Molecular evolution of Phytocyanin gene and analysis of expression at different coloring periods in apple (Malus domestica).

BACKGROUND: PC (phytocyanin) is a class of copper-containing electron transfer proteins closely related to plant photosynthesis, abiotic stress responses growth and development in plants, and regulation of the expression of some flavonoids and phenylpropanoids, etc., however, compared with other plants, the PC gene family has not been systematically characterized in apple. RESULTS: A total of 59 MdPC gene members unevenly distributed across 12 chromosomes were identified at the genome-wide level. The proteins of the MdPC family were classified into four subfamilies based on differences in copper binding sites and glycosylation sites: Apple Early nodulin-like proteins (MdENODLs), Apple Uclacyanin-like proteins (MdUCLs), Apple Stellacyanin-like proteins (MdSCLs), and Apple Plantacyanin-like proteins (MdPLCLs). Some MdPC members with similar gene structures and conserved motifs belong to the same group or subfamily. The internal collinearity analysis revealed 14 collinearity gene pairs among members of the apple MdPC gene. Interspecific collinearity analysis showed that apple had 31 and 35 homologous gene pairs with strawberry and grape, respectively. Selection pressure analysis indicated that the MdPC gene was under purifying selection. Prediction of protein interactions showed that MdPC family members interacted strongly with the Nad3 protein. GO annotation results indicated that the MdPC gene also regulated the biosynthesis of phenylpropanoids. Chip data analysis showed that (MdSCL3, MdSCL7 and MdENODL27) were highly expressed in mature fruits and peels. Many cis-regulatory elements related to light response, phytohormones, abiotic stresses and flavonoid biosynthetic genes regulation were identified 2000 bp upstream of the promoter of the MdPC gene, and qRT-PCR results showed that gene members in Group IV (MdSCL1/3, MdENODL27) were up-regulated at all five stages of apple coloring, but the highest expression was observed at the DAF13 (day after fruit bag removal) stage. The gene members in Group II (MdUCL9, MdPLCL3) showed down-regulated or lower expression in the first four stages of apple coloring but up-regulated and highest expression in the DAF 21 stage. CONCLUSION: Herein, one objective of these findings is to provide valuable information for understanding the structure, molecular evolution, and expression pattern of the MdPC gene, another major objective in this study was designed to lay the groundwork for further research on the molecular mechanism of PC gene regulation of apple fruit coloration.

The asymmetric expression of plasma membrane H+-ATPase family genes in response to pulvinus-driven leaf phototropism movement in Vitis vinifera.

Phototropism movement is crucial for plants to adapt to various environmental changes. Plant P-type H+-ATPase (HA) plays diverse roles in signal transduction during cell expansion, regulation of cellular osmotic potential and stomatal opening, and circadian movement. Despite numerous studies on the genome-wide analysis of Vitis vinifera, no research has been done on the P-type H+-ATPase family genes, especially concerning pulvinus-driven leaf movement. In this study, 55 VvHAs were identified and classified into nine distinct subgroups (1 to 9). Gene members within the same subgroups exhibit similar features in motif, intron/exon, and protein tertiary structures. Furthermore, four pairs of genes were derived by segmental duplication in grapes. Cis-acting element analysis identified numerous light/circadian-related elements in the promoters of VvHAs. qRT-PCR analysis showed that several genes of subgroup 7 were highly expressed in leaves and pulvinus during leaf movement, especially VvHA14, VvHA15, VvHA16, VvHA19, VvHA51, VvHA52, and VvHA54. Additionally, we also found that the VvHAs genes were asymmetrically expressed on both sides of the extensor and flexor cell of the motor organ, the pulvinus. The expression of VvHAs family genes in extensor cells was significantly higher than that in flexor cells. Overall, this study serves as a foundation for further investigations into the functions of VvHAs and contributes to the complex mechanisms underlying grapevine pulvinus growth and development.

Mechanism of the Pulvinus-Driven Leaf Movement: An Overview.

Leaf movement is a manifestation of plant response to the changing internal and external environment, aiming to optimize plant growth and development. Leaf movement is usually driven by a specialized motor organ, the pulvinus, and this movement is associated with different changes in volume and expansion on the two sides of the pulvinus. Blue light, auxin, GA, H+-ATPase, K+, Cl-, Ca2+, actin, and aquaporin collectively influence the changes in water flux in the tissue of the extensor and flexor of the pulvinus to establish a turgor pressure difference, thereby controlling leaf movement. However, how these factors regulate the multicellular motility of the pulvinus tissues in a species remains obscure. In addition, model plants such as Medicago truncatula, Mimosa pudica, and Samanea saman have been used to study pulvinus-driven leaf movement, showing a similarity in their pulvinus movement mechanisms. In this review, we summarize past research findings from the three model plants, and using Medicago truncatula as an example, suggest that genes regulating pulvinus movement are also involved in regulating plant growth and development. We also propose a model in which the variation of ion flux and water flux are critical steps to pulvinus movement and highlight questions for future research.

The Genome-Wide Identification of the Dihydroflavonol 4-Reductase (DFR) Gene Family and Its Expression Analysis in Different Fruit Coloring Stages of Strawberry.

Dihydroflavonol 4-reductase (DFR) significantly influences the modification of flower color. To explore the role of DFR in the synthesis of strawberry anthocyanins, in this study, we downloaded the CDS sequences of the DFR gene family from the Arabidopsis genome database TAIR; the DFR family of forest strawberry was compared; then, a functional domain screen was performed using NCBI; the selected strawberry DFR genes were analyzed; and the expression characteristics of the family members were studied by qRT-PCR. The results showed that there are 57 members of the DFR gene family in strawberry, which are mainly expressed in the cytoplasm and chloroplast; most of them are hydrophilic proteins; and the secondary structure of the protein is mainly composed of α-helices and random coils. The analysis revealed that FvDFR genes mostly contain light, hormone, abiotic stress, and meristem response elements. From the results of the qRT-PCR analysis, the relative expression of each member of the FvDFR gene was significantly different, which was expressed throughout the process of fruit coloring. Most genes had the highest expression levels in the full coloring stage (S4). The expression of FvDFR30, FvDFR54, and FvDFR56 during the S4 period was 8, 2.4, and 2.4 times higher than during the S1 period, indicating that the DFR gene plays a key role in regulating the fruit coloration of strawberry. In the strawberry genome, 57 members of the strawberry DFR gene family were identified. The higher the DFR gene expression, the higher the anthocyanin content, and the DFR gene may be the key gene in anthocyanin synthesis. Collectively, the DFR gene is closely related to fruit coloring, which lays a foundation for further exploring the function of the DFR gene family.

Genome-wide identification of grape ANS gene family and expression analysis at different fruit coloration stages.

BACKGROUND: Anthocyanin synthase (ANS) is the enzyme downstream of the anthocyanins synthesis pathway and the rate-limiting enzyme of the synthesis pathway. It catalyzes the conversion of colorless anthocyanins to anthocyanins and plays an important role in plant color presentation and stress resistance. However, ANS gene is rarely studied in grapes. RESULTS: In this study, 121 VvANS genes were identified and distributed on 18 chromosomes, VvANS family members were divided into 8 subgroups. Secondary structure prediction showed mainly irregular coils and α-helices, and subcellular localization indicated that VvANS gene family is mainly located in chloroplast, cytoplasm and nucleus. The promoter region of the VvANS gene family contains multiple cis-acting elements that are associated with light, abiotic stress, and hormones. Intraspecific collinearity analysis showed that there were 13 pairs of collinearity between VvANS genes. Interspecific collinearity analysis showed that there was more collinearity between grape, apple and Arabidopsis, but less collinearity between grape and rice. Microarray data analysis showed that VvANS17, VvANS23 and VvANS75 had higher expression levels in flesh and peel, while VvANS25, VvANS64 and VvANS106 had higher expression levels in flower. The results of qRT-PCR analysis showed that VvANS genes were expressed throughout the whole process of fruit coloring, such as VvANS47 and VvANS55 in the green fruit stage, VvANS3, VvANS64 and VvANS90 in the initial fruit color turning stage. The expression levels of VvANS21, VvANS79 and VvANS108 were higher at 50% coloring stage, indicating that these genes play an important role in the fruit coloring process. VvANS4, VvANS66 and VvANS113 had the highest expression levels in the full maturity stage. CONCLUSIONS: These results indicated that different members of VvANS gene family played a role in different coloring stages, and this study laid a foundation for further research on the function of ANS gene family.

Genome-Wide Identification and Expression Analysis of ANS Family in Strawberry Fruits at Different Coloring Stages.

To elucidate the structural characteristics, phylogeny and biological function of anthocyanin synthase (ANS) and its role in anthocyanin synthesis, members of the strawberry ANS gene family were obtained by whole genome retrieval, and their bioinformatic analysis and expression analysis at different developmental stages of fruit were performed. The results showed that the strawberry ANS family consisted of 141 members distributed on 7 chromosomes and could be divided into 4 subfamilies. Secondary structure prediction showed that the members of this family were mainly composed of random curls and α-helices, and were mainly located in chloroplasts, cytoplasm, nuclei and cytoskeletons. The promoter region of the FvANS gene family contains light-responsive elements, abiotic stress responsive elements and hormone responsive elements, etc. Intraspecific collinearity analysis revealed 10 pairs of FvANS genes, and interspecific collinearity analysis revealed more relationships between strawberries and apples, grapes and Arabidopsis, but fewer between strawberries and rice. Chip data analysis showed that FvANS15, FvANS41, FvANS47, FvANS48, FvANS49, FvANS67, FvANS114 and FvANS132 were higher in seed coat tissues and endosperm. FvANS16, FvANS85, FvANS90 and FvANS102 were higher in internal and fleshy tissues. Quantitative real-time PCR (qRT-PCR) showed that the ANS gene was expressed throughout the fruit coloring process. The expression levels of most genes were highest in the 50% coloring stage (S3), such as FvANS16, FvANS19, FvANS31, FvANS43, FvANS73, FvANS78 and FvANS91. The expression levels of FvANS52 were the highest in the green fruit stage (S1), and FvANS39 and FvANS109 were the highest in the 20% coloring stage (S2). These results indicate that different members of the FvANS gene family play a role in different pigmentation stages, with most genes playing a role in the expression level of the rapid accumulation of fruit coloring. This study lays a foundation for further study on the function of ANS gene family.

Identification of Flavanone 3-Hydroxylase Gene Family in Strawberry and Expression Analysis of Fruit at Different Coloring Stages.

The color of strawberry fruit is an important appearance quality index that affects the marketability of fruit, and the content and type of anthocyanin are two of the main reasons for the formation of fruit color. At present, the research on anthocyanin synthesis mainly focuses on the phenylpropane metabolic pathway, and the F3H gene family is an important member of this metabolic pathway. Therefore, in order to clarify the role of flavanone 3-hydroxylase (F3H) in regulating anthocyanin accumulation in strawberry, we identified F3H gene family members in strawberry and analyzed their bioinformatics and expression at different fruit color stages. The results showed that the strawberry F3H family contains 126 members, which are distributed on seven chromosomes and can be divided into six subgroups. The promoter region of strawberry F3H gene family contains light response elements, abiotic stress response elements and hormone response elements. Intraspecic collinearity analysis showed that there were six pairs of collinearity of the F3H gene. Interspecific collinearity analysis showed that there were more collinearity relationships between strawberry and apple, grape and Arabidopsis, but less collinearity between strawberry and rice. Via tissue-specific expression analysis, we found that the expression levels of FvF3H48, FvF3H120 and FvF3H74 were higher in the stages of germination, growth, flowering and fruit setting. The expression levels of FvF3H42 and FvF3H16 were higher in seeds. The expression levels of FvF3H16 and FvF3H11 were higher in the ovary wall of stage 1, stage 2, stage 3 and stage 5. FvF3H15 and FvF3H48 were highly expressed in the pericardium, anther, receptacle and anther. Real-time fluorescence quantitative PCR showed the expression changes in F3H in the fruit coloring process. The results indicate that the expression levels of most members were higher during the S3 stage, such as FvF3H7, FvF3H16, FvF3H32, FvF3H82, FvF3H89, FvF3H92 and FvF3H112. FvF3H63 and FvF3H104 exhibited particularly high expression levels during the S1 stage, with some genes also showing elevated expression during the S4 stage, including FvF3H13, FvF3H27, FvF3H66 and FvF3H103. FvF3H58, FvF3H69, FvF3H79 and FvF3H80 showed higher expression levels during the S2 stage. These findings lay the groundwork for elucidating the biological functions of the strawberry F3H gene family and the selection of related genes.

High-level hemicellulosic arabinose predominately affects lignocellulose crystallinity for genetically enhancing both plant lodging resistance and biomass enzymatic digestibility in rice mutants.

Rice is a major food crop with enormous biomass residue for biofuels. As plant cell wall recalcitrance basically decides a costly biomass process, genetic modification of plant cell walls has been regarded as a promising solution. However, due to structural complexity and functional diversity of plant cell walls, it becomes essential to identify the key factors of cell wall modifications that could not much alter plant growth, but cause an enhancement in biomass enzymatic digestibility. To address this issue, we performed systems biology analyses of a total of 36 distinct cell wall mutants of rice. As a result, cellulose crystallinity (CrI) was examined to be the key factor that negatively determines either the biomass enzymatic saccharification upon various chemical pretreatments or the plant lodging resistance, an integrated agronomic trait in plant growth and grain production. Notably, hemicellulosic arabinose (Ara) was detected to be the major factor that negatively affects cellulose CrI probably through its interlinking with ß-1,4-glucans. In addition, lignin and G monomer also exhibited the positive impact on biomass digestion and lodging resistance. Further characterization of two elite mutants, Osfc17 and Osfc30, showing normal plant growth and high biomass enzymatic digestion in situ and in vitro, revealed the multiple GH9B candidate genes for reducing cellulose CrI and XAT genes for increasing hemicellulosic Ara level. Hence, the results have suggested the potential cell wall modifications for enhancing both biomass enzymatic digestibility and plant lodging resistance by synchronically overexpressing GH9B and XAT genes in rice.

An integrated genomic and metabolomic framework for cell wall biology in rice.

BACKGROUND: Plant cell walls are complex structures that full-fill many diverse functions during plant growth and development. It is therefore not surprising that thousands of gene products are involved in cell wall synthesis and maintenance. However, functional association for the majority of these gene products remains obscure. One useful approach to infer biological associations is via transcriptional coordination, or co-expression of genes. This approach has proved useful for several biological processes. Nevertheless, combining co-expression with other large-scale measurements may improve the biological inferences. RESULTS: In this study, we used a combined approach of co-expression and cell wall metabolomics to obtain new insight into cell wall synthesis in rice. We initially created a weighted gene co-expression network from publicly available datasets, and then established a comprehensive cell wall dataset by determining cell wall compositions from 29 tissues that almost cover the whole life cycle of rice. We subsequently combined the datasets through the conversion of co-expressed gene modules into eigen-vectors, representing expression profiles for the genes in the modules, and performed comparative analyses against the cell wall contents. Here, we made three major discoveries. First, we confirmed our approach by finding primary and secondary wall cellulose biosynthesis modules, respectively. Second, we found co-expressed modules that strongly correlated with re-organization of the secondary cell walls and with modifications and degradation of hemicellulosic structures. Third, we inferred that at least one module is likely to play a regulatory role in the production of G-rich lignification. CONCLUSIONS: Here, we integrated transcriptomic associations and cell wall metabolism and found that certain co-expressed gene modules are positively correlated with distinct cell wall characteristics. We propose that combining multiple data-types, such as coordinated transcription and cell wall analyses, may be a useful approach to glean new insight into biological processes. The combination of multiple datasets, as illustrated here, can further improve the functional inferences that typically are generated via a single type of datasets. In addition, our data extend the typical co-expression approach to allow deeper insight into cell wall biology in rice.

Expression analysis of the apple HSP70 gene family in abiotic stress and phytohormones and expression validation of candidate MdHSP70 genes.

Heat shock protein 70 (HSP70) is one kind of molecular chaperones which are widely found in organisms, and its members are highly conserved among each other, with important roles in plant growth and development. In this study, 56 HSP70 genes were identified from the apple genome database. Analysis of gene duplication events showed that tandem and segmental duplication events play an important role in promoting the amplification of the MdHSP70 gene family. Collinearity analysis showed that HSP70 family members of apple were more closely related to HSP70 family members of Arabidopsis, tomato and soybean. The promoter region of the apple HSP70 genes contains a large number of cis-acting elements in response to hormones and stress. Tissue-specific expression analysis showed that some of the genes were associated with various stages of the apple growth process. Codon preference analysis showed small differences between codon bases 1 and 3 in the apple HSP70 genome, and the codon base composition had a small effect on codon usage preference. The multiple expression patterns of the MdHSP70 gene suggested that MdHSP70 gene members play important roles in growth and development and in response to hormonal and abiotic stresses. The yeast two-hybrid (Y2H) demonstrated that MdHSP70-53 interacts with MdDVH24_032563. The qRT-PCR analysis showed that most MdHSP70 members' hormonal and abiotic stresses (MdHSP70-6, MdHSP70-26 and MdHSP70-45) appeared to be highly expressed. To further elucidate the function of MdHSP70 (6, 26, 45), we introduced them into tobacco to confirm subcellular locations and noted that these genes are located in the cytoplasm and cell membrane. This study serves as a theoretical basis for further studies of the MdHSP70 gene and helps to further investigate the functional characterization of MdHSP70 gene.

DNA methylation supports intrinsic epigenetic memory in mammalian cells.

We have investigated the role of DNA methylation in the initiation and maintenance of silenced chromatin in somatic mammalian cells. We found that a mutated transgene, in which all the CpG dinucleotides have been eliminated, underwent transcriptional silencing to the same extent as the unmodified transgene. These observations demonstrate that DNA methylation is not required for silencing. The silenced CpG-free transgene exhibited all the features of heterochromatin, including silencing of transcriptional activity, delayed DNA replication, lack of histone H3 and H4 acetylation, lack of H3-K4 methylation, and enrichment in tri-methyl-H3-K9. In contrast, when we tested for transgene reactivation using a Cre recombinase-mediated inversion assay, we observed a marked difference between a CpG-free and an unmodified transgene: the CpG-free transgene resumed transcription and did not exhibit markers of heterochromatin whereas the unmodified transgene remained silenced. These data indicate that methylation of CpG residues conferred epigenetic memory in this system. These results also suggest that replication delay, lack of histone H3 and H4 acetylation, H3-K4 methylation, and enrichment in tri-methyl-H3-K9 are not sufficient to confer epigenetic memory. We propose that DNA methylation within transgenes serves as an intrinsic epigenetic memory to permanently silence transgenes and prevent their reactivation.

The human beta-globin locus control region can silence as well as activate gene expression.

Using recombinase-mediated cassette exchange to test multiple transgenes at the same site of integration, we demonstrate a novel chromatin context-dependent silencer activity of the beta-globin locus control region (LCR). This silencer activity requires DNase I hypersensitive sites HS2 and HS3 but not HS4. After silencing, the silenced cassettes adopt a typical closed chromatin conformation (histone H3 and H4 deacetylation, histone H3-K4 methylation, DNA methylation, and replication in late S phase). In the absence of the LCR at the same site of integration, the chromatin remains decondensed. We demonstrate that the LCR is necessary but not sufficient to trigger these chromatin changes. We also provide evidence that this novel silencing activity is caused by transcriptional interference triggered by activation of transcription in the flanking sequences by the LCR.

Silica distinctively affects cell wall features and lignocellulosic saccharification with large enhancement on biomass production in rice.

Rice is a typical silicon-accumulating crop with enormous biomass residues for biofuels. Silica is a cell wall component, but its effect on the plant cell wall and biomass production remains largely unknown. In this study, a systems biology approach was performed using 42 distinct rice cell wall mutants. We found that silica levels are significantly positively correlated with three major wall polymers, indicating that silica is associated with the cell wall network. Silicon-supplied hydroculture analysis demonstrated that silica distinctively affects cell wall composition and major wall polymer features, including cellulose crystallinity (CrI), arabinose substitution degree (reverse Xyl/Ara) of xylans, and sinapyl alcohol (S) proportion in three typical rice mutants. Notably, the silicon supplement exhibited dual effects on biomass enzymatic digestibility in the mutant and wild type (NPB) after pre-treatments with 1% NaOH and 1% H2SO4. In addition, silicon supply largely enhanced plant height, mechanical strength and straw biomass production, suggesting that silica rescues mutant growth defects. Hence, this study provides potential approaches for silicon applications in biomass process and bioenergy rice breeding.

Purification of Protein Disulfide-isomerase from Human Liver and Preparation of Its Antiserum.

Protein disulfide-isomerase has been isolated from human liver. The preparative procedure involved heat treatment, (NH(4))(2)SO(4) precipitation, CM-Sephadex C50 and DEAE-fast flow chromatography. The enzyme was homogenous and had a molecular mass of 60 kD or 120 kD as determined by sodium dodecy1 sulphate electro-phoresis and gel filtration respectively, indicating that the enzyme was a 120 kD dimmer with a subunit with molecular mass of 60 kD. The enzyme activity was as high as 830 U/g.protein as measured by the reactivation of "scrambled" ribonuclease. The antiserum of high titer was prepared by immunizing New Zealand rabbit with a mixture of the protein disulfide-isomerase and adjuvant.

A Micro-RNA Connection in BRaf(V600E)-Mediated Premature Senescence of Human Melanocytes.

Recent high-throughput-sequencing of the cancer genome has identified oncogenic mutations in BRaf genetic locus as one of the critical events in melanomagenesis. In normal cells, the activity of BRaf is tightly regulated. Gain-of-function mutations like those identified in melanoma frequently lead to enhanced cell-survival and unrestrained growth. The activating mutation of BRaf will also induce the cells to senesce. However, the mechanism by which the oncogenic BRaf induces the senescent barrier remains poorly defined. microRNAs have regulatory functions toward the expression of genes that are important in carcinogenesis. Here we show that expression of several microRNAs is altered when the oncogenic version of BRaf is introduced in cultured primary melanocytes and these cells undergo premature cellular senescence. These include eight microRNAs whose expression rates are significantly stimulated and three that are repressed. While most of the induced microRNAs have documented negative effects on cell cycle progression, one of the repressed microRNAs has proven oncogenic functions. Ectopic expression of some of these induced microRNAs increased the expression of senescence markers and induced growth arrest and senescence in primary melanocytes. Taken together, our results suggest that the change in microRNA expression rates may play a vital role in senescence induced by the oncogenic BRaf.