Pak. J. Bot., 52(4): 1121-1129, 2020. DOI: http://dx.doi.org/10.30848/PJB2020-4(4) LncRNAs PARTICIPATE IN SALT TOLERANCE RELATED PATHWAYS BY REGULATING TARGET GENES IN WHEAT YAN YAN Ɵ , XIN LIN Ɵ , FAN WU, SHAOSHUAI SONG, YUMEI ZHANG AND PING MU * College of Agronomy, Qingdao Agricultural University, Qingdao, Shandong, China 266109 Ɵ These authors contributed equally to this work *Corresponding author’s email: [email protected]; Ph: 86-532-88030050 Abstract Long non-coding RNAs (lncRNAs) are key regulatory elements that play important roles in plant development as well as stress responses in plants. A genome-wide analysis of lncRNA expression in salt tolerant (Xiaoyan60) and salt sensitive (Lumai21) wheat cultivars under salt stress conditions was performed. We identified a total of 746 differentially expressed lncRNAs under salt stress that 675 were expressed in Xiaoyan60 and 592 in Lumai21. Gene ontology enrichment analysis indicated that differentially expressed genes were enriched in biological process, cellular component and molecular function. KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis revealed that the distinct lncRNAs were involved in salt tolerance-related pathways, including plant hormone signal transduction and 27 other pathways. We identified lncRNAs that were significantly upregulated (i.e., lnc_521, lnc_593 and lnc_743) in Xiaoyan60 when compared with Lumai21 after salt stress. These results indicated that lncRNAs were involved in salt tolerance, and our findings provided an important insight regarding wheat adaptation to salt stress. Key words: Wheat; Salt stress; lncRNA; Target genes; Up-regulation; Down-regulation. Introduction Salt stress is a major abiotic factor that affects photosynthesis, growth and development in plants; and high soil salinity is a major global problem (Ma et al., 2016; Sun et al., 2016). Salinity is both an osmotic and ionic stressor. Plant survival in a saline environment depends upon protective adaptations including osmolyte synthesis, increased expression of key response genes and ion compartmentation (Yeo et al., 2017; Fu et al., 2013; Munns & Tester, 2008; Hirayama & Shinozaki , 2010; Rajendran et al., 2009). Plant response to salinity is mediated by multiple approaches, including calcium signaling and other metabolic pathways (Alsahli et al., 2019). Long noncoding RNAs (lncRNA) are a class of >200nt endogenously expressed non-coding RNAs that are transcribed primarily by RNA polymerase II. Two other lncRNA classes are transcribed by RNA polymeraseIV and RNA polymeraseV, respectively. They are required for regulation of gene expression by gene silencing and epigenetic controls (Wierzbicki et al., 2008; Zhang & Chen, 2013). LncRNAs are transcribed from multiple locations in the genome including introns, exons and intergenic regions (Shumayla et al., 2017). They are key regulators of gene expression and function at both transcriptional and post-transcriptional levels (Heo et al., 2013; Zhang et al., 2014). The regulatory mechanisms that employ lncRNAs are complex, and they act both directly and indirectly (Wilusz et al., 2009). LncRNA functions in plants are largely unknown but available evidence associates them with plant development and stress responses (Xin et al., 2011;Wang et al., 2017). A previous study of lncRNAs in Arabidopsis identified 1832 of these molecules that regulate plants responses to drought, cold, high-salt, abscisic acid (ABA) and bacterial elongation factor Ef-Tu (Liu et al., 2012). In addition, 13 salt-responsive lncRNAs in Arabidopsis were identified and validated by qRT-PCR (Di et al., 2014). The over expression of npc536 in Arabidopsis showed a heightened root growth in salt stress (Amor et al., 2009). In Medicagotruncatula, 2477 lncRNAs were found up-regulated during salt stress (Wang et al., 2015). TE-LincRNA11195, alncRNA associated with transposable elements was reportedly involved in abiotic stress responses including salt treatments in plants (Wang et al., 2017). However, lncRNAs in wheat have not been fully cataloged, although some associated with wheat stripe rust and powdery mildew have been identified (Zhang et al., 2013; Zhang et al., 2016). In addition, 125 putative wheat stress-responsive long non- protein coding RNAs responsible to powdery mildew infection and heat stress have been identified (Xin et al., 2011). An analysis of 52 RNA-Seq data sets in Triticumaestivum indicated that lncRNAs were regulated during tissue development and under abiotic stress (Shumayla et al., 2017). The co-expression of lncRNA with other regulatory mRNAs indicated that lncRNAs are involved in numerous biological processes such as ABA biosynthesis and some acted as target mimics of known miRNAs (Shumayla et al., 2017). Further studies on lncRNAs and their molecular mechanisms in response to salt stress in wheat are needed. In the current work, we studied expression patterns of the salt stress-responsive lncRNAs of the salt-tolerant (Xiaoyan60) and salt-sensitive (Lumai21) wheat cultivars using a high-throughput sequencing approach. The salt- responsive wheat lncRNA candidates were predicted and their target genes, biological processes and significant metabolic pathways were assessed using bioinformatics tools. We further analyzed the salt-stress-responsive pathways of lncRNAs participation. We identified a total of 746 candidate lncRNAs that were both up and down- regulated and validated expression levels of 6 lncRNAsusing qRT-PCR.
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Pak. J. Bot., 52(4): 1121-1129, 2020. DOI: http://dx.doi.org/10.30848/PJB2020-4(4)
LncRNAs PARTICIPATE IN SALT TOLERANCE RELATED PATHWAYS BY
REGULATING TARGET GENES IN WHEAT
YAN YANƟ, XIN LINƟ, FAN WU, SHAOSHUAI SONG, YUMEI ZHANG AND PING MU*
College of Agronomy, Qingdao Agricultural University, Qingdao, Shandong, China 266109 ƟThese authors contributed equally to this work
Long non-coding RNAs (lncRNAs) are key regulatory elements that play important roles in plant development as well
as stress responses in plants. A genome-wide analysis of lncRNA expression in salt tolerant (Xiaoyan60) and salt sensitive
(Lumai21) wheat cultivars under salt stress conditions was performed. We identified a total of 746 differentially expressed
lncRNAs under salt stress that 675 were expressed in Xiaoyan60 and 592 in Lumai21. Gene ontology enrichment analysis
indicated that differentially expressed genes were enriched in biological process, cellular component and molecular function.
KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis revealed that the distinct lncRNAs were involved in salt
tolerance-related pathways, including plant hormone signal transduction and 27 other pathways. We identified lncRNAs that
were significantly upregulated (i.e., lnc_521, lnc_593 and lnc_743) in Xiaoyan60 when compared with Lumai21 after salt
stress. These results indicated that lncRNAs were involved in salt tolerance, and our findings provided an important insight
regarding wheat adaptation to salt stress.
Key words: Wheat; Salt stress; lncRNA; Target genes; Up-regulation; Down-regulation.
Introduction
Salt stress is a major abiotic factor that affects
photosynthesis, growth and development in plants; and
high soil salinity is a major global problem (Ma et al.,
2016; Sun et al., 2016). Salinity is both an osmotic and
ionic stressor. Plant survival in a saline environment
depends upon protective adaptations including
osmolyte synthesis, increased expression of key
response genes and ion compartmentation (Yeo et al.,
2017; Fu et al., 2013; Munns & Tester, 2008;
Hirayama & Shinozaki, 2010; Rajendran et al., 2009).
Plant response to salinity is mediated by multiple
approaches, including calcium signaling and other
metabolic pathways (Alsahli et al., 2019).
Long noncoding RNAs (lncRNA) are a class
of >200nt endogenously expressed non-coding RNAs that
are transcribed primarily by RNA polymerase II. Two
other lncRNA classes are transcribed by RNA
polymeraseIV and RNA polymeraseV, respectively. They
are required for regulation of gene expression by gene
silencing and epigenetic controls (Wierzbicki et al., 2008;
Zhang & Chen, 2013). LncRNAs are transcribed from
multiple locations in the genome including introns, exons
and intergenic regions (Shumayla et al., 2017). They are
key regulators of gene expression and function at both
transcriptional and post-transcriptional levels (Heo et al.,
2013; Zhang et al., 2014). The regulatory mechanisms
that employ lncRNAs are complex, and they act both
directly and indirectly (Wilusz et al., 2009). LncRNA
functions in plants are largely unknown but available
evidence associates them with plant development and
stress responses (Xin et al., 2011;Wang et al., 2017).
A previous study of lncRNAs in Arabidopsis
identified 1832 of these molecules that regulate plants
responses to drought, cold, high-salt, abscisic acid (ABA)
and bacterial elongation factor Ef-Tu (Liu et al., 2012).
In addition, 13 salt-responsive lncRNAs in Arabidopsis
were identified and validated by qRT-PCR (Di et al.,
2014). The over expression of npc536 in Arabidopsis
showed a heightened root growth in salt stress (Amor et
al., 2009). In Medicagotruncatula, 2477 lncRNAs were
found up-regulated during salt stress (Wang et al., 2015).
TE-LincRNA11195, alncRNA associated with
transposable elements was reportedly involved in abiotic
stress responses including salt treatments in plants
(Wang et al., 2017). However, lncRNAs in wheat have
not been fully cataloged, although some associated with
wheat stripe rust and powdery mildew have been
identified (Zhang et al., 2013; Zhang et al., 2016). In
addition, 125 putative wheat stress-responsive long non-
protein coding RNAs responsible to powdery mildew
infection and heat stress have been identified (Xin et al.,
2011). An analysis of 52 RNA-Seq data sets in
Triticumaestivum indicated that lncRNAs were regulated
during tissue development and under abiotic stress
(Shumayla et al., 2017). The co-expression of lncRNA
with other regulatory mRNAs indicated that lncRNAs
are involved in numerous biological processes such as
ABA biosynthesis and some acted as target mimics of
known miRNAs (Shumayla et al., 2017). Further studies on lncRNAs and their molecular
mechanisms in response to salt stress in wheat are needed. In the current work, we studied expression patterns of the salt stress-responsive lncRNAs of the salt-tolerant (Xiaoyan60) and salt-sensitive (Lumai21) wheat cultivars using a high-throughput sequencing approach. The salt-responsive wheat lncRNA candidates were predicted and their target genes, biological processes and significant metabolic pathways were assessed using bioinformatics tools. We further analyzed the salt-stress-responsive pathways of lncRNAs participation. We identified a total of 746 candidate lncRNAs that were both up and down-regulated and validated expression levels of 6 lncRNAsusing qRT-PCR.
Table 2. Expression level of selected LncRNAs based on sequencing results.
LncRNA XY1 XY2 XY3 XY4 LM1 LM2 LM3 LM4
364 0.00 0.00 6.80 8.88 0.00 0.00 0.00 7.86
521 0.00 3.43 0.00 0.00 0.00 0.00 3.06 0.00
593 0.00 2.90 0.00 0.00 0.00 2.97 0.00 0.00
693 0.00 6.33 9.33 7.55 0.00 3.71 5.99 4.57
726 0.00 0.00 3.01 9.52 0.00 0.00 5.32 11.01
743 0.00 0.00 5.46 3.24 0.00 0.00 0.00 0.00
Fig. 2. The number of total assembled transcripts and potential lncRNAtranscripts.
a: The numbers of total assembled transcripts from 8 samples; and b: The numbers of potential lncRNA transcripts screened.
Note: XY1, XY2, XY 3 and XY4 indicated Xiaoyan 60 (XY) under salt stress for 0, 3, 12 and 24 hours; LM1, LM 2 LM3 and LM4
indicated Lumai 21 (LM) under salt stress for 0, 3, 12 and 24 hours.
GO term enrichment of differential genes between XY and LM: The differential genes of test modules XY and LM were assigned and enriched to different GO terms and covered3 domains including BP, CC and MF. Functional enrichments of 488 significant XY genes were associated with photosynthesis. These enrichments of genes included photosystem I (GO:0009522), light harvesting (GO:0009765), chlorophyll binding (GO:0016168), plastid thylakoid (GO:0031976), photosynthesis (GO:0015979), electron transport chain (GO:0022900), thylakoid part (GO:0044436), and chloroplast thylakoid (GO:0009534). However, 2185 significant genes of LM were enriched in items related to cellular structure, secondary metabolism, and redox. For example, intrinsic to membrane (GO:0031224), integral to membrane (GO:0016021), secondary metabolite biosynthetic process (GO:0044550), and oxidoreductase activity (GO:0016491) were all enriched in LM (Table S2). Pathways analysis of stress-responsive lncRNA participation: To examine the function of lncRNAs related to salt stress, we analyzed relationships between gene function and trans-lncRNAs using KEGG analysis. We identified 52 paths of significant enrichment with 25 and 30 pathways significantly enriched in XY and LM, respectively. In cultivar XY, at least 98 differentially expressed lncRNAs were involved in the significant pathways and 116 for LM (Fig. 4). We also identified lncRNA target genes associated with salt stress. In the plant hormone signal transduction category, the results were only significant at the start of the experiments (3 h). However, we identified 31 lncRNAs in XY (29 up and 2 down) and 25 lncRNAs in LM (22 up and 3 down) (Table S3).
In cultivar XY, we identified 5 pathways associated
with the salt stress response at 12 and 24 h with
correlation pathways including photosynthesis (5
lncRNAs, 4 up and 1 down), oxidative phosphorylation
(17 lncRNAs, 8up and 9 down), glycine, serine and
threonine metabolism (21 lncRNAs, 20 up and 1 down) in
XY3. Photosynthesis (7 lncRNAs, 4 up and 3 down),
oxidative phosphorylation (20 lncRNAs, 13 up and 7
down), flavonoid biosynthesis (21 lncRNAs, 18 up and 3
down), and photosynthesis-antenna proteins (1 up
lncRNA) correlated with the salt stress in XY4 (Table S3).
There were4 significant pathways in LM. Only the
glutathione metabolism pathway (43 lncRNAs, 40 up and
3 down) was related to salt stress (12 h). The related
pathways are plant hormone signal transduction (20
lncRNAs, 10 up and 10 down), peroxisome (10 lncRNAs,
5up and 5 down), glutathione metabolism (15 lncRNAs,
12 up and 3 down) and arginine and proline metabolism
(6 lncRNAs, 5 up and 1 down) in LM4.
Significantly enriched pathways of lncRNA
participation in two cultivars: We compared the different
expression levels of lncRNA of LM vs XY, and then
identified 4 commonly up-regulated lncRNAs. A total of
111 differentially expressed lncRNAs were involved in
significant KEGG pathways. We found 28 pathways that
were significantly enriched in LM, including systemic