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Aloe is widely used as a cosmetic and medicinal plant. Numerous studies have reported that aloe gel extract has antioxidant, anticancer, antidiabetic, immunity, and skin antiaging properties. However, few studies have investigated the properties of fermentation products of aloe processing byproducts. Aloe stalks and leaves remain as byproducts after the aloe beverage manufacturing process. This study evaluated whether fermentation products of blender and press extracts of aloe processing byproducts (BF and PF, respectively) that remain after beverage manufacturing were useful as functional biomaterial by investigating their effects on adipocyte differentiation, hyaluronic acid (HA) production, tyrosinase activity, and antioxidant activity. Co-fermentation of G. xylinus and S. cerevisiae was conducted for fermentation of aloe processing byproducts. The BF and PF products did not induce observable cytotoxicity effects. However, BF and PF products did inhibit a 3T3-L1 adipocyte differentiation compared with control, with the BF product displaying greater inhibition of 3T3-L1 adipocyte differentiation than the PF product. HA production increased in HaCaT cell cultures as the concentration of the MF product increased, as compared with the untreated control. The levels of tyrosinase inhibition, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, and superoxide dismutase (SOD)-like activity also depended on the MF product concentration. This study indicates that the fermented products of aloe processing byproducts have biological potential for applications in the manufacturing of cosmetics, pharmaceuticals, and beverages. These laboratory bench results provide the foundation for future studies of scaling and practical applications at the industrial level.

Dart1-24, one of the 37 autonomous DNA transposon Dart1s, was heritably activated by the demethylation of the 5' region following 5-azaC treatment of rice seeds. Transposons are controlled by epigenetic regulations. To obtain newly activated autonomous elements of Dart1, a DNA transposon, in rice, seeds of a stable pale yellow leaf (pyl-stb) mutant caused by the insertion of nDart1-0, a nonautonomous element in OsClpP5, were treated with 5-azaC, a demethylating agent. In the 5-azaC-treated M1 plants, 60-70% of the plants displayed variegated pale yellow leaf (pyl-v) phenotype, depending on the concentration of 5-azaC used, suggesting that inactivated Dart1 might become highly activated by 5-azaC treatment and nDart1-0 was excised from OsClpP5 by the activated Dart1s. Although the M2 plants derived from most of these pyl-v plants showed stable pyl phenotypes, some variegated M1 plants generated pyl-v M2 progeny. These results indicated that most M1 pyl-v phenotypes at M1 were not heritable. Dart1-24, 1-27 and 1-28 were expressed in the M2 pyl-v plants, and mapping analysis confirmed that Dart1-24 was newly activated. Further, the transgenerational activation of Dart1-24 was demonstrated to be caused by the demethylation of nucleotides in its 5' region.

We identified a peel-specific expressed gene in Citrus unshiu fruits by differentially expressed gene (DEG) analysis, which showed a homology with carotenoid isomerase-like genes identified from other plants and, therefore, designated as CuCRTISO-like. Here we determined the promoter sequence of CuCRTISO-like and analyzed histochemical GUS activity using transgenic Arabidopsis plants harboring CuCRTISO-like promoter-GUS gene constructs (pCRTL-Prom1~pCRTL-Prom5 lines). The promoter activity of CuCRTISO-like was detected in the cotyledon at 5 and 10 days after germination (DAG), young leaf, and anther, but not in the cotyledon at 15 DAG and mature leaf. Several cis-acting elements involved in hormones and abiotic stresses are located on the CuCRTISO-like promoter. Salicylic acid and ethylene treatments induced the GUS activity in the pCRTL-prom1 and pCRTL-Prom4 line, respectively. Treatment of drought and wounding stress induced the GUS activity in the pCRTL-Prom4 and pCRTL-Prom3 line, respectively. Heat stress treatment induced GUS activity more strongly as the promoter length decreased except for no GUS activity in the pCRTL-Prom5 line. The CuCRTISO-like expression during fruit maturation of C. unshiu showed a peel-specific expression pattern. Our results suggest that CuCRTISO-like promoter activity is regulated in a developmental and organ-specific manner, and responds to hormones and abiotic stresses.

Citrus fruits are rich sources of different phytochemicals for human health due to their high anti-oxidant capacity. However, the anti-aging effect of citrus fruits has not been well understood. In this study, methanol extracts taken at various developmental stages from tissues of Citrus unshiu was used to investigate its anti-aging effect by an elastase/collagenase inhibition assay, and a gas chromatography-mass spectrometry (GC-MS) analysis was carried out to identify the potential anti-aging compositions. The elastase/collagenase inhibitory activity was greatest in the flesh of immature green fruit (i.e., early July flesh (EJF)), and four candidate compounds were selected by GC-MS and evaluated by a collagenase inhibition assay. Three of the four candidate compounds (heptadecanoic acid, D-allose, and 5-hydroxymethyl-2-furaldehyde (HMF)) showed anti-aging activity, and the activity was highest in heptadecanoic acid, followed by D-allose and HMF. The total phenolic content (TPC), total flavonoid content (TFC), and anti-oxidant activity (DPPH and ferric reducing anti-oxidant power (FRAP) assay) were also investigated. Interestingly, the patterns of the total phenolic/flavonoid content and the anti-oxidant activity were different from that of the elastase/collagenase inhibitory activity. Flowers had the most anti-oxidant activity followed by immature fruit, and the fruit peels had more anti-oxidant activity than its flesh at all stages of development. This study demonstrated that the flesh of immature fruit and flowers of C. unshiu could be sources of anti-aging and anti-oxidant agents for human health, respectively.

Recent methylome analyses of the entire Arabidopsis thaliana genome using various mutants have provided detailed information about the DNA methylation pattern and its function. However, information about DNA methylation in other plants is limited, partly because of the lack of mutants. To study DNA methylation in rice (Oryza sativa) we applied homologous recombination-mediated gene targeting to generate targeted disruptants of OsDRM2, a rice orthologue of DOMAINS REARRANGED METHYLASE 1 and 2 (DRM1/2), which encode DNA methyltransferases responsible for de novo and non-CG methylation in Arabidopsis. Whereas Arabidopsis drm1 drm2 double mutants showed no morphological alterations, targeted disruptants of rice OsDRM2 displayed pleiotropic developmental phenotypes in both vegetative and reproductive stages, including growth defects, semi-dwarfed stature, reductions in tiller number, delayed heading or no heading, abnormal panicle and spikelet morphology, and complete sterility. In these osdrm2 disruptants, a 13.9% decrease in 5-methylcytosine was observed by HPLC analysis. The CG and non-CG methylation levels were reduced in RIRE7/CRR1 retrotransposons, and in 5S rDNA repeats. Associated transcriptional activation was detected in RIRE7/CRR1. Furthermore, de novo methylation by an RNA-directed DNA methylation (RdDM) process involving transgene-derived exogenous small interfering RNA (siRNA) was deficient in osdrm2-disrupted cells. Impaired growth and abnormal DNA methylation of osdrm2 disruptants were restored by the complementation of wild-type OsDRM2 cDNA. Our results suggest that OsDRM2 is responsible for de novo, CG and non-CG methylation in rice genomic sequences, and that DNA methylation regulated by OsDRM2 is essential for proper rice development in both vegetative and reproductive stages.

Carotenoid isomerase (CRTISO) catalyzes the isomerization of prolycopene to all-trans-lycopene in the carotenoid biosynthetic pathway. We isolated a promoter region of CuCRTISO from Citrus unshiu. We determined whether the promoter encoded organ-specific or developmental-specific expression and identified possible cis-acting promoter elements. The promoter and two truncated versions were fused to the β-glucuronidase (GUS) gene and transformed into Arabidopsis thaliana. Transgenic lines expressing the 2501-bp promoter (pCiso-Prom1) and truncated promoters (pCiso-Prom2 and pCiso-Prom3) showed the same developmental and organ-specific activity. GUS expression was detected in the cotyledon and root at 5 and 10 days after germination, mature leaf, and anther. The CuCRTISO promoter contained several cis-acting elements involved in hormonal and environmental stress. Drought stress or abscisic acid treatment did not induce GUS expression in any transgenic lines. Heat stress induced GUS expression in the pCiso-Prom1 line; this promoter construct contains the heat stress-responsive element. Ethylene and cold stress treatments induced GUS expression only in the pCiso-Prom3 line, although all transgenic lines contained the same cis-acting ethylene and low-temperature response elements, which could indicate the existence of unknown repressor element(s) in the CuCRTISO promoter. The CuCRTISO expression of Citrus unshiu during fruit maturation showed that in the green stage, it was highly expressed and then subsequently decreased after the stage. These studies indicate that CuCRTISO promoter activity is regulated in a developmental and organ-specific manner that responds to heat, cold, and ethylene. These results provide new insights into the role of cis-acting element(s) in CuCRTISO promoter activity.

In contrast to angiosperms, some gymnosperms form well-development suspensors during somatic embryogenesis. This feature is highly useful to studies of suspensor biology. In cell cultures of Japanese larch ( Larix leptolepis Gordon), somatic embryos rarely formed when the initial cell density was lower than 0.1 ml packed cell volume (PCV) l −1 . However, in the presence of phytosulfokine (PSK), a peptidyl plant growth factor, mitotic activity during somatic embryogenesis was stimulated, even when the cell density was lower than 0.1 ml PCV l −1 , particularly, the development of the suspensor. Manual separation of somatic embryos into the embryo proper and the suspensor arrested further development, but when embryos proper lacking a suspensor were treated with PSK, the suspensor regenerated and development proceeded to maturity. Because no division of the suspensor cells was observed, the cells of the regenerated suspensor probably originated from basal cells of the embryo proper. In contrast, PSK treatment of suspensors lacking the embryo proper did not stimulate further development. These results indicate that PSK stimulates not only cell division of embryo proper but also development of the suspensor.

In carrot (Daucus carota L.) somatic embryos, desiccation tolerance is induced by treatment with abscisic acid (ABA). Six cDNA clones that showed ABA-enhanced expression were isolated from carrot by differential screening with ABA-treated and ABA-untreated somatic embryos, and they were named the CAISE (carrot ABA-induced in somatic embryos) genes. Five of the clones encode late embryogenesis abundant (LEA) proteins, and the other clone encodes a glucose and ribitol dehydrogenase. The expression of the CAISE genes was detected in maturing seeds, embryogenic cells, and ABA-treated somatic embryos in which exhibit desiccation tolerance induced by endogenous or exogenous ABA. These results indicate that ABA-induced desiccation tolerance in carrot somatic embryos may be induced by the LEA proteins and the glucose and ribitol dehydrogenase encoded by these ABA-inducible genes.

Phytosulfokine-α (PSK-α), a peptidyl plant growth factor that was originally isolated from conditioned medium derived from asparagus mesophyll cell culture, stimulates cell proliferation in some plants in suspension culture. However, the physiological relationship between phytohormones and this growth factor in plants is not well understood. Using carrot ( Daucus carota L.) non-embryogenic cell culture, we investigated the stimulatory effects of PSK-α on the cell proliferation induced by different combinations and concentrations of auxin [2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), or naphthaleneacetic acid (NAA)] and PSK-α. Cell proliferation was not stimulated by PSK-α at high auxin concentrations, but was promoted at low auxin concentrations. There was no proliferation in the absence of auxin. These results indicate that PSK-α stimulates cell proliferation when cell division activity is low, and that auxin is required for this process. We also evaluated PSK-α production using medium derived from non-embryogenic cell culture with high or low concentrations of auxin (2,4-D, IAA or NAA) in a competition ELISA system. More PSK-α was produced at high auxin concentrations than at low auxin concentrations. These results suggest that auxin participates in PSK-α production.

Cells in culture with a high cell density can proliferate in medium that contains phytohormones, but cells at low Phytosulphokine-a (PSK-a) is the first chemically char- cell density cannot. However, cells at a low cell density acterized peptide that acts as a plant growth factor. can grow in the presence of conditioned medium (Stuart It stimulates the proliferation of asparagus and rice and Street, 1969; Somers et al., 1987; Birnberg et al., cells, but no information is yet available on its effects 1988; Bellincampi and Morpurgo, 1987, 1989; Huang on plant morphogenesis. The effects of PSK-a on et al., 1990; Bellincampi et al., 1993). None of the somatic embryogenesis in carrot (Daucus carota L.) phytohormones characterized to date is able to substitute were examined. PSK-a, when added to the induction for the conditioned medium. Thus, it was postulated medium for somatic embryogenesis, increased the that certain factors accumulate in conditioned medium number of somatic embryos. The chemical analogues and promote the division of cells cultured at low cell [2‐5]PSK-a and tyrosine sulphate ester (Tyr-SO 3 H), density. Matsubayashi and Sakagami (1996) isolated which have been used as negative controls in other phytosulphokine-a (PSK-a) as such a factor from the systems, had no effect. Moreover, the proliferation conditioned medium of a culture of mesophyll cells of of cells during somatic embryogenesis was also Asparagus oYcinalis L. PSK-a was determined to be a enhanced by PSK-a. These results indicate that PSK- sulphated pentapeptide: H-Tyr(SO