The number of peroxisomes in a cell can change rapidly in response to changing environmental and physiological conditions. Pexophagy, a type of selective autophagy, is involved in peroxisome degradation, but its physiological role remains to be clarified. Here, we report that cells of the cucumber anthracnose fungus Colletotrichum orbiculare undergo peroxisome degradation as they infect host plants. We performed a random insertional mutagenesis screen to identify genes involved in cucumber pathogenesis by C. orbiculare. In this screen, we isolated a homolog of Pichia pastoris ATG26, which encodes a sterol glucosyltransferase that enhances pexophagy in this methylotrophic yeast. The C. orbiculare atg26 mutant developed appressoria but exhibited a specific defect in the subsequent host invasion step, implying a relationship between pexophagy and fungal phytopathogenicity. Consistent with this, its peroxisomes are degraded inside vacuoles, accompanied by the formation of autophagosomes during infection-related morphogenesis. The autophagic degradation of peroxisomes was significantly delayed in the appressoria of the atg26 mutant. Functional domain analysis of Atg26 suggested that both the phosphoinositide binding domain and the catalytic domain are required for pexophagy and pathogenicity. In contrast with the atg26 mutant, which is able to form appressoria, the atg8 mutant, which is defective in the entire autophagic pathway, cannot form normal appressoria in the earlier steps of morphogenesis. These results indicate a specific function for Atg26-enhanced pexophagy during host invasion by C. orbiculare.

Gonocytes are primitive germ cells that reside in the seminiferous tubules of neonatal testes and give rise to spermatogonia, thereby initiating spermatogenesis. Due to a lack of specific markers, the isolation and culture of these cells has proven to be difficult in the pig. In the present study, we show that a lectin, Dolichos biflorus agglutinin (DBA), which has specific affinity for primordial germ cells (PCGs) in the genital ridge, binds specifically to gonocytes in neonatal pig testes. The specific affinity of DBA for germ cells was progressively lost with age. This suggests that DBA binds strongly to primitive germ cells, such as gonocytes, weakly to primitive spermatogonia, and not at all to spermatogonia. The presence of alkaline phosphatase (AP) activity in the germ cells of neonatal pig testis confirmed the existence of primitive germ cells. Gonocytes from neonatal pig testis were purified, and a cell population that consisted of approximately 70% gonocytes was obtained, as indicated by the DBA binding assay. Purified gonocytes were cultured in DMEM/F12 supplemented with 10% FBS in the absence of any specific growth factors for 7 days. The cells remained viable and proliferated actively in culture. Initially, the gonocytes grew as focal colonies that transformed to three-dimensional colonies by 7 days of culture. Cultured germ cells expressed SSEA-1, a marker for embryonic stem (ES) cells, and were negative for the expression of somatic cell markers. These results should help to establish a male germ cell line that could be used for studying spermatogenesis in vitro and for genetic modification of pigs.

The porcine antral follicles, 3-6 mm in diameter, were dissected from the ovaries of mature pigs, and then granulosa and cumulus cells were isolated from each follicle. In atretic follicles, high activity of neutral Ca2+/Mg2+-dependent endonuclease and DNA ladder formation, estimated by electrophoresis, were noted in granulosa cells but not in cumulus cells. Extremely low activity of the endonuclease and no DNA ladder formation were observed in both types of cells obtained from healthy follicles. Moreover, apoptotic cells were observed histochemically among granulosa cells only. A good correlation (r = 0.987) between the endonuclease activity of granulosa cells and the progesterone/estradiol ratio of follicular fluid in each follicle was found. These results suggest that apoptosis occurs in granulosa cells but not cumulus cells in the atretic antral follicles in pigs.

In Arabidopsis (Arabidopsis thaliana), malate released into the rhizosphere has various roles, such as detoxifying rhizotoxic aluminum (Al) and recruiting beneficial rhizobacteria that induce plant immunity. ALUMINUM-ACTIVATED MALATE TRANSPORTER1 (AtALMT1) is a critical gene in these responses, but its regulatory mechanisms remain unclear. To explore the mechanism of the multiple responses of AtALMT1, we profiled its expression patterns in wild-type plants, in transgenic plants harboring various deleted promoter constructs, and in mutant plants with defects in signal transduction in response to various inducers. AtALMT1 transcription was clearly induced by indole-3-acetic acid (IAA), abscisic acid (ABA), low pH, and hydrogen peroxide, indicating that it was able to respond to multiple signals, while it was not induced by methyl jasmonate and salicylic acid. The IAA-signaling double mutant nonphototropic hypocotyls4-1; auxin-responsive factor19-1 and the ABA-signaling mutant aba insensitive1-1 did not respond to auxin and ABA, respectively, but both showed an Al response comparable to that of the wild type. A synthetic microbe-associated molecular pattern peptide, flagellin22 (flg22), induced AtALMT1 transcription but did not induce the transcription of IAA-and ABA-responsive biomarker genes, indicating that both Al and flg22 responses of AtALMT1 were independent of IAA and ABA signaling. An in planta b-glucuronidase reporter assay identified that the ABA response was regulated by a region upstream (2317 bp) from the first ATG codon, but other stress responses may share critical regulatory element(s) located between 2292 and 2317 bp. These results illustrate the complex regulation of AtALMT1 expression during the adaptation to abiotic and biotic stresses.

The methylation pathway, which consists of two metabolic cycles of nutrients, i.e., the methionine and folate cycles, generates S-adenosylmethionine, the methyl donor for the methylation of DNA and histones. Using reverse transcription-polymerase chain reaction, we examined the gene expression patterns of the methylation pathway enzymes during bovine oocyte maturation and preimplantation embryonic development up to the blastocyst stage. Bovine oocytes were demonstrated to have the mRNA of all methylation pathway enzymes examined, namely, methionine adenosyltransferase 1A (MAT1A), MAT2A, MAT2B, S-adenosylhomocysteine hydrolase (AHCY), 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), betaine-homocysteine methyltransferase (BHMT), serine hydroxymethyltransferase 1 (SHMT1), SHMT2, and 5,10-methylenetetrahydrofolate reductase (MTHFR). All the transcripts were consistently expressed throughout all developmental stages, except for MAT1A, which was not detected from the 8-cell stage onward and BHMT, which was not detected in the 8-cell stage. Immunofluorescence analysis of MAT1A protein revealed the relatively higher expression in oocytes and early cleavage stage embryos up to the 8-cell stage compared with the morula and blastocyst stage. Further, to investigate the effects of methylation pathway disruption during the earliest stages of embryonic development, the effects of exogenous homocysteine on preimplantation development and DNA methylation of bovine embryos were investigated in vitro. As results, high concentrations of homocysteine induced hypermethylation of genomic DNA as well as developmental retardation in bovine embryos. These results provide a new insight into nutrient-sensitive epigenetic regulation and perturbation at the earliest stage of our life.