Identification of Candidate Genes and Regulation Mechanism for Drought Stress in Maize. Jie Xu

1. Jie Xu
Sichuan Agricultural University, China
Oct. 8th - 10th, 2018
Ludhiana, India
Identification of Candidate Genes and
Regulation Mechanism for Drought
Stress in Maize
13th Asian Maize Conference

2. Maize is one of the most important crops
and drought is the main environmental
constrain for maize production in both the
world and China.
Therefore, studies on maize drought
tolerance related issues and development
of drought tolerance varieties are very
important in terms of both theoretical and
applied implications.
Background

3. Schnable PS, et al.. Science 2009；Y. Jiao et al.,. Nature 2017；
N. M. Springer et al., Nature genetics 2018; S. Sun et al., Nature
genetics 2018;
The publicly available maize genomes and development of next-
generation sequencing (NGS) technologies, provide direct
insight into the genome and transcriptome variations among
inbreds and facilitate maize drought tolerance research.
Background

4. Identification of candidate genes for drought tolerance by
whole-genome resequencing in maize (Resequencing strategy)
Background
Comprehensive analysis of maize natural-antisense
transcripts pairs and their regulations in response to drought
stress (Transcriptome sequencing strategy)
Experimental validationGenome-wide association study

5.  16 maize inbreds were selected based selection criteria such as grain yield, ASI
and leaf senescence under WW and WS.
 Whole-genome resequencing was performed on Illumina Hiseq 2000 platform
 4.6 billion (407 gigabases) sequence reads were aligned against B73 genome
 The non-synonymous SNPs (nsSNPs) within the genes were filtered.
Common Variants (CV) strategy
SVD cluster strategy
nsSNP
Inbreds： 3 extremely drought-tolerant + 3 extremely drought-sensitive
+ 4 moderate drought-tolerant + 6 moderate drought-sensitive
1. Identification of candidate genes for drought
tolerance by whole-genome resequencing in maize
Detection of
nsSNPs and
candidate genes in
response to
drought stress

6. The densities of candidate nsSNPs by both CV and cluster analyses and
reported QTL on chromosome 1 for drought tolerance
Clustering
CV
QTL
Bin1.07
Bin 1.07 region harbored six reported QTL for flowering time
and grain yield under water-stress condition with high
phenotypic variation explained.
J. Xu et al., BMC plant biology 14, 83 (2014).

7. Borsani O, Zhu J-K, Cell. 2005
Natural antisense transcripts (NATs) was one prominent and
complex class of regulatory RNA. By definition, NAT are
transcribed from the strand opposite to that of the sense
transcripts of either protein coding or non-protein-coding genes.
2. Comprehensive analysis of NAT pairs and their
regulations in reponse to drought stress

8. Traits: total root length, root surface area, root volume and tips number
AC7643 AC7729/TZSRW Well Water Water Stress
20% PEG; 24 h
Maize lines selected for RNA-seq and NAT identified for
drought tolerance (over 200 maize lines were phenotyped)

9. NATs identified in maize under WW and WS
 More NAT pairs were detected
only in the RILs (85.00%) than
were expressed only in parental
lines (58.76%)
 RILs shared a larger set of NAT
pairs (47.26%) than did the
parental lines (28.27%)
 A significantly higher percentage
of NAT pairs were expressed in
WS (83.28%) than in WW
(75.66%)
Venn diagram of detectable NATs among
different material and water conditions
χ2 test P value < 0.001

10.  In 615 NAT pairs (37.80%, 1,627), both sense and antisense
transcripts were drought-responsive in at least one of the four test
lines, with a log2 fold change > 1 or < -1 and FDR < 0.05.
 Of these, the sense and antisense transcripts in 488 pairs were
concordantly regulated (both up or down) under WS
Concordant Discordant
AC7643/AC7729/TZSRWR/RIL208/RIL
64 -sense/antisense
A heat map of fold change in gene expression
revealed consistently concordant or
discordant response to drought
Expression correlation of sense and
antisense transcription in response to water
stress

11. Translational efficiency of NAT pairs under
drought stress
Translation efficiency was compared
between sense and antisense
transcripts using ribosome release
scores (RRS) generated by ribosome
profiling from B73 seedlings before and
after drought stress (data from Lai lab,
The Plant Journal, 84, 1206-1218)
3'
3'UTR
CDS
UTR
CDS
Ribosome
mRNA
Reads
Reads
Reads
Reads
RRS
 
 
 

 
 
 
Correlation of gene expression and
translational efficiency.

12. SmRNA enrichment in NATs and regulation by Leafbladeless 1
smRNA enrichment 1 kb upstream and downstream of the transcription start
and termination sites, as well as within the genes
 The number of smRNA reads per kb was 19.74 in non-NAT genes, but 25.82
and 24.50 in NAT genes and the corresponding sense genes.
 The average coverage was 67.39 (bp per kb) in non-NAT pools, but 160.38 and
164.21 in sense and antisense transcripts.
 Coverage was highest at 222.29 in overlapping regions of NAT pairs

13. Hypomethylation and chromatin modification in NAT pairs
DNA methylation levels in NAT pairs The degree of enrichment of histone marks
plotted along transcription start site (TSS)
and transcription termination site (TTS)

14. Genotype
368 inbreds SNPs（RNA-seq）
Phenotype
Potential functions of NAT pairs responsive to drought stress in
the association and bi-parental populations
Survival rate in drought stress;
Flowering time and seed oil
content in well water condition
(368 lines)
19 RIL populations (GBS) Grain yield, plant height and
flowering time in WW and WS
environments (19 RIL pops)
To infer the contributions of NAT pairs genes made to drought tolerance in maize,
the association between the SNPs existed in each NAT pairs genes and target
traits was evaluated using 368 maize inbreds and 19 RIL populations

15.  SNPs within 85.12% (2,711 of 3,815) of NAT pairs were informative, of which 14.09%
(382 of 2,711) were significantly associated with drought survival rate.
 NAT pairs were not specifically enriched for SNPs associated with kernel oil content or
days to tassel under normal conditions.
 These SNPs were more heavily concentrated in drought-responsive NATs than in
drought-insensitive NATs
In the association populations (368 maize inbreds)

16. In bi-parental populations
 The 19 biparental populations consisting of 3,273 lines derived from crosses
among 23 tropical maize inbreds (flowering, plant height, and grain yield
under WS).
 On average, there were about 2.51 SNPs per kb in each NAT pair gene
linked to drought response, but only 1.79 in non-NAT genes

17. Experimental validation of maize NAT pairs
Strand specific RT-PCR and SYBR Green-based
quantitative real-time PCR

18. Four NAT pairs (eight genes)
were selected for validation.
All NATs were successfully
detected and changes in
expression were consistent
with strand-specific RNA-
sequencing.
Experimental validation of maize NAT pairs

19. RNA-FISH of ZmNAC48 and cis-NATZmNAC48 in maize roots
WW
WS
ZmNAC48 cis-NATZmNAC48
nucleus (blue)
3. The antisense transcript-mediated mechanism
of drought response induced by ZmNAC48 in
maize

20. Subcellular localization of ZmNAC48 and cis-NATZmNAC48
ZmNAC48 mostly localized in the nucleus and cell membrane
CaMV35S-cis-NATZmNAC48-
eGFP
GFP

21. The expression of ZmNAC48 and cis-NATZmNAC48 in vitro
50um 20um
The expression of ZmNAC48
was significantly reduced
when cis-NATZmNAC48 was
over-expressed in maize
protoplasts, and vice versa
The fluorescence intensity
weakened and the number of
cells with fluorescence reduced
when sense and antisense
constructs co-expressed

22. SiRNAs expression level under WW and WS
siRNAs generated from the
overlapped regions of ZmNAC48
and cis-NATZmNAC48 were detected
in maize roots and transgenic
Arabidopsis, and the expression
of which were in accordance with
the cis-NATs expression.
A:The gene structure of ZmNAC48, cis-
NATZmNAC48 and smRNAs mapping in sense/
antisense overlap of ZmNAC48.
B and C:The expression of siRNA8 and siRNA9
in the root of the tolerant maize inbred line
AC7643 by qRT-PCR analysis
D and E:The expression of siRNA8 and siRNA9
in the root of maize drought-sensitive inbred line
AC7729/TZSRW
F:The expression of ZmNAC48, cis-NATZmNAC48,
siRNA8 and siRNA9 in WT and transgenic
Arabidopsis with ZmNAC48 and cis-NATZmNAC48

23. Over-expression of ZmNAC sense /antisense in Arabidopsis
Over-expression of ZmNAC48
resulted in less tolerance to drought
in Arabidopsis by the shorten root
length and inhibition the growth of
plant erial part
The transgenic Arabidopsis with
expression of cis-NATZmNAC48
had no significant difference
with WT

24. Acknowlegement
 National Natural Science
Foundation of China
 The youth fund of
SiChuan province, China
 CIMMYT/CGIAR Research Program/Asian Maize Conference/Maize-Asia
Youth Innovators Awards
 Prof. Tingzhao Rong/Prof. Yanli Lu, Sichuan Agricultural University
 Prof. Micheal Freeling, University of California, Berkeley
 Prof. Damon Lisch, Purdue University, West Lafayette

25. Thank you
Jie Xu (jiexu28@gmail.com)
Sichuan Agricultural University, China