Lamium galeobdolon, (L.) L.

Hannemann, Laura, Lucaciu, Calin Rares, Sharma, Sapna, Rattei, Thomas, Mayer, Klaus F. X., Gierl, Alfons & Frey, Monika, 2018, A promiscuous beta-glucosidase is involved in benzoxazinoid deglycosylation in Lamium galeobdolon, Phytochemistry 156, pp. 224-233 : 225

2.2. De novo assembly identifies four of L. galeobdolon BGlu genes

Leaf, flower and adventitious root tissue were used for isolation of total RNA. Equal amounts of the RNA were combined for generation of a normalised random primed cDNA library. Contigs were assembled from 18 to 20 million paired-end reads. To estimate the quality of the data we screened for the presence of the previously isolated L. galeobdolon genes LgIgl1 and LgIgl2 ( Schullehner et al., 2008) and found the sequences fully and without mistakes represented in the sequence library. Based on published BGLU sequences from monocots and dicots ( Dick et al., 2012; Rouyi et al., 2014) the data were screened for putative BGlu genes. Four full size-genes termed LgGlu1 to LgGlu4 (Supplementary data Table S2) were identified that have in BLAST analysis E -values below 1e-100 for query BGLU amino acid sequences (Supplementary data Table S3). Lg GLU3 differs from the other enzymes by the mannosidase signature around the catalytic glutamate residue ( Czjzek et al., 2000; Xu et al., 2004). In addition, Lg GLU3 has two in frame potential start codons and can possibly encode two proteins that differ by 18 amino acids at the amino-terminus. For the longer version localisation in mitochondria or plastids is predicted by iPSORT and WoLF PSORT ( Bannai et al., 2002; Horton et al., 2007). The shorter protein displays a putative signal peptide for channelling into the secretion pathway or the vacuole. The secretion pathway is clearly predicted by the programs SignalP-4.1 and iPSORT for the other three BGLU enzymes. WoLF PSORT indicates location at the endoplasmatic reticulum for Lg GLU4.

Expression of L. galeobdolon BGlu genes was analysed by quantitative RT-PCR ( Fig. 1 , see Experimental). Transcripts of all four genes were detectable in leaf, flower and root ( Fig. 1A ). Compared to the housekeeping gene GAP C the steady state expression levels in leaves were moderate comprising 2–10% thereof. An exception is LgGlu2, for which extreme variation of transcript levels was displayed, reaching amounts equal to GAP C in single biological replicates. What is causing this variability in leaves is unknown. For other organs, the LgGlu2 values are relatively constant and the lowest expression level was determined for the root. LgGlu3 steady state transcript levels are higher for flower and root than for leaves. LgGlu1 and LgGlu4 had similar low mRNA levels in all organs. To investigate a potential impact of physical damage on gene expression we crushed leaves (see Experimental) and isolated RNA at different time points ( Fig. 1B ). Only for LgGlu4 an impact of wounding on mRNA levels was detected. The RNA amount increased transiently, peaking at 6 h after damage reaching about 20- fold elevated amounts.

Figures

Fig. 1. Steady state RNA levels of LgGlus. (A) Expression in leaves (white column), flower (light grey column) and root (dark grey column). (B) Expression after wounding. Light grey columns, controls; dark grey columns, wounded leaves. All values are normalised to GAP C expression. The mean values of three biological replicates for flower and root, and nine replicates for leaves are displayed. The standard deviation is indicated. t1: 2 min, t2: 30 min, t3: 60 min, t4: 240 min, t5: 480 min. Significant differences with respect to tissue steady state levels are demonstrated for LgGlu2 (p <0.05). Expression in leaves is higher for LgGlu2 compared to the other genes (p <0.001 LgGlu1, LgGlu4, 0 <0.01 for LgGlu3). LgGlu3 has the highest mRNA concentration in the flower, the difference is significant (p <0.05) in comparison with LgGlu1 and LgGlu4. Root transcription levels are highest for LgGlu3, the difference is significant at p <0.05 with respect to the other genes. Wounding does only cause significant differences in the case of LgGlu4. The increase of transcript level after wounding at the time point t4 and t5 is significant (p <0.001) compared to all other induced and control plants. Statistical analysis was by ANOVA tests with RSTudio. Pairwise t-tests were performed for statistical significant results.