Transcript Results

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
1
Introduction
2
Materials and methods
3
Results
4
Discussion
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 methods
seeds germination and plant growth
salinity treatment was
applied by submerging
the roots of the plants
with a 200 mM NaCl
for 12 h, and the second and third pairs of
fully expanded leaves
below the terminal bud
were collected.
RNA-Seq analysis
salinity treatment
chilling and heat treatment
drought stress
ABA and SA treatment
Collected the second
and third pairs of fully expanded leaves
below the terminal
bud from each treated samples.
Cotyledons were collected
after the seeds germinated;
leaves, stems, and roots were
collected from eight-true
leaves stage seedlings. C.lavandulifolium specimens with
eight- true leaves were transferred into a short-day artificial clima-te chamber for
flowering, and then the flower buds, flower petals and
seeds were collected, frozen
in liquid nitrogen and preserved.
RT-PCR analysis
RT-PCR adn qRT-PCR analysis
Materials and methods
Full-length amplification of candidate genes and bioinformatics analysis
• 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.
Results
Salt-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 significantly enriched in the leaves of C.lavandulifolium.
• A large number of transcription factors in the leaves
of C. lavandulifolium responded to the NaCl treatment (see Appendix S4 in Supporting information).
Results
44 genes belong to NAC family
NaCl treatment
14 genes displayed
unchanged expression
6 genes ≥2
28 genes displayed upregulated expression
8 genes ≥4
6 genes ≥8
2 genes displayed downregulate expression
2 genes ≥16
6 genes ≥30
Results
Expression 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 fluorescence absolute quantification of electrophoretic bands was performed
by Image Quant 5.2 software. The
colors indicate expression intensity
(red, high expression; black, no
expression). 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 figure
legend, 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 expression 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 unchanged in RNA-Seq, but up-regulated in RT-PCR) showed different expression patterns between the two technologies, suggesting 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 chosen as candidate genes to perform further study.
Results
Phylogenetic 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 factorlike family, TERN tobacco elicitor-responsive NAC protein-like family, TIP turnip crinkle virus interacting protein-like family, ONAC001
Oryza 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
Transcription factors in C. lavandulifolium responded to salt stress
• In C. lavandulifolium, except for four gene families(MYB (Myeloblastosis), HSF (Heat-Shock Factor), AP2/EREBP (Apetala-2/EREBP) 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.
Discussion
• Members of the plant NAC gene family display tissue-specific
expression and are involved in various processes of plant growth 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. lavandulifolium 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 but
were not regulated by ABA
Discussion
Candidate stress-responsive NAC genes in C. lavandulifolium
Subfamily
ATAF
Members
functions
ClNAC3, ClNAC17, OsNAC5 and 6
stress-responsive genes
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 1
ClNAC21, ANAC021/22, GmNAC11
and GmNAC20
SNAC AtNAC3
increase the number of
lateral roots of transgenic
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.