ORIGINAL RESEARCH
published: 28 January 2019
doi: 10.3389/fpls.2019.00024
RNA Sequencing Characterizes
Transcriptomes Differences in Cold
Response Between Northern
and Southern Alternanthera
philoxeroides and Highlight
Adaptations Associated With
Northward Expansion
Edited by:
TingFung Chan,
The Chinese University of Hong Kong,
China
Reviewed by:
Gonzalo Gajardo,
University of Los Lagos, Chile
Chiara Campoli,
University of Dundee, United Kingdom
*Correspondence:
Dasheng Liu
liu_sdiep@126.com
David Horvath
david.horvath@ars.usda.gov
† These
authors have contributed
equally to this work
Specialty section:
This article was submitted to
Evolutionary and Population Genetics,
a section of the journal
Frontiers in Plant Science
Received: 19 September 2018
Accepted: 09 January 2019
Published: 28 January 2019
Citation:
Liu D, Horvath D, Li P and Liu W
(2019) RNA Sequencing
Characterizes Transcriptomes
Differences in Cold Response
Between Northern and Southern
Alternanthera philoxeroides
and Highlight Adaptations Associated
With Northward Expansion.
Front. Plant Sci. 10:24.
doi: 10.3389/fpls.2019.00024
Dasheng Liu 1* † , David Horvath 2* † , Peng Li 1 and Wenmin Liu 3
1
Shandong Institute of Environmental Science, Jinan, China, 2 USDA-ARS, Sunflower and Plant Biology Research Unit,
Fargo, ND, United States, 3 College of Life Sciences, Shandong Normal University, Jinan, China
Alternanthera philoxeroides recently expanded its range northwards in China. It is
unknown if the range expansion has a genetic and/or epigenetic basis, or merely an
environmental basis due to a warming climate. To test these possibilities, we used an
RNAseq approach with a common greenhouse design to examine gene expression
in individuals from the northern edge and central portion of alligator weed range from
China to determine if there were differences in their responses to cold temperatures.
We hypothesized that if the recent range expansion was primarily environmental,
we would observe few differences or only differences unrelated to low-temperature
adaptations. We assembled over 75,000 genes of which over 65,000 had long
open reading frames with similarity to sequences from arabidopsis. Differences in
expression between northern and southern populations that were both exposed to low
temperatures showed similar expression among genes in the C-REPEAT/DRE BINDING
FACTOR (CBF) regulon. However, gene set and sub-network enrichment analysis
indicated differences in the response of photosynthetic processes and oxidative stress
responses were different between the two populations and we relate these differences
to cold adaptation. The transcriptome differences in response to cold between the
individuals from the two populations is consistent with adaptations potentiating or
resulting from selection after expansion into colder environments and may indicate that
genetic changes have accompanied the recent northward expansion of A. philoxeroides
in China. However, we cannot rule out the possibility of epigenetic changes may have a
role in this expansion.
Keywords: invasive plant, Alternanthera philoxeroides, local adaptation, cold hardiness, RNA sequencing, range
expansion, North China
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Cold Response of Alternanthera philoxeroides
INTRODUCTION
(Diao, 1990), and cultivated widely as a forage in southern
China in the 1950–60s. It subsequently escaped cultivation and
currently inflicts serious damage to agriculture (Yin, 1992; Liu
and Huang, 2002), causing annual losses of 600 million Chinese
Yuan Renminbi (equivalent to 98 million US dollars) (Li and
Xie, 2002). This weed is on the first shortlist of the invasive
species requiring special control in China (State Environmental
Protection Administration of China and Chinese Academy of
Sciences, 2003). A recent survey indicated that A. philoxeroides
has now invaded the Xiaqing River in Jinan (N36.6, E117.1),
500 km north of the Shanghai in northern China (Liu et al.,
2006, 2012). This invasion is more extensive than the 32◦ north
latitude northern range limit predicted for A. philoxeroides in
China (Julien et al., 1995). It is unknown if the recent ability of
this weed to survive winters in this northern region is due to
selection for genes that have increased the cold hardiness of this
species, or if the range expansion was due primarily to a warming
climate.
Increased cold hardiness can result from either avoidance
mechanisms such as production of underground propagules or
repression of ice nucleation and crystal growth, or from resistance
mechanisms such as changes in expression of genes involved
in altering membrane lipid saturation, resistance to oxidative
stress by reducing reactive oxygen species (ROS) production
through reduced photosynthesis from unadapted thylakoid
membranes, or induction of free radical scavenging enzymes
such as APX1, SOD1, NPX1, etc. (Gusta and Wisniewski,
2013). Many cold tolerant plant species can cold acclimate in
response to chilling temperatures (Gusta and Wisniewski, 2013).
Induction of these protective responses are orchestrated through
several cold-responsive ‘regulons’ (Fowler and Thomashow,
2002). Specific transcription factors such as the C-REPEAT/DRE
BINDING FACTOR (CBF) family of transcription factors
and the transcription factor RESPONSIVE TO HIGH LIGHT
41 (ZAT12) are often induced upon exposure to chilling
temperatures (Thomashow, 2010). These in turn alter the
expression of downstream genes required for modifying the
cellular physiology to withstand cold. Such co-regulated clusters
of genes and their regulators are often referred to as regulons. The
best characterized is the CBF2 regulon which can work with or
without integrating information from hormones such as abscisic
acid (ABA). Another regulon associated with and augmented by
subset of cold responses is controlled by the ABSCISIC ACID
RESPONSIVE ELEMENTS-BINDING FACTOR (ABRE) family
of transcription factors (Shinozaki and Yamaguchi-Shinozaki,
2007). Both the CBF and ABRE regulons are responsive to, and
provides feedback to transcription factors that control circadian
responses in plants (Dong et al., 2011; Cao et al., 2005). Likewise,
ZAT12 regulates a smaller but still important set of genes
(including CBF2) (Vogel et al., 2005). ZAT12 is also regulated by
circadian genes and in particular blue light signals through CYR1
and CRY2 (Kilian et al., 2007). ZAT12 has been specifically linked
to responses involved in high light stress responses (Davletova
et al., 2005).
Many of the studies described above utilized microarray
analysis for gene expression resulting from cold treatments.
However, the reduced cost of next generation sequencing has
Biological invasions are becoming a major global environmental
and economic problem (Cohen and Carlton, 1998), and
intentional or unintentional human activities have increasingly
resulted in the introduction of invasive species far outside their
native or naturalized range. The fact that invasive species are
introduced to new areas that differ from their native range
provide a valuable insights into how ecological and evolutionary
processes are influenced under novel environmental conditions
(Sax et al., 2007; Lachmuth et al., 2010). Understanding the
mechanisms driving successful adaptation and invasion in
novel environments is a key issue in ecology, evolution, and
conservation biology (Vandepitte et al., 2014). Physiological and
phenotypic plasticity, evolution, and admixtures have all been
linked to local adaptation resulting in range expansion and
invasiveness (Bock et al., 2014). Next-generation sequencing
is an ideal tool for examining adaptive responses and genetic
differences resulting in range expansion and is uniquely suited
for non-model species (Prentis et al., 2010; Stapley et al., 2010;
Ekblom and Galindo, 2011; Puzey and Vallejo-Marin, 2014).
However, sequence evidence of adaptation during the invasion
process is currently scant for most invasive plants due to the
lack of genomic resources in weedy species (Stewart et al., 2009;
Vandepitte et al., 2014).
Recently, the invasive plant, Alternanthera philoxeroides
(alligator weed), has recently expanded it range northward by
nearly 5◦ of latitude beyond what was its predicted range in
China (Liu et al., 2012). This expansion occurred despite evidence
that A. philoxeroides in China has limited genetic variation
(Xu et al., 2003). Thus, this observation offers an opportunity
to look for genetic and/or epigenetic factors associated with this
recent northward range expansion.
Alternanthera philoxeroides weed (A. philoxeroides) belongs to
family Amaranthaceae (Mabberley, 1997), order Caryophyllales,
subclass Caryophyllidae (Cronquist, 1988), and it is an invasive
semi-aquatic weed (Julien and Bourne, 1988). It originated in the
Parana River region of South America (Maddox, 1968; Vogt et al.,
1979), and was spread to the other areas of South America, North
America, Asia, Australia and some adjacent island countries
(Julien et al., 1995). It is a very difficult to manage invasive
weed, and in all cases A. philoxeroides has become invasive in its
introduced habitats (Julien et al., 1995). This weed grows in both
aquatic and terrestrial habitats and can also invade farm lands.
Its stems are hollow, buoyant, and its floating mats can expand
over surfaces of all types of waterways making them difficult to
navigate, clogging drainage canals and waterways, reducing water
flow (Buckingham, 2002), and disrupting economies (Holm et al.,
1997). A. philoxeroides is a polyploid species with a genome size
that varies on the ploidy level between 2 and 6 n (Telesnicki
et al., 2011). Yet, despite its world-wide invasiveness, less than
100 genes have been sequenced and made public1 .
Alternanthera philoxeroides was introduced to China
mainland in 1940 by the Japanese in Shanghai (N31.4, E121.5)
1
http://www.ncbi.nlm.nih.gov/gquery/?term=Alternanthera+philoxeroides
(accessed December 02, 2014)
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Cold Response of Alternanthera philoxeroides
respectively, under natural light condition and photoperiod
for 4 weeks prior to treatment. Plants were first subjected to
2 day of interim temperatures (11–19◦ C night/day) in a nontemperature controlled greenhouse with natural light condition
and photoperiod in the Jan 2014, and then moved to an unheated
open-air concrete building and subjected to 2 weeks of cold
temperatures (daytime 6–13 C, nighttime 4–9 C) with natural
lighting supplemented during the day with incandescent lighting
to increase ambient light intensity within the building. Three
representative individual A. philoxeroides plants were selected
from each location, and the top 2nd and 3rd leaf pairs were
excised between 10:00 am–12:00 am and immediately frozen in
liquid nitrogen for future RNA extraction and sequencing library
construction.
made it possible to combine transcriptomics analysis with gene
sequencing and discovery in non-model systems such as wild
invasive weeds. Indeed, recent studies on cold stress in tea
(Camellia sinensis) (Wang et al., 2013), and Chrysanthemum
nankingense (Ren et al., 2014) highlight the power of this
new technology for identifying cold-responsive genes. Because
such studies produce large numbers of gene sequences, they
also provide a source of gene sequences from non-model
plants suitable for phylogenetic analyses (Puzey and VallejoMarin, 2014). Additionally, such sequence databases provide
a rich source of genetic markers including potential simple
sequence repeats (SSRs) and single nucleotide polymorphisms
(SNPs).
Here we examine the differences in cold-responsive gene
expression in A. philoxeroides between three individuals each
from two populations, one from the northern edge of its range
in Jinan, North China and the other from the central portion of
its range in Shanghai, South China. If the recent range expansion
was primarily environmental, we would expect few differences in
gene expression between cold-treated plants from the northern
and central populations, or only differences unrelated to lowtemperature adaptations. We test this hypothesis by looking for
differences in transcriptome responses indicative with a response
to cold using a common garden experimental design. Thus,
any such differences, if observed, would be attributed to genetic
and/or epigenetic differences between the individuals from the
two populations. Additionally, we expected to develop a database
of expressed sequences tags (ESTs) for A. philoxeroides and
eventually identify polymorphisms that have potential as genetic
markers for more cold-adapted populations.
RNAseq Library Construction and
Sequencing
A total amount of 3 µg RNA per sample was used as input
material for the RNA sample preparations. Sequencing libraries
were generated using NEBNextUltra RNA Library Prep Kit
for Illumina (NEB, United States) following manufacturer’s
recommendations and index codes were added to attribute
sequences to each sample. Briefly, mRNA was purified from total
RNA using poly-T oligo-attached magnetic beads. Fragmentation
was carried out using divalent cations under elevated temperature
in NEBNext First Strand Synthesis Reaction Buffer (5X). First
strand cDNA was synthesized using random hexamer primer and
M-MuLV Reverse Transcriptase RNaseH-. Second strand cDNA
synthesis was subsequently performed using DNA polymerase I
and RNase H. Remaining overhangs were converted into blunt
ends via exonuclease/polymerase activities. After adenylation
of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin
loop structure were ligated to prepare for hybridization. In
order to select cDNA fragments of preferentially 150∼200 bp
in length, the library fragments were purified with AMPure XP
system (Beckman Coulter, Beverly, United States). Then 3 µl
USER Enzyme (NEB, United States) was used with size-selected,
adaptor-ligated cDNA at 37◦ C for 15 min followed by 5 min at
95◦ C before PCR. Then PCR was performed with Phusion HighFidelity DNA polymerase, Universal PCR primers and Index (X)
Primer. At last, PCR products were purified (AMPure XP system)
and library quality was assessed on an Agilent Bioanalyzer 2100
system.
The clustering of the index-coded samples was performed on a
cBot Cluster Generation System using TruSeq SR Cluster Kit v3cBot-HS (Illumina) according to the manufacturer’s instructions.
After cluster generation, the library preparations were sequenced
on an Illumina Hiseq 2000/2500 platform and 100 bp paired-end
reads were generated.
MATERIALS AND METHODS
Plant Material
50–60 individual wild A. philoxeroides plants were collected in
November 2013 from local rivers of Jinan and Shanghai. Jinan,
the provincial capital of Shandong, is located in North China
in the lower reaches of the Yellow River, the second longest
river in China. The average annual temperature in Jinan is
14.7◦ C with an average of −0.4◦ C in January and 27.5◦ C in July.
The average annual rainfall is 672.7 mm. Its location is N36.6,
E117.1. Shanghai is located in South China in the lower reaches
of the Yangtze River, the longest river in China. The average
annual temperature in Shanghai is 16.6◦ C with an average of
4.7◦ C in January and 28.0◦ C in July. The average annual rainfall
is 1184.4 mm. Its location is N31.4, E121.5. The mentioned
meteorological data from China Meteorological Data Sharing
Service System (cdc. cma.gov. cn), and also see our paper (Liu
et al., 2012).
The plants were collected from each location and established
in a common garden plot of greenhouse in Jinan, Shandong
province, North China. Plants were grown hydroponically in
the 67 × 43 × 7 cm pots containing 50% concentration
Hoagland’s solution and watered every 2–3 days with same
solution (Hoagland, 1938). Plants were allowed to grow in
the greenhouse with night and day temperatures of 16–31◦ C,
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Sequencing Analysis and Bioinformatics
Primer sequences were trimmed from raw sequence reads
and the resulting files were trimmed for high quality reads
using the program Sickle-quality-based-trimming (Joshi and
Fass, 2011) in the iPlant discovery environment (DE) (Oliver
et al., 2013). The trimming parameters were: quality scores
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Cold Response of Alternanthera philoxeroides
TABLE 1 | Numbers or raw and trimmed reads from the six different libraries.
greater than 20 with a minimum trimmed read length of
70 bases. The trimmed reads from each end for all six samples
(from both populations) were concatenated and the two files
(one from each paired end) were kmer normalized using the
Trinity_normalize_by_kmer_coverage (Grabherr et al., 2011) in
the iPlant DE with parameters of 30 reads per kmer. The resulting
two files were assembled using the program Trinity (Robertson
et al., 2011) in the iPlant DE using the default parameters.
The assembled contigs were checked for quality by assessing
contig length and coverage and for completeness using the
program CEGMA (Parra et al., 2007) in the iPlant DE. Likewise,
the reads from each of the three individuals from the two
populations were similarly assembled and assessed separately.
BlastX against the arabidopsis peptide database was used to
annotate the assembled contigs with a minimum blast hit of
E-value < 10−5 .
The program Bowtie (Langmead et al., 2009) was used
to map the reads from the individual samples back to the
Trinity assembled reference contig file. Likewise, the reads were
also mapped separately to the Trinity assembled reference files
for both the central and northern populations accordingly.
Differential expression analysis was accomplished using the
program RSEM (Li and Dewey, 2011) with the program EBseq
to identify differentially expressed genes (Leng et al., 2012).
Programs were run on the iPlant Atmosphere resource using
an Ubuntu 12.04.5 - iPlant Base interface and scripts with
options are noted in Supplementary Table S1. The differentially
expressed genes and contigs were additionally annotated by
BlastX against the non-redundant database (downloaded May 23,
2013) using the Blast 2.26 stand-alone program (McGinnis and
Madden, 2004). Open reading frames were identified using the
program Transcript decoder 1.0 in the iPlant DE. Gene set and
sub-network analysis of the resulting normalized expression vales
of the genes was done using the program Pathway Studio 9.0
(Bogner et al., 2011). Only expression data from contigs with >10
transcripts per million (TPM) in all three biological replicates
from at least one of the treatment groups, and which had
significant similarity (as above) to arabidopsis genes were used for
gene set enrichment analysis or sub-network enrichment analysis
(GSEA or SNEA). Raw sequence data, normalized expression
data, and transcript assemblies are all available through the
Gene Expression Omnibus (accession # GSE63585) and links
therein.
Library
Trimmed reads
Northern 1
37394696
34672020
Northern 2
31662866
29386538
Northern 3
33400004
31161772
Central 1
36996538
34551382
Central 2
37294423
34744435
Central 3
36135432
33623100
212883959
198139247
Total
the program Trinity resulted in over 700,000 contigs (transcript
equivalents) representing over 360,000 components (gene
equivalents) with an N50 of over 1300 bases and a maximum
length of over 16,000 bases (Table 2). A comparison to a list
of highly conserved eukaryotic genes using the program Core
Eukaryotic Genes Mapping Approach (CEGMA) indicated
that over 93% were represented as full length transcripts in the
assembly with over 99% represented at least by partial sequences
(Table 3).
Similarly, assemblies were performed using the transcripts
from each of the two populations separately. Both separate
assemblies resulted in over 570,000–580,000 contigs representing
about 290,000 components each, with N50s and maximum
contig lengths very similar to the combined assembly (Table 2).
Likewise, results from the program CEGMA indicated similar
completeness of coverage of the conserved genes obtained for
both separate assemblies (Table 3).
Annotation of the Assemblies
Contigs were examined to identify those containing long open
reading frames indicative of functional genes. We identified
330,642 transcripts with reading frames over 90 amino acids in
length in the combined 745,348 contigs assembly. These 330,642
transcripts represented about 75,000 genes. Of these, 58,000
were also assembled from both population-specific sequences as
determined by aligning the resulting assembled contigs using
BlastN of. Overlap between the different the contig assembles
can be visualized in the Venn diagram (Figure 1). Of the 75,000
open reading frame-containing transcripts, 64,000 had BlastX
hits (E < 10−5 ) to the arabidopsis TAIR10 database. These blast
hits were used for the primary functional annotation of the
transcripts (Supplementary Table S1). BlastX against the nonredundant database was performed for differentially expressed
transcripts and genes (Supplementary Tables S2, S3) as well
as from a random set of 4000 open reading frame-containing
contigs. Of the 5,429 differentially-expressed transcripts for
which a source organism was identified by BlastX against the nonredundant database, 4241(77%) had the top hit corresponding
to a known plant gene, 267 had a top hit corresponding to
an animal gene (247 of which were to arthropods), 11 were
likely bacterial genes, 6 were from plasmodium 3 from protists,
and 1 from fungi. If the gene list was trimmed to include only
those transcripts that had expression values greater than 10
transcripts per million (TPM) in all three biological replicates
of either of the two treatments (populations), over 99% of the
RESULTS
Sequencing and Assembly of Transcripts
Our study presents the first transcription sequence for
A. philoxeroides. Over 212 million one hundred base, paired
end RNA sequencing reads were obtained from 3 cold-treated
individuals from both central and northern populations.
Sequences ranging from 29 and 48 million fragments were
obtained after trimming the raw reads based on sequence
quality scores with a minimum read length of 70 bases.
Fewer than 8% of the fragments were removed by the quality
trimming (Table 1). Assembly of the combined sequences by
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Raw reads
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TABLE 2 | Assembly statistics from the three assemblies.
Count
sum_len
N50
min_len
max_len
med_len
ave_len
sd_len
Data source
745348
634400797
1387
201
16184
507
851
850
Combined populations
574621
485190870
1395
201
16710
495
844
845
Northern population only
582659
488823500
1382
201
16946
493
839
843
Central population only
Count = number of contigs, sum_len = total number of bases assembled, N50 = the size in bases above which half of the transcriptome is represented in contigs equal
to or larger than the indicated value, min_len = size of smallest contig length, max len = size of largest contig, med_len = median contig length, ave_len = average contig
length, sd_len = the standard deviation of contig lengths, data source = the data set used to assemble the contigs.
Gene Set Enrichment Analysis
differentially accumulating transcripts with species information
were annotated as plant genes (Supplementary Tables S2, S3).
Far fewer non-plant hits with only 2% of non-plant origin
(data not shown) were observed in a random set of 1000
open-reading-frame-containing transcripts with hits to the nonredundant database than from the differentially expressed
set.
Gene set enrichment analysis (GESA) was used to identify
over-represented terms from the AraCyc (Müller et al., 2003)
and GO (Ashburner et al., 2000) ontologies for those genes
with arabidopsis homologues and with expression values greater
than 10 TPM in all three biological replicates from either
treatment group. A full list of all the enriched terms with p
values < 0.05 can be found in Supplementary Table S4. We
identified 28 significantly over-represented terms (p < 0.001)
associated with biochemical pathways from the AraCyc database
when the entire gene set was subjected to GSEA (Figure 2A).
All but one of these (phenylpropanoid biosynthesis) had median
expression values that suggested they were more highly expressed
in the central population. Likewise 19 and 26 over-represented
AraCyc terms were identified when genes were filtered for
those that were preferentially expressed in the northern or
Differential Gene Expression
Differential gene expression was examined using the program
RSEM to quantify and normalize the expression of both
transcripts and genes using each of the assembled transcript
files as reference transcriptomes. 6,200 differentially expressed
transcripts and 851 differentially expressed genes were identified
when the combined assembly was used as the reference
(Supplementary Tables S2, S3). Of the significantly differentially
expressed sequences, 46% of the contigs and 33% of the
genes were more highly expressed in the northern populations
in response to cold temperatures. We also examined the
differential expression using separate assemblies from either
the northern or central populations (Supplementary Table S1).
When fragments were mapped to the assembly constructed
only from sequences derived from the central population, we
observed 4,566 differentially expressed genes with only 66
genes also identified as being differentially expressed when the
transcripts were mapped to the combined assembly. Likewise,
7,318 genes were differentially expressed when transcripts were
mapped to the northern population assembly of which 1,653
were also significantly differentially expressed in the combined
assembly. Of the 66 genes common to the mapping to both the
central population assembly and the combined assembly, only
18 were found to be differentially expressed when mapped to all
three assemblies. Among the differentially expressed sequences,
approximately 251 transcripts and 12 genes encoded putative
transcription factors.
TABLE 3 | Results of completeness study using the CEGMA program.
Assembly
% complete
% partial
Combined
93.95
100
Northern only
96.37
100
Central only
97.58
FIGURE 1 | Venn diagram showing the numbers of uniquely assembled
contigs in each of the three assemblies and the number of contigs which were
assembled in more than one assembly process. The numbers in parenthesis
or brackets indicate the number of commonly assembled contigs identified
from reverse BlastN analysis. The differences in numbers for each comparison
is because some individual contigs had best matches multiple contigs in the
various analyses.
99.6
Statistics of the completeness of the genome based on the percentage of 248
Conserved Eukaryotic Genes that are represented among assembled transcripts
as either full length (complete) or as fragments of transcripts (partial).
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FIGURE 2 | (A,B) Graphic showing the median fold change of genes associated with significantly over-represented AraCyc pathways identified from (A) either the
entire dataset, or (B) selected genes that were preferentially expressed in either the central population relative to the northern population (positive values) or genes
that were preferentially expressed in the northern population (negative values).
central populations, respectively (Figure 2B). An analysis of
GO terms associated with biological processes identified 45
significantly over-represented terms for which 33 had median
expression values of the associated genes indicating preferential
expression in the central population (Figure 3A). Again, 25 and
29 biological process terms were over-represented among genes
that were preferentially expressed in only the northern or central
populations, respectively (Figure 3B).
DISCUSSION
Sequencing and Assembly Characterize
Numerous Genes From Invasive
A. philoxeroides
We performed an RNAseq analysis following a cold treatment
on three individuals of two A. philoxeroides populations. One
population from Jinan in northern China and appear to have
gained the ability to flourish in a more northern climates
previously uninhabitable by this weed. The other individuals were
from a central population near Shanghai in the center of its
established range. The goals of these experiments were to develop
a sequence database of genes from A. philoxeroides, and to
determine if these two populations differ in their transcriptomic
response to cold temperature treatments. The cold treatments
did not appear to result in obvious physical differences between
the two populations, but transcriptome differences were detected
between the tested individuals from the two populations. Further
studies are needed to determine if either population is better
able to survive and reproduce in the more northerly latitudes.
Sub-Network Analysis
The pathway studio program maintains a database of
transcription factor targets, interacting proteins, and proteins/
chemicals regulating cellular processes (Supplementary
Table S5). The results of the sub-network analysis identified a
limited number of regulatory or protein interaction networks
associated with differences in response to cold stress between
the two populations (Figures 4A,B). Even with a stringent
significance cut off of p < 0.01, we identified 12, 6, and 5 terms
when all of the genes were analyzed or when the genes were
limited to those preferentially expressed in the central and
northern populations, respectively.
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FIGURE 3 | (A,B) Graphic showing the median fold change of genes associated with significantly over-represented biological processes identified from (A) either the
entire dataset, or (B) selected genes that were preferentially expressed in either the central population relative to the northern population (positive values) or genes
that were preferentially expressed in the northern population (negative values ).
transcripts were from non-plant sources (Mantello et al., 2014).
However, the percentage of non-plant genes identified from
differentially expressed contigs, as was done in our case, is likely
to be over-represented. Low abundance genes such as those
likely coming from contaminating organisms such as insects,
bacteria and fungi, are more likely to be classified as differentially
expressed (Trapnell et al., 2013). This possibility is supported by
an assessment of non-plant transcripts from a random sample
of 1000 open-reading-frame-containing transcripts with hits to
the non-redundant database from the combined assembly in
which only 2% were suspected as coming from non-plant sources.
Regardless, our results provide a rich set of gene sequences from
this invasive weed which could be used as source for SSR or SNP
markers for further population genetic analyses.
Although a majority of the genes from the combined assembly
were represented in assemblies generated from the individual
However, differences in gene expression were observed between
the individuals from these two populations and the changes in
gene expression are consistent with cold resistance mechanisms.
These observations are most consistent with the hypothesis that
the northward expansion of A. philoxeroides into Northern China
is not simply the result of global warming, but is likely due to
evolution of adaptations to colder environments.
We identified over 75,000 genes with long open reading
frames of which >85% were significantly similar to known
arabidopsis genes. BlastX searches against the non-redundant
database of the differentially expressed transcripts (5,429)
indicated that ∼25% might result from contamination of the
samples from other organisms (mostly arthropods). A search
of the literature suggests that most RNAseq assemblies contain
about 1–3% non-plant sequences, although one study in rubber
tree (Heveabrasiliensis) indicated that 17% of the assembled
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Cold Response of Alternanthera philoxeroides
FIGURE 4 | (A,B) Graphic showing the median fold change of genes associated with significantly over-represented Sub-network ontologies identified from (A) either
the entire dataset, or (B) selected genes that were preferentially expressed in either the central population relative to the northern population (positive values) or
genes that were preferentially expressed in the northern population (negative values).
128 had expression levels >10 TMP in all biological replicates of
either treatment group (Supplementary Table S1). Surprisingly,
258 of these 1978 transcripts were classified as differentially
expressed with 4 transcripts expressed at >10 TPM in all
three biological replicates from the northern population. The
later observation suggests that even though these genes were
not assembled in the northern population assembly, a modest
populations, there were clearly unique contigs that were
assembled from a single population. Some of these are likely to be
poorly represented sequences, perhaps from contaminating DNA
from non-Alternanthera philoxeroides sources or from otherwise
low abundant sequences. This hypothesis is bolstered by the fact
that, of the 1978 contigs from the complete assembly that were
represented only in the assembly of the central population, only
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Liu et al.
Cold Response of Alternanthera philoxeroides
These processes have been associated with growth inhibition in
Arabidopsis and other plants (Besseau et al., 2007; Rivas-San
Vicente and Plasencia, 2011). Conversely, processes linked
to photosynthesis and growth such as cellulose biosynthesis,
photosynthesis - light reaction, and fatty acid biosynthesis
were over-represented among genes preferentially expressed
in the tested central population individuals. As noted above,
photosynthesis during cold treatment is associated with
production of damaging oxidative radicals (Wise, 1995).
Likewise, continued growth in the presence of oxidative
radicals could result in increased damage to DNA (Cadet et al.,
2003). Thus, although no obvious CBF regulon modification
are indicated, a potentially protective down-regulation in
photosynthetic activity and an increase in pathways that are
protective to reactive oxygen species are observed in the
northern population individuals but not the central population
individuals.
number of RNAseq reads from the northern samples still mapped
to the assembled contigs. This observation underscores the
potential problem of false positives resulting from differential
mapping rather than true differential expression when comparing
gene expression among two different accessions.
Differential Expression Suggests Genetic
Changes Provide Enhanced Cold
Tolerance of Individuals From Northern
Populations
Because of the observed high number of differentially expressed
non-plant transcripts we limited further GSEA and SNEA to
a subset of genes with >10 TPM and which has reliable
BlastX hits to known arabidopsis genes. A large number of
genes were identified as differentially expressed (q-value < 0.05)
between the central and northern populations in response to the
cold treatment. The most notable differences were involved in
photosynthesis, which appear repressed in the tested individuals
from northern populations. This observation is supported from
both an examination of the most differentially expressed genes
and from the gene set and sub-network enrichment analyses.
One of the primary causes of damage due to chilling stress is
the production of oxidative radicals produced when the electron
transport chain is disrupted by altered membrane fluidity that
prevent proper association of the proteins involved in the
electron transport in the chloroplast stroma (Wise, 1995). One
mechanism through which plants survive cold treatments is
by modifying or repressing the photosynthetic apparatus under
chilling conditions. In another study, two isogenic lines of wheat
in which a single point mutation resulted in reduced cold
tolerance (Wells et al., 1969) were used in a transcriptomics
analysis analogous to our study on A. philoxeroides (Sutton
et al., 2009). A comparison of the GSEA results from differences
between the two lines following cold treatment also implicated
altered photosynthesis as potentially underlying the differences
in cold tolerance (Karki et al., 2013).
In our study, very little evidence points to alterations in the
CBF regulon between the northern and central populations.
Neither “neighbors of CBF” or “response to cold” were indicated
as over-represented ontologies by SNEA or GSEA. Likewise, no
known targets of CBF were indicated as significantly differentially
expressed – despite the presence of putative sequences of targets
(such as a COR47-like gene) among the contigs and genes that
were assembled. However, we observed up-regulation of other
cold-responsive regulons in the northern population. Genes such
as a COR27-like and RAV1-like that in arabidopsis are modulated
through circadian clock and ABA signaling (deMontaigu et al.,
2010) were found to be significantly up-regulated in northern
populations. This observation is consistent with alterations in
upstream genes involved in responses that integrate ABA and
circadian signaling required for non-CBF-regulated cold-induced
gene expression in the northern populations.
In addition to non-CBF regulated cold inducible circadianresponsive genes, GSEA identified numerous genes in biotic
defense responses, such as salicylic acid and flavonoid
biosynthesis, as up-regulated in the northern population.
Frontiers in Plant Science | www.frontiersin.org
CONCLUSION
We have demonstrated there are differences in gene expression
associated with response to cold treatments between tested
individuals from populations of A. philoxeroides in the central
portion of its range in South China and individuals from a
population in North China which has recently expanded its
range northward. These results are most consistent with the
development and/or selection of genetic and/or epigenetic
differences that enhance the cold-tolerance of the northern
populations. Further work using a population genetics
approach will be needed to determine if the differences
observed in these individuals is representative of the whole
population, and to identify loci associated with these differences.
However, the differences we observed in gene expression point
towards alteration in the cold-responsive regulon controlling
circadian/ABA signaling rather than alterations in the CBF
regulon. Importantly, our results also provide the first largescale database resource of A. philoxeroides gene and transcript
sequences. Such sequences should serve as a rich source for
markers needed to examine the population genetics of a species
that has proven invasive on at least three continents.
AUTHOR CONTRIBUTIONS
LD, LP, and LW performed the experiments and collected
samples. DH performed primary data analysis and carried out
bioinformatics analysis. LD and DH designed the experiments
and wrote the manuscript. All authors read and approved the
final manuscript.
FUNDING
We would like to thank for Shandong Science & Technology
Program (2009GG10008014) and Shandong Environmental
Protection Research and Development Program (2010 Xiaoqing
River Ecological Monitoring) for funding this project.
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Cold Response of Alternanthera philoxeroides
ACKNOWLEDGMENTS
TABLE S1 | Annotation and expression analysis of all assembled contigs from
both the combined assembly and from the assembly of the transcriptome from
the three plants collected from the northern and southern populations separately.
We thank Profs. Han Fai, Du Xihua, and Dr. Fan
Weijuan for their assistance. Thanks also Dr. Katrina
Dlugosch for critical review of this manuscript prior to
submission.
TABLE S2 | Annotation and gene expression analysis of the assembled contigs of
transcripts determined to be differentially expressed between the northern and
southern populations.
TABLE S3 | Annotation and gene expression analysis of the clustered contigs of
genes determined to be differentially expressed between the northern and
southern populations.
SUPPLEMENTARY MATERIAL
TABLE S4 | Gene set enrichment analysis from the indicated sub-populations of
genes.
The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fpls.2019.00024/
full#supplementary-material
TABLE S5 | Sub-network enrichment analysis from the indicated sub-populations
of genes.
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Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Copyright © 2019 Liu, Horvath, Li and Liu. This is an open-access article distributed
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