Transcriptome analysis of NAC family in Populus simonii×P. nigra
The mRNA abundance of each gene in each sample was profiled as fragment per kilo bases per million reads (FPKM). The FPKM information of all 289 NAC members in the roots, stems and leaves of Populus simonii×P. nigra was retrieved from RNA-seq data (Supplemental Excel 1). There were a total of 231 NAC genes detected by RNA-Seq. Based on FPKM ≥ 4 in at least one tissue, 126 out of the 231 genes were screened to count the expression of NAC genes. Out of the 126 genes, there were 115, 123, 118 differentially expressed genes in the comparison pairs between leaves and stems, roots and stems, leaves and roots, respectively. As many as 110 NAC genes were differentially expressed in the three tissues. The heatmap of the 110 genes showed the expression pattern in the leaves and stems can be clustered together, which indicated the genes have similar expression pattern in the two tissues (Fig. 1).
Phylogenetic analysis of NAC15
The 1,257 bp coding sequence of NAC15 gene from Populus simonii × P. nigra (Potri.001G448400.1) contains an ORF encoding 418 amino acids. Amino acids sequence blasts indicated that NAC15 from poplar shared 86%, 77%, 75%, 77%, 72%, 66%, 66%, 66%, 65%, 65% sequence similarity with Salix purpurea (SapurV1A.0131s0060.3), Ricinus communis (30068.m002591), Manihot esculenta (Manes.02G001600.1), Theobroma cacao (Thecc1EG015621t1), Gossypium raimondii (Gorai.004G129200.1), Prunus persica (Prupe.5G131900.1), Malus domestica (MDP0000762302), Fragaria vesca (mrna01881.1-v1.0-hybrid), Eucalyptus grandis (Eucgr.E01053.1), and Vitis vinifera (GSVIVT01019670001), respectively. Multiple amino acids alignment showed that above proteins shared a highly conserved domain of 160 amino acids, namely NAC domains, which can be divided into A-E sub-domains (Fig. 2A). The phylogenetic tree with the top 10 identical protein sequences indicated that NAC15 from poplar had relatively high homology with the proteins from willow, cassava and castor-oil plant, while had relatively low homology with those from wild-strawberry, peach tree and apple tree (Fig. 2B).
Localization of NAC15 protein
As shown in Fig. 3, the fluorescence signal of NAC15-GFP (green fluorescent protein) fusion was detected in the nucleus while the control was fully expressed in the cell, which revealed NAC15 protein was localized to the nucleus. To confirm the result, the NAC15-GFP-transfected onion cells were stained with DAPI and observed under immunofluorescence microscope. The combined fluorescence signal of DAPI and GFP was consistently in the nuclei (Supplemental Figure 1), which exactly proved nuclear localization of NAC15.
Expression pattern analysis of NAC15 gene
NAC15 gene was differentially expressed in the leaves, stems and roots, and its mRNA abundance was the highest in the stems, followed by leaves and roots based on RNA-Seq. The relative expression level of NAC15 gene in different tissues at different developmental stage was quantified by RT-qPCR. The results indicated the expression pattern of NAC15 gene was hugely diverse at different tissues and displayed a rapid decrease from xylems and leaves to cambiums and roots. The highest expression level appeared in the secondary xylems and the lowest was in the roots, which was in accordance with RNA-Seq results in trend. And the highest expression level was about 173 times higher than the lowest (Fig. 4). The relative expression level of NAC15 gene was also significantly different during developing stages. For example, it was higher in the secondary xylems than that in the primary and crude xylems of poplar (Fig. 4). In conclusion, the expression of NAC15 gene had spatio-temporal specificity, and its expression pattern may play a pivotal role in the temporal and spatial regulation of wood-associated genes in the process of wood formation.
Generation of transgenic tobacco overexpressing NAC15 gene
We obtained 18 transgenic tobacco lines including 12 TLs and 6 CLs. The transgenic tobacco was confirmed by PCR and RT-PCR. As shown in Fig. 5, the expected bands were amplified in the TLs, but not in the CLs and wild type (WT) plants, which proved successful integration of NAC15 gene in tobacco.
Gene expression analysis of lignin- and cellulose-related genes by RT-qPCR
A few lignin- and cellulose-related genes, such as CesA (Cellulose synthase), C4H (Cinnamate 4-hydroxylase), CAD (Cinnamyl alcohol dehydrogenase), PAL (Phe ammonia-lyase), CL (Coumarate: coenzyme A ligase), CCOMT (Caffeoyl-CoA O-methyltransferase) etc. (Supplemental Table 1) were required for secondary wall biosynthesis in plants [23, 24]. Taken CesA as an example, PtoCesA3 was highly expressed during primary cell wall formation and was proved to be associated with growth and wood properties of Populus tomentosa [25]. PAL1 and PAL2 were identified to have relationship with tissue-specific lignin synthesis [26]. RT-qPCR was conducted to detect the relative expression level of lignin- and cellulose-related genes in the transgenic plants. The results indicated the relative expression level of CesA, CAD, PAL, 4CL, and C4H etc. in the TLs was significantly higher than that in the CLs (Fig. 6).
Histological analysis of transgenic tobacco overexpressing NAC15 gene
There are three types of polymers (hemicelluloses, cellulose and lignin) in the secondary cell wall of plants [23, 27]. Cellulose is the most abundant polysaccharide in plants and its microfibrils can form a main load-bearing network. Hemicellulose mainly consists of xylans, glucans, and mannans. Lignin affects ‘waterproofing’ capacity, mechanical strength, rigidity and environmental protection of plants [23, 27]. The relative content of hemicellulose, cellulose and lignin was determined to compare wood properties between TLs and CLs. The results showed that the relative content of hemicellulose, cellulose and lignin in the TLs was 1.09-1.38, 1.29-1.40, 1.31-1.58 times higher than that in the CLs, respectively (Fig. 7).
Phloroglucinol-HCl staining
Phloroglucinol-HCl staining method is commonly used for the characterization of plant lignifications [28]. Therefore, phloroglucinol-HCl staining was conducted to compare wood properties between TLs and CLs in the study. The result showed there was darker staining in the vascular bundles of TLs, compared to the CLs (Fig. 8). It showed three levels of stem lignifications based on the staining color in the TL1, TL3 and TL5, which was in accordance with the relative content of lignin and relative expression level of lignin-related genes.