Fig. 3
PtoUBC34 is expressed ubiquitously and responds to stress factors. a. qRT-PCR analysis of PtoUBC34 expression in various poplar tissues, using Actin6 (Potri.001G309500), eIF-5A (Potri.018G107300), and UBQ (Potri.014G115100) genes as internal controls, according to Wang et al. [94]. The expression level of PtoUBC34 in leaves was set as 1. b. The expression levels of PtoUBC34 in mature leaves in response to heat shock treatment by qRT-PCR, using Actin6 (Potri.001G309500) and EF1-beta (Potri.009G018600) genes as internal controls, which were validated in an evaluation assay of eight reference genes during heat shock treatment in P. tomentosa (unpublished data). Plants were treated at 37 °C for 1 h (HS1), allowed to recover for 2 h at 24 °C (HSR), and then were subjected to 42 °C for 2.5 h (HS2). The fold-expression was normalized relative to the 24 °C control (CK). c. The expression levels of PtoUBC34 in mature leaves in response to 300 mM sodium chloride (NaCl) treatment were analyzed by qRT-PCR, using UBQ (Potri.014G115100) and TUB (Potri.003G126800) genes as internal controls, which were validated in an evaluation assay of eight reference genes during salt stress in P. tomentosa (unpublished data). The fold-expression was normalized relative to the non-NaCl-treatment control. Data are mean of three biological samples from three plants, respectively, with three technical replicates for each one. Error bar represents standard deviation of the three biological samples. Significance was tested with one-way ANOVA to evaluate the effect of heat shock and NaCl treatment on UBC34 expression. The different alphabets above the bar indicates statistically significant differences with p value < 0.05, while the same alphabet mean no significant differences. d. UBC34 from P. trichocarpa was expressed constitutively in distinct differentiation stages across wood formation. The expression data are derived from the ASPWOOD project (http://aspwood.popgenie.org/aspwood-v3.0/) [58]. Phloem, cambium, developing xylem, and mature xylem were collected from stems through longitudinal continuous sections using a cryo-microtome. The tissue type was characterized by examining the images of cross-sections during sampling. All of the samples were collected during the current growth year. Poplar CesA8-B, specifically expressed during SCW thickening, was used as a reference