Virus-Induced Flowering by Apple Latent Spherical Virus Vector: Effective Use to Accelerate Breeding of Grapevine.

Apple latent spherical virus (ALSV) was successfully used in promoting flowering (virus-induced flowering, VIF) in apple and pear seedlings. In this paper, we report the use of ALSV vectors for VIF in seedlings and in vitro cultures of grapevine. After adjusting experimental conditions for biolistic inoculation of virus RNA, ALSV efficiently infected not only progeny seedlings of Vitis spp. 'Koshu,' but also in vitro cultures of V. vinifera 'Neo Muscat' without inducing viral symptoms. The grapevine seedlings and in vitro cultures inoculated with an ALSV vector expressing the 'florigen' gene (Arabidopsis Flowering locus T, AtFT) started to set floral buds 20-30 days after inoculation. This VIF technology was successfully used to promote flowering and produce grapes with viable seeds in in vitro cultures of F1 hybrids from crosses between V. ficifolia and V. vinifera and made it possible to analyze the quality of fruits within a year after germination. High-temperature (37 °C) treatment of ALSV-infected grapevine disabled virus movement to newly growing tissue to obtain ALSV-free shoots. Thus, the VIF using ALSV vectors can be used to shorten the generation time of grapevine seedlings and accelerate breeding of grapevines with desired traits.

Rapid identification of apple (Malus×domestica Borkh.) S alleles using sequencing-based DNA marker APPLid.

All apple cultivars harbor the trait called self-incompatibility. Self-incompatibility represents that the pistils of the flowers are not successfully fertilized with own, the same cultivar's pollens. Compatibility or incompatibility of apple flowers are determined by S alleles. For example, the most popular apple cultivar 'Fuji' possesses the S1 and S9 alleles (S1S9 ). Thus, 'Fuji' is incompatible with S1S9 cultivars, but is compatible with the cultivars possessing different combinations of S alleles such as S2S7 and S1S7 . Apple S alleles have been identified by performing a series of allele-specific PCR amplifications, to detect more than ten different S alleles separately. Here, we developed a new type of sequencing-based DNA marker of the apple S-RNase gene, which identifies S alleles. This DNA marker was named APPLid (apple S-allele identifier). A 53-base region in the first coding sequence of S-RNase is the target of APPLid sequencing. Variation in nucleotide sequences in this APPLid sequence enables allele identifications. This region is amplified from apple genomic DNA by using a pair of degenerate primers. The forward primer is attached with 'DS5 adaptor.' After PCR amplification, electrophoresis and gel extraction of 177-bp DNA fragments, APPLid sequence is determined by direct sequencing with a sequencing primer. The APPLid sequences of 20 apple cultivars completely matched their S alleles, which include triploid cultivars. In conclusion, APPLid identifies apple S alleles (S1 , S2 , S3 , S4 , S5 , S7 , S9 , S10 , S20 , S24 , S25 , S26 , S27 and S28 , so far) just by a single sequencing analysis.

A sustained increase in the intracellular Ca²âº concentration induces proteolytic cleavage of EAG2 channel.

Voltage-gated EAG2 channel is abundant in the brain and enhances cancer cell growth by controlling cell volume. The channel contains a cyclic nucleotide-binding homology (CNBH) domain and multiple calmodulin-binding motifs. Here we show that a raised intracellular Ca(2+) concentration causes proteolytic digestion of heterologously expressed and native EAG2 channels. A treatment of EAG2-expressing cells with the Ca(2+) ionophore A23187 for 1h reduces the full-length protein by ∼80% with a concomitant appearance of 30-35-kDa peptides. Similarly, a treatment with the Ca(2+)-ATPase inhibitor thapsigargin for 3h removes 30-35-kDa peptides from ∼1/3 of the channel protein. Moreover, an incubation of the isolated rat brain membrane with CaCl2 leads to the generation of fragments with similar sizes. This Ca(2+)-induced digestion is not seen with EAG1. Mutations in a C-terminal calmodulin-binding motif alter the degrees and positions of the cleavage. Truncated channels that mimic the digested proteins exhibit a reduced current density and altered channel gating. In particular, these shorter channels lack a rapid activation typical in EAG channels with more than 20-mV positive shifts in voltage dependence of activation. The truncation also eliminates the ability of EAG2 channel to reduce cell volume. These results suggest that a sustained increase in the intracellular Ca(2+) concentration leads to proteolytic cleavage at the C-terminal cytosolic region following the CNBH domain by altering its interaction with calmodulin. The observed Ca(2+)-induced proteolytic cleavage of EAG2 channel may act as an adaptive response under physiological and/or pathological conditions.

Purification, biochemical characterization, and cloning of phospholipase D from Streptomyces racemochromogenes strain 10-3.

We previously isolated Streptomyces racemochromogenes strain 10-3, which produces a phospholipase D (PLD) with high transphosphatidylation activity. Here, we purified and cloned the PLD (PLD103) from the strain. PLD103 exerted the highest hydrolytic activity at a slightly alkaline pH, which is in contrast to the majority of known Streptomyces PLDs that have a slightly acidic optimum pH. PLD103 shares only 71-76% amino acid sequence identity with other Streptomyces PLDs that have a slightly acidic optimum pH; thus, the diversity in the primary structure might explain the discrepancy observed in the optimum pH. The purified PLD displayed high transphosphatidylation activity in the presence of glycerol, L: -serine, and 2-aminoethanol hydrochloride with a conversion rate of 82-97% in a simple one-phase system, which was comparable to the rate of other Streptomyces PLDs in a complicated biphasic system.