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Multicopper oxidase-1 is required for iron homeostasis in Malpighian tubules of Helicoverpa armigera.

Liu X, Sun C, Liu X, Yin X, Wang B, Du M, An S - Sci Rep (2015)

Bottom Line: HaMCO1 was also found to be highly abundant in Malpighian tubules.HaMCO1 knockdown caused a significant decrease in iron accumulation and also significantly reduced transferrin and ferritin transcript expression.Therefore, HaMCO1 is coordinately regulated by 20E and JH and is required for iron homeostasis in Malpighian tubules.

View Article: PubMed Central - PubMed

Affiliation: State key Laboratory of Wheat and Maize Crop Science/College of Plant Protection, Henan Agricultural University, Zhengzhou 450002 P.R. China.

ABSTRACT
Multicopper oxidases (MCOs) are enzymes that contain 10 conserved histidine residues and 1 cysteine residue. MCO1 has been extensively investigated in the midgut because this MCO is implicated in ascorbate oxidation, iron homeostasis and immune responses. However, information regarding the action of MCO1 in Malpighian tubules is limited. In this study, Helicoverpa armigera was used as a model to investigate the function of MCO1 in Malpighian tubules. Sequence analysis results revealed that HaMCO1 exhibits typical MCO characteristics, with 10 histidine and 1 cysteine residues for copper ion binding. HaMCO1 was also found to be highly abundant in Malpighian tubules. Temporal expression patterns indicated that HaMCO1 is mainly expressed during larval molting stages. Hormone treatments [the molting hormone 20-hydroxyecdysone (20E) and juvenile hormone (JH)] revealed that 20E inhibits HaMCO1 transcript expression via its heterodimer receptor, which consists of ecdysone receptor (EcR) and ultraspiracle (USP), and that JH counteracts the action of 20E to activate HaMCO1 transcript expression via its intracellular receptor methoprene-tolerant (Met). HaMCO1 knockdown caused a significant decrease in iron accumulation and also significantly reduced transferrin and ferritin transcript expression. Therefore, HaMCO1 is coordinately regulated by 20E and JH and is required for iron homeostasis in Malpighian tubules.

No MeSH data available.


Regulation by 20E of HaMCO1 transcript expression.(A) The effect of 20E treatment on the expression of HaMCO1 transcript. (B) qPCR analysis of ECR and USP dsRNAi efficiency on ECR and USP transcript levels, respectively. (C) The effect of ECR and USP knockdown on HaMCO1 transcript level. The 18S rRNA was used as the housekeeping gene for normalization in all qPCR analyses. The data represent the mean ± SD of three biological replicates. The significance of comparisons were determined by Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001).
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f6: Regulation by 20E of HaMCO1 transcript expression.(A) The effect of 20E treatment on the expression of HaMCO1 transcript. (B) qPCR analysis of ECR and USP dsRNAi efficiency on ECR and USP transcript levels, respectively. (C) The effect of ECR and USP knockdown on HaMCO1 transcript level. The 18S rRNA was used as the housekeeping gene for normalization in all qPCR analyses. The data represent the mean ± SD of three biological replicates. The significance of comparisons were determined by Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001).

Mentions: Considering the observed developmental expression patterns, we investigated the effects of two hormones on HaMCO1 transcript expression. The results showed that HaMCO1 transcript expression was significantly inhibited after treatment with 20E. The inhibitory effect was rapid: 20E treatments for 0.5 h significantly inhibited HaMCO1 transcript expression. This inhibitory effect continued until 6 h after 20E treatment was administered (Fig. 6A). The results suggested that 20E inhibits HaMCO1 transcript expression. 20E-triggered cascades of insect molting and metamorphosis are mediated by its heterodimer receptor, which consists of ecdysone receptor (EcR) and ultraspiracle (USP). Furthermore, RNAi-mediated knockdown of USP via injection of USP dsRNA resulted in a significant decrease in USP mRNA at 24, 48 and 72 h (Fig. 6B). Similarly, RNAi-mediated knockdown of ECR also caused a significant reduction in the ECR mRNA level at 24, 48 and 72 h after ECR dsRNA was injected (Fig. 6B). After 20E receptor (ECR or USP) expression was successfully inhibited, 20E treatments significantly increased the expression of HaMCO1 mRNA in comparison with EGFP dsRNA treatments (Fig. 6C). These results confirmed that 20E inhibits HaMCO1 transcript expression via its heterodimer receptor, specifically USP and ECR.


Multicopper oxidase-1 is required for iron homeostasis in Malpighian tubules of Helicoverpa armigera.

Liu X, Sun C, Liu X, Yin X, Wang B, Du M, An S - Sci Rep (2015)

Regulation by 20E of HaMCO1 transcript expression.(A) The effect of 20E treatment on the expression of HaMCO1 transcript. (B) qPCR analysis of ECR and USP dsRNAi efficiency on ECR and USP transcript levels, respectively. (C) The effect of ECR and USP knockdown on HaMCO1 transcript level. The 18S rRNA was used as the housekeeping gene for normalization in all qPCR analyses. The data represent the mean ± SD of three biological replicates. The significance of comparisons were determined by Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4593997&req=5

f6: Regulation by 20E of HaMCO1 transcript expression.(A) The effect of 20E treatment on the expression of HaMCO1 transcript. (B) qPCR analysis of ECR and USP dsRNAi efficiency on ECR and USP transcript levels, respectively. (C) The effect of ECR and USP knockdown on HaMCO1 transcript level. The 18S rRNA was used as the housekeeping gene for normalization in all qPCR analyses. The data represent the mean ± SD of three biological replicates. The significance of comparisons were determined by Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001).
Mentions: Considering the observed developmental expression patterns, we investigated the effects of two hormones on HaMCO1 transcript expression. The results showed that HaMCO1 transcript expression was significantly inhibited after treatment with 20E. The inhibitory effect was rapid: 20E treatments for 0.5 h significantly inhibited HaMCO1 transcript expression. This inhibitory effect continued until 6 h after 20E treatment was administered (Fig. 6A). The results suggested that 20E inhibits HaMCO1 transcript expression. 20E-triggered cascades of insect molting and metamorphosis are mediated by its heterodimer receptor, which consists of ecdysone receptor (EcR) and ultraspiracle (USP). Furthermore, RNAi-mediated knockdown of USP via injection of USP dsRNA resulted in a significant decrease in USP mRNA at 24, 48 and 72 h (Fig. 6B). Similarly, RNAi-mediated knockdown of ECR also caused a significant reduction in the ECR mRNA level at 24, 48 and 72 h after ECR dsRNA was injected (Fig. 6B). After 20E receptor (ECR or USP) expression was successfully inhibited, 20E treatments significantly increased the expression of HaMCO1 mRNA in comparison with EGFP dsRNA treatments (Fig. 6C). These results confirmed that 20E inhibits HaMCO1 transcript expression via its heterodimer receptor, specifically USP and ECR.

Bottom Line: HaMCO1 was also found to be highly abundant in Malpighian tubules.HaMCO1 knockdown caused a significant decrease in iron accumulation and also significantly reduced transferrin and ferritin transcript expression.Therefore, HaMCO1 is coordinately regulated by 20E and JH and is required for iron homeostasis in Malpighian tubules.

View Article: PubMed Central - PubMed

Affiliation: State key Laboratory of Wheat and Maize Crop Science/College of Plant Protection, Henan Agricultural University, Zhengzhou 450002 P.R. China.

ABSTRACT
Multicopper oxidases (MCOs) are enzymes that contain 10 conserved histidine residues and 1 cysteine residue. MCO1 has been extensively investigated in the midgut because this MCO is implicated in ascorbate oxidation, iron homeostasis and immune responses. However, information regarding the action of MCO1 in Malpighian tubules is limited. In this study, Helicoverpa armigera was used as a model to investigate the function of MCO1 in Malpighian tubules. Sequence analysis results revealed that HaMCO1 exhibits typical MCO characteristics, with 10 histidine and 1 cysteine residues for copper ion binding. HaMCO1 was also found to be highly abundant in Malpighian tubules. Temporal expression patterns indicated that HaMCO1 is mainly expressed during larval molting stages. Hormone treatments [the molting hormone 20-hydroxyecdysone (20E) and juvenile hormone (JH)] revealed that 20E inhibits HaMCO1 transcript expression via its heterodimer receptor, which consists of ecdysone receptor (EcR) and ultraspiracle (USP), and that JH counteracts the action of 20E to activate HaMCO1 transcript expression via its intracellular receptor methoprene-tolerant (Met). HaMCO1 knockdown caused a significant decrease in iron accumulation and also significantly reduced transferrin and ferritin transcript expression. Therefore, HaMCO1 is coordinately regulated by 20E and JH and is required for iron homeostasis in Malpighian tubules.

No MeSH data available.