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Identification and characterization of miRNAs in ripening fruit of Lycium barbarum L. using high-throughput sequencing.

Zeng S, Liu Y, Pan L, Hayward A, Wang Y - Front Plant Sci (2015)

Bottom Line: Of the novel miRNAs, 36 were specific to L. barbarum fruits compared with L. chinense.These include miR156 targeting of fructokinase and 1-deoxy-D-xylulose-5-phosphate synthase and miR164 targeting of beta-fructofuranosidase.In sum, valuable information revealed by small RNA sequencing in this study will provide a solid foundation for uncovering the miRNA-mediated mechanism of fruit ripening and quality in this nutritional food.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences Guangzhou, China ; Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences Guangzhou, China.

ABSTRACT
MicroRNAs (miRNAs) are master regulators of gene activity documented to play central roles in fruit ripening in model plant species, yet little is known of their roles in Lycium barbarum L. fruits. In this study, miRNA levels in L. barbarum fruit samples at four developmental stages, were assayed using Illumina HiSeqTM2000. This revealed the presence of 50 novel miRNAs and 38 known miRNAs in L. barbarum fruits. Of the novel miRNAs, 36 were specific to L. barbarum fruits compared with L. chinense. A number of stage-specific miRNAs were identified and GO terms were assigned to 194 unigenes targeted by miRNAs. The majority of GO terms of unigenes targeted by differentially expressed miRNAs are "intracellular organelle," "binding," "metabolic process," "pigmentation," and "biological regulation." Enriched KEGG analysis indicated that nucleotide excision repair and ubiquitin mediated proteolysis were over-represented during the initial stage of ripening, with ABC transporters and sulfur metabolism pathways active during the middle stages and ABC transporters and spliceosome enriched in the final stages of ripening. Several miRNAs and their targets serving as potential regulators in L. barbarum fruit ripening were identified using quantitative reverse transcription polymerase chain reaction. The miRNA-target interactions were predicted for L. barbarum ripening regulators including miR156/157 with LbCNR and LbWRKY8, and miR171 with LbGRAS. Additionally, regulatory interactions potentially controlling fruit quality and nutritional value via sugar and secondary metabolite accumulation were identified. These include miR156 targeting of fructokinase and 1-deoxy-D-xylulose-5-phosphate synthase and miR164 targeting of beta-fructofuranosidase. In sum, valuable information revealed by small RNA sequencing in this study will provide a solid foundation for uncovering the miRNA-mediated mechanism of fruit ripening and quality in this nutritional food.

No MeSH data available.


Related in: MedlinePlus

A hypothetical model underlying of miRNA modulation of fruit ripening in Lycium barbarum L. The black and blue “T” signs denote the interactions among miRNA-target gene investigated and predicted in this study, respectively. Dashed arrows indicate the potential function of LbCNR and LbNAC homologous to SlNAC4 in controlling carotenoid biosynthesis. In tomato, both SlCNR (Fraser et al., 2001) and SlNAC4 (Zhu et al., 2014) are evidenced to be positive regulators in carotenoid biosynthesis while SlCOP1like (Liu et al., 2004) and SlDDB1 (Wang et al., 2008) are negative regulators in fruit pigmentation. LbFK, L. barbarum fructokinase; LbDXS, L. barbarum 1-deoxy-D-xylulose-5-phosphate synthase; LbβFFase, L. barbarum beta-fructofuranosidase; L. barbarum P-glycoprotein; LbMAP3K1, L. barbarum mitogen-activated protein kinase kinase kinase 1; LbE2H, L. barbarum ubiquitin-conjugating enzyme E2H.
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Figure 8: A hypothetical model underlying of miRNA modulation of fruit ripening in Lycium barbarum L. The black and blue “T” signs denote the interactions among miRNA-target gene investigated and predicted in this study, respectively. Dashed arrows indicate the potential function of LbCNR and LbNAC homologous to SlNAC4 in controlling carotenoid biosynthesis. In tomato, both SlCNR (Fraser et al., 2001) and SlNAC4 (Zhu et al., 2014) are evidenced to be positive regulators in carotenoid biosynthesis while SlCOP1like (Liu et al., 2004) and SlDDB1 (Wang et al., 2008) are negative regulators in fruit pigmentation. LbFK, L. barbarum fructokinase; LbDXS, L. barbarum 1-deoxy-D-xylulose-5-phosphate synthase; LbβFFase, L. barbarum beta-fructofuranosidase; L. barbarum P-glycoprotein; LbMAP3K1, L. barbarum mitogen-activated protein kinase kinase kinase 1; LbE2H, L. barbarum ubiquitin-conjugating enzyme E2H.

Mentions: The L. barbarum homologs for two additional genes involved in carotenoid synthesis and fruit pigmentation during ripening, COP1like and DDB1, were also targetted by ripening stage-specific miRNAs in this study; miR2111a-5p (S2-specific) and miR6022 (specific in stages from S1 to S3), respectively (Table S5). DDB1 RNAi knockouts in tomato show enhanced numbers of plastids and pigment accumulation (Wang et al., 2008). Furthermore, repression of SlCOP1like results in increased carotenoid content in transgenic tomato fruits, suggesting that SlCOP1like functions as a negative regulator of fruit pigmentation (Liu et al., 2004). In tomato, the cnr mutation results in fruit-specific low levels of total carotenoid (Fraser et al., 2001). In this study, miR156/157 was also predicted to target LbCNR, suggesting that LbCNR may affect carotenoid biosynthesis in L. barbarum fruit. As such, the Lycium homologs to genes and their regulatory miRNAs in tomato might fulfill similar functions to determine fruit pigmentation. The glycolysis and sucrose metabolism-related genes FK, PK, and βFFase were predicted targets of miR156, miR157, and miR164, suggesting that these miRNAs may be involved in modulating fruit quality. Overall, miRNAs are master modulators, orchestrating certain metabolic, developmental or physiological processes by regulating both secondary regulators (i.e., TFs) and other related genes (i.e., biosynthetic genes) involved in the same biological event, for instance fruit ripening (Figure 8).


Identification and characterization of miRNAs in ripening fruit of Lycium barbarum L. using high-throughput sequencing.

Zeng S, Liu Y, Pan L, Hayward A, Wang Y - Front Plant Sci (2015)

A hypothetical model underlying of miRNA modulation of fruit ripening in Lycium barbarum L. The black and blue “T” signs denote the interactions among miRNA-target gene investigated and predicted in this study, respectively. Dashed arrows indicate the potential function of LbCNR and LbNAC homologous to SlNAC4 in controlling carotenoid biosynthesis. In tomato, both SlCNR (Fraser et al., 2001) and SlNAC4 (Zhu et al., 2014) are evidenced to be positive regulators in carotenoid biosynthesis while SlCOP1like (Liu et al., 2004) and SlDDB1 (Wang et al., 2008) are negative regulators in fruit pigmentation. LbFK, L. barbarum fructokinase; LbDXS, L. barbarum 1-deoxy-D-xylulose-5-phosphate synthase; LbβFFase, L. barbarum beta-fructofuranosidase; L. barbarum P-glycoprotein; LbMAP3K1, L. barbarum mitogen-activated protein kinase kinase kinase 1; LbE2H, L. barbarum ubiquitin-conjugating enzyme E2H.
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Figure 8: A hypothetical model underlying of miRNA modulation of fruit ripening in Lycium barbarum L. The black and blue “T” signs denote the interactions among miRNA-target gene investigated and predicted in this study, respectively. Dashed arrows indicate the potential function of LbCNR and LbNAC homologous to SlNAC4 in controlling carotenoid biosynthesis. In tomato, both SlCNR (Fraser et al., 2001) and SlNAC4 (Zhu et al., 2014) are evidenced to be positive regulators in carotenoid biosynthesis while SlCOP1like (Liu et al., 2004) and SlDDB1 (Wang et al., 2008) are negative regulators in fruit pigmentation. LbFK, L. barbarum fructokinase; LbDXS, L. barbarum 1-deoxy-D-xylulose-5-phosphate synthase; LbβFFase, L. barbarum beta-fructofuranosidase; L. barbarum P-glycoprotein; LbMAP3K1, L. barbarum mitogen-activated protein kinase kinase kinase 1; LbE2H, L. barbarum ubiquitin-conjugating enzyme E2H.
Mentions: The L. barbarum homologs for two additional genes involved in carotenoid synthesis and fruit pigmentation during ripening, COP1like and DDB1, were also targetted by ripening stage-specific miRNAs in this study; miR2111a-5p (S2-specific) and miR6022 (specific in stages from S1 to S3), respectively (Table S5). DDB1 RNAi knockouts in tomato show enhanced numbers of plastids and pigment accumulation (Wang et al., 2008). Furthermore, repression of SlCOP1like results in increased carotenoid content in transgenic tomato fruits, suggesting that SlCOP1like functions as a negative regulator of fruit pigmentation (Liu et al., 2004). In tomato, the cnr mutation results in fruit-specific low levels of total carotenoid (Fraser et al., 2001). In this study, miR156/157 was also predicted to target LbCNR, suggesting that LbCNR may affect carotenoid biosynthesis in L. barbarum fruit. As such, the Lycium homologs to genes and their regulatory miRNAs in tomato might fulfill similar functions to determine fruit pigmentation. The glycolysis and sucrose metabolism-related genes FK, PK, and βFFase were predicted targets of miR156, miR157, and miR164, suggesting that these miRNAs may be involved in modulating fruit quality. Overall, miRNAs are master modulators, orchestrating certain metabolic, developmental or physiological processes by regulating both secondary regulators (i.e., TFs) and other related genes (i.e., biosynthetic genes) involved in the same biological event, for instance fruit ripening (Figure 8).

Bottom Line: Of the novel miRNAs, 36 were specific to L. barbarum fruits compared with L. chinense.These include miR156 targeting of fructokinase and 1-deoxy-D-xylulose-5-phosphate synthase and miR164 targeting of beta-fructofuranosidase.In sum, valuable information revealed by small RNA sequencing in this study will provide a solid foundation for uncovering the miRNA-mediated mechanism of fruit ripening and quality in this nutritional food.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences Guangzhou, China ; Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences Guangzhou, China.

ABSTRACT
MicroRNAs (miRNAs) are master regulators of gene activity documented to play central roles in fruit ripening in model plant species, yet little is known of their roles in Lycium barbarum L. fruits. In this study, miRNA levels in L. barbarum fruit samples at four developmental stages, were assayed using Illumina HiSeqTM2000. This revealed the presence of 50 novel miRNAs and 38 known miRNAs in L. barbarum fruits. Of the novel miRNAs, 36 were specific to L. barbarum fruits compared with L. chinense. A number of stage-specific miRNAs were identified and GO terms were assigned to 194 unigenes targeted by miRNAs. The majority of GO terms of unigenes targeted by differentially expressed miRNAs are "intracellular organelle," "binding," "metabolic process," "pigmentation," and "biological regulation." Enriched KEGG analysis indicated that nucleotide excision repair and ubiquitin mediated proteolysis were over-represented during the initial stage of ripening, with ABC transporters and sulfur metabolism pathways active during the middle stages and ABC transporters and spliceosome enriched in the final stages of ripening. Several miRNAs and their targets serving as potential regulators in L. barbarum fruit ripening were identified using quantitative reverse transcription polymerase chain reaction. The miRNA-target interactions were predicted for L. barbarum ripening regulators including miR156/157 with LbCNR and LbWRKY8, and miR171 with LbGRAS. Additionally, regulatory interactions potentially controlling fruit quality and nutritional value via sugar and secondary metabolite accumulation were identified. These include miR156 targeting of fructokinase and 1-deoxy-D-xylulose-5-phosphate synthase and miR164 targeting of beta-fructofuranosidase. In sum, valuable information revealed by small RNA sequencing in this study will provide a solid foundation for uncovering the miRNA-mediated mechanism of fruit ripening and quality in this nutritional food.

No MeSH data available.


Related in: MedlinePlus