ORANGE interplays with TCP7 to regulate endoreduplication and leaf size.

Leaf size is a crucial agronomic trait directly affecting crop yield, which is mainly determined by coordinated cell proliferation, growth, and differentiation. Although endoreduplication is known to be correlated with the onset of cell differentiation and leaf size, the underlying molecular mechanisms are largely unclear. The DnaJ-like zinc finger domain-containing protein ORANGE (OR) was initially demonstrated to confer the massive accumulation of carotenoids in cauliflower curds. However, the cauliflower or mutant also possesses other phenotypes such as smaller curds, smaller leaves with elongated petioles, and delayed flowering. Here, we demonstrated that OR physically interacts with the transcription factor TCP7, which promotes endoreduplication by inducing the expression of the cell cycle gene CYCLIN D 1;1 (CYCD1;1). Overexpression of OR resulted in smaller rosette leaves, whereas the OR-silencing plants had larger rosette leaves than wild-type plants. Our microscopic observations and flow cytometry analysis revealed that the variation in leaf size was a result of different endoreduplication levels. Genetic analyses showed that OR functions antagonistically with TCP7 in regulating the endoreduplication levels in leaf cells. While the expression of OR is induced by TCP7, OR represses the transactivation activity of TCP7 by affecting its binding capability to the TCP-binding motif in the promoter region of CYCD1;1. Through this interaction, OR negatively regulates the expression of CYCD1;1 and reduces the nuclear ploidy level in rosette leaf cells. Our findings provide new insights into the regulatory network of leaf size and also reveal a regulatory circuit controlling endoreduplication in leaf cells.

Prolactin upregulates amino acids uptake in dairy cow mammary epithelial cells via LAT1.

The uptake of AA in mammary tissues is affected by prolactin (PRL). To investigate whether PRL-induced AA uptake is involved in L-type AA transporter 1 (LAT1), we analyzed the changes of AA in the medium of dairy cow mammary epithelial cells in the presence of PRL or PRL plus BCH, an inhibitor of LAT1. Then Western blot and luciferase assay were used to detect the regulation mechanism of PRL on LAT1 expression and function. Our results showed that Thr, Val, Met, Ile, Leu, Tyr, Lys, Phe, and His are LAT1 substrates and could be transported into mammary epithelial cells via LAT1. PRL stimulation increased the uptake of most AA into mammary epithelial cells of dairy cows, however, inhibition of LAT1 transport activity reduced PRL-induced AA uptake, suggesting that the effect of PRL on AA transport depends on LAT1 expression and function. PRL stimulation upregulated LAT1 expression and plasma membrane location not only in dairy cow mammary epithelial cells, but also in mouse mammary epithelial cell line HC11. Western blot showed that PI3K-AKT-mTOR signaling could be activated in PRL-stimulated mammary epithelial cells. Treatment of cells with LY294002 decreased PI3K-AKT-mTOR activation, as well LAT1 expression, that in turn decreased milk protein synthesis. Luciferase assay showed PRL treatment increased the promoter activity of LAT1 promoter fragment -419∼-86 bp. Treatment of cells with LY294002, an inhibitor of PI3K, or SC79, an activator of AKT abolished or promoted the transcriptional activity of this promoter fragment in the presence of PRL. These results suggested that the -419∼-86 bp fragment of LAT1 promoter mediates the action of PI3K-AKT-mTOR signaling on LAT1 transcription in mammary epithelial cells of dairy cows, which in turn increased LAT1 expression and AA uptake.

Screening and Identification of Candidate GUN1-Interacting Proteins in Arabidopsis thaliana.

Chloroplasts are semi-autonomous organelles governed by the precise coordination between the genomes of their own and the nucleus for functioning correctly in response to developmental and environmental cues. Under stressed conditions, various plastid-to-nucleus retrograde signals are generated to regulate the expression of a large number of nuclear genes for acclimation. Among these retrograde signaling pathways, the chloroplast protein GENOMES UNCOUPLED 1 (GUN1) is the first component identified. However, in addition to integrating aberrant physiological signals when chloroplasts are challenged by stresses such as photooxidative damage or the inhibition of plastid gene expression, GUN1 was also found to regulate other developmental processes such as flowering. Several partner proteins have been found to interact with GUN1 and facilitate its different regulatory functions. In this study, we report 15 possible interacting proteins identified through yeast two-hybrid (Y2H) screening, among which 11 showed positive interactions by pair-wise Y2H assay. Through the bimolecular fluorescence complementation assay in Arabidopsis protoplasts, two candidate proteins with chloroplast localization, DJC31 and HCF145, were confirmed to interact with GUN1 in planta. Genes for these GUN1-interacting proteins showed different fluctuations in the WT and gun1 mutant under norflurazon and lincomycin treatments. Our results provide novel clues for a better understanding of molecular mechanisms underlying GUN1-mediated regulations.

Prolactin regulates LAT1 expression via STAT5 (signal transducer and activator of transcription 5) signaling in mammary epithelial cells of dairy cows.

The l-type amino acid transporter 1 (LAT1; also known as SLC7A5) is a transporter that allows the uptake of large neutral amino acids into mammalian cells. In dairy cows, LAT1 is highly expressed in lactating mammary tissues and involved in milk protein synthesis. Prolactin (PRL) has a lactogenic role and is capable of inducing milk production in ruminants. However, the relationship between PRL stimulation and LAT1 expression in dairy cow mammary gland has not been well understood. In this study, we showed that PRL stimulation increased expression of LAT1 and ß-casein in mammary epithelial cells of dairy cows. The stimulatory effect of PRL on milk protein production was inhibited by LAT1-specific inhibitor or LAT1 knockdown, suggesting that PRL-induced milk protein production is involved in LAT1 expression. To determine whether the PRL signaling pathway participates in regulation of LAT1 expression, PRLR (PRL receptor) or STAT5 (signal transducer and activator of transcription 5) was knocked down by short interfering (si)RNA in mammary epithelial cells of dairy cows. Western blot results showed that knockdown of PRLR or STAT5 with siRNA markedly decreased PRL-stimulated LAT1 expression. In addition, we observed a marked increase in plasma membrane expression of LAT1 in PRL-stimulated cells compared with control cells. These observations indicated that PRL signaling can regulate LAT1 expression and activity in mammary epithelial cells of dairy cows, contributing to increased amino acid availability and milk protein synthesis in mammary gland of dairy cow.

Examination of methionine stimulation of gene expression in dairy cow mammary epithelial cells using RNA-sequencing.

In this research communication, a cell model with elevated ß-CASEIN synthesis was established by stimulating bovine mammary epithelial cells with 0.6 mM methionine, and the genome-wide gene expression profiles of methionine-stimulated cells and untreated cells were investigated by RNA sequencing. A total of 458 differentially expressed genes (DEGs; 219 upregulated and 239 downregulated) were identified between the two groups. Gene Ontology (GO) analysis showed that the two highest-ranked GO terms in 'molecular function' category were 'binding' and 'catalytic activity', suggesting that milk protein synthesis in methionine-stimulated cells requires induction of gene expression to increase metabolic activity. Kyoto Encyclopedia of Genes and Genomes analysis revealed that within the 'environmental information processing' category, the subcategory that is most highly enriched for DEGs was 'signal transduction'. cGMP-PKG, Rap1, calcium, cAMP, PI3K-AKT, MAPK, and JAK-STAT are the pathways with the highest number of DEGs, suggesting that these signaling pathways have potential roles in mediating methionine-induced milk protein synthesis in bovine mammary epithelial cells. This study provides valuable insights into the physiological and metabolic adaptations in cells stimulated with methionine. Understanding the regulation of this transition is essential for effective intervention in the lactation process.

Functional analysis of the dairy cow mammary transcriptome between early lactation and mid-dry period.

In this research communication we used digital gene expression (DGE) analysis to identify differences in gene expression in the mammary glands of dairy cows between early lactation and the mid-dry period. A total of 741 genes were identified as being differentially expressed by DGE analysis. Compared with their expression in dry cows, 214 genes were up-regulated and 527 genes were down-regulated in lactating cow mammary glands. Gene Ontology analysis showed that lactation was supported by increased gene expression related to metabolic processes and nutrient transport and was associated with decreased gene expression related to cell proliferation. Pathway mapping using the Kyoto Encyclopedia of Genes and Genomes showed that 579 differentially expressed genes had pathway annotations related to 204 pathways. Metabolic pathway-related genes were the most significantly enriched. Genes and pathways identified by the present study provide insights into molecular events that occur in the mammary gland between early lactation and mid-dry period, which can be used to facilitate further investigation of the mechanisms underlying lactation and mammary tissue remodeling in dairy cows.

ORANGE negatively regulates flowering time in Arabidopsisthaliana.

Floral transition is an important process in plant development, which is regulated by at least four flowering pathways: the photoperiod, vernalization, autonomous, and gibberellin (GA)-dependent pathways. The DnaJ-like zinc finger domain-containing protein ORANGE (OR) was originally cloned from the cauliflower or mutant, which has distinct phenotypes of the carotenoid-accumulating curd, the elongated petioles, and the delayed-flowering time. OR has been demonstrated to interact with phytoene synthase for carotenoid biosynthesis in plastids and with eukaryotic release factor 1-2 (eRF1-2) in the nucleus for the first two phenotypes, respectively. In this study, we showed that overexpression of OR in Arabidopsis thaliana resulted in a delayed-flowering phenotype resembling the cauliflower or mutant. Our results indicated that OR negatively regulates the expression of the flowering integrator genes FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1). Both GA3 and vernalization treatments could not rescue the delayed-flowering phenotype of the OR-overexpressing seedlings, suggesting the repression of floral transition by OR does not depend on SOC1-mediated vernalization or GA-dependent pathways. Moreover, our analysis revealed that transcripts of OR and FT fluctuated in opposite directions diurnally, and the overexpression of OR repressed the accumulation of CONSTANS (CO), FT, and SOC1 transcripts in a 16 h/8 h light/dark long-day cycle. Our results indicated the possibility that OR represses flowering through the CO-FT-SOC1-mediated photoperiodic flowering pathway.

Transcriptomic Analysis Suggests a Coordinated Regulation of Carotenoid Metabolism in Ripening Chili Pepper (Capsicum annuum var. conoides) Fruits.

Carotenoids are not only photosynthetic and photoprotective pigments in plants, but also essential antioxidative nutrients for human health. The fruit is the main plant organ that synthesizes and sequestrates carotenoids. Fruit ripening is a complicated developmental process, during which the rewiring of the metabolic network is tightly coordinated with the re-organization of cellular and organellular structures. Chili pepper (Capsicum annuum) is one of the major crops that accumulates a distinct level of carotenoids, especially capsanthin, in their ripened fruits. To elucidate how different metabolic and developmental scenarios are regulated in ripening chili pepper fruits, we analyzed the carotenoid profiles and transcriptomes of fruits at different ripening stages. Our pigment analysis indicated an opposite correlation between the contents of carotenoid species with ß,ß-structures (e.g., ß-carotene, zeaxanthin, and capsanthin) and of lutein with the ß,ε-structure, whereas lutein displayed a high correlation with chlorophylls during ripening. From the chili pepper Zunla-1 genome, a full repertoire of 38 homologous genes encoding enzymes in the carotenoid biosynthetic pathway was identified. The fluctuations in their transcript abundances during ripening suggested different involvement of these genes in the regulation of carotenoid biosynthesis. We further searched genes of which the expression showed high correlations with the accumulation of ß-carotene during the ripening process. Moreover, from the transcriptomic analysis, a total of 17 transcription factors that co-expressed with different groups of carotenoid biosynthetic genes were identified.

Nutrigenomic Role of Acetate and ß-Hydroxybutyrate in Bovine Mammary Epithelial Cells.

Acetate and ß-hydroxybutyrate (BHBA) are the predominant substrates for de novo fatty acid (FA) synthesis in mammary gland of dairy cow. To investigate the nutrigenomic role of acetate and BHBA in bovine mammary epithelial cells during milk fat production, RNA sequencing (RNA-seq) transcriptomic analysis was used to identify differentially expressed genes (DEGs) between acetate- and BHBA-treated cells (high-milk fat cells) and control cells. A total of 625 DEGs (358 upregulated and 267 downregulated) were identified between the high-milk fat cells and control cells. Gene ontology enrichment analysis revealed that the upregulated genes in high-milk fat cells were mainly involved in lipid biosynthetic process, steroid biosynthetic process, oxidation-reduction process, receptor binding, and vesicle and small molecule biosynthetic process. The downregulated genes were mainly associated with immune response, cytokine production, negative regulation of biological process, and peptidyl-threonine modification. Pathway analysis indicated that FA metabolism and steroid biosynthesis were significantly enriched for the upregulated genes in the high-milk fat cells, while apoptosis was enriched for the downregulated genes. This work provides a profile of gene expression changes that occur during acetate- and BHBA-induced milk fat synthesis in bovine mammary epithelial cells, which furthers our understanding of the molecular regulation of lipid metabolism.