PtomtAPX is an autonomous lignification peroxidase during the earliest stage of secondary wall formation in Populus tomentosa Carr

Table 1. Specific activity of PtomtAPX -Pr toward monolignol substratesTable 2. Lignin profiles of the stems of 3-month-old WT and transgenic P. tomentosa.the significance (*P < 0.05, **P < 0.01, one-sided Student’s t-test)Figure 1. Western blotting. Proteins were extracted from 2-month-old seedings, positive control protein expressed by E.coli, total proteins extracted from whole plant, mitochondrial proteins from leaves of the plant, and cell wall proteins from stem xylem. Anti-PtomtAPX antibody, anti-Tom20 antibody, anti-CESA8 antibody, anti-CAT1 antibody or anti-PtosAPX antibody was used as primary antibody, respectively.  Protein stained with Ponceau S served as the loading control. CK, positive control; T, total protein; M, mitochondrial proteins; CWP, xylem cell wall proteins.Figure 2.Western blot analysis of mitochondrial protein and cell wall protein extracts from suspended P. tomentosa cells. PtomtAPX relocated from mitochondria to cell wall along with 100mM MV treatment. Anti-PtomtAPX antibody, anti-Tom20 antibody or anti-CESA8 antibody was used as the primary antibody, respectively, for detection of each protein. Proteins stained with Ponceau S served as the loading control. CK, positive control (PtomtAPX expressed in E. coli); M, mitochondrial proteins; CWP, cell wall proteins.Figure 4. qRT-PCR for PtomtAPX expression in different transgenic lines. WT, wild type P. tomentosa; OX-1, OX-2, OX-3, OX-4, OX-5, OX-6, OX-7 and OX-8, overexpression PtomtAPX transgenic lines; anti-3, anti-9, anti-14 and anti-24, antisense PtomtAPX transgenic lines. Data are mean?±?SD of 3 independent plants from each line (n?=?3 biological replicates); The asterisks indicate the significance (*P < 0.05, **P < 0.01, one-sided Student’s t-test).Plant heights of the WT, anti-24, and OX-3 lines during the three developmental stages. Data are mean?±?SD of 6 independent plants from each line (n?=?6 biological replicates); The asterisks indicate the significance (**P < 0.01, one-sided Student’s t-test).Xylem thickness of the fourth internode from the base upward in the WT, anti-24, and OX-3 lines during the three developmental stages. Data are mean?±?SD of 3 independent plants from each line (n?=?3 biological replicates); The asterisks indicate the significance (**P < 0.01, one-sided Student’s t-test).Figure 5.Fiber cell wall thickness of different internodes from the base upward in the WT, anti-24, and OX-3 lines during the three developmental stages. Data are mean?±?SD of 60 independent cells from each line (n?=?60 biological replicates). The asterisks indicate the significance (*P < 0.05, **P < 0.01, one-sided Student’s t-test). Raman images of lignin distribution within transverse stem sections of wild-type and transgenic P. tomentosa at 2 months (a–c) and 3 months (d–f) of age. Bars = 2 μm. (a, d) Wild type, (b, e) antisense, and (c, f) overexpression plants. All the microcopy observations were validated in at least three independent experiments.Figure 6.The relative expression levels of PtomtAPX in different developmental stages. Data are mean?±?SD of 3 independent plants from each developmental stage (n?=?3 biological replicates); The asterisks indicate the significance (**P < 0.01,  one-sided Student’s t-test). Immunoblot detection of PtomtAPX protein in different developmental stages. Expressed PtomtAPX from E. coli was as positive control. CK, positive control; WT-1M, WT-2M and WT-3M is the stage of 1-month-old, 2-month-old and 3-month-old, respectively. Western blots were validated in at least three independent experiments.