Genome-Wide Investigation of the NAC Gene Family and Its Potential Association with the Secondary Cell Wall in Moso Bamboo

Abstract

NAC (NAM, ATAF, and CUC) transcription factors (TFs) are implicated in the transcriptional regulation of diverse processes and have been characterized in a number of plant species. However, NAC TFs are still not well understood in bamboo, especially their potential association with the secondary cell wall (SCW). Here, 94 PeNACs were identified and characterized in moso bamboo (Phyllostachys edulis). Based on their gene structures and conserved motifs, the PeNACs were divided into 11 groups according to their homologs in Arabidopsis. PeNACs were expressed variously in different tissues of moso bamboo, suggesting their functional diversity. Fifteen PeNACs associated with the SCW were selected for co-expression analysis and validation. It was predicted that 396 genes were co-expressed with the 15 PeNACs, in which 16 and 55 genes were involved in the lignin catabolic process and cellulose biosynthetic process respectively. As the degree of lignification in the growing bamboo shoots increased, all 15 PeNACs were upregulated with a trend of rising first and then decreasing except PeNAC37, which increased continuously. These results indicated that these PeNACs might play important roles in SCW biosynthesis and lignification in bamboo shoots. Seven of 15 PeNACs had been found positively co-expressed with seven PeMYBs, and they had similar expression patterns with those of the PeMYBs in bamboo shoots. The targeted sites of miR164 were found in 16 PeNACs, of which three PeNACs associated with SCW were validated to have an opposite expression trend to that of miR164 in growing bamboo shoots. In addition, three PeNACs were selected and verified to have self-activation activities. These results provide comprehensive information of the NAC gene family in moso bamboo, which will be helpful for further functional studies of PeNACs to reveal the molecular regulatory mechanisms of bamboo wood property.

Keywords: NAC identification; expression analysis; moso bamboo; secondary cell wall.

Figure 1

Exon–intron structures of PeNACs in moso bamboo. The black boxes represented exons and spaces between the black boxes corresponded to introns.

Figure 2

Schematic representation of the conserved motifs in PeNACs. Motifs were identified using the MEME online tool. Each motif was indicated by different colored blocks (motif 1–motif 8). The position and length of each colored box represented the actual motif size.

Figure 3

Phylogenetic tree based on the proteins of moso bamboo and Arabidopsis. The tree was constructed using the neighbor-joining (N–J) method with 1000 bootstrap replicates as implemented in MEGA6.0. The names of clade were shown outside of the circle. Geometric figures of different colors and shapes were used to mark the NAC members from different species.

Figure 4

Expression patterns of PeNACs in different tissues/organs of moso bamboo based on the transcriptome data. Heatmap represented for the expression of PeNACs in leaves, early panicles, advanced panicles, rhizomes, roots, 20 cm shoots, and 50 cm shoots. Color scale represented log₂ expression values, with the color from blue to red indicating low to high expression abundance.

Figure 5

Gene Ontology (GO) enrichment analysis of the genes co-expressed with 15 PeNACs associated with SCW. (A): GO term of the co-expression genes with PeNACs of Clade1; (B): GO term of the co-expression genes with PeNACs of Clade1. The x-axis was log₁₀ (p-value) and y-axis was the GO term.

Figure 6

Positive co-expression network of PeNACs and PeMYBs. The green and orange circles represent PeNACs and PeMYBs respectively.

Figure 7

Diagram of the secondary wall NAC binding element (SNBE) sequences in the 2.0-kb promoters of PeMYBs. The consensus nucleotides in the SNBEs were shaded and the consensus SNBE sequence was shown at the bottom.

Figure 8

Expression analysis of 15 PeNACs in different height shoots using qRT-PCR. PeNTB was used as the reference gene. Average and error bars represented standard deviation of three biological replicates. Asterisks indicated significant difference compared to the transcription level of control groups (* p < 0.05, and ** p < 0.01). 1: 1.0 m shoots; 2: 2.0 m shoots; 3: 4.0 m shoots; 4: 6.0 m shoots; and 5: 8.0 m shoots.

Figure 9

Expression analysis of 7 PeMYBs in different height shoots using qRT-PCR. PeNTB was used as the reference gene. Average and error bars represented standard deviation of three biological replicates. Asterisks indicated significant difference compared to the transcription level of control groups (* p <0.05, and ** p <0.01). 1: 1.0 m shoots; 2: 2.0 m shoots; 3: 4.0 m shoots; 4: 6.0 m shoots; and 5: 8.0 m shoots.

Figure 10

Transverse sections of vascular bundle in bamboo shoots with different heights. (A): 1.0 m shoots; (B): 2. 0 m shoots; (C): 4.0 m shoots; (D): 6.0 m shoots; and (E): 8.0 m. Scale bar: 100 μm.

Figure 11

Assay of PeNACs transcriptional activation activity in yeast. The positive constructs, negative constructs, and PeNACs fused constructs were transformed into AH109 strains respectively, and successively incubated in SD/-Trp media and SD/-Trp/-His/-Ade supplemented with X-α-Gal.