identifier	taxonID	type	CVterm	format	language	title	description	additionalInformationURL	UsageTerms	rights	Owner	contributor	creator	bibliographicCitation
3018404A9E07FFBB806DFF6240B8E832.text	3018404A9E07FFBB806DFF6240B8E832.taxon	http://purl.org/dc/dcmitype/Text	http://rs.tdwg.org/ontology/voc/SPMInfoItems#GeneralDescription	text/html	en	Digitalis lanata L.	<div><p>2.4. BAHD-type malonyltransferase genes from Digitalis lanata</p><p>Focusing once again on the role of 21- O -malonylation in cardenolide formation in Digitalis, we performed a function-based search for BAHD-type malonyltransferases in the transcriptome database of D. purpurea (https://medicinalplantgenomics.msu.edu). This search resulted in 9 hits. The respective sequences were annotated as either putative malonyl-CoA:anthocyanin 5- O -glucoside-6 ′′ - O -malonyltransferases or putative quercetin 3- O -glucoside 6 ′′ - O -malonyltransferases. The sequences were checked for completeness by protein BLASTs using the NCBI databases (Altschul et al., 1990). Finally, two promising candidates were identified, both annotated as quercetin 3- O -glucoside-6 ′′ - O -malonyltransferases, encoding for proteins with molecular masses of 47 kDa and 50 kDa, respectively.</p><p>The first sequence isolated from D. lanata, termed Dlmat1 (GenBank MT992078), contained 1374 nucleotides coding for 457 amino acids and had a molecular mass of 51.0 kDa. Dlmat1 was 94% identical to a sequence in the D. purpurea genome annotated as quercetin 3- O -glucoside-6 ′′ - O -malonyltransferase. BLAST searches also revealed high sequence identities with BAHD-type malonyltransferases from Dorcoceras hygrometricum Bunge and Sesamum indicum L. It was confirmed that the four motifs conserved among all members of the BAHD enzyme superfamily (D’ Auria, 2006; Tuominen et al., 2011) are also present in Dl MaT1. The clade-Ia-specific motif LTFFD (Tuominen et al., 2011) as well as the anthocyanin metabolism related motif NYFGNC (Yu et al., 2009) were also identified. Based on this isolated sequence we were able to isolate three further genes coding for putative BAHD-type malonyltransferases (Dlmat2, GenBank MW013542; Dlmat3, GenBank MW013543; Dlmat4, GenBank MW013544), sharing nucleotide sequence identities ranging from 49.6% to 92.1% and amino acid sequence identities from 32.1% to 93.4% with each other and Atpmat1. The protein sequences were further analyzed using SignalP (Nielsen, 2017) and DeepLoc-1.0 (Almagro Armenteros et al., 2017). All of them were predicted to be ‘soluble’ proteins as already described for several BAHD-type enzymes (D’ Auria 2006; Bontpart et al., 2015).</p></div>	https://treatment.plazi.org/id/3018404A9E07FFBB806DFF6240B8E832	Public Domain	No known copyright restrictions apply. See Agosti, D., Egloff, W., 2009. Taxonomic information exchange and copyright: the Plazi approach. BMC Research Notes 2009, 2:53 for further explanation.		Plazi	Tropper, Marina;H, Stephanie;ohn;Wolf, Laura-Sophie;Fritsch, Julia;Kastner-Detter, Nina;Rieck, Christoph;Munkert, Jennifer;Meitinger, Nadine;Lanig, Harald;Kreis, Wolfgang	Tropper, Marina, H, Stephanie, ohn, Wolf, Laura-Sophie, Fritsch, Julia, Kastner-Detter, Nina, Rieck, Christoph, Munkert, Jennifer, Meitinger, Nadine, Lanig, Harald, Kreis, Wolfgang (2021): 21 - Hydroxypregnane 21 - O-malonylation, a crucial step in cardenolide biosynthesis, can be achieved by substrate-promiscuous BAHD-type phenolic glucoside malonyltransferases from Arabidopsis thaliana and homolog proteins from Digitalis lanata. Phytochemistry (112710) 187: 1-10, DOI: 10.1016/j.phytochem.2021.112710, URL: http://dx.doi.org/10.1016/j.phytochem.2021.112710
3018404A9E05FFB9806DFA264710EFF1.text	3018404A9E05FFB9806DFA264710EFF1.taxon	http://purl.org/dc/dcmitype/Text	http://rs.tdwg.org/ontology/voc/SPMInfoItems#GeneralDescription	text/html	en	Digitalis lanata subsp. leaves L.	<div><p>4.6. Protein purification from fresh young Digitalis lanata leaves</p><p>Crude extracts were prepared from fresh young leaves of D. lanata homogenized in an extraction buffer of 50 mM sodium phosphate, 30 mM β- mercaptoethanol, 20% polyvinylpolypyrrolidone (w/w), pH 7.5 at a ratio of 1:4 (w/V). The extract was stirred for 30 min and subsequently filtered through 4 layers of Miracloth (Merck KGaA, Darmstadt, Germany) before centrifugation at 48,000× g for 20 min.</p><p>Ammonium sulfate was added to the crude extracts and stirred for 30 min to obtain precipitates of 0–15%, 15–40%, 40–55% and 55–100% salt saturation. After centrifugation at 48,000× g for 20 min, the pellets were dissolved in phosphate buffer (pH 7.5, 50 mM, 30 mM β- mercaptoethanol).</p><p>The 40–55% and 55–100% precipitates were applied to hydrophobic interaction chromatography on Phenylsepharose FF 16/10 (low sub) (GE Healthcare Bio-Sciences AB, Sweden) equilibrated with 4 column volumes of buffer A (50 mM sodium phosphate, 30 mM β- mercaptoethanol, 1 M (NH 4) 2 SO 4, pH 7.5). Fractions of 10 ml were collected at a flow rate of 2.0 ml min 1. Unbound proteins were flushed with 7 column volumes of buffer A and bound proteins were eluted by a decreasing salinity gradient of 0–100% of buffer B (50 mM sodium phosphate, 30 mM β- mercaptoethanol) followed by 7 column volumes of 100% buffer B.</p><p>Fractions were tested for enzyme activity and the most active fractions were pooled and applied consecutively to ion exchange chromatography on Resource Q 1, ml (GE Healthcare Bio-Sciences AB, Sweden) at pH 8.0 and pH 6.5 and to Resource S, 1 ml (GE Healthcare Bio-Sciences AB, Sweden) at pH 5.0 for purification of the 55–100% precipitate. The precipitate containing 40–55% salt saturation was applied to Resource Q 1 ml (GE Healthcare Bio-Sciences AB, Sweden) at pH 8.0 and pH 8.5. The buffer of the protein solutions was exchanged before each chromatographic step using Amicon Ultra Centrifugal filter devices 1000 MWCO 20 ml (Millipore Carrightwohill, Ireland). The columns were equilibrated with 4 column volumes of appropriate buffer A (Table S1). The flow rate was 4.0 ml min 1 and eluates were collected in fractions of 0.5 ml. The column was washed with 10 column volumes of buffer A, and bound proteins were eluted by a gradient of 0–50% 1 M NaCl over 15 column volumes followed by 8 column volumes of 100% 1 M NaCl.Prior to SEC on Superdex 75 10/300_GL (GE Healthcare Bio-Sciences AB, Sweden), the protein solution was concentrated to a sample volume of 200 μl using Amicon Ultra Centrifugal filter devices 1000 MWCO 20 ml (Millipore Carrightwohill, Ireland). The column was equilibrated with 4 column volumes of sodium phosphate buffer (50 mM, 30 mM β- mercaptoethanol, 0.15 M NaCl, pH 7.5). The flow rate was set to 0.4 ml min 1 and fractions of 250 μl were collected. Proteins were eluted isocratically with 1.5 column volumes of buffer.</p></div>	https://treatment.plazi.org/id/3018404A9E05FFB9806DFA264710EFF1	Public Domain	No known copyright restrictions apply. See Agosti, D., Egloff, W., 2009. Taxonomic information exchange and copyright: the Plazi approach. BMC Research Notes 2009, 2:53 for further explanation.		Plazi	Tropper, Marina;H, Stephanie;ohn;Wolf, Laura-Sophie;Fritsch, Julia;Kastner-Detter, Nina;Rieck, Christoph;Munkert, Jennifer;Meitinger, Nadine;Lanig, Harald;Kreis, Wolfgang	Tropper, Marina, H, Stephanie, ohn, Wolf, Laura-Sophie, Fritsch, Julia, Kastner-Detter, Nina, Rieck, Christoph, Munkert, Jennifer, Meitinger, Nadine, Lanig, Harald, Kreis, Wolfgang (2021): 21 - Hydroxypregnane 21 - O-malonylation, a crucial step in cardenolide biosynthesis, can be achieved by substrate-promiscuous BAHD-type phenolic glucoside malonyltransferases from Arabidopsis thaliana and homolog proteins from Digitalis lanata. Phytochemistry (112710) 187: 1-10, DOI: 10.1016/j.phytochem.2021.112710, URL: http://dx.doi.org/10.1016/j.phytochem.2021.112710
