Comprehensive analysis of NAC domain transcription factor gene family in Populus trichocarpa

Abstract

Background: NAC (NAM, ATAF1/2 and CUC2) domain proteins are plant-specific transcriptional factors known to play diverse roles in various plant developmental processes. NAC transcription factors comprise of a large gene family represented by more than 100 members in Arabidopsis, rice and soybean etc. Recently, a preliminary phylogenetic analysis was reported for NAC gene family from 11 plant species. However, no comprehensive study incorporating phylogeny, chromosomal location, gene structure, conserved motifs, and expression profiling analysis has been presented thus far for the model tree species Populus.

Results: In the present study, a comprehensive analysis of NAC gene family in Populus was performed. A total of 163 full-length NAC genes were identified in Populus, and they were phylogenetically clustered into 18 distinct subfamilies. The gene structure and motif compositions were considerably conserved among the subfamilies. The distributions of 120 Populus NAC genes were non-random across the 19 linkage groups (LGs), and 87 genes (73%) were preferentially retained duplicates that located in both duplicated regions. The majority of NACs showed specific temporal and spatial expression patterns based on EST frequency and microarray data analyses. However, the expression patterns of a majority of duplicate genes were partially redundant, suggesting the occurrence of subfunctionalization during subsequent evolutionary process. Furthermore, quantitative real-time RT-PCR (RT-qPCR) was performed to confirm the tissue-specific expression patterns of 25 NAC genes.

Conclusion: Based on the genomic organizations, we can conclude that segmental duplications contribute significantly to the expansion of Populus NAC gene family. The comprehensive expression profiles analysis provides first insights into the functional divergence among members in NAC gene family. In addition, the high divergence rate of expression patterns after segmental duplications indicates that NAC genes in Populus are likewise to have been retained by substantial subfunctionalization. Taken together, our results presented here would be helpful in laying the foundation for functional characterization of NAC gene family and further gaining an understanding of the structure-function relationship between these family members.

Figure 1

Joined phylogenetic tree of NAC domain-containing proteins from Populus, Arabidopsis and rice. The deduced full-length amino acid sequences of 163 Populus, 105 Arabidopsis and 140 rice NAC genes were aligned by Clustal X 1.83 and the phylogenetic tree was constructed using MEGA 4.0 by the Neighbor-Joining (NJ) method with 1,000 bootstrap replicates. Each NAC subfamily was indicated in a specific color. Members of NAC protein from Populus were denoted in red.

Figure 2

Chromosomal locations of Populus NAC genes. Only 120 NAC genes are mapped to the 19 Linkage Groups (LG), while the other 43 genes reside on unassembled scaffolds. The schematic diagram of genome-wide chromosome organization arisen from the salicoid genome duplication event in Populus was adapted from Tuskan et al., (2006) [5]. Segmental duplicated homeologous blocks are indicated with the same color. Only the duplicated regions containing NAC genes are connected with lines in shaded colors. Tandemly duplicated genes are indicated with red lines. Scale represents a 5 Mb chromosomal distance.

Figure 3

Phylogenetic relationships, gene structure and motif compositons of Populus NAC genes. A. Multiple alignments of 163 full-length amino acids of NAC genes from Populus were executed by Clustal X 1.83 and the phylogenetic tree was constructed using MEGA 4.0 by the Neighbor-Joining (NJ) method with 1,000 bootstrap replicates. The percentage bootstrap scores higher than 50% are indicated on the nodes. The ten major phylogenetic subfamilies designated as I to X are marked with different color backgrounds. B. Exon/intron structures of NAC genes from Populus. Exons and introns are represented by green boxes and black lines, respectively. The sizes of exons and introns can be estimated using the scale at bottom. C. Schematic representation of the conserved motifs in the NAC proteins from Populus elucidated by MEME. Each motif is represented by a number in the colored box. The black lines represent the non conserved sequences. Refer to Additional file 7 for the details of individual motif.

Figure 4

In silico EST analysis of Populus NAC genes. EST frequency of 74 Populus NAC genes was obtained by screening the EST datasets from various libraries across a set of 14 tissues. Expression of NAC genes was plotted as counts of corresponding ESTs for particular gene in the database.

Figure 5

EST profiles of Populus NAC genes in wood-forming tissues. EST distribution of 36 Populus NAC genes was obtained by searching the EST datasets originated from libraries of wood-forming tissues. Expression of NAC genes was plotted as counts of corresponding ESTs for particular gene in the database.

Figure 6

Expression profiles of Populus NAC genes in wood-forming tissues. Microarray data were obtained from PopGenIE ftp://aspnas.fysbot.umu.se/[84]. The expression data were gene-wise normalized and hierarchical clustered with average linkage. Each row corresponds to the relative expression levels normalized against the maximum value. Color scale at the top of each dendrogram represents relative expression levels: green represents low level and red indicates high level. A. Dynamic expression levels of 11 NAC genes in different states of cambiums. YR1 to YR9 represent the nine sampling time points from 20 April (before bud break) until 13 December (cambium dormant). B. Expression patterns of 19 NACs in active and dormant cambiums. C, D. Hierarchical clustering of expression profiles of 19 NAC genes in the cambial region. Phl, phloem; A, meristematic cells; B, early expansion; C, late expansion; D, secondary wall formation; E, late cell maturation. CS2A1, CS2A2, CS2B1, CS2B2 and CS2B4, phloem; CS2A3, CS2A4, CS2B6 and CS2B7, cambium region; CS2A5, CS2A7, CS2A8 and CS2B8, xylem. E. Expression pattern of three NAC genes in cambium regions. The tissues are the same as depicted in C and D. F. Expression profiles of 14 NACs in vascular tissues. Phl, phloem; C, cambium; EX, expanding xylem; MX0, initiation of secondary cell-wall deposition stage; MX1-3, maturing xylem. G. Deferential expressions of 21 NAC genes during the xylem differentiation. EX, early xylem; LX, late xylem.

Figure 7

Hierarchical clustering of expression profiles of Populus NAC gene across different tissues. The genome-wide microarray data generated by Wilkins and coworker were re-analyzes [61]. The expression data were gene-wise normalized and hierarchical clustered based on Pearson correlation. The genes marked in the same color indicate duplicated gene pairs. The relative expression level of particular gene in each row is normalized against the maximum value. Color scale at the top of each dendrogram represents log2 expression values, green represents low level and red indicates high level of transcript abundances. CL, continuous light-grown seedling; DL, etiolated dark-grown seedling transferred to light for 3h; DS, dark-grown seedlings; YL, young leaf; ML, mature leaf; R, root; DX, differentiating xylem; FC, female catkins; MC, male catkins.

Figure 8

Expression analysis of selected NAC genes using quantitative real-time RT-PCR (RT-qPCR). The relative mRNA abundance of 25 selected NAC genes was normalized with respect to reference gene UBQ10 in different tissues. The bars are standard deviations (SD) of three technical repeats. SA, shoot apices; L, leaf (4~6 internodes); Phl, phloem; DX, differentiating xylem; OR, old root; RT, root tip; C, cortex.