Transcriptome-wide survey and expression analysis of stress-responsive NAC genes in Chrysanthemum lavandulifolium

汇报人：刘晨指导老师：寇晓虹

Contents

Introduction1

Materials and methods2

Results3

Discussion4

Introduction

• Plants respond to various environmental stress using three primary strategies.

• Under high salt conditions, a variety of genes are induced to express.

• Genes encoding transcription factors (TFs) are the most highly regulated,which include members of the WRKY,bZIP, MYB (Myeloblastosis), AP2/EREBP (Apetala-2/EREBP) and NAC (NAM, ATAF, and CUC) families . These genes often function together as a large gene family in plants, with differentmembers of the same gene family participating in differentstress responses.

Introduction

• There is relationship between structure and function of NAC transcription factors

• Many members of NAC gene family responded to adversities and hormonal treatments, and some showed tissue-specific expression patterns.

• Most of the stress-responsive NACs belong to the ATAF and AtNAC3 subfamilies.

• Ten candidate ClNAC genes in C.lavandulifolium responded to multiple-stress and hormoral treatments.

Materials and methodsseeds germination and plant growth

salinity treatment was applied by submergingthe roots of the plants with a 200 mM NaCl for 12 h, and the seco-nd and third pairs of fully expanded leaves below the terminal bud were collected.

RNA-Seq analysis

salinity treatmentchilling and heat tr-eatmentdrought stressABA and SA treat-ment

Cotyledons were collected after the seeds germinated; leaves, stems, and roots were collected from eight-true leaves stage seedlings. C.lav-andulifolium specimens with eight- true leaves were tran-sferred into a short-day art-ificial clima-te chamber for flowering, and then the flow-er buds, flower petals and seeds were collected, frozen in liquid nitrogen and prese-rved.

RT-PCR analysis

Collected the secondand third pairs of fu-lly expanded leaves below the terminalbud from each treat-ed samples.

RT-PCR adn qRT-PCR analysis

Materials and methods

• Amplifed the candidate genes(ClNAC2, -3, -4, -9, -11, -12, -17, -21, -39, -44) with genes engineering.

• The phylogenetic tree was constructed using MEGA4.0 software and the neighbor-joining method [37] with the following parameters: Poisson correction, pairwise deletion, and bootstrap (1000 replicates; random seed). Multiple sequence alignments were performed using the default parameters for ClustalW [38]. The 10 ClNAC genes were classified into the corresponding subfamilies according to Ooka et al. and Fang et al. . We used the TMpred server to predict of the membrane-bound ClNAC members encoded by the 10 candidate genes.

Full-length amplification of candidate genes and bioinformatics analysis

ResultsSalt-induced expression of transcription factors in the leaves of C. lavandulifolium

Fig. 1. Distribution of “molecular function” GO)terms of the C. lavandulifolium unigenes in leaves subject to salt between 0 and 12 h. GO annotation categories assigned to fewer than 30 unigenes were not shown. The x-axis refers to number of unigenes differentially expressed in RNA-Seq analysis, and the y-axis represents functional categories (P < 0.01, FDR < 0.05).

Results

• Activities of transcription regulators were significant-ly enriched in the leaves of C.lavandulifolium.

• A large number of transcription factors in the leaves of C. lavandulifolium responded to the NaCl treatme-nt (see Appendix S4 in Supporting information).

Results44 genes belong to NAC family

14 genes displayed unchanged expression

28 genes displayed up-regulated expression

2 genes displayed down-regulate expression

NaCl treatment

6 genes ≥2 8 genes ≥4 6 genes ≥8 2 genes ≥16 6 genes ≥30

ResultsExpression pattern of ClNACs in different organs

Fig. 2. Heat map representation of tissue-specific expression of 44 ClNACs. Expression patterns of 44 ClNACs genes were analyzed using RT-PCR, and the fluorescen-ce absolute quantification of elec-trophoretic bands was performed by Image Quant 5.2 software. The colors indicate expression intensity (red, high expression; black, noexpression). SE, seeds; CO, cotyledon; LE, leaves; ST, stems; BU, flower buds; FP, flower petals; RO, roots. (For interpretation of the references to color in this figurelegend, the reader is referred to the web version of the article.)

Expression pattern of ClNACs under abiotic stresses and hormone treatments

Fig. 3. Heat map representation of the expression data of 44 ClNACs in response to diverse environmental stress stimuli and hormonal treat-ments. Expression data for the ClNAC genes were calculated based on RT-PCR expression values in the leaves of treated samples at different time points.

Fig. 4. Expression patterns for the ClNAC genes in response to multiple environmental stimuli and hormonal treatments were extracted from RT-PCR. Expression patterns of ClNAC genes under multiple-treatments are presented with white squares, plus or minus signs.

Results

• we compared the results of RNA-Seq and RT-PCR after 12-h salt treatment. Among the 44 ClNACs, only ClNAC11 (whose expres-sion was down-regulated in RNA-Seq, but unchanged in RT-PCR), ClNAC14 (whose expressi-on was upregulated in RNA-Seq, but unchanged in RT-PCR) and ClNAC15 (whose expression was un-changed in RNA-Seq, but up-regulated in RT-PCR) showed diffe-rent expression patterns between the two technologies, suggest-ing the RNA-Seq was a useful and powerful tool to monitor gene expression.

• Ten ClNACs(ClNAC2,-3,-17,-4,-9,-11,-21,-12,-39 and -44) were ch-osen as candidate genes to perform further study.

ResultsPhylogenetic analysis of candidate NAC genes

Fig. 5. Phylogenetic relationship among candidate ClNAC genes and other plant NAC homologous genes. Phylogenetic tree of NAC domain-containing proteins from C. lavandulifolium, Arabidopsis and Oryza sativa. Amino acid sequences were aligned using ClustalW and a neighbor-joining tree was constructed with a 1000-bootstrap replication support. The subfamilies within the NAC family, as designated by Ooka et al. [8] were grouped as indicated. Abbreviations for the name of the subfamilies are as follows: ATAF Arabidopsis transcription factor-like family, NAP NAC-like activated by APETALA3/PISTILLATA family, SENU5 tomato senescence up-regulated 5-like family, NAC2 Arabidopsis thaliana NAC protein 2-like family, NAM no apical meristem transcription factor-like family, NAC1 Arabidopsis thaliana NAC protein 1-like family, ANAC001 Arabidopsis NAC protein 001 transcription factor-like family, TERN tobacco elicitor-responsive NAC protein-like family, TIP turnip crinkle virus interacting protein-like family, ONAC001Oryza sativa NAC protein 001 transcription factor-like family, ONAC002 Oryza sativa NAC protein 002 transcription factor-like family, ONAC022 Oryza sativa NAC protein 022 transcription factor-like family. Accession numbers of NAC sequences from other plant species used in the analysis are in listed in Supplement 5.

Results

Results

Discussion

• In C. lavandulifolium, except for four gene families(MYB (Myelo-blastosis), HSF (Heat-Shock Factor), AP2/EREBP (Apetala-2/ER-EBP) and WRKY (named after the WRKY amino acid motif)), we found that 28 ClNACs were up-regulated in C. lavandulifolium, making it the most highly induced TF family in this species.

Transcription factors in C. lavandulifolium responded to salt stress

Discussion

• Members of the plant NAC gene family display tissue-specific expression and are involved in various processes of plant grow-th and development.

Gene Express tissue Biological process

22 ClNAC(Group C-F) genes buds and petals flowering

5 ClNAC(Group D) genes cotyledons seed gernination

9 ClNAC genes roots later root formation and development

Discussion

Mechanisms of C. lavandulifolium NAC genes in response to abiotic stresses

Expression analyses showed that there were15 NAC genes in C. lavandu-lifolium that were up-regulated by ABA (see Groups III and V in Figs. 3 and 4), suggesting the existence of other ABA-dependent stress resistance pathways in plants that may be regulated by NAC TFs.

ClNACs in Groups I, II and VI responded to abiotic stresses butwere not regulated by ABA

DiscussionCandidate stress-responsive NAC genes in C. lavandulifolium

Subfamily Members functions

ATAF ClNAC3, ClNAC17, OsNAC5 and 6 stress-responsive genes

AtNAC3 ClNAC2, ANAC019, ANAC055 and ANAC072/RD26 in A. thaliana stress-responsive genes

NAP ClNAC12, -39 and -44 stress-responsive genes

SENU5 ClNAC9, CarNAC1 in chickpea functions in the process of plants’ response to aging

ONAC022 ClNAC11, LOV1 and ONAC063 in rice

NAC 1ClNAC21, ANAC021/22, GmNAC11and GmNAC20

increase the number of lateral roots of transgenic

SNAC

Summary

• ATAF-type gene ClNAC17 and the NAC 1-type gene ClNAC21 can respond to six kinds of treatments.

• Further studies of these 2 genes are needed in order to provide tress-responsive genetic resources for the Chrysanthemum abiotic resistant transgenic breeding.