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transcriptome analysis provides new insights into the transcriptional regulation of methyl jasmonate-induced flavonoid biosynthesis in pear calli | bmc plant biology | full text

Flavonoid biosynthesis is strongly influenced by phytohormones. For example, methyl jasmonate (MeJA) enhances the flavonoid accumulation in pear. However, the molecular mechanism underlying the MeJA-induced flavonoid biosynthesis in pear is largely uncharacterized. Therefore, the transcriptome of pear calli treated with MeJA was analyzed to elucidate the mechanism regulating MeJA-mediated flavonoid biosynthesis.

The application of exogenous MeJA significantly enhanced flavonoid accumulation, especially anthocyanin, in pear calli. A weighted gene co-expression network analysis identified the differentially expressed genes associated with MeJA-induced flavonoid biosynthesis. The MeJA treatment upregulated the expression of the flavonoid biosynthesis pathway structural genes (PcCHS, PcCHI, PcF3H, PcDFR, PcANS, PcANR2a, and PcLAR1). The MYB family members were the main transcription factors regulating the MeJA-induced flavonoid biosynthesis, but the bHLH, AP2-EREBP, NAC, WRKY, and TIFY families were also involved. In addition to PcMYB10, which is a known positive regulator of anthocyanin biosynthesis in pear, several novel MYB candidates that may regulate flavonol and proanthocyanidin biosynthesis were revealed. Yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated that PcMYB10 and PcMYC2 can directly interact with each other and bind to JAZ repressors (PcJAZ1 and PcJAZ2).

The PcMYB10PcMYC2 molecular complex is likely involved in the regulation of jasmonate-mediated flavonoid biosynthesis at the transcript level. The data generated in this study may clarify the transcriptional regulatory network associated with the MeJA-induced flavonoid accumulation in pear calli and provide a solid foundation for future studies.

Flavonoids are a group of secondary metabolites that are extensively distributed in plants. They have been divided into several major subgroups such as anthocyanins, proanthocyanidins, flavonols, flavones, and isoflavones [1]. These metabolites play important biological roles specifically related to plant development and defense. Anthocyanins are water soluble pigments that are mainly involved in flower and fruit coloration. Therefore, anthocyanins are important for attracting pollinators and they also influence seed dispersal [2]. Additionally, anthocyanins are natural antioxidants [3]. Proanthocyanidins are condensed tannins and are primarily concentrated in seeds, but they also affect fruit flavor [4]. Flavonols, flavones, flavanones, and isoflavones help protect plants from ultraviolet radiation and pathogens [5]. Furthermore, flavonoids are essential for plant adaptations to biotic and abiotic stresses [6].

The flavonoid biosynthesis pathway is a branch of the phenylpropanoid pathway [7] and requires several enzymes. For example, genes encoding PAL (phenylalanine ammonia lyase), CHS (chalcone synthase), CHI (chalcone isomerase), and F3H (flavanone 3-hydroxylase) are the early biosynthetic genes (EBGs) that produce common precursors in the early steps of the pathway [8]. The late biosynthetic genes (LBGs) contribute to a later stage, during which specific flavonoid products are synthesized such as anthocyanins, proanthocyanidins, and flavonols. The LBGs include those encoding DFR (dihydroflavonol 4-reductase), ANS (anthocyanin synthase), and UFGT (UDP-glucose:flavonoid 3-glucosyltransferase), which are specifically involved in anthocyanin biosynthesis [9]. In contrast, LAR (leucoanthocyanidin reductase) and ANR (anthocyanin reductase) are key enzymes mediating proanthocyanidin biosynthesis [10]. Additionally, FLS (flavonol synthase) is specific for flavonol biosynthesis [11]. The structural genes of the flavonoid biosynthesis pathway are transcriptionally controlled by the MYBbHLHWDR (MBW) complex comprising a MYB transcription factor, a basic helix-loop-helix (bHLH), and a WD-repeat protein [12].

Flavonoid biosynthesis is affected by various factors, including light [13], temperature [14], water deficit [15], and nutrient deficiency [16]. Moreover, phytohormones are among the most important regulators of the biosynthesis of flavonoid compounds in plants. The effects of plant hormones, such as jasmonate [17, 18], abscisic acid [19, 20], auxin [21], ethylene [22], cytokinin [23], and gibberellin [24], on flavonoid accumulation have been widely studied.

Jasmonates are oxylipins (oxygenated fatty acids) synthesized by the octadecanoid/hexadecanoid pathways [25]. Jasmonic acid can be metabolized to several derivatives, including methyl jasmonate (MeJA), jasmonoyl-isoleucine (JA-Ile), jasmonyl-1-aminocyclopropane-1-carboxylic acid (JA-ACC), glucosylated derivatives of JA (e.g., JA-O-Glc), and cis-jasmone. However, of these derivatives, only MeJA and JA-Ile have been well characterized [26]. Multiple studies have revealed that MeJA application induces flavonoid biosynthesis in different fruit species such as apple (Malus domestica) [27], grape [28], blueberry [29], and strawberry (Fragaria ananassa) [30]. In pear, the post-harvest application of MeJA induces anthocyanin accumulation in the fruit peel under UV-B/Vis irradiation [31]. In addition to anthocyanin, Ni et al. [22] reported that MeJA increases the accumulation of other flavonoid derivatives, including flavone and isoflavone, in pear fruit.

The molecular mechanism underlying jasmonate-induced anthocyanin accumulation has been clarified in Arabidopsis thaliana (Arabidopsis) and apple [17, 32, 33]. Jasmonate ZIM-domain proteins (JAZs) are substrates of the SCFCOI1 complex and negatively regulate the jasmonate signaling pathway [34, 35]. The JAZ proteins can directly interact with MYB and bHLH and disrupt the formation of the MBW complex [32, 36]. After the jasmonate signal is perceived, JAZ proteins are recruited by COI1 to the SCFCOI1 complex for ubiquitination and are subsequently degraded by the 26S proteasome pathway [32]. This triggers the release of MYB and bHLH transcription factors and the formation of the MBW complex to activate the expression of flavonoid biosynthesis pathway structural genes [18, 33]. The expression levels of MYB and bHLH transcription factor genes are upregulated by MeJA in Arabidopsis and apple, suggesting these transcription factors are regulated by the jasmonate signaling pathway. However, the molecular mechanism associated with MeJA-induced flavonoid biosynthesis in pear is largely unknown. Therefore, in the present study, pear calli treated with MeJA underwent a comprehensive transcriptome analysis to identify the differentially expressed genes (DEGs) between the MeJA-treated and untreated control pear calli. Moreover, a co-expression network was constructed to detect the transcripts specifically related to MeJA-induced flavonoid biosynthesis. This study generated a pool of candidate genes that should be analyzed in greater detail to clarify the molecular mechanism associated with MeJA-induced flavonoid biosynthesis in pear. Specifically, we examined pear calli because of their lack of seasonal restrictions and the ease in which their gene effects can be observed in a homogeneous system, which can substantially accelerate the study of gene functions in pear.

To assess the effect of MeJA on flavonoid biosynthesis, pear calli were transferred to Murashige and Skoog (MS) medium containing 50mol/L MeJA, whereas control calli were transferred to MS medium with 1% methanol. Distinct phenotypic differences between the MeJA-treated and control pear calli were observed at 48h after the treatment (Fig. 1a). Additionally, red coloration was detected in the MeJA-treated pear calli. The anthocyanin and flavonoid contents of the MeJA-treated pear calli increased substantially after 48h and continued to increase for the duration of the treatment period (Fig. 1b and c).

Effects of a MeJA treatment on anthocyanin and flavonoid accumulation in pear calli. a Phenotypic comparison of the pear calli treated with MeJA (50mol/L) and the control calli (1% methanol). b Anthocyanin contents of the MeJA-treated and control pear calli. c Flavonoid contents of the MeJA-treated and control pear calli

Total RNA was extracted from pear calli sampled at 0, 12, and 48h after the MeJA treatment and from the corresponding control samples for an RNA sequencing (RNA-seq) analysis. The number of raw reads for each library ranged from 44.39 to 62.10 million. After the quality filtering process, 43.43 to 60.77 million clean reads were generated for each library. Additionally, the Q20 and Q30 values for all libraries were96.48% and90.9%, respectively, confirming the high quality of the RNA and sequencing data that were used for further analyses of gene expression. The total reference genome mapping rate varied from 70 to 72.67%, with 63.9 to 65.79% of the reads uniquely mapped (Additional file 4: Table S1).

To determine the differences in gene expression between MeJA-treated and control pear calli, gene expression levels were normalized based on the fragments per kilobase per million (FPKM) values (Additional file 5: Table S2). All uniquely mapped reads were used to calculate the gene FPKM values. The DEGs were identified and filtered according to the following criteria: adjusted p-value <0.005 and log2 (fold-change) value >1. At 12h after the treatment, the expression levels of 4228 and 3410 genes were upregulated and downregulated, respectively, in the MeJA-treated pear calli relative to the corresponding control levels. Furthermore, 2583 and 1659 gene expression levels were upregulated and downregulated, respectively, in the MeJA-treated pear calli compared with the control levels at 48h after the treatment (Additional file 1: Fig. S1).

All unigenes were functionally annotated based on the Gene Ontology (GO) database. The predicted genes were grouped into the three main categories (biological process, molecular function, and cellular component). (Additional file 2: Fig. S2). Additionally, the enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways among the unigenes were determined to elucidate the biological pathways activated by the MeJA treatment. The functional analysis revealed that the flavonoid biosynthesis (mdm0094) pathway was significantly enhanced in the MeJA-treated pear calli at 12 and 48h after the treatment. In addition to flavonoid biosynthesis, plant hormone signal transduction (mdm04075), biosynthesis of secondary metabolites (mdm01110), and phenylalanine, tyrosine, and tryptophan biosynthesis (mdm00400) were also significantly enhanced in the MeJA-treated pear calli compared with the control (Fig. 2).

Enriched KEGG pathways among the DEGs more highly expressed in MeJA-treated pear calli than in control calli (1% methanol) after 12 and 48h. The y-axis and x-axis present the KEGG pathways and the Rich factors, respectively. Dot size corresponds to the number of distinct genes, whereas dot color reflects the q-value

Several jasmonate signaling factors were identified after annotating DEGs associated with the jasmonate signal transduction pathway (Fig. 3a). For example, Pbr021060.1 was differentially expressed and annotated as PcJAR1. The PcJAR transcript level was lower in MeJA-treated pear calli than in the untreated control. Two PcCOI1 genes (Pbr011349.1 and Pbr009479.1) were differentially expressed, with expression levels that were upregulated in response to the MeJA treatment. Furthermore, 10 JAZ genes belonging to the TIFY family had upregulated expression levels. The transcript abundance of the differentially expressed JAZ genes in pear calli was lower at 48h than at 12h after the MeJA treatment. Additionally, Pbr018411.1, Pbr042466.1, and Pbr037679.1, which were annotated as PcMYC2, were more highly expressed in the control calli than in the MeJA-treated calli. The relative expression of selected genes was further analyzed by quantitative real-time (qRT)-PCR to verify the sequencing data. The expression levels of the selected jasmonate signaling factor genes were consistent with the RNA-seq data (Fig. 3b).

Jasmonate signal transduction pathway. a Transcriptional profiles of DEGs associated with the JA signaling pathway. The log10 (FPKM +1) values for the DEGs were calculated based on three biological replicates of pear calli for each time-point. The progression of the color scale from blue to red represents an increase in the FPKM values. b Verification of the expression of differentially expressed genes in the JA pathway by qRT-PCR analysis

In addition to jasmonate, several DEGs were revealed to be involved in signal transduction pathways related to other plant hormones, including cytokinin, ethylene, auxin, abscisic acid, and brassinosteroid (Additional file 6: Table S3).

A gene co-expression network was constructed via a weighted gene co-expression network analysis (WGCNA) to identify the DEGs associated with MeJA-induced flavonoid biosynthesis. The individual branches of the dendrogram represent the clusters of interconnected genes (i.e., modules). Hierarchical clustering identified eight co-expressed WGCNA modules (Fig. 4a). Each module was analyzed regarding their co-expression related to the trait phenotype (anthocyanin and flavonoid contents). The largest (3139 genes) and smallest (77 genes) modules were lavenderblush and plum, respectively. An analysis of the moduletrait relationships revealed that the green module was highly positively correlated with the pear calli anthocyanin (r=0.99, p=21012) and flavonoid (r=0.94, p=2107) contents (Fig. 4b). The green module comprised 1334 genes, and their overall expression patterns are presented in Fig. 4c. This module was selected for further analyses because it was the module most positively correlated with flavonoid biosynthesis in pear calli.

Weighted gene co-expression network analysis of the DEGs identified in the MeJA-treated pear calli. a Dendrogram with co-expressed gene modules. b Moduletrait correlations and p-values (in parentheses). The color scale on the right presents the moduletrait correlations from 1 (blue) to 1 (red). The Anthocyanin and Flavonoid panels represent anthocyanin biosynthesis and flavonoid biosynthesis as traits. c Heat map presenting the expression patterns of the DEGs in the ME green module. Clustering applied the log10 (FPKM +1) value. Red and blue denote genes with high and low expression levels, respectively

Several structural genes were identified after identifying the DEGs related to the flavonoid biosynthesis pathway. For example, PcCHS (Pbr020913.1 and Pbr020914.1), PcCHI (Pbr038148.1 and Pbr032289.1), and PcF3H (Pbr034840.1) were identified as EBGs. Additionally, Pbr020145.1 and Pbr005931.1 were annotated as PcDFR. The Pbr001543.1 (PcANS) gene was identified as specifically involved in anthocyanin biosynthesis, whereas Pbr013248.1 (PcLAR1) and Pbr032454.1 (PcANR2a) were revealed to affect proanthocyanidin biosynthesis. We mapped the selected structural genes of the flavonoid biosynthesis pathway and determined their expression patterns (Fig. 5a). The EBGs and LBGs were positively correlated with anthocyanin and flavonoid accumulation in pear calli, with expression levels that were significantly upregulated in the MeJA-treated pear calli, especially at 48h after the treatment. The relative expression levels of selected structural genes were further analyzed by qRT-PCR to verify the sequencing data. The results indicated PcDFR expression was approximately 2-fold higher in MeJA-treated pear calli than in the control calli at 48h after the treatment. The PcANS expression level was upregulated by the MeJA treatment to about 7-fold higher than that in the control calli (Fig. 5b).

Flavonoid biosynthesis pathway. a Transcriptional profiles of differentially expressed structural genes in the flavonoid biosynthesis pathway. The log10 (FPKM +1) values for the DEGs were calculated based on three biological replicates of pear calli for each time-point. The progression of the color scale from blue to red represents an increase in the FPKM values. b Verification of the expression of differentially expressed structural genes in the flavonoid biosynthesis pathway by qRT-PCR analysis

Transcription factor families potentially involved in MeJA-induced flavonoid accumulation were identified through the WGCNA (Table1). The MYB family members were the predominant transcription factor genes regulating flavonoid biosynthesis, followed by the bHLH and AP2-EREBP genes. Additionally, NAC and WRKY family genes were also differentially expressed. Moreover, the TIFY and zinc finger protein (C2H2, C3H, and C2C2-Dof) transcription factor families were also identified as related to the jasmonate signal transduction pathway. A total of 108 differentially expressed transcription factor genes were included in the green module following the WGCNA. The transcriptional profiles of differentially expressed transcription factor genes in the green module are presented in Additional file 3: Fig. S3. Most of these genes were highly expressed in response to the MeJA treatment. In addition to the MYB transcription factors, the bHLH genes, such as Pbr006544.1, Pbr017127.1, Pbr017379.1, and Pbr030521.1, exhibited significantly upregulated expression in the MeJA-treated calli relative to the control levels. Moreover, Pbr029330.1, Pbr023747.1, and Pbr008278.1 were highly expressed WRKY transcription factor genes following the MeJA treatment.

We identified 21 green module genes encoding candidate MYB transcription factors involved in MeJA-induced flavonoid biosynthesis in pear calli. A phylogenetic tree was constructed based on known flavonoid regulatory MYB transcription factors in pear (Pyrus spp.), apple (M. domestica), and Arabidopsis (Fig. 6a). For example, Pbr016663.1 (MYB10) was identified as a known anthocyanin regulatory MYB transcription factor in pear. Additionally, several novel candidate MYB transcription factors were identified. Both Pbr015228.1 (PcMYB79) and Pbr024492.1 (PcMYB173) were phylogenetically related to flavonol regulators such as PbMYB12b in pear and flavonol regulatory MYB transcription factors in Arabidopsis (AtMYB12, AtMYB111, and AtMYB11). Furthermore, four candidate MYB transcription factors [Pbr031682.1 (PcMYB134), Pbr019902.1 (PcMYB142), Pbr015230.1 (PcMYB78), and Pbr024978.1 (PcMYB62)] were identified as potential regulators of proanthocyanidin biosynthesis. In contrast, Pbr034465.1 (PcMYB176) was revealed as a potential repressor because it was grouped with flavonoid biosynthesis repressors in apple and Arabidopsis.

Analysis of differentially expressed MYB transcription factor genes related to flavonoid biosynthesis. a Phylogenetic tree with flavonoid regulatory MYB transcription factors in pear (Pyrus spp.), apple (Malus domestica), and Arabidopsis (Arabidopsis thaliana). b Heat map presenting the expression patterns of differentially expressed MYB transcription factor genes in the ME green module of the weighted gene co-expression network. The progression of the color scale from blue to red represents an increase in the FPKM values. c Verification of the expression of differentially expressed MYB transcription factor genes by qRT-PCR analysis

The transcriptional profiles of the candidate MYB transcription factor genes indicated that most of these genes were more highly expressed in the MeJA-treated pear calli than in the control calli, with expression levels that were positively correlated with anthocyanin and flavonoid biosynthesis (Fig. 6b). The relative expression levels of selected MYB transcription factor genes determined by qRT-PCR were consistent with the FPKM values based on the sequencing data (Fig. 6c).

The physical interactions of PcMYB10 and PcMYC2 with selected JAZ proteins were analyzed in yeast two-hybrid (Y2H) assays. The results demonstrated that PcMYB10 and PpMYC2 can physically interact with each other and with PcJAZ1 as well as PcJAZ2 (Fig. 7a). These interactions were verified in bimolecular fluorescence complementation (BiFC) assays, in which fluorescence was undetectable in the negative controls (Fig. 7b). However, consistent with the Y2H results, a strong green fluorescent protein signal was observed in the nuclei when PcMYB10-2YN was co-expressed with PcMYC2-2YC, PcJAZ1-2YC, and PcJAZ2-2YC. Additionally, fluorescence was detected in samples co-infiltrated with PcMYC2-2YN and PcJAZ1-2YC as well as PcJAZ2-2YC. These findings indicate that PcMYB10 and PcMYC2 can physically interact with each other and with PcJAZ1 and PcJAZ2.

Interactions between PcMYB10 and JA signaling factors. a Yeast two-hybrid analyses of the interactions of PcMYB10 with PcMYC2, PcJAZ1, and PcJAZ2. b Bimolecular fluorescence complementation assay presenting the interaction of PcMYB10 with PcMYC2, PcJAZ1, and PcJAZ2 in Nicotiana benthamiana leaves

In plants, jasmonates are essential signaling molecules [37] that promote the biosynthesis of secondary metabolites, especially flavonoids [38]. Flavonoids are important determinants of fruit quality and economic value because of their effects on color, aroma, astringency, and antioxidant properties [39]. Therefore, over the last few decades, numerous studies have been performed to develop strategies to increase fruit flavonoid contents via jasmonate treatments [22, 27, 30, 31]. In the present study, the application of exogenous MeJA activated the jasmonate signaling pathway in pear calli. The expression levels of many DEGs were upregulated in the MeJA-treated pear calli relative to the corresponding levels in the untreated control (Additional file 1: Fig. S1). Additionally, several important jasmonate signaling factors (JAR1, COI1, JAZ, and MYC2) were annotated as part of the jasmonate signal transduction pathway (Fig. 3).

We used pear calli to study the effects of MeJA on flavonoid biosynthesis because they can be continuously and uniformly produced to efficiently use the available space. We observed that MeJA significantly enhanced the biosynthesis of flavonoids in pear calli, especially anthocyanin (Fig. 1). Similarly, previous studies concluded that MeJA promotes anthocyanin and proanthocyanidin accumulation in apple calli [18, 20, 33]. Furthermore, efficient in vitro systems reportedly can produce high quality anthocyanins on a commercial scale [40].

In this study, the MeJA treatment upregulated the expression of EBGs, such as PcCHS, PcCHI, and PcF3H, which are involved in the early stages of the flavonoid biosynthesis pathway. Additionally, the LBGs, which are specifically involved in anthocyanin and proanthocyanidin biosynthesis, were more highly expressed in the MeJA-treated pear calli than in the control calli (Fig. 5). Both DFR and ANS are considered key enzymes for anthocyanin biosynthesis [9]. Consistent with our results, Shan et al. [17] reported that jasmonate strongly upregulates the expression of AtDFR in Arabidopsis seedlings, thereby regulating anthocyanin accumulation. Furthermore, the expression levels of other flavonoid biosynthetic genes, including AtPAL, AtCHS, AtCHI, AtF3H, and AtF3H, also increased in response to jasmonate, although the expression levels were still relatively low. Sun et al. [20] demonstrated that the application of exogenous MeJA enhances the anthocyanin accumulation in red-fleshed apple calli because of the associated upregulated MdCHS, MdF3H, and MdUFGT expression. In addition to anthocyanin biosynthesis-related genes, the expression of key genes involved in proanthocyanidin biosynthesis (PcANR2a and PcLAR1) was also upregulated by MeJA (Fig. 5). Moreover, a KEGG analysis revealed that the flavonoid biosynthesis pathway was significantly enhanced in the MeJA-treated pear calli compared with the untreated control (Fig. 2).

Transcription factors regulate the expression of flavonoid biosynthesis pathway structural genes. For example, MYB, bHLH, and WDR proteins form the MBW complex that regulates flavonoid biosynthesis in many plant species [12]. Previous studies indicated that MYB transcription factors are the key elements in the regulatory networks controlling specific gene expression patterns during flavonoid biosynthesis [1]. In pear, PpMYB10 was initially identified as a R2R3-MYB transcription factor that positively regulates anthocyanin biosynthesis [41]. Additionally, PbMYB10b and PbMYB9 were characterized as positive regulators of anthocyanin and proanthocyanidin biosynthesis in pear [42]. Earlier investigations determined that PpMYB114 and PpbHLH3 can co-regulate anthocyanin biosynthesis in pear fruit [43], whereas PbMYB12b was functionally annotated as a flavonol regulator in pear [44]. However, MYB transcription factors involved in jasmonate-mediated flavonoid biosynthesis have not been specifically characterized in pear. Nevertheless, in apple, MdMYB9 and MdMYB11 reportedly interact with MdbHLH3 and MdTTG1 to form a MBW complex that regulates jasmonate-mediated anthocyanin and proanthocyanidin accumulation [33]. Recently, MdMYB24L was overexpressed in apple calli and functionally characterized as a gene encoding a jasmonate-responsive MYB transcription factor contributing to MeJA-induced anthocyanin accumulation in apple. Other studies proved that jasmonate-induced anthocyanin accumulation in Arabidopsis is mediated by MYB transcription factors, including PAP1 (MYB75), PAP2 (MYB90), and GL3, that upregulate the expression of anthocyanin biosynthetic genes [17, 32].

RNA sequencing is an effective tool for clarifying the transcriptional regulation of essential genes in secondary metabolite biosynthesis pathways [45]. On the basis of a WGCNA, we identified 21 candidate MYB transcription factor genes whose expression levels were significantly positively correlated with MeJA-induced flavonoid biosynthesis in pear calli. These genes included Pbr016663.1 (PcMYB10), which encodes a known MYB transcription factor that positively regulates anthocyanin biosynthesis in pear. Interestingly, we detected several novel MYB candidates as potential regulators of proanthocyanidin and flavonol biosynthesis in pear (Fig. 6).

Genes encoding other candidate transcription factors belonging to bHLH, AP2-EREBP, NAC, WRKY, and TIFY families were also revealed as differentially expressed based on our transcriptome data (Additional file 3: Fig. S3). Several bHLH transcription factors that regulate jasmonate-responsive anthocyanin accumulation in plants have been reported. A previous study proved that MYC2, which belongs to the bHLH family, regulates diverse jasmonate responses in Arabidopsis, including anthocyanin biosynthesis, wound responses, root growth inhibition, and oxidative stress adaptations [17]. Additionally, MYC3 and MYC4, which are close homologs of MYC2, function additively with MYC2 in the jasmonate signaling pathway [46]. Moreover, GL3, EGL3, and TT8 bHLH transcription factors are also positive regulators of jasmonate-responsive anthocyanin accumulation in Arabidopsis [32]. In apple, MdMYC2 has been functionally characterized as a positive regulator of jasmonate-induced anthocyanin biosynthesis [47]. However, bHLH transcription factors involved in the pear jasmonate-mediated flavonoid biosynthesis have yet to be reported. In the current study, we identified several candidate bHLH transcription factors, including PcMYC2, that might participate in the jasmonate-induced flavonoid biosynthesis in pear calli (Fig. 3 and Additional file 3: Fig. S3).

The molecular mechanism underlying jasmonate-induced anthocyanin biosynthesis has been thoroughly characterized in Arabidopsis [17, 32, 35, 48]. The JAZ proteins are believed to repress the jasmonate signaling pathway. The JAZ repressors directly interact with MYB transcription factors (PAP1, PAP2, and GL1) and bHLH transcription factors (GL3, EGL3, and TT8) of the MBW complex to inhibit transcription and subsequently repress anthocyanin biosynthesis in Arabidopsis. Following the jasmonate signal-induced degradation of JAZ proteins, the MBW complex is activated to induce the expression of the downstream structural genes and mediate jasmonate-induced anthocyanin biosynthesis [32]. Moreover, Shan et al. [17] revealed that the F-box protein COI1 is essential for the expression of transcription factor genes, including those encoding PAP1, PAP2, and GL3. In apple, an earlier study concluded that MdJAZ2 inhibits the recruitment of MdbHLH3 to the MdMYB9 and MdMYB1 promoters. After jasmonate signals are perceived, MdbHLH3 is released to form the MBW complex involved in activating the downstream genes related to flavonoid biosynthesis [33]. However, the molecular mechanism associated with jasmonate-mediated flavonoid biosynthesis remains largely unknown in pear.

In the present study, we observed that PcMYB10 and PcMYC2 can directly interact with each other and with JAZ proteins (PcJAZ1 and PcJAZ2) (Fig. 7). In contrast, our transcriptome data indicated that 10 JAZ genes were more highly expressed in the MeJA-treated calli than in the control calli (Fig. 3). Although the transcription of JAZ genes may be upregulated, it may not be consistent with the high translated protein levels because of post-translational regulatory activities [49]. In a previous study on apple, MdJAZ8 and MdJAZ11 were observed to form a complex with the MdMYB24-like protein to weaken the transcriptional activity of the MYBMYC2 complex, and in response to a jasmonate application, MdJAZ8 and MdJAZ11 were degraded to release MdMYC2 and MdMYB24L, thereby promoting JA-mediated anthocyanin accumulation [18]. Consequently, the findings of the present suggest that the PcMYB10PcMYC2 molecular complex may be involved in the transcriptional regulation of jasmonate-mediated flavonoid biosynthesis in pear. However, this will need to be experimentally confirmed in future studies.

In the current study, the application of exogenous MeJA activated the jasmonate signaling pathway. Moreover, MeJA induced the accumulation of flavonoids, especially anthocyanin, in pear calli by upregulating the expression of structural genes (PcCHS, PcCHI, PcF3H, PcDFR, PcANS, and PcLAR1) in the flavonoid biosynthesis pathway. The MYB family was prominently involved in the transcriptional regulation of flavonoid biosynthesis, with the bHLH, AP2-EREBP, NAC, WRKY, and TIFY family members also contributing. Additionally, protein interaction assays suggested the PcMYB10PcMYC2 molecular complex might influence the transcriptional regulation of jasmonate-mediated flavonoid biosynthesis in pear calli. Our comprehensive transcriptome analysis revealed a set of candidate transcription factors that may be relevant for future functional studies related to the transcriptional regulation of MeJA-mediated flavonoid biosynthesis in pear.

Pear calli were induced from the flesh of young Clapps Favorite (Pyrus communis) pear fruit in our in vitro laboratory according to a published protocol [50]. The pear fruit sample was kindly provided by Prof. Yuanjun Li from the Yantai Academy of Agricultural Sciences, Yantai, Shandong, China. Briefly, pear calli were grown in darkness on MS solid medium supplemented with sucrose (30g/L), 6-benzylaminopurine (0.5mg/L), and 2,4-dichlorophenoxyacetic acid (1.0mg/L). The calli were sub-cultured every 3weeks. They were then transferred to MS medium containing MeJA, which had been diluted to 50mol/L in methanol. Accordingly, control calli were transferred to MS medium with 1% methanol. The treated and control calli were incubated under continuous white light (2000lx) at 24C. Calli samples were collected at 0, 12, 24, 48, 96, and 144h after the treatment. At each time-point, samples were collected from three calli plates (three replicates). The experiment was conducted according to a completely randomized design. The collected calli samples were immediately frozen in liquid nitrogen and stored at 80C until analyzed.

Flavonoids were analyzed with the Cominbio Plant Flavonoid Extraction kit (Suzhou Keming Biotechnology Co., Ltd., Suzhou, China). Specifically, flavonoids were extracted with 60% ethanol and then complexed with an aluminum ion in an alkaline nitrite solution. The absorbance (at 530nm) of the sample extract was measured with the DU800 spectrophotometer (Beckman Coulter, Brea, CA, USA), after which the flavonoid content was calculated with the following formula:

Anthocyanins were extracted from the pear calli as previously described [14]. Briefly, 0.2g frozen pear calli were treated with 1mL methanol:acetic acid (99:1 volume) overnight in darkness at 4C. The absorbance (at 530, 620, and 650nm) was measured with the DU800 spectrophotometer. The total anthocyanin content was calculated with the following formula:

Total RNA was extracted from the collected samples according to the cetyltrimethylammonium bromide method [51]. The qRT-PCR analysis was performed as previously described [52]. Details regarding the qRT-PCR primers are listed in Additionalfile7: Table S4.

The pear calli sampled at 0, 12, and 48h after the MeJA treatment as well as the corresponding control samples (1% methanol) were used for the RNA-seq analysis, which was completed as described by Bai et al. [50]. The libraries were prepared and then sequenced with the HiSeq X system (Illumina, San Diego, CA, USA) by Novogene (Beijing, China). The clean reads were mapped to the Pyrus bretschneideri genome sequence (http://gigadb.org/dataset/100083) with the default parameters of HISAT2.

The GO enrichment analysis of DEGs was completed with the goseq R package, in which the gene length bias was corrected. The GO terms with a corrected p-value less than 0.05 were considered significantly enriched. The KOBAS software was used to identify the significantly enriched KEGG pathways among the DEGs (http://www.genome.jp/kegg/) [53].

A WGCNA was performed with the R package according to a published method [54]. The sequences of flavonoid regulatory MYB transcription factors in pear (Pyrus spp.), apple (M. domestica), and Arabidopsis were downloaded from the NCBI database. Protein accessions are provided in Additionalfile8: Table S5. A phylogenetic tree was generated with the neighbor-joining method (500 bootstrap replicates) of the MEGA 6.0 program.

Yeast two-hybrid assays were performed with the Matchmaker Gold Yeast Two-Hybrid System Kit (TaKaRa, Dalian, China). The full-length coding sequences encoding the prey and bait proteins were cloned into the pGADT7 (AD) and pGBKT7 (BD) vectors, respectively. First, the full-length PcMYB10-BD and PcMYC2-BD plasmids were inserted into Y2HGold cells with an empty AD vector, after which a lack of self-activation was confirmed for PcMYB10-BD and PcMYC2-BD. Y2HGold competent cells were co-transformed with the recombinant gene-AD and gene-BD plasmids and spread agar-solidified SD/Leu/Trp medium. To evaluate potential physical interactions, the co-transformed colonies were selected on SD medium lacking adenine, histidine, leucine, and tryptophan, supplemented with X--gal.

The BiFC assays were conducted as previously described [52]. Briefly, the PcMYB10, PcMYC2, PcJAZ1, and PcJAZ2 coding sequences without terminator codons were amplified and cloned into the p2YN and p2YC vectors. The resulting recombinant plasmids were inserted into Agrobacterium tumefaciens strain GV3101 cells, which were then infiltrated into Nicotiana benthamiana leaves. At 48h after the infiltration, fluorescence was detected in the transformed leaves with a confocal laser scanning microscope (Nikon, Japan).

A phylogenetic tree was generated with the neighbor-joining method of the MEGA 6.0 program. The tree included bootstrap values from 1000 replications next to the branch nodes and a bar indicating an evolutionary distance of 0.1%. The accession numbers of the flavonoid regulatory MYB transcription factors in pear (Pyrus spp.), apple (M. domestica), and Arabidopsis that were included in the phylogenetic analysis are listed in Additional file 8: Table S5.

Experiments were performed according to a completely randomized design. Significant differences (*p<0.05, **p<0.01, and ***p<0.001) between two independent treatments were determined with Students t-test. All data were analyzed with the SPSS software (version 25) (SPSS Inc., Chicago, IL, USA).

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This study was financially supported by the National Natural Science Foundation of China (Grant Nos. 31901986 to JN and 31772272 to SB) and the Earmarked Fund for China Agriculture Research System (CARS-28 to YT). The funding bodies were not involved in the design of the study, data collection, interpretation of data, or in writing the manuscript.

YT conceived the study. ATP performed the experiments. ATP, SB, JN, and JS analyzed the transcriptome data. ATP, YT, JN, and SB wrote the manuscript. All authors read and approved the final manuscript.

. Gene ontology (GO) classification of differentially expressed upregulated unigenes in the MeJA-treated pear calli after 12 and 48h. The x-axis and y-axis present the enriched GO terms and the number of differentially expressed genes, respectively.

Heat map presenting the expression patterns of differentially expressed transcription factor genes in the ME green module of the weighted gene co-expression network. The progression of the color scale from blue to red represents an increase in the FPKM values.

Accession numbers of the flavonoid regulatory MYB transcription factors in pear (Pyrus spp.), apple (Malus domestica), and Arabidopsis (Arabidopsis thaliana) that were included in the phylogenetic analysis.

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Premathilake, A.T., Ni, J., Shen, J. et al. Transcriptome analysis provides new insights into the transcriptional regulation of methyl jasmonate-induced flavonoid biosynthesis in pear calli. BMC Plant Biol 20, 388 (2020). https://doi.org/10.1186/s12870-020-02606-x

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production system: concept and models | industries | production management

The production system is a part of a larger system the business firm. The production system can be viewed as a framework or skeleton of activities within which the creation of value can occur. Briefly, the difference between the value of inputs and the value of outputs represents the value created through production activities. At one end of the production system are the inputs and at the other end are outputs. Connecting the inputs and outputs are a series of operations or processes, storages and inspections. Fig. 2.1 represents a simplified production system.

The concept of production system is applicable to both production of components and production of services as well. The production of any component or service can be viewed in terms of a production system. For example, the manufacture of furniture involves such inputs as wood, glue, nails, screws, paints, sand paper, saws, workers etc. After these inputs are acquired, they must be stored until ready for use.

Then several operations, such as sawing, nailing, sanding and painting can occur through which inputs are converted into such outputs as chairs, tables, etc. After the finishing operation, a final inspection occurs. Then the outputs are held in stock rooms until they are shipped to the customers.

It is one of the basic models of the production system. A production system is the set of interconnected input-output elements and is made up of three component parts namely inputs, process and outputs (Fig. 2.3). A wide variety of inputs are transformed so that they give out a set of outputs. The transforming process can be complicated and the design of an actual input and output system for manufacturing may be expensive and difficult.

= Output/Input 1, a system with output equal to input is considered to be ideal. But in a system of Production Management this definition of efficiency means utter failure and ultimately the end of the business. In economic system, the efficiency has to be greater than one which means a state of profit. A production management system comprehends and integrates both engineering and economic criteria in its activities.

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production worker job description [updated for 2021]

A Production Worker, or General Production Worker, is responsible for helping assemble and prepare products for shipment. Their duties include placing raw materials or products into manufacturing machines to aid the assembly process, packaging finished products and organizing them for shipments and completing checks on equipment and products to ensure quality production.

The ideal candidate will have some mechanical and production experience (although not required) and excellent communication skills. You will be responsible for operating production line equipment, finishing products and reporting any issues with equipment directly to maintenance supervisors.

We are a growing internet nutritional supplement company located on Eastgate Rd in Henderson. We are looking for an individual to join our manufacturing team. Your job duties will include, but not limited to, packaging our powdered dietary supplements and cleaning of manufacturing equipment.

Perdue Farms, Inc. is an Equal Opportunity / Affirmative Action employer. All qualified applicants will receive consideration for employment without regard to race, color, religion, sex, sexual orientation, gender identity, national origin, disability, or protected veteran status.

Production Workers typically work for factories or manufacturing plants to help assemble products and monitor manufacturing equipment for product defects. They work closely with other Production Workers to check product quality and complete assembly tasks by set deadlines. Their job is to adhere to production quotas and complete cleaning activities at the end of each shift. They may also be responsible for using warehouse equipment like forklifts to organize and retrieve products ahead of shipments.

A minimum of a high school diploma is often required to work as an entry-level Production Worker. Many Production Workers will begin in an entry-level position and work toward a higher position. Much of the training for a Production Worker will be on-the-job, where Production Workers can learn the individual requirements and work expectations of their employer. Many Production Workers will work on a team, often under the direct supervision of a Lead Production Worker, while they learn the processes of the warehouse.

Experience requirements for Production Workers will vary from employer to employer. Some hiring managers may prefer to hire an entry-level Production Worker and then train them on the individual needs of the company whereas others prefer candidates with previous experience. Additionally, some hiring managers may require specific Production Worker skills, like the operation or upkeep or warehouse equipment. Industry-specific skills can dictate a higher expected salary as a Production Worker. Production Workers who have leadership or management experience tend to be more in demand and can often expect a higher salary.

Both Production Workers and Machine Operators work in manufacturing plants or factory settings to meet production quotas. However, their qualifications and specific job duties differ. For example, Production Workers only need a high school diploma and on-site job training to assemble and package products or use factory equipment.

In contrast, Machine Operators usually need a high school diploma followed by an associate degree or professional certification from a trade school. Their additional education allows them to operate manufacturing equipment like milling machines or grinders. Machine Operators and Production Workers may work closely together to complete specific tasks. One example of this would be when Production Workers load raw materials on an assembly line, which slowly feeds them into milling machines. From here, Machine Operators use machine controls to manipulate raw materials into products.

On a typical day, a Production Worker starts by sitting in on meetings with the production team. During these meetings, they learn about changes to manufacturing procedures and shipment schedules before receiving their assembly quota for the day. Throughout their shift, they move between different stations like assembling products, packaging products and organizing products on warehouse shelves. They take short breaks throughout the day to preserve their energy and eat meals. At the end of each shift, they clean their work area and report their quotas to their direct Supervisor.

A good Production Worker is someone who is committed to workplace safety. This quality motivates them to read over workplace safety procedures and complete their work within those guidelines. Their commitment to a safe work environment also means they voice their concerns when they see coworkers misusing equipment and notify Managers when unsafe activities occur. Further, a good Production Worker knows how to stay energized and alert throughout their shift to assemble products correctly and catch potential product defects.

A Production Worker usually reports to the Production Supervisor at their workplace. The Production Supervisor oversees all assembly activities during a particular shift. Production Workers communicate with Supervisors to determine quota needs and how to assemble products correctly. In the absence of a Production Supervisor, Production Workers may report to the Production Manager. When Production Workers experience advanced machine defects, they may also report indirectly to a Maintenance Technician or a Maintenance Supervisor.

enhanced production of astaxanthin without decrease of dha content in aurantiochytrium limacinum by overexpressing multifunctional carotenoid synthase gene | springerlink

Aurantiochytrium limacinum produces both docosahexaenoic acid (DHA) and astaxanthin, respectively. Organisms that produce these industrially important materials more efficiently than microalgae are currently needed. In this study, we overexpressed a putative homolog of CarS, which is involved in synthesizing the astaxanthin precursor, -carotene, in A. limacinum to increase carotenoid synthesis with the goal of obtaining strains that produce large amounts of both DHA and carotenoids. AlCarS transformants #1 and #18 produced significantly increased amounts of astaxanthin as assessed according to culture (up to 5.8-fold) and optical density (up to 9.3-fold). The improved astaxanthin production of these strains did not affect their DHA productivity. Additionally, their CarS expression levels were higher than those of the wild-type strain, suggesting that CarS overexpression enhanced -carotene production, which in turn improved astaxanthin productivity. Although cell yields were slightly decreased, these features will be valuable in health food, medical care, and animal feed fields.

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Watanabe, K., Arafiles, K. H. V., Higashi, R., Okamura, Y., Tajima, T., Matsumura, Y., Nakashimada, Y., Matsuyama, K., & Aki, T. (2018). Isolation of high carotenoid-producing Aurantiochytrium sp. mutants and improvement of astaxanthin productivity using metabolic information. Journal of Oleo Science, 67, 571578.

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Sakaguchi, K., Matsuda, T., Kobayashi, T., Ohara, J. I., Hamaguchi, R., Abe, E., Nagano, N., Hayashi, M., Ueda, M., Honda, D., Okita, Y., Sugimoto, S., Okino, N., & Ito, M. (2012). Versatile transformation system that is applicable to both multiple transgene expression and gene targeting for Thraustochytrids. Applied and Environmental Microbiology, 78, 31933202.

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Li, Y., Han, D., Hu, G., Sommerfeld, M., & Hu, Q. (2010). Inhibition of starch synthesis results in overproduction of lipids in Chlamydomonas reinhardtii. Biotechnology and Bioengineering, 107, 258268.

Tokunaga, S., Sanda, S., Uraguchi, Y., Nakagawa, S., & Sawayama, S. (2019). Overexpression of the DOF-type transcription factor enhances lipid synthesis in Chlorella vulgaris. Applied Biochemistry and Biotechnology, 189, 116128.

Yuki Kubo: investigation, methodology, writingoriginal draft. Mai Shiroi: investigation, methodology. Tokuhiro Higashine: methodology. Yuki Mori: methodology. Daichi Morimoto: investigation, writingoriginal draft, review, and editing. Satoshi Nakagawa: methodology, supervision. Shigeki Sawayama: conceptualization, supervision, writingreview and editing.

Kubo, Y., Shiroi, M., Higashine, T. et al. Enhanced Production of Astaxanthin without Decrease of DHA Content in Aurantiochytrium limacinum by Overexpressing Multifunctional Carotenoid Synthase Gene. Appl Biochem Biotechnol 193, 5264 (2021). https://doi.org/10.1007/s12010-020-03403-w

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soft ferrites | manufacture | spinel

Our manufacturing facilities occupy an area of more than 200,000 square meters and yield production of over 1,000 models of magnetic cores of over 20 materials, with a total annual production of 12,000 tons. In addition, Spinel is completing an intelligent factory project which will be capable of optimized production, digitized production, visualized workshops, dashboard management and intelligent, automated production.

The first step of the manufacturing process is powdering. The entire powdering workshop covers 6,000 square meters and operates a dry-type production line which has been developed based on our 40+ years experience. This line provides the functions of automatic accurate electronic scaling, automatic transfer and central control. Powders manufactured by our company are feature precise formula and excellent formability.

Forming is the second step in magnetic core production. We have acquired over 80 sets of pressing machines of various tonnages. The presses have been imported from Japan and are able to meet the production standards of less than 200-tonnage pressure. The forming workshop produces 3 million magnetic products daily and 100 million every month.

The third step of ferrite core production is sintering, which plays a vital role in determining the products properties. Spinel is equipped with 10 Toshiba fully-automatic nitrogen protection 19-channel kilns, 2 sets of fully-automatic bell-type 8-workstation furnaces and 1 set of 4-workstation furnace. These kilns and furnaces ensure a daily production of more than 30 tons. Additionally, we have implemented a fully-automatic monitoring system that provides continuous 24-hour testing and is the first fully-automatic monitoring system in the industry.

After sintering, all products must undergo grinding, in order to reach clients requirements for size and inductance. We have introduced more than 20 grinding machines from Japan, which perform different standard GAP grinding of products.

Sorting is the final process. Only qualified products are packed and sent to clients. Spinel develops many automatic size sorting machines and fully-automatic visual sorting system to replace traditional manual sorting, vastly improving efficiency and product quality. The fully-automatic visual sorting system accomplishes the sorting work of 120 products every minute and guarantees 100% quality.

SPINELs manufacturing facilities cover an area of more than 200,000 square meters and yield production of over 1,000 models of magnetic cores of over 20 materials, with a total annual production of 12,000 tons.

SPINELs intelligent factory project is intended for increased efficiency. We undertake this project in order to improve technology for both MES system and production lines. This system can achieve a wide range of management modes, such as optimized production, digitized production, visualized workshops, dashboard management, intelligent production, etc.

Features of the rotary kiln: 1. Indirect heating of natural gas 2. Three-stages preheating 3. The 1st stage heating area: Exhaust gas is used to preheat feedstock for heat recycling and energy savings. 4. The main heating area: Lengthened design results in a fully preheating process. 5. The cooling of the end region is separated from the heating area, which can fulfill the technical cooling requirements.

The powder that has been preheated is roughly ground and then mixed with a preset amount of water. A sand mill, as seen in the picture, is used to grind the powder into very small particles. It can realize the accurate measurement of water, powder and batches, with a high level efficiency and zero waste. We also utilize pipeline to transport our fluids.

Forming is the second step in magnetic core production. According to customers requirements, we can produce magnetic cores in a variety of shapes. Our company has acquired over 80 sets of pressing machines of various tonnages. The presses have been imported from Japan and are able to meet the production standards of less than 200-tonnage pressure. The rotary pressing machine, as seen in this picture, features high efficiency, stable performance and great capacity.

The third step of ferrite core production is sintering, which plays a vital role in determining the products properties. Our company has introduced fully-automated bell-type furnaces and fully-automated nitrogen protection kilns. These kilns and furnaces ensure a daily production of more than 30 tons.

The bell-type furnace is a piece of intelligent equipment which automatically performs sintering in a closed loop. It offers a wider adjustment range and a higher level of stability. We have 3 bell-type furnaces available for meeting your top-grade product sintering requirements.

We have introduced more than 20 diverse grinding machines from Japan and Taiwan, which perform different standard GAP grinding of products. These grinding machines are designed exclusively for magnetic cores. Their prominent features include high precision and easy adjustment.

Sorting is the final process in which we sort out our products according to different sizes and appearance. Those products that have been carefully selected will be packed and warehoused. We have developed many automatic size sorting machines and fully-automatic visual sorting system to replace traditional manual sorting, vastly improving efficiency and product quality.

This system can complete the size or appearance sorting process in a fully automated mode. Our product sorting process is adapted to specific requirements. This results in high production efficiency and low failure rate.

Add.: Five-Star Industrial Park, Donggang Town, Xishan District, Wuxi, Jiangsu province, China. Tel.: Mobile: Fax: Contact Person: Huang Yufeng E-mail: [email protected]

pretreatment filters and uv sterilizer with ozone generator for beverage production line supplier - wenzhou hengtong water treatment co.,ltd

WTRO series reverse osmosis system possesses the characteristics of high quality, rich experience and reasonable price. Using the international advanced RO membrane, the high efficiency and low noise high pressure pump and correct meter compose and independent reverse osmosis system, so that the whole system unit is on an independent stainless steel support, meanwhile, combines with a clever PLC control system, which can automatically instruct the high pressure pump start or stop and high or low voltage protection, automatically low energy cleaning function.

This RO system is mainly used to get pure water and pre-treatment water for all kinds of beverage. It can effectively get rid of suspended matters and smell and color in raw source water,also filter substances such as organics,microorganisms,chloride,colloidal particles,residual chlorine so as to make it reach to standard of drinking water.