Epigenetic regulation of <Emphasis Type="Italic">MdMYB1</Emphasis> is associated with paper bagging-induced red pigmentation of apples

Main conclusion

Paper-bagging treatment can transform non-transcribed MdMYB1 - 2 and MdMYB1 - 3 alleles into transcribed alleles through epigenetic regulations, resulting in the red pigmentation of a normally non-red apple cultivar ‘Mutsu.’ Anthocyanin biosynthesis in apples is regulated by MdMYB1/A/10, an R2R3-Type MYB gene. ‘Mutsu,’ a triploid apple cultivar harboring non-transcribed MdMYB1-2 and MdMYB1-3 alleles, retains green skin color under field conditions. However, it can show red/pink pigmentation under natural or artificial ultraviolet-B (UV-B) light exposure after paper-bagging and bag removal treatment. In the present study, we found that in ‘Mutsu,’ paper bagging-induced red pigmentation was due to the activation of non-transcribed MdMYB1-2/-3 alleles, which triggered the expression of downstream anthocyanin biosynthesis genes in a UV-B-dependent manner. By monitoring the epigenetic changes during UV-B-induced pigmentation, no significant differences in DNA methylation and histone modifications in the 5′ upstream region of MdMYB1-2/-3 were recorded between the UV-B-treated fruit skin (red) and the fruit skin treated only by white light (green). In contrast, bag treatment lowered the DNA methylation in this region of MdMYB1-2/-3 alleles. Similarly, higher levels of histone H3 acetylation and trimethylation of H3 tail at lysine 4, and lower level of trimethylation of H3 tail at lysine 27 were observed in the 5′ upstream region of MdMYB1-2/-3 in the skin of the fruit immediately after bag removal. These results suggest that bagging treatment can induce epigenetic changes, facilitating the binding of trans factor(s) to MdMYB1-2/-3 alleles, resulting in the activation of these MYBs after bag removal.

     

Cerebral primitive neuroectodermal tumor in an adult male. A case report

BACKGROUND: Primitive neuroectodermal tumors (PNETs) are very rare. Malignant tumors of the cerebrum in young individuals are composed predominantly of undifferentiated cells, with moderate differentiation along either neuronal or glial lines. To our knowledge, cerebral PNETs in adults are extraordinarily rare and have been reported in only 11 cases, with little cytologic documentation in the literature. The cytopathologic, immunohistochemical and ultrastructural features of cerebral PNET arising in an adult male are presented. CASE: A cystic tumor, on computed tomography and magnetic resonance imaging, arose from the left frontal lobe in a 39-year-old man and contained histopathologic features of PNET. Specimens obtained from surgery revealed the presence of an undifferentiated type of PNET with moderate neuronal and glial differentiation and mild characteristic findings of peripheral PNET. The cytologic and histologic specimens showed evidence of a scattered pattern of blastic and undifferentiated tumor cells and a neural arrangement with Homer-Wright-like rosettes. Immunohistochemically, the tumor cells were glial fibrillary acidic protein, neuron-specific enolase, synaptophysin and CD-99 positive and epithelial membrane antigen, S-100 protein and vimentin negative. Ultrastructurally, neither microtubular structures nor intermediate filaments, except neurosecretory granules, were found in the tumor cells. CONCLUSION: Both immunohistochemical and ultrastructural studies on cytologic and histologic slides were important for the diagnosis of PNET because of establishing not only undifferentiated tumor cells but also neural and glial differentiation.     

Linkage maps of the apple ( Malus × domestica Borkh.) cvs ‘Ralls Janet’ and ‘Delicious’ include newly developed EST markers

Two apple genetic linkage maps were constructed using amplified fragment length polymorphisms (AFLPs), simple sequence repeats (SSRs), random amplified polymorphic DNAs (RAPDs), and expressed sequence tag (EST)-derived markers in combination with a pseudo-testcross mapping strategy in which the cultivars ‘Ralls Janet’ and ‘Delicious’ were used as the respective seed parents. Mitsubakaido (Malus sieboldii) was used as the pollen parent for each of the segregating F₁ populations. Expressed sequence tag data were obtained from the random sequencing of cDNA libraries constructed from in vitro cultured shoots and maturing fruits of cv ‘Fuji’, which is the offspring of a cross between ‘Ralls Janet’ and ‘Delicious’. In addition, a number of published gene sequences were used to develop markers for mapping. The ‘Ralls Janet’ map consisted of 346 markers (178 AFLPs, 95 RAPDs, 54 SSRs, 18 ESTs, and the S locus) in 17 linkage groups, with a total length of 1082 cM, while that of ‘Delicious’ comprised 300 markers (120 AFLPs, 81 RAPDs, 64 SSRs, 32 ESTs, and the S, Rf, and MdACS-1 loci) on 17 linkage groups spanning 1031 cM. These maps are amenable to comparisons with previously published maps of ‘Fiesta’ and ‘Discovery’ (Liebhard et al., Mol Breed 10:217–241, 2002; Liebhard et al., Theor Appl Genet 106:1497–1508, 2003a) because several of the SSRs (one to three markers per linkage group) were used in all of the maps. Distorted marker segregation was observed in three and two regions of the ‘Ralls Janet’ and ‘Delicious’ maps, respectively. These regions were localized in different parts of the genome from those in previously reported apple linkage maps. This marker distortion may be dependent on the combinations of cultivars used for map construction.     

Direct Transfer of Plasmid DNA from Intact Yeast Spheroplasts into Plant Protoplasts

We developed a polyethylene glycol (PEG)-mediated direct DNAtransfer method from intact Saccharomyces cerevisiae spheroplastsinto Arabidopsis thaliana protoplasts. To monitor the DNA transferfrom yeast to plant cells, ß-glucuronidase (GUS) reportergene in which a plant intron was inserted was used as a reporter.This intron-GUS reporter gene on a 2µm-based plasmid vectorwas not expressed in yeast transformants, while it expressedGUS activity when the plasmid DNA was introduced into plantcells. When a mixture of 1 x 10⁸ of S. cerevisiae spheroplastsharboring the plasmid and 2 x 10⁶ of A. thaliana protoplastswas treated with PEG and high pH-high Ca²⁺ solution (0.4 M mannitol,50 mM CaCl₂, 50 mM glycine-NaOH pH 10.5), GUS activity was detectedin the extract of the plant cells after a three-day culture.The GUS activity was higher than that of a reconstitution experimentin which the mixture of 1 x 10⁸ of S. cerevisiae spheroplastswhich did not carry the reporter gene, 2 x 10⁶ of A. thalianaprotoplasts and the same amount of the reporter plasmid DNAas that contained in 1 x 10⁸ of S. cerevisiae spheroplasts,was treated with PEG and high pH-high Ca²⁺ solution. Moreover,the GUS gene expression was resistant to micrococcal nucleasetreatment before and during PEG treatment. From these results,we concluded that plasmid DNA can be directly transferred fromintact yeast spheroplasts to plant protoplasts by a nuclease-resistantprocess, possibly by the cell fusion. ²Deceased on September 15, 1992.     

Effects of ethynylestradiol on vitellogenin synthesis and sex differentiation in juvenile grey mullet (Mugil cephalus) persist after long-term exposure to a clean environment

We investigated the continuing effects of exposure to ethynylestradiol (EE₂) in juvenile grey mullet after transfer to a clean environment. Eleven-month-old juvenile fish containing immature phenotype gonad were fed dry diets; the low and high EE₂-treated groups were fed diets with 0.04 and 4 μg EE₂/g body weight for 4 weeks, respectively. After treatment, they were transferred to clean seawater, and reared with an EE₂ free diet for 350 days. Vitellogenin (VTG) was not detected in the serum of the control group throughout the experimental period. However, in both treatment groups, abnormal values of serum VTG were detected until approximately 100 days after transfer to a clean environment. In the control group, sex differentiation was not confirmed until 206 days after transfer to a clean environment. However, some of the fish in the 0.04 μg EE₂-treated group had ovarian cavity and oocytes at 26 days. In most of the fish in the 4 μg EE₂-treated group, the ovarian cavity had already appeared at the end of EE₂ treatment (0 day), and oocytes were observed at 26 days, suggesting that EE₂ accelerates ovarian differentiation. These results suggest that previous exposure to EE₂ has long-term effects on VTG synthesis and gonadal development.     

Four TFL1/CEN-Like Genes on Distinct Linkage Groups Show Different Expression Patterns to Regulate Vegetative and Reproductive Development in Apple (Malusxdomestica Borkh.)

Recent molecular analyses in several plant species revealedthat TERMINAL FLOWER1 (TFL1) and CENTRORADIALIS (CEN) homologsare involved in regulating the flowering time and/or maintainingthe inflorescence meristem. In apple (Malusxdomestica Borkh.),four TFL1/CEN-like genes, MdTFL1, MdTFL1a, MdCENa and MdCENb,were found and mapped by a similar position on putatively homoeologouslinkage groups. Apple TFL1/CEN-like genes functioned equivalentlyto TFL1 when expressed constitutively in transgenic Arabidopsisplants, suggesting that they have a potential to complementthe TFL1 function. Because MdTFL1 and MdTFL1a were expressedin the vegetative tissues in both the adult and juvenile phases,they could function redundantly as a flowering repressor anda regulator of vegetative meristem identity. On the other hand,MdCENa was mainly expressed in fruit receptacles, cultured tissuesand roots, suggesting that it is involved in the developmentof proliferating tissues but not in the control of the transitionfrom the juvenile to the adult phase. In contrast, MdCENb wassilenced in most organs probably due to gene duplication bythe polyploid origin of apple. The expression patterns of MdTFL1and MdCENa in apple were also supported by the heterologousexpression of β-glucuronidase fused with their promoterregions in transgenic Arabidopsis. Our results suggest thatfunctional divergence of the roles in the regulation of vegetativemeristem identity may have occurred among four TFL1/CEN-likegenes during evolution in apple.