the presence of monovalent cations. These results indicate that the former intermediate was a Na+-activated ATPase, similar to Na+,K+-ATPases from animals, and the latter was similar to H+,K+-ATPases from higher plants. The physiological significance of the two kinds of ATPase in the plasma membrane of marine algae.

Changes of endogenous 9, 10-ketol-octadecadienoic acid (KODA) concentrations, which is synthesized from linolenic acid by 9-lipoxygenase, were analyzed in apple [Malus xsylvestris (L.) Mill. var. domestica (Borkh.) Mansf.] buds. In addition, the effects of 15,16-chloro, hydroxy-9-hydroxy-10-oxo-12(Z)-octadecenoic acid (CKODA) application, which is an analog of KODA, on flower bud formation and the expression of MdTFLl (terminal flower 1) and MdFTl (flowering locus 11) genes in apple buds were investigated in heavy-crop treatment (HCT) and under shade. An increase of endogenous KODA in the buds in the fruit-thinning treatment, which resulted in a higher proportion of flower bud formation than in HCT, was observed at 63 days after full bloom, but no such increase was found in HCT. In the shade-treated and heavy-crop trees, the expression of MdTFLl in the buds to which CKODA was applied was lower than that in untreated buds. In contrast, under shade, the expression of MdFTl in the CKODA-treated buds was higher than that in untreated buds. These results suggest that endogenous KODA may be associated with flower bud formation, and its application may be effective at improving the proportion of flower bud formation through its effect on MdTFLl and MdFTl. [ABSTRACT FROM AUTHOR]