Recombinant Citrus maxima Flavanone 7-O-glucoside 2''-O-beta-L-rhamnosyltransferase (C12RT1)
Product Specs
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Target Background
Involved in the biosynthesis of the bitter neohesperidosides in citrus. Exhibits strict specificity for UDP-rhamnose as the donor substrate.
KEGG: ag:AAL06646
Q&A
Basic Research Questions
What is the biochemical role of C12RT1 in citrus flavonoid biosynthesis?
C12RT1 catalyzes the rhamnosylation of flavanone 7-O-glucosides to form flavanone disaccharides, a critical step in citrus flavonoid diversification . This enzyme transfers a rhamnose moiety from UDP-rhamnose to the 2''-position of the glucose group attached to flavanones, producing nonbitter rutinosides (e.g., narirutin) or bitter neohesperidosides depending on substrate specificity . Methodologically, its activity is confirmed via in vitro assays using recombinant protein and HPLC-MS to analyze products .
How is C12RT1 activity experimentally detected and quantified?
Key protocols include:
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Recombinant protein expression: Heterologous expression in E. coli or yeast systems with affinity tag purification .
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Enzyme assays: Incubation of purified C12RT1 with substrates (e.g., naringenin-7-O-glucoside) and UDP-rhamnose, followed by HPLC-MS to detect rhamnosylated products .
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Kinetic analysis: Measurement of K<sub>m</sub> and k<sub>cat</sub> values using varying substrate concentrations under optimized pH (6.0–7.5) and temperature (30–37°C) .
How does C12RT1 function differ across Citrus species?
Functional divergence is observed in substrate specificity:
Advanced Research Questions
What experimental strategies resolve contradictions in C12RT1 substrate specificity reports?
Discrepancies arise from:
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Tissue-specific isoforms: Transient overexpression in pummelo leaves vs. in vitro assays may yield differing activities due to post-translational modifications .
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Allelic variations: Type A alleles of 1,6RhaT genes (e.g., CgUGT89AK1) show functional activity, while B/C types are nonfunctional . Validate via allele-specific CRISPR editing or chimeric protein studies.
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Co-factor requirements: NADPH enhances rhamnosylation in cell-free extracts, suggesting auxiliary metabolic interactions .
How can C12RT1 be engineered to modulate flavonoid profiles for metabolic studies?
Approaches include:
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Site-directed mutagenesis: Target residues in the PSPG motif (e.g., W352, Q354) to alter UDP-sugar binding specificity .
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Promoter-swapping: Use tissue-specific promoters (e.g., fruit peel-specific) in transgenic citrus to study spatial regulation of flavonoid biosynthesis .
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Multi-enzyme cascades: Co-express C12RT1 with upstream glucosyltransferases (e.g., CgUGT89AK1) in N. benthamiana for pathway reconstitution .
What role does C12RT1 play in citrus-pathogen interactions?
C12RT1-derived flavanones are implicated in Huanglongbing (HLB) resistance. HLB-infected pummelo shows upregulated CitUGT89AK1 (a glucosyltransferase upstream of C12RT1) and increased flavanone glycosides, suggesting a defense-oriented metabolic shift . Methodological validation involves:
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Pathogen challenge assays: Quantify C12RT1 expression (qRT-PCR) and flavonoid accumulation (LC-MS) post-Candidatus Liberibacter infection .
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RNAi silencing: Knock down C12RT1 in citrus to assess susceptibility phenotypes .
