The PpMYB75-PpDFR module reveals the difference between 'SR' and its bud variant 'RMHC' in peach red flesh.

'Red Meat Honey Crisp (RMHC)' has been widely cultivated by growers in recent years due to its early maturity, and red meat type characteristics. As a bud variant of 'Super Red (SR)' peach, red flesh is the most distinctive characteristic of 'Red Meat Honey Crisp (RMHC)'. However, the mechanism of red flesh formation in 'RMHC' remains unclear. In this study, 79 differentially produced metabolites were identified by metabolomics analysis. The anthocyanin content in 'RMHC' was significantly higher than that in 'SR' during the same period, such as cyanidin O-syringic acid and cyanidin 3-O-glucoside. Other flavonoids also increased during the formation of red flesh, including flavonols (6-hydroxykaempferol-7-O-glucoside, hyperin), flavanols (protocatechuic acid, (+)-gallocatechin), and flavonoids (chrysoeriol 5-O-hexoside, tricetin). In addition, transcriptomic analysis and RT-qPCR showed that the expression levels of the flavonoid synthesis pathway transcription factor MYB75 and some structural genes, such as PpDFR, PpCHS, PpC4H, and PpLDOX increased significantly in 'RMHC'. Subcellular localization analysis revealed that MYB75 was localized to the nucleus. Yeast single hybridization assays showed that MYB75 bound to the cis-acting element CCGTTG of the PpDFR promoter region. The MYB75-PpDFR regulatory network was identified to be a key pathway in the reddening of 'RMHC' flesh. Moreover, this is the first study to describe the cause for red meat reddening in 'RMHC' compared to 'SR' peaches using transcriptomics, metabolomics and molecular methods. Our study identified a key transcription factor involved in the regulation of the flavonoid synthetic pathway and contributes to peach breeding-related efforts as well as the identification of genes involved in color formation in other species.

MdWRKY15 improves resistance of apple to Botryosphaeria dothidea via the salicylic acid-mediated pathway by directly binding the MdICS1 promoter.

Isochorismate synthase (ICS) plays an essential role in the accumulation of salicylic acid (SA) and plant disease resistance. Diseases caused by Botryosphaeria dothidea affect apple yields. Thus, it is important to understand the role of ICS1 in disease resistance to B. dothidea in apple. In this study, SA treatment enhanced the resistance to B. dothidea. MdICS1 was induced by B. dothidea and enhanced the resistance to B. dothidea. MdICS1 promoter analysis indicated that the W-box was vital for the response to B. dothidea treatment. MdWRKY15 was found to interact with the W-box using yeast one-hybrid screening. Subsequently, the interaction was confirmed by EMSA, yeast one-hybrid, ChIP-PCR, and quantitative PCR assays. Moreover, luciferase and GUS analysis further indicated that MdICS1 was transcriptionally activated by MdWRKY15. Finally, we found the function of MdWRKY15 in the resistance to B. dothidea was partially dependent on MdICS1 from the phenotype of transgenic apples and calli. In summary, we revealed that MdWRKY15 activated the transcription of MdICS1 by directly binding to its promoter to increase the accumulation of SA and the expression of disease-related genes, thereby resulting in the enhanced resistance to B. dothidea in the SA biosynthesis pathway.

MdWRKY46-Enhanced Apple Resistance to Botryosphaeria dothidea by Activating the Expression of MdPBS3.1 in the Salicylic Acid Signaling Pathway.

Salicylic acid (SA) is closely related to disease resistance of plants. WRKY transcription factors have been linked to the growth and development of plants, especially under stress conditions. However, the regulatory mechanism of WRKY proteins involved in SA production and disease resistance in apple is not clear. In this study, MdPBS3.1 responded to Botryosphaeria dothidea and enhanced resistance to B. dothidea. Electrophoretic mobility shift assays, yeast one-hybrid assays, and chromatin immunoprecipitation and quantitative PCR demonstrated that MdWRKY46 can directly bind to a W-box motif in the promoter of MdPBS3.1. Glucuronidase transactivation and luciferase analysis further showed that MdWRKY46 can activate the expression of MdPBS3.1. Finally, B. dothidea inoculation in transgenic apple calli and fruits revealed that MdWRKY46 improved resistance to B. dothidea by the transcriptional activation of MdPBS3.1. Viral vector-based transformation assays indicated that MdWRKY46 elevates SA content and transcription of SA-related genes, including MdPR1, MdPR5, and MdNPR1 in an MdPBS3.1-dependent way. These findings provide new insights into how MdWRKY46 regulates plant resistance to B. dothidea through the SA signaling pathway.

MdHIR4 transcription and translation levels associated with disease in apple are regulated by MdWRKY31.

KEY MESSAGE: Here we describe that the regulation of MdWRKY31 on MdHIR4 in transcription and translation levels associated with disease in apple. The phytohormone salicylic acid (SA) is a main factor in apple (Malus domestica) production due to its function in disease resistance. WRKY transcription factors play a vital role in response to stress. An RNA-seq analysis was conducted with 'Royal Gala' seedlings treated with SA to identify the WRKY regulatory mechanism of disease resistance in apple. The analysis indicated that MdWRKY31 was induced. A quantitative real-time polymerase chain reaction (qPCR) analysis demonstrated that the expression of MdWRKY31 was induced by SA and flg22. Ectopic expression of MdWRKY31 in Arabidopsis and Nicotiana benthamiana increased the resistance to flg22 and Pseudomonas syringae tomato (Pst DC3000). A yeast two-hybrid screen was conducted to further analyze the function of MdWRKY31. As a result, hypersensitive-induced reaction (HIR) protein MdHIR4 interacted with MdWRKY31. Biomolecular fluorescence complementation, yeast two-hybrid, and pull-down assays demonstrated the interaction. In our previous study, MdHIR4 conferred decreased resistance to Botryosphaeria dothidea (B. dothidea). A viral vector-based transformation assay indicated that MdWRKY31 evaluated the transcription of SA-related genes, including MdPR1, MdPR5, and MdNPR1 in an MdHIR4-dependent way. A GUS analysis demonstrated that the w-box, particularly w-box2, of the MdHIR4 promoter played a major role in the responses to SA and B. dothidea. Electrophoretic mobility shift assays, yeast one-hybrid assay, and chromatin immunoprecipitation-qPCR demonstrated that MdWRKY31 directly bound to the w-box2 motif in the MdHIR4 promoter. GUS staining activity and a protein intensity analysis further showed that MdWRKY31 repressed MdHIR4 expression. Taken together, our findings reveal that MdWRKY31 regulated plant resistance to B. dothidea through the SA signaling pathway by interacting with MdHIR4.

Ultrafast dynamics of exciton-polariton fluids at non-zero momenta.

In this study, we have explored the ultrafast formation and decay dynamics of exciton-polariton fluids at non-zero momenta, non-resonantly excited by a small-spot femtosecond pump pulse in a ZnO microcavity. Using the femtosecond angle-resolved spectroscopic imaging technique, multidimensional dynamics in both the energy and momentum degrees of freedom have been obtained. Two distinct regions with different decay rate in the energy dimension and various decay-channels in the momentum dimension can be well-resolved. Theoretical simulations based on the generalized Gross-Pitaevskii equation can reach a qualitative agreement with the experimental observations, demonstrating the significance of the initial potential barrier induced by the pump pulse during the decay process. The finding of our study can provide additional insights into the fundamental understanding of exciton-polariton condensates, enabling further advancements for controlling the fluids and practical applications.

MdCOP1 ubiquitin E3 ligases interact with MdMYB1 to regulate light-induced anthocyanin biosynthesis and red fruit coloration in apple.

MdMYB1 is a crucial regulator of light-induced anthocyanin biosynthesis and fruit coloration in apple (Malus domestica). In this study, it was found that MdMYB1 protein accumulated in the light but degraded via a ubiquitin-dependent pathway in the dark. Subsequently, the MdCOP1-1 and MdCOP1-2 genes were isolated from apple fruit peel and were functionally characterized in the Arabidopsis (Arabidopsis thaliana) cop1-4 mutant. Yeast (Saccharomyces cerevisiae) two-hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays showed that MdMYB1 interacts with the MdCOP1 proteins. Furthermore, in vitro and in vivo experiments indicated that MdCOP1s are necessary for the ubiquitination and degradation of MdMYB1 protein in the dark and are therefore involved in the light-controlled stability of the MdMYB1 protein. Finally, a viral vector-based transformation approach demonstrated that MdCOP1s negatively regulate the peel coloration of apple fruits by modulating the degradation of the MdMYB1 protein. Our findings provide new insight into the mechanism by which light controls anthocyanin accumulation and red fruit coloration in apple and even other plant species.

Molecular cloning and functional analysis of a blue light receptor gene MdCRY2 from apple (Malus domestica).

KEY MESSAGE: MdCRY2 was isolated from apple fruit skin, and its function was analyzed in MdCRY2 transgenic Arabidopsis. The interaction between MdCRY2 and AtCOP1 was found by yeast two-hybrid and BiFC assays. Cryptochromes are blue/ultraviolet-A (UV-A) light receptors involved in regulating various aspects of plant growth and development. Investigations of the structure and functions of cryptochromes in plants have largely focused on Arabidopsis (Arabidopsis thaliana), tomato (Solanum lycopersicum), pea (Pisum sativum), and rice (Oryza sativa). However, no data on the function of CRY2 are available in woody plants. In this study, we isolated a cryptochrome gene, MdCRY2, from apple (Malus domestica). The deduced amino acid sequences of MdCRY2 contain the conserved N-terminal photolyase-related domain and the flavin adenine dinucleotide (FAD) binding domain, as well as the C-terminal DQXVP-acidic-STAES (DAS) domain. Relationship analysis indicates that MdCRY2 shows the highest similarity to the strawberry FvCRY protein. The expression of MdCRY2 is induced by blue/UV-A light, which represents a 48-h circadian rhythm. To investigate the function of MdCRY2, we overexpressed the MdCRY2 gene in a cry2 mutant and wild type (WT) Arabidopsis, assessed the phenotypes of the resulting transgenic plants, and found that MdCRY2 functions to regulate hypocotyl elongation, root growth, flower initiation, and anthocyanin accumulation. Furthermore, we examined the interaction between MdCRY2 and AtCOP1 using a yeast two-hybrid assay and a bimolecular fluorescence complementation assay. These data provide functional evidence for a role of blue/UV-A light-induced MdCRY2 in controlling photomorphogenesis in apple.

Apple-marigold intercropping improves soil properties by changing soil metabolomics and bacterial community structures.

Marigold can protect crops against soil-borne diseases. However, the effects of intercropping with marigold on apple rhizosphere soils are not known. In this study, we investigated the metabolite profiles and bacterial community structures in rhizosphere soils of the apple-marigold intercropping system by high-throughput sequencing and soil metabolomics. The results show that intercropping marigold could significantly enhance soil moisture, nitrogen, and enzyme activities compared with clean tillage. The soil metabolite profiles and the soil bacterial community structures in the rhizosphere soils were different between the inter-and mono-cropping systems. Among nine metabolites, carbohydrates were more increased in the intercropping system than in the monocropping system. Pathway enrichment analysis revealed that the greatest differential, in terms of metabolic pathway, was starch and sucrose metabolism. Moreover, intercropping marigold significantly increased the relative abundance of plant growth promoting bacteria in rhizosphere soils, such as Rhizobiales, Pseudomonadales, and Bacillales. These results indicate that marigold intercropping positively affected the apple orchard's soil quality and may provide a new intercropping technique to improve soil fertility in orchards and promote plant growth.

Three Chitin Deacetylase Family Members of Beauveria bassiana Modulate Asexual Reproduction and Virulence of Fungi by Mediating Chitin Metabolism and Affect Fungal Parasitism and Saprophytic Life.

As an important chitin-modifying enzyme, chitin deacetylase (CDA) has been characterized in many fungi, but its function in the entomopathogenic fungus Beauveria bassiana remains unclear. Three CDAs with conserved domains of the carbohydrate esterase 4 (CE-4) family were identified in B. bassiana. Disruption of CDA1 resulted in growth restriction of the fungus on medium with chitin as a carbon source or without a carbon source. Deletion of CDA1 and CDA2 led to defects in fungal conidial formation and conidial vitality compared with those of the wild type (WT), and the conidial yield decreased by 25.81% to 47.68%. Inactivation of three CDA genes resulted in a decrease of 20.23% to 27% in the blastospore yield. ΔCDA1 and ΔCDA3 showed 29.33% and 23.34% reductions in cuticular infection virulence, respectively. However, the CDA family may not contribute to hemocoel infection virulence. Additionally, the sporulation of the insect carcass showed that the three gene deletion mutants were 68.45%, 63.84%, and 56.65% less than WT. Penetration experiments with cicada wings and enzyme activity assays were used to further explore the effect of the fungus on chitin metabolism after gene deletion. Although the three gene deletion mutants penetrated the cicada wings successfully and continued to grow on the underlying medium, their colony sizes were reduced by 29.12% to 47.76%. The CDA enzyme activity of ΔCDA1 and ΔCDA3 decreased by 84.76% and 83.04%, respectively. These data showed that members of the CDA family play a different role in fungal growth, conidial quality, and virulence. IMPORTANCE In this study, we report the roles of CDA family in entomopathogenic fungus B. bassiana. Our results indicated that CDA modulates asexual development and regulates fungal virulence by altering chitin deacetylation and metabolic capacity. CDA affected the biological control potential and life history of B. bassiana by affecting its parasitic and saprophytic life. These findings provide novel insights into the roles of multiple CDA paralogues existing in fungal biocontrol agents.

Ada2 and Ada3 Regulate Hyphal Growth, Asexual Development, and Pathogenicity in Beauveria bassiana by Maintaining Gcn5 Acetyltransferase Activity.

The histone acetyltransferase (HAT) Gcn5 ortholog is essential for a variety of fungi. Here, we characterize the roles of Ada2 and Ada3, which are functionally linked to Gcn5, in the insect-pathogenic fungus Beauveria bassiana. Loss of Ada2 and Ada3 led to severe hyphal growth defects on rich and minimal media and drastic decreases in blastospore yield and conidiation capacity, with abnormal conidia-producing structures. ΔAda2 and ΔAda3 exhibited a delay in conidial germination and increased sensitivity to multiple chemical stresses and heat shock. Nearly all their pathogenicity was lost, and their ability to secrete extracellular enzymes, Pr1 proteases and chitinases for cuticle degradation was reduced. A yeast two-hybrid assay demonstrated that Ada2 binds to Ada3 and directly interacts with Gcn5, confirming the existence of a yeast-like Ada3-Ada2-Gcn5 HAT complex in this fungus. Additionally, deletion of the Ada genes reduced the activity of Gcn5, especially in the ΔAda2 strain, which was consistent with the acetylation level of histone H3 determined by Western blotting. These results illustrate the dependence of Gcn5 enzyme activity on Ada2 and Ada3 in fungal hyphal growth, asexual development, multiple stress responses, and pathogenicity in B. bassiana. IMPORTANCE The histone acetyltransferase Gcn5 ortholog contributes significantly to the growth and development of various fungi. In this study, we found that Ada2 and Ada3 have critical regulatory effects on Gcn5 enzyme activity and influence the acetylation of histone H3. Deletion of Ada2 or Ada3 decreased the fungal growth rate and asexual conidial yield and increased susceptibility to multiple stresses in Beauveria bassiana. Importantly, Ada genes are vital virulence factors, and their deletion caused the most virulence loss, mainly by inhibiting the activity of a series of hydrolytic enzymes and the dimorphic transition ability. These findings provide a new perspective on the function of the Gcn5 acetyltransferase complex in pathogens.

Clarifying the mechanisms of the light-induced color formation of apple peel under dark conditions through metabolomics and transcriptomic analyses.

Many studies have demonstrated that anthocyanin synthesis in apple peel is induced by light, but the color of bagged apple peel continues to change under dark conditions after light induction has not been characterized. Here, transcriptional and metabolic changes associated with changes in apple peel coloration in the dark after different light induction treatments were studied. Apple pericarp can achieve a normal color under complete darkness followed by light induction. Metabolomics analysis indicated that the expression levels of cyanidin-3-O-galactoside and cyanidin-3-O-glucoside were high, which might be associated with the red color development of apple peel. Transcriptome analysis revealed high expression levels of MdUFGTs, MdMYBs, and MdNACs, which might play a key role in light-induced anthocyanin accumulation under dark conditions. 13 key genes related to dark coloring after light induction was screened. The results of this study provide new insights into the mechanism of anthocyanin synthesis under dark conditions.

High temperature increased lignin contents of poplar (Populus spp) stem via inducing the synthesis caffeate and coniferaldehyde.

Lignin contributes to plant resistance to biotic and abiotic stresses and is dominantly regulated by enzymes which catalyze the generation of metabolites intermediates in lignin synthesis. However, the response of lignin and its key regulatory factors to high temperature stress are poorly understood. Here, this finding revealed that the content of lignin in poplar (Populus spp) stem increased after 3 days of high temperature stress treatment. In fourteen metabolic intermediates of lignin biosynthetic pathway with targeted metabolomics analysis, caffeate and coniferaldehyde increased evidently upon heat stress. C3'H (p-Coumaroylshikimate 3-hydroxylase) and CCR (Cinnamoyl-CoA reductase) are recognized to catalyze the formation of caffeate and coniferaldehyde, respectively. Transcriptome data and RT-qPCR (reverse transcription-quantitative real-time polymerase chain reaction) analysis uncovered the high transcriptional level of PtrMYBs (PtrMYB021, PtrMYB074, PtrMYB85, PtrMYB46), PtrC3'H1 (Potri.006G033300) and PtrCCR2 (Potri.003G181400), suggesting that they played the vital role in the increase of lignin and its metabolic intermediates were induced by high temperature. The discovery of key regulators and metabolic intermediates in lignin pathway that respond to high temperature provides a theoretical basis for quality improvement of lignin and the application of forest resources.

The MdWRKY31 transcription factor binds to the MdRAV1 promoter to mediate ABA sensitivity.

The phytohormone abscisic acid (ABA) is a major element involved in apple (Malus domestica) production because of its role in seed germination and early seedling development. The WRKY family, which is one of the largest families of transcription factors, plays an important role in ABA signaling in plants. However, the underlying molecular mechanisms of WRKY-mediated ABA sensitivity in apple are poorly understood. A genome-wide transcriptome analysis indicated that MdWRKY31 is a key factor induced by ABA. Quantitative real-time PCR showed that MdWRKY31 is induced by ABA in response to PEG4000, which is used to simulate drought. As a transcription factor, MdWRKY31 is localized in the nucleus. Ectopic expression of MdWRKY31 in Arabidopsis and Nicotiana benthamiana enhanced plant sensitivity to ABA. Overexpression of MdWRKY31 in apple roots and apple calli increased sensitivity to ABA, whereas repression of MdWRKY31 reduced sensitivity to ABA in the roots of 'Royal Gala'. Electrophoretic mobility shift assays, chromatin immunoprecipitation PCR, and yeast one-hybrid assays indicated that MdWRKY31 directly binds to the promoter of MdRAV1. Expression analyses of transgenic apple calli containing MdWRKY31 and pMdRAV1::GUS implied that MdWRKY31 represses the expression of MdRAV1. We also found that MdRAV1 binds directly to the promoters of MdABI3 and MdABI4 and repressed their expression. Our findings reveal a new important regulatory mechanism of MdWRKY31-MdRAV1-MdABIs in the ABA signaling pathway in apple.

Functional identification of apple on MdHIR4 in biotic stress.

In plants, hypersensitive-induced reaction (HIR) proteins are involved in stress responses, especially biotic stress. However, the potential molecular mechanisms of HIR-mediated biotic resistance in plants are rarely reported. We found that apple (Malus domestica) MdHIR4 was localized in the cell nucleus and membrane similar to AtHIR1 in Arabidopsis. Moreover, salicylic acid and the bacterial flagellin flg22 (a conserved, 22-amino acid motif), which are relevant to biotic stress, could induce MdHIR4 expression. Additionally, the transcription level of MdHIR4 was increased by Methyl jasmonate treatment. Ectopic expression of MdHIR4 in Arabidopsis and Nicotiana benthamiana reduced sensitivity to Methyl jasmonate and enhanced resistance to the bacterial pathogen Pst DC3000 (Pseudomonas syringae tomato DC3000). The interaction between MdHIR4 and AtJAZs proteins (AtJAZ3, AtJAZ4, and AtJAZ9) implied that MdHIR4 participated in the jasmonic acid (JA) signaling pathway. We found the expression of JA-related genes and PRs to change in transgenic plants, further demonstrating that MdHIR4 mediated biotic stress through the JA signaling pathway. Repressing the expression of MdHIR4 in apple leaves and calli increased resistance to Botryosphaeria dothidea by influencing the transcription of resistance-related genes. Our findings reveal the resistant function to biotic stress of MdHIR4 in transgenic plants, including Arabidopsis, tobacco, and apple, and identify the regulating mechanism of MdHIR4-related biotic resistance.

MdHIR proteins repress anthocyanin accumulation by interacting with the MdJAZ2 protein to inhibit its degradation in apples.

In higher plants, jasmonate ZIM-domain (JAZ) proteins negatively regulate the biosynthesis of anthocyanins by interacting with bHLH transcription factors. However, it is largely unknown if and how other regulators are involved in this process. In this study, the apple MdJAZ2 protein was characterized in regards to its function in the negative regulation of anthocyanin accumulation and peel coloration. MdJAZ2 was used as a bait to screen a cDNA library using the yeast two-hybrid method. The hypersensitive induced reaction (HIR) proteins, MdHIR2 and MdHIR4, were obtained from this yeast two-hybrid. The ZIM domain of MdJAZ2 and the PHB domain of the MdHIR proteins are necessary for their interactions. The interactions were further verified using an in vitro pull-down assay. Subsequently, immunoblotting assays demonstrated that MdHIR4 enhanced the stability of the MdJAZ2-GUS protein. Finally, a viral vector-based transformation method showed that MdHIR4 inhibited anthocyanin accumulation and fruit coloration in apple by modulating the expression of genes associated with anthocyanin biosynthesis.

Molecular cloning and functional analysis of a UV-B photoreceptor gene, MdUVR8 (UV Resistance Locus 8), from apple.

UVR8 (UV Resistance Locus 8) is an ultraviolet-B (UV-B; 280-315nm) light receptor that is involved in regulating many aspects of plant growth and development. UV-B irradiation can increase the development of flower and fruit coloration in many fruit trees, such as grape, pear and apple. Previous investigations of the structure and functions of UVR8 in plants have largely focused on Arabidopsis. Here, we isolated the UVR8 gene from apple (Malus domestica) and analyzed its function in transgenic Arabidopsis. Genomic and protein sequence analysis showed that MdUVR8 shares high similarity with the AtUVR8 protein from Arabidopsis, including the conserved seven-bladed ß-propeller, the C27 region, the 3 "GWRHT" motifs and crucial amino-acid residues (14 Trps, 2 Args). A point mutation prediction and three-dimensional structural analysis of MdUVR8 indicated that it has a similar structure to AtUVR8 and that the crucial residues are also important in MdUVR8. In terms of transcript levels, MdUVR8 expression was up-regulated by UV-B light, which suggests that its expression follows a 24-h circadian rhythm. Using heterologous expression of MdUVR8 in both uvr8-1 mutant and wild-type (WT) Arabidopsis, we found that MdUVR8 regulates hypocotyl elongation and gene expression under UV-B light. These data provide functional evidence for a role of MdUVR8 in controlling photomorphogenesis under UV-B light and indicate that the function of UVR8 is conserved between Arabidopsis and apple. Furthermore, we examined the interaction between MdUVR8 and MdCOP1 (constitutive photomorphogenic1) using a yeast two-hybrid assay and a co-immunoprecipitation assay. This interaction provides a direction for investigating the regulatory mechanisms of the UV-B-light pathway in apple.

A low-calcemic vitamin D analog (Ro 25-4020) inhibits the growth of LNCaP human prostate cancer cells with increased potency by producing an active 24-oxo metabolite (Ro 29-9970).

In this study, we have characterized a novel less-calcemic vitamin D analog Ro 25-4020 (1alpha, 25 dihydroxy-16-ene-5,6-trans-vitamin D3) and investigated the mechanisms underlying its enhanced growth inhibitory properties. We found that Ro 25-4020 (IC50 = 0.3 nM) exhibited greater inhibitory activity than 1,25(OH)2D3 (IC50 = 1 nM) on LNCaP human prostate cancer cell growth. However, Ro 25-4020 was tenfold less active than 1,25(OH)2D3 in receptor-binding assays, ligand-induced heterodimerization and transactivation assays using VDR. HPLC and GC-MS analyses revealed that 1,25(OH)2D3 is converted to a 24-hydroxy metabolite, which has been shown to be less potent than 1,25(OH)2D3. In contrast, Ro 25-4020 was converted to a major 24-oxo metabolite that was more stable. Ligand-binding assays reveal that both Ro 25-4020 and its 24-oxo metabolite have similar affinity for VDR. Synthetic 24-oxo-Ro 25-4020, however, inhibited LNCaP cell proliferation as potently as 1,25(OH)2D3 and was more potent in transactivation of two out of three vitamin D target genes tested. These results suggest that conversion of Ro 25-4020 into an active and more stable 24-oxo metabolite with longer half-life contributes significantly to its potent antiproliferative actions on the LNCaP cells.

Molecular cloning of cryptochrome 1 from apple and its functional characterization in Arabidopsis.

Cryptochromes are blue-light photoreceptors involved in regulating many aspects of plant growth and development. Investigations of cryptochromes in plants have largely focused on Arabidopsis (Arabidopsis thaliana), tomato (Solanum lycopersicum), rice (Oryza sativa) and pea (Pisum sativum). Here, we isolated the cryptochrome 1 gene from apple (Malus domestica) (MdCRY1) and analyzed its function in transgenic Arabidopsis. The predicted MdCRY1 protein was most closely homologous to strawberry CRY1. In terms of transcript levels, MdCRY1 expression was up-regulated by light. The function of MdCRY1 was analyzed through heterologous expression in Arabidopsis. Overexpression of MdCRY1 in Arabidopsis is able to rescue the cry1 mutant phenotype, inhibit hypocotyl elongation, promote root growth, and enhance anthocyanin accumulation in wild-type seedlings under blue light. These data provide functional evidence for a role of MdCRY1 in controlling photomorphogenesis under blue light and indicate that CRY1 function is conserved between Arabidopsis and apple. Furthermore, we found that MdCRY1 interacts with AtCOP1 in both yeast and onion cells. This interaction may represent an important regulatory mechanism in blue-light signaling pathway in apple.