Unrooted phylogenetic tree of the GTases based on amino acid sequence similarity. GenBank accession numbers and sources for the respective protein sequences are: CsGT45 (FJ194947) from Crocus sativus; flavonoid 3-O-glucosyltransferases from Arabidopsis thaliana (AAD17392), AtUGT73B4 and (At5G17050), At GT; Zea mays (X13502), ZmF3GT; from Vitis vinifera (AAB81682), VvF3GT; from Fragaria × ananassa (BAA12737), GtF3GT; from Dianthus caryophyllus (BAD52005), DicGT3 and (BAD52003), DicGT1; At3RhaT, flavonol 3-O-rhamnosyltransferase from Arabidopsis thaliana (At1g30530); At7RhaT, flavonol 7-O-rhamnosyltransferase from Arabidopsis thaliana (NP_563756); flavonoid 7-O-glucosyltransferases from Scutellaria baicalensis (BAA83484), ScbF7GT; Pyrus communis (AAY27090), PcF7GT; from Nicotiana tabacum (BAB88935), NtF7GT from Arabidopsis thaliana (AAR01231), AtF7GT; NtSalGT, salicylic acid glucosyltransferase from Nicotiana tabacum (AAF61647); AtUGT73B3, pathogen-responsive glucosyltransferase from Arabidopsis thaliana (AAD17393); DicGT4, chalcononaringenin 2′-O-glucosyltransferase (BAD52006) from Dianthus caryophyllus; DbBet5GT, betanidin-5-O-glucosyltransferase from Dorotheanthus bellidiformis (CAB56231); UGT74F1, UGT74F2, and UGT73B1, flavonoid glucosyltransferases from Arabidopsis thaliana (AAB64022.1), (AAB64024.1) and (At4g34138); Letwi1, wound-inducible glucosyltransferase from Solanum lycopersicum (CAA59450); NtIS5a, immediate-early salicylate-induced glucosyltransferase from Nicotiana tabacum (AAB36653); FaGT7, multi-substrate flavonol-O-glucosyltransferase (ABB92749); AtF3GTb, putative flavonol 3-O-glucosyltransferases from Arabidopsis thaliana (NP_180535.1), AtF3GTc and (NP_180534.1), from Petunia hybrida (AAD55985), PhF3GT; from Gentiana triflora (BAA12737), GtF3GT; from Dianthus caryophyllus (BAD52004), DicF3GT; DbBET6GT, betanidin 6-O-glucosyltransferase from Dorotheanthus bellidiformis (AAL57240); UGT71B6, glucosyltransferase from Arabidopsis thaliana (AB025634); FaGT3 and FaGT7, flavonol-O-glucosyltransferases from Fragaria × ananassa (AAU09444) and (ABB92748); NtGT1a and NtGT1b, broad substrate specificity glucosyltransferases from Nicotiana tabacum (BAB60720) and (BAB60721); AtA5GT, glucosyltransferase from Arabidopsis thaliana (AAM91686); anthocyanin 5-O-glucosyltransferases from Torenia hybrida (BAC54093), ThA5GT; from Verbena hybrida (BAA36423), VhA5GT; from Perilla frutescens (BAA36421), PfA5GT; from Petunia hybrida (BAA89009), PhA5Gt; UGT71F1, regioselective 3,7 flavonoid glucosyltransferase from Beta vulgaris (AY526081); UGT73A4, regioselective 4′,7 flavonoid glucosyltransferases from Beta vulgaris (AY526080); UGT71G1, triterpene glucosyltransferase from Medicago truncatula (AAW56092). The horizontal scale shows the number of differences per 100 residues derived from the ClustalW alignment.
Because C. sativus is a triploid, we employed in silico screening of a large stigma cDNA EST database http://www.saffrongenes.org/ as an effective method for identification of potential CsGT45 alleles. We identified three EST clones with 98% identity in 611 bp (EX147039.1), 98% identity in 264 bp (EX144545.1) and 84% identity in 426 bp (EX148389.1). The first two ESTs correspond to CsGT45, and the third could correspond to a CsGT45 allele.
The carboxyl terminal of the protein contained the plant secondary product glycosyltransferase (PSPG) box signature motif. Analysis of CsGT45 sequence for N-terminal targeting signal or C- terminal membrane anchor signal using SignalP and TMpred web-based programmes predicted CsGT45 to be non-secretory with an absence of predicted signal peptides or transmembrane signals .
Findings presented here suggest that CsGT45 is an active enzyme that plays a role in the formation of flavonoid glucosides in C. sativus.
Profile of flavonols accumulation during stigma tissue development
Stigmas at the time of anthesis were ground in liquid nitrogen. The fine powder obtained was extracted with methanol (500 μl), centrifuged and the supernatant analysed by LC-ESI-MS using a C18 Ascentis column 15 × 2.1, particle size 3 um (Supelco, Sigma-Aldrich) and following the method previously described . For flavonoid analysis from stigmas at different developmental stages (yellow to +3da), three stigmas of each stage were collected and freeze-dried. The powder obtained from one stigma was extracted with 500 μl of methanol containing 0.2 mg/ml 4-methylumbelliferyl β-D-glucuronide as an internal standard. The samples were centrifuged (5,000 g, 10 min), and the supernatant evaporated and treated as described . Samples in triplicate were analysed by HPLC as described  using a C18 Ascentis, 25 × 4.6, particle size 5 um column (Supelco, Sigma-Aldrich).
Cheng H, Qin L, Lee S, Fu X, Richards DE, Cao D, et al. Gibberellin regulates Arabidopsis floral development via suppression of DELLA protein function. Development. 2004;131(5):1055–64. https://doi.org/10.1242/dev.00992.
BioProject PRJNA285604. Sequencing and analysis of Lupinus luteus transcriptome. https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA285604. Accessed 28 Oct 2020.
Katarzyna Marciniak & Krzysztof Przedniczek
In yellow lupine, GA3 likely participates in the regulation of LlGAMYB by controlling the LlMIR159 expression level. The LlGAMYB and LlMIR159 transcriptional activity in four selected phases of LAD, as well as after GA3 and PAC application, were examined (Fig. 6). The LlGAMYB expression was almost identical with or without GA3 treatment, but the lack of GA3 due to PAC application resulted in increased LlGAMYB transcripts, especially in the first and second LAD stages (Fig. 6A). These results suggest that GA3 possibly indirectly regulates LlGAMYB expression. Therefore, we examined the LlMIR159 expression profile (Fig. 6B). GA3 treatment increased LlMIR159 mRNA levels, and importantly, PAC application significantly decreased the expression of LlMIR159. By analysing the natural conditions without the application of any compounds, it can be concluded that LlGAMYB expression is high in the first two stages of LAD and then decreases. This negatively correlates with the transcriptional activity of LlMIR159, which was significantly increased in the second phase of LAD. This is probably the cause of the reduced mRNA content of LlGAMYB in the third and fourth LAD stages. The results suggest that LlGAMYB is coexpressed with LlMIR159 in yellow lupine anthers. It also follows that the LlGAMYB transcript level is potentially regulated by miR159 in the anthers of yellow lupine.
Sanders PM, Bui AQ, Le BH, Goldberg RB. Differentiation and degeneration of cells that play a major role in tobacco anther dehiscence. Sex Plant Reprod. 2005;17(5):219–41. https://doi.org/10.1007/s00497-004-0231-y.