Genome-wide survey of switchgrass NACs family provides new insights into motif and structure arrangements and reveals stress-related and tissue-specific NACs

Abstract

NAC proteins comprise of a plant-specific transcription factor (TF) family and play important roles in plant development and stress responses. Switchgrass (Panicum virgatum) is the prime candidate and model bioenergy grass across the world. Excavating agronomically valuable genes is important for switchgrass molecular breeding. In this study, a total of 251 switchgrass NAC (PvNACs) family genes clustered into 19 subgroups were analyzed, and those potentially involved in stress response or tissue-specific expression patterns were pinpointed. Specifically, 27 PvNACs were considered as abiotic stress-related including four membrane-associated ones. Among 40 tissue-specific PvNACs expression patterns eight factors were identified that might be relevant for lignin biosynthesis and/or secondary cell wall formation. Conserved functional domains and motifs were also identified among the PvNACs and potential association between these motifs and their predicted functions were proposed, that might encourage experimental studies to use PvNACs as possible targets to improve biomass production and abiotic stress tolerance.

Figure 1

Phylogenetic relationships show the PvNACs that are orthologous to annotated AB- or stress- responsive NAC genes in rice and Arabidopsis. (a) PvNAC249 and PvNAC216 were orthologous to Os11g03310.1. (b) PvNAC41 was orthologous to Os08g339910.1. (c) PvNAC208 and PvNAC185 were orthologous to Os11g03300.1 and Os12g03040.1. (d) PvNAC219 was orthologous to Os07g48450.1. (e) PvNAC141 was orthologous to Os03g60080.1. (f) PvNAC239 and PvNAC174 were orthologous to ANAC002, ANAC032, Os05g34830.1, and Os01g66120.1.

Figure 2

Phylogenetic relationship of NAC MTFs from switchgrass, rice, and Arabidopsis. Multiple sequences alignment of NAC MTFs was conducted using ClustalX, and MEGA5.0 was used to construct phylogenetic tree by using Neighbor-joining method with 1000 bootstrap replicates and p-distance method, and bootstrap values are shown next to the branch.

Figure 3

Chromosomal locations of 163 PvNACs. Duplications generated by allotetraploidy were connected by full lines, while tandem duplications were connected by thick red lines. The numbers listed along each chromosome were locations of PvNACs, and the smaller/larger number indicated the PvNACs were closer to start/end point of chromosome. The number below each chromosome was the whole length.

Figure 4

The special expression profiles for several tissues of PvNACs. (a–e) Special expression patterns of PvNACs in root, lignified, leave, flower, and seed tissues. AP13_ Seed0d, AP13_ Seed5d, AP13_ Seed10d, AP13_ Seed15d, AP13_ Seed20d, AP13_ Seed25d, AP13_ Seed30d represent whole flowers at anthesis stage, whole seeds 5 days post fertilization, whole seeds with visible caryopsis, whole seeds at the milk stage, whole seeds at the soft dough stage, whole seeds at the hard dough stage, whole seeds at the physiological maturity stage, respectively. Inflo-meristem: Inflorescence meristem (0.5–3.0 mm). Inflo-floret: Floret of inflorescence when glumes are 10–20 mm. Inflo-REL: Rachis and branch elongation of inflorescence (50–150 mm). Inflo-PEM: Panicle emergence of inflorescence (>200 mm). E4-LFB: Pooled leaf blade from plant. E4-LSH: Pooled leaf sheath. E4i3m: Middle 1/5 fragment of the 3rd internode. E4i3mVB: Vascular bundle isolated from 1/5 fragment of the 3rd internode. E4i4b: Bottom 1/5 fragment of the 4th internode. E4i4t: Top 1/5 fragment of the 4th internode. E4i4m: Middle 1/5 fragment of the 4th internode 4. E4-root: Whole root system. E4-crown: Whole crown. E4-node: Pooled nodes. The genes with red frames were chosen for qRT-PCR in further step.

Figure 5

The expression analysis of nine selected PvNACs relating to ABA and drought, salt, and cold stresses via qRT-PCR. Relative expression of these PvNACs were normalized in relation to reference gene UCE2 in different stresses. The bars represent error bar. The black * represents the expression for treatment group is more than twice as control group (0 d), while red * represents treatment group is below half of the control group (0 d).

Figure 6

The expression analysis of three selected PvNACs based on array data relating to ABA and drought, salt, cold, and heat stresses through qRT-PCR. Relative expression of these PvNACs were normalized in relation to reference gene UCE2 in different stresses. The bars represent error bar. The black * represents the expression for treatment group is more than twice as control group (0 d), while red * represents treatment group is below half of the control group (0 d).

Figure 7

The expression analysis of selected six tissue specific PvNACs via qRT-PCR. Relative expression of these PvNACs were normalized in relation to reference gene UCE2 in different tissues (Root, stem, leaf, flower, spikelet, seed). The bars represent error bar.

Figure 8

Schematic representation of the conserved motifs in N-terminal regions of PvNACs for displayed by MEME analysis. (a) Motifs in N-terminal regions. The colored boxes represent each types of motif, and black lines indicate the non-conserved sequences. (b) The logo of motifs 1–10 in N-terminal regions.