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Investigation of the multifunctional gene AOP3 expands the regulatory network fine-tuning glucosinolate production in Arabidopsis.

Jensen LM, Kliebenstein DJ, Burow M - Front Plant Sci (2015)

Bottom Line: In this study, we use transgenic plants in combination with natural variation to investigate the regulatory role of the AOP3 gene found in GS-AOP locus previously suggested to contribute to the regulation of glucosinolate defense compounds.Phenotypic analysis and QTL mapping in F2 populations with different AOP3 transgenes support that the enzymatic function and the AOP3 RNA both play a significant role in controlling glucosinolate accumulation.Furthermore, we find different loci interacting with either the enzymatic activity or the RNA of AOP3 and thereby extend the regulatory network controlling glucosinolate accumulation.

View Article: PubMed Central - PubMed

Affiliation: DNRF Center DynaMo, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen Frederiksberg, Denmark ; Copenhagen Plant Science Centre, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen Frederiksberg, Denmark.

ABSTRACT
Quantitative trait loci (QTL) mapping studies enable identification of loci that are part of regulatory networks controlling various phenotypes. Detailed investigations of genes within these loci are required to ultimately understand the function of individual genes and how they interact with other players in the network. In this study, we use transgenic plants in combination with natural variation to investigate the regulatory role of the AOP3 gene found in GS-AOP locus previously suggested to contribute to the regulation of glucosinolate defense compounds. Phenotypic analysis and QTL mapping in F2 populations with different AOP3 transgenes support that the enzymatic function and the AOP3 RNA both play a significant role in controlling glucosinolate accumulation. Furthermore, we find different loci interacting with either the enzymatic activity or the RNA of AOP3 and thereby extend the regulatory network controlling glucosinolate accumulation.

No MeSH data available.


The effect of introducing different versions of AOP3 into Col-0 and Gie-0. (A) Overview of the different AOP3 constructs for expression in planta and their potential to give rise to AOP3 RNA, protein (Prot.), and enzymatic activity (Enz.). (B) Aliphatic glucosinolates with different chain-lengths; C3, C4, and LC. Col-0 WT (black), Col-0 AOP3 FL (light gray), Col-0 AOP3 NF (medium gray), or Col-0 AOP3 UT (dark gray). (C) Gie-0 WT (black), Gie-0 AOP3 FL (light gray), Gie-0 AOP3 NF (medium gray), or Gie-0 AOP3 UT (dark gray). (D) 3mtp, 3msp, and 3ohp levels in Gie-0 WT (black), Gie-0 AOP3 FL (light gray), Gie-0 AOP3 NF (medium gray), or Gie-0 AOP3 UT (dark gray). Data represent means and standard error for lines carrying the same construct. Differences were tested for significance by ANOVA, * indicates P < 0.05, for additional information see methods and Supplementary Data. FW, fresh weight.
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Figure 3: The effect of introducing different versions of AOP3 into Col-0 and Gie-0. (A) Overview of the different AOP3 constructs for expression in planta and their potential to give rise to AOP3 RNA, protein (Prot.), and enzymatic activity (Enz.). (B) Aliphatic glucosinolates with different chain-lengths; C3, C4, and LC. Col-0 WT (black), Col-0 AOP3 FL (light gray), Col-0 AOP3 NF (medium gray), or Col-0 AOP3 UT (dark gray). (C) Gie-0 WT (black), Gie-0 AOP3 FL (light gray), Gie-0 AOP3 NF (medium gray), or Gie-0 AOP3 UT (dark gray). (D) 3mtp, 3msp, and 3ohp levels in Gie-0 WT (black), Gie-0 AOP3 FL (light gray), Gie-0 AOP3 NF (medium gray), or Gie-0 AOP3 UT (dark gray). Data represent means and standard error for lines carrying the same construct. Differences were tested for significance by ANOVA, * indicates P < 0.05, for additional information see methods and Supplementary Data. FW, fresh weight.

Mentions: To focus on the effects of AOP3 and simultaneously test if its regulatory function solely requires the enzymatic activity, we introduced three different versions of the gene into two accessions not expressing a functional AOP2 or AOP3 in leaves and varying in the accumulation of C3 and C4 glucosinolates. We chose Col-0 and Gie-0 based on their difference in their major SC glucosinolate, i.e., Col-0 accumulating C4 due to expression of a functional MAM1 and Gie-0 accumulating the AOP3 substrate, 3msp, due to MAM2 expression. We introduced different versions of AOP3 driven by a 35S promoter (Figure 3A). Accessions that express AOP3 in leaves use the AOP2 promoter that has previously been shown to be at least as strong as the 35S promoter (Wentzell et al., 2007; Chan et al., 2010; Kerwin et al., 2011). In addition to an enzymatically functional full-length genomic version of AOP3, we constructed a version with a mutation abolishing the active site of AOP3 (Hogan et al., 2000), which generates a non-active enzyme but still expresses a protein allowing us to test for the importance of the enzymatic activity. Generation of the third version included introduction of a stop codon as the third codon of the transcript and chancing the subsequent two potential start codons in frame, i.e., a construct that is unable to generate any AOP3 protein but only the transcript enabling us to test the function of the AOP3 RNA. Together, these three different versions of AOP3 allowed us to systematically test whether its regulatory capacity in any of the two accessions varying in GS-ELONG relies on its enzymatic activity, the protein, or the RNA (Figure 3A).


Investigation of the multifunctional gene AOP3 expands the regulatory network fine-tuning glucosinolate production in Arabidopsis.

Jensen LM, Kliebenstein DJ, Burow M - Front Plant Sci (2015)

The effect of introducing different versions of AOP3 into Col-0 and Gie-0. (A) Overview of the different AOP3 constructs for expression in planta and their potential to give rise to AOP3 RNA, protein (Prot.), and enzymatic activity (Enz.). (B) Aliphatic glucosinolates with different chain-lengths; C3, C4, and LC. Col-0 WT (black), Col-0 AOP3 FL (light gray), Col-0 AOP3 NF (medium gray), or Col-0 AOP3 UT (dark gray). (C) Gie-0 WT (black), Gie-0 AOP3 FL (light gray), Gie-0 AOP3 NF (medium gray), or Gie-0 AOP3 UT (dark gray). (D) 3mtp, 3msp, and 3ohp levels in Gie-0 WT (black), Gie-0 AOP3 FL (light gray), Gie-0 AOP3 NF (medium gray), or Gie-0 AOP3 UT (dark gray). Data represent means and standard error for lines carrying the same construct. Differences were tested for significance by ANOVA, * indicates P < 0.05, for additional information see methods and Supplementary Data. FW, fresh weight.
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Related In: Results  -  Collection

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Figure 3: The effect of introducing different versions of AOP3 into Col-0 and Gie-0. (A) Overview of the different AOP3 constructs for expression in planta and their potential to give rise to AOP3 RNA, protein (Prot.), and enzymatic activity (Enz.). (B) Aliphatic glucosinolates with different chain-lengths; C3, C4, and LC. Col-0 WT (black), Col-0 AOP3 FL (light gray), Col-0 AOP3 NF (medium gray), or Col-0 AOP3 UT (dark gray). (C) Gie-0 WT (black), Gie-0 AOP3 FL (light gray), Gie-0 AOP3 NF (medium gray), or Gie-0 AOP3 UT (dark gray). (D) 3mtp, 3msp, and 3ohp levels in Gie-0 WT (black), Gie-0 AOP3 FL (light gray), Gie-0 AOP3 NF (medium gray), or Gie-0 AOP3 UT (dark gray). Data represent means and standard error for lines carrying the same construct. Differences were tested for significance by ANOVA, * indicates P < 0.05, for additional information see methods and Supplementary Data. FW, fresh weight.
Mentions: To focus on the effects of AOP3 and simultaneously test if its regulatory function solely requires the enzymatic activity, we introduced three different versions of the gene into two accessions not expressing a functional AOP2 or AOP3 in leaves and varying in the accumulation of C3 and C4 glucosinolates. We chose Col-0 and Gie-0 based on their difference in their major SC glucosinolate, i.e., Col-0 accumulating C4 due to expression of a functional MAM1 and Gie-0 accumulating the AOP3 substrate, 3msp, due to MAM2 expression. We introduced different versions of AOP3 driven by a 35S promoter (Figure 3A). Accessions that express AOP3 in leaves use the AOP2 promoter that has previously been shown to be at least as strong as the 35S promoter (Wentzell et al., 2007; Chan et al., 2010; Kerwin et al., 2011). In addition to an enzymatically functional full-length genomic version of AOP3, we constructed a version with a mutation abolishing the active site of AOP3 (Hogan et al., 2000), which generates a non-active enzyme but still expresses a protein allowing us to test for the importance of the enzymatic activity. Generation of the third version included introduction of a stop codon as the third codon of the transcript and chancing the subsequent two potential start codons in frame, i.e., a construct that is unable to generate any AOP3 protein but only the transcript enabling us to test the function of the AOP3 RNA. Together, these three different versions of AOP3 allowed us to systematically test whether its regulatory capacity in any of the two accessions varying in GS-ELONG relies on its enzymatic activity, the protein, or the RNA (Figure 3A).

Bottom Line: In this study, we use transgenic plants in combination with natural variation to investigate the regulatory role of the AOP3 gene found in GS-AOP locus previously suggested to contribute to the regulation of glucosinolate defense compounds.Phenotypic analysis and QTL mapping in F2 populations with different AOP3 transgenes support that the enzymatic function and the AOP3 RNA both play a significant role in controlling glucosinolate accumulation.Furthermore, we find different loci interacting with either the enzymatic activity or the RNA of AOP3 and thereby extend the regulatory network controlling glucosinolate accumulation.

View Article: PubMed Central - PubMed

Affiliation: DNRF Center DynaMo, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen Frederiksberg, Denmark ; Copenhagen Plant Science Centre, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen Frederiksberg, Denmark.

ABSTRACT
Quantitative trait loci (QTL) mapping studies enable identification of loci that are part of regulatory networks controlling various phenotypes. Detailed investigations of genes within these loci are required to ultimately understand the function of individual genes and how they interact with other players in the network. In this study, we use transgenic plants in combination with natural variation to investigate the regulatory role of the AOP3 gene found in GS-AOP locus previously suggested to contribute to the regulation of glucosinolate defense compounds. Phenotypic analysis and QTL mapping in F2 populations with different AOP3 transgenes support that the enzymatic function and the AOP3 RNA both play a significant role in controlling glucosinolate accumulation. Furthermore, we find different loci interacting with either the enzymatic activity or the RNA of AOP3 and thereby extend the regulatory network controlling glucosinolate accumulation.

No MeSH data available.