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Received: 18 December 2023
|
Accepted: 5 February 2024
DOI: 10.1002/ajb2.16310
SPECIAL ISSUE ARTICLE
Halophytes and heavy metals: A multi‐omics approach
to understand the role of gene and genome duplication
in the abiotic stress tolerance of Cakile maritima
Shawn K. Thomas
1,2,3
|Kathryn Vanden Hoek
4
|Tasha Ogoti
5
|
Ha Duong
3,4
|Ruthie Angelovici
1,3
|J. Chris Pires
6
|
David Mendoza‐Cozatl
3,7
|Jacob Washburn
3,8
|Craig A. Schenck
3,4
1
Division of Biological Sciences, University
of Missouri, Columbia, MO 65211, USA
2
Bioinformatics and Analytics Core, University
of Missouri, Columbia, MO 65211, USA
3
Interdisciplinary Plant Group, University
of Missouri, Columbia, MO 65211, USA
4
Department of Biochemistry, University
of Missouri, Columbia, MO 65211, USA
5
Department of Computer Science, University
of Missouri, Columbia, MO 65211, USA
6
Soil and Crop Sciences, Colorado State
University, Fort Collins, CO 80523‐1170, USA
7
Division of Plant Sciences and Technology,
University of Missouri, Columbia, MO
65211, USA
8
Plant Genetics Research Unit, USDA‐ARS,
Columbia, MO 65211, USA
Correspondence
Shawn K. Thomas, Division of Biological Sciences,
University of Missouri, Columbia, MO 65211
USA.
Email: shawnkt4@gmail.com
Craig A. Schenck, Department of Biochemistry,
University of Missouri, Columbia, MO 65211
USA.
Email: caschenck@missouri.edu
This article is part of joint special issues of the
American Journal of Botany and Applications in
Plant Sciences:“Twice as Nice: New Techniques
and Discoveries in Polyploid Biology.”
Abstract
Premise: The origin of diversity is a fundamental biological question. Gene
duplications are one mechanism that provides raw material for the emergence of
novel traits, but evolutionary outcomes depend on which genes are retained and how
they become functionalized. Yet, following different duplication types (polyploidy and
tandem duplication), the events driving gene retention and functionalization remain
poorly understood. Here we used Cakile maritima, a species that is tolerant to salt and
heavy metals and shares an ancient whole‐genome triplication with closely related
salt‐sensitive mustard crops (Brassica), as a model to explore the evolution of abiotic
stress tolerance following polyploidy.
Methods: Using a combination of ionomics, free amino acid profiling, and
comparative genomics, we characterize aspects of salt stress response in C. maritima
and identify retained duplicate genes that have likely enabled adaptation to salt and
mild levels of cadmium.
Results: Cakile maritima is tolerant to both cadmium and salt treatments through
uptake of cadmium in the roots. Proline constitutes greater than 30% of the free
amino acid pool in C. maritima and likely contributes to abiotic stress tolerance. We
find duplicated gene families are enriched in metabolic and transport processes and
identify key transport genes that may be involved in C. maritima abiotic stress
tolerance.
Conclusions: These findings identify pathways and genes that could be used to
enhance plant resilience and provide a putative understanding of the roles of
duplication types and retention on the evolution of abiotic stress response.
KEYWORDS
abiotic stress, amino acid, gene duplication, ionomics, polyploidy
Gene duplications are a source of evolutionary novelty. In
plants, gene duplication events are abundant and have
spurred the evolution of diverse adaptive responses to
extreme environments (Vanneste et al., 2014; Panchy
et al., 2016). However, evolutionary outcomes depend on
which genes are retained, with most duplicated genes being
lost. Although rare, retention of duplicates appears to be a
non‐random process (Rody et al., 2017). Retention and
functional divergence depend on the type of duplication
event and the original function of the gene that was
Am J Bot. 2024;e16310. wileyonlinelibrary.com/journal/AJB
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https://doi.org/10.1002/ajb2.16310
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the
original work is properly cited.
© 2024 The Authors. American Journal of Botany published by Wiley Periodicals LLC on behalf of Botanical Society of America.
duplicated (Rody et al., 2017; Birchler and Yang, 2022). The
two major mechanisms for duplicating genes are polyploidy
events resulting in whole‐genome duplications and triplica-
tions (WGD, WGT) and small‐scale duplications (SSD)
(Edger and Pires, 2009; Freeling, 2009; Conant et al., 2014;
Conant, 2014; Vance and McLysaght, 2023). Retention of
duplicate genes and functional divergence drive gene family
expansions, which can lead to novel traits (Vanneste
et al., 2014; Panchy et al., 2016; DeGiorgio and Assis, 2021).
After polyploidy, redundancy, increased dosage, and rewir-
ing of gene networks can lead to novel metabolic,
regulatory, or developmental pathways key in stress biology
(Van de Peer et al., 2021; Tossi et al., 2022).
Two major abiotic stresses impacting agriculture are
salinization of soils and accumulation of heavy metals
(Minhas et al., 2017;Guletal.,2022). Halophytes (plants
tolerant to salt) and heavy metal accumulators employ diverse
strategies to mitigate stresses and thrive in inhospitable soils
(Hasanuzzaman et al., 2014; Kazachkova et al., 2018). Some of
these strategies include exclusion, uptake, and sequestration
of salt and/or heavy metals, salt glands to exude excess ions,
and alteration of metabolite profiles to cope with stress
(Mendoza‐Cózatl et al., 2011; Hasanuzzaman et al., 2014;Oh
et al., 2015; Kazachkova et al., 2018;Yanetal.,2020).
However, most crop species are glycophytes; that is, they are
plants susceptible to salt stress (Hasanuzzaman et al., 2014).
Since crop wild relatives can be locally adapted to diverse
environments, some of which are impacted by salt and heavy
metals (Lopes et al., 2015;Millaetal.,2018;Melinoand
Tester, 2023), they provide a source of genetic variation that
may be useful in crop improvement strategies. Metabolically
stress‐adapted plants often accumulate osmoprotectants, such
as the amino acid proline and sugars, before stress or in
response to stress (Slama et al., 2015). Halophytes display a
wide range of salt concentration tolerances, and depending
on the threshold to define a halophyte, there are estimated to
be anywhere from around 350–2600 halophytes distributed
across the green plant phylogeny (Flowers and Colmer, 2008;
Flowers et al., 2010; Bromham, 2015) Often, strategies for
abiotic stress tolerance have evolved following gene duplica-
tion and neofunctionalization or subfunctionalization of
duplicated genes (Schranz et al., 2012). Natural variation
within crop wild relatives can be used to identify mechanisms
by which these plants cope with abiotic stress and provide
genetic tools to enhance crop resilience.
Cakile maritima Scop. (Brassicaceae) is an facultative
halophyte (Arbelet‐Bonnin et al., 2018,2019)nativeto
coastal dunes in the Mediterranean and has invaded
coastlines across the globe. It is a crop wild relative that is
closely related to canola and other Brassica crops (Arias and
Pires, 2012; Walden et al., 2020; Hendriks et al., 2023). Cakile
maritima exhibits typical halophytic behavior in response to
salt stress such as exclusion, uptake, and sequestration
(Hamed‐Louati et al., 2016;Arbelet‐Bonnin et al., 2018,2019)
and has been shown to be tolerant to heavy metals (Taamalli
et al., 2014). Yet how C. maritima has evolved to tolerate
abiotic stress is not well known. Cakile maritima shares the
recent whole‐genome triplication (WGT) with Brassica crops
called Br‐⍺WGT (Lysak et al., 2005,2007; Tang et al., 2012;
Emery et al., 2018; Qi et al., 2021) and is closely related to
other salt‐tolerant Brassicaceae including Eutrema salsugi-
neum and Schrenkiella parvula that do not share the more
recent Br‐⍺WGT (Figure 1A) (Walden et al., 2020; Hendriks
et al., 2023). Thus, C. maritima is well positioned
phylogenetically to investigate the role of duplication events
(SSD and WGT) on evolution of stress tolerance.
In this study, we investigated the role of retained
duplicate genes in stress adaptation from the Br‐⍺WGT
and SSD events in C. maritima using a combination of
ionomics, free amino acid profiling, and comparative
genomics. We found that C. maritima can take up ions
and overaccumulate stress metabolites. We identified
expanded gene families, some of which show evidence of
functional divergence, and hypothesized that these may
contribute to the abiotic stress adaptation of C. maritima.
These findings provide insight into the roles of duplication
on abiotic stress tolerance.
A
B
FIGURE 1 Cakile maritima is tolerant to salt and heavy metals and
shares the Br‐⍺WGT with Brassica rapa. (A) Cladogram depicting
relationships between species used in this study with representative images.
Ancient polyploidy events are indicated by the four‐and six‐pointed stars.
C. maritma and B. rapa images are from the public domain (licensed under
https://creativecommons.org/publicdomain/zero/1.0/). Schrenkiella
parvula image provided by Dr. Pramod Pantha. Eutrema salsugineum
image observed in Russia by Сергей (licensed under https://
creativecommons.org/licenses/by-nc/4.0/). Arabidopsis thaliana image
observed in Germany by Wolfgang Jauch (licensed under https://
creativecommons.org/licenses/by/4.0/) (B) A. thaliana, B. rapa, and C.
maritima were grown for 6 days on ½ MS plates and moved to plates
containing 150 mM NaCl (salt stress), 20 μM cadmium (Cd
2+
heavy metal
stress), or control plates, then grown for 5 days. Root length was measured
using ImageJ. Bars represent mean root length ± SEM of N ≥6 biological
replicates. *P< 0.01 in unpaired t‐test.
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MATERIALS AND METHODS
Plant growth on plates
Seeds of Arabidopsis thaliana (L.) Heyn. Col‐0 (Brassicaceae),
Brassica rapa L. R500 (Brassicaceae), and Cakile maritima
(henceforth referred to as Arabidopsis, Brassica, and Cakile;
accession information is available in Appendix S1)were
surface‐sterilized with 90% v/v ethanol for 1 min, 15% v/v
bleach for 12 min, and rinsed five times for 6 min each with
sterilized water. Sterilized seeds were placed on plates
containing half‐strength Murashige and Skoog (½x MS)
agar plus 1% w/v sucrose for germination. After 6 days,
seedlings were moved to plates to induce different stresses: ½x
MS agar + 0.5% sucrose supplemented with 150 mM NaCl for
salt stress, ½x MS agar + 0.5% sucrose supplemented with
20 µM CdCl
2
for heavy metal stress, or ½x MS agar + 0.5%
sucrose for the control (Tran et al., 2022). The NaCl and Cd
2+
concentrations were chosen becausetheywouldimposea
stress, but would not kill sensitive plants within the time
frames of the experiments (Tran et al., 2022). Plants were
grown at 23°C with a photosynthetic photon flux density of
~140 mM m
–2
s
–1
and 16 h light/8 h dark. After 5 days, plants
on the plates were imaged with a camera (Canon PowerShot
SX540 HS), and root length was measured using ImageJ
software of N≥6 biological replicates (Figure 1;AppendixS2).
Hydroponics and ionomics
Cakile seeds were surface‐sterilized and germinated on ½x
Hoagland modified basal salt agar in plastic cups (236.5 mL
[8 oz]) with dome lids to increase humidity. Once the
seedlings were approximately 2.5 cm tall, the lids were
removed, and the cups were placed in a flat tray filled with
water. When seedlings had adapted to lower humidity levels
(after approximately 6 days), they were transferred to
hydroponics containers.
To house the hydroponics solution, we used black
plastic cans (3.79 L [1 gallon]) with lids. In the lid, three
large holes were cut to house a foam sponge with a slit
downthemiddle.Cakileseedlingswereplacedinsidethe
foam sponge, and the containers were filled with ½x
Hoagland solution that had been aerated and adjusted to
pH 5.6 as described by Nguyen et al. (2016). The solution
was changed every 2 weeks. Hydroponics containers with
plants were kept in a growth chamber set to 22–23°C,
55% humidity, photosynthetic photon flux density of
120–140 mM m
–2
s
–1
,anda16hlight/8hdark.Cakile
plants were grown for 4 weeks before stress treatments.
Cakile plants were transferred to new hydroponics
solutions containing 150 mM NaCl, 20 µM CdCl
2
, and
150 mM NaCl + 20 µM CdCl
2
or ½x MS+0.5% sucrose
control solution. Leaf and root tissue were collected at 0, 24,
and 48 h of stress treatment to identify elemental changes
occurring rapidly during stress treatment. Tissue samples
were washed, processed, and analyzed using inductively
coupled plasma optical emission spectroscopy (ICP‐OES)
following methods from Chen et al. (2006) for data on K
+
,
Na
+
,Fe
2+
,Zn
2+
, and Cd
2+
concentrations (µg/mg dry mass)
(Figure 2). We monitored changes in these metals because
K
+
and Zn
2+
are essential nutrients and their levels can be
affected by Na
+
(during salt stress) and Cd
2+
, respectively
(Hauser and Horie, 2010; Mendoza‐Cózatl et al., 2011).
Maintenance of high K
+
/Na
+
ratios is an important
phenotype in salt‐tolerant plants (Hauser and Horie, 2010),
and Cd
2+
can disrupt Zn
2+
transporter activity (Mendoza‐
Cózatl et al., 2011). Fe
2+
was also measured because it is
linked to stress‐induced reactive oxygen species (ROS)
accumulation, which leads to iron‐sulfur cluster degrada-
tion (Zandalinas et al., 2020).
Amino acid extraction, detection,
and quantification
Tissue was collected from plants grown in a growth
chamber for analysis of amino acids without stress
(Figure 3A; Appendices S3–S5). Whole seedlings were used
from salt treatments on plates (Figure 3B; Appendix S6). All
plants were grown with 16 h light/8 h dark at 22°C with a
photosynthetic photon flux density at ~140 mM m
–2
s
–1
.
Tissue was harvested and flash frozen in liquid nitrogen and
lyophilized for 72 h. Lyophilized tissue was pulverized with
3‐mm glass beads using a Spex SamplePrep 2010 Geno/
Grinder (Cole‐Parmer, Metuchen, NJ, USA), and free
amino acids were extracted, detected, and quantified by
liquid chromatography‐tandem mass spectrometry (LC‐
MS/MS) as previously described (Angelovici et al., 2013;
Yobi et al., 2020).
Identification of expanding and contracting
gene families
The primary coding sequence (CDS) and amino acid
sequences from Arabidopsis (Lamesch et al., 2012), Brassica
(Lou et al., 2020), Cakile (Brassicales Map Alignment Project,
DOE‐JGI, http://bmap.jgi.doe.gov/), Eutrema salsugineum
(Pall.) Al‐Shehbaz & Warwick (Brassicaceae) (Yang et al.,
2013) (henceforth referred to as Eutrema) and Schrenkiella
parvula (Shrenk) D.A. German & Al‐Shehbaz (Brassicaceae)
(Dassanayake et al., 2011; Oh et al., 2014)(henceforth
referred to as Schrenkiella) were obtained from Phytozome
and the Brassicales Map Alignment Project (BMAP) project
(with permission from the project's principal collaborators;
data sources can be found in Appendix S1). Orthologous
gene families (orthogroups) were inferred using amino acid
sequences in OrthoFinder version 2.5.4 (Emms and
Kelly, 2019) with parameters ‐t20and‐a20touse20
parallel analysis threads for sequence search, MSA and tree
inference (‐t 20), and internal, RAM intensive tasks (‐a20).
All other parameters were left as default. Using the inferred
orthogroups from Orthofinder, we examined gene family
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expansion and contraction of orthogroups using CAFE5
(Mendes et al., 2021). Gene counts per species for all
orthogroups were extracted from the Orthofinder output,
and orthogroups with more than 100 gene copies in one or
more species were removed. Two hypotheses were tested to
account for variation in evolutionary rates (λ)acrossthe
phylogeny: First, λis constant across the tree and, second,
λchanges after the Br‐⍺WGT event. In the first hypothesis,
A
B
C
FIGURE 2 Elemental profile for Cakile maritima after abiotic stress. Plants grown hydroponically for 4 weeks, then grown under control conditions
(‐Salt/‐Cd), (A) +Salt/‐Cd (150 mM NaCl), (B) ‐Salt/+Cd (20 uM Cd
2+
) or (C) +Salt/+Cd (150 mM NaCl + 20 uM Cd
2+
). Following stress treatment, root
and shoot tissue samples were harvested at 0, 24, and 48 h. Tissue samples were washed, processed, and elemental concentrations K
+
,Na
+
,Fe
2+
,Zn
2+
, and
Cd
2+
were measured using ICP‐OES. Each graph shows measurements over time per each tissue and ion combination in stress treatments; red = +Salt/‐Cd,
blue = ‐Salt/+Cd, and purple = +Salt/+Cd) vs the control (grey = ‐Salt/‐Cd). Points represent the mean (N= 3); error bars show standard error. Significant
differences in the experimental treatments by time compared to the control (‐Salt/‐Cd) are denoted by an asterisk (*P< 0.05; **P< 0.01, Kruskal–Wallis test).
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one λparameter was inferred for the whole tree, and in the
second hypothesis, two separate λparameters were inferred,
one for the Br‐⍺WGT clade (including Cakile and Brassica
branches; Figures 1A, 4A) and another for the rest of the tree.
The model with a separate λparameter for the Br‐⍺clade had
ahigherlikelihood(final likelihood, –lnL: 234,507) than the
model with constant λparameter (final likelihood, –lnL:
244,248) and was used for downstream analyses. The
λparameters estimated for the Br‐⍺WGT clade and
the rest of the tree were ~0.022 and ~0.004, respectively.
The cutofffor significant gene family expansions or
contractions was set as P< 0.05. Orthogroups with significant
FIGURE 3 Proline accumulates in Cakile maritima before salt stress. (A) Proline composition of Arabidopsis thaliana,Brassica rapa, and C. maritima
tissues grown under standard conditions. Pro levels were higher in C. maritima before stress in all tissues analyzed compared to A. thaliana and B. rapa. Bars
are means ± SEM of N≥4 biological replicates. (B) Amino acid (aa) changes during salt stress. A. thaliana,B. rapa, and C. maritima were grown for 6 days
on ½ MS plates and moved to plates containing 150 mM NaCl; roots and leaves from whole seedlings were analyzed for amino acids after 0, 2, 24, and 72 h
of salt stress. Select amino acids are shown (full amino acid profiles: Appendix S6). Bars represent means ± SEM of N≥4 biological replicates.
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expansions at the Cakile branch and the branch shared by
Brassica and Cakile (Br‐⍺branch) were binned into three
sets: Cakile specific expansions, Br‐⍺specific expansions, and
expansions shared at both branches.
Identification and classification of duplicate
gene classes
Orthogroups in which all five species (Cakile, Brassica,
Arabidopsis, Eutrema, Schrenkiella) retained sequences were
classified into duplication classes: single copy (SC), small‐scale
duplication (SSD), whole‐genome triplication (WGT), and
whole‐genome triplication and small‐scale duplication (WGT
+SSD). This filtering was based on Cakile duplications
enriched at branches of ortholog gene trees (Figure 4). The
SC group represents genes maintained in single copies in all
five species. The SSD group represents orthogroups enriched
for duplications only at the Cakile branch in gene trees. The
WGT group represents orthogroups enriched for duplications
only at the branch shared by Brassica and Cakile (Br‐⍺
branch). The WGT+SSD group represents orthogroups that
are enriched for duplications at both the Cakile and the Br‐⍺
branch. This approach for classifying duplicate genes in Cakile
has some limitations as differential retention of duplicates in
various lineages could skew the expected patterns described
above. If gene loss is not consistent across species, differential
retention of genes could make WGT genes appear as SSD as
A
rabidopsis
Eutrema
Schrenkiella
Brassica
Cakile
+549/-133
+28/-67
+13/-127
+928/-176
+550/-448
+184/-1309
+356/-1106
+300/-1122
A
rabidopsis
Eutrema
Schrenkiella
Brassica
Cakile
A
rabidopsis
Eutrema
Schrenkiella
Brassica
Cakile
Cakile
A
rabidopsis
Eutrema
Schrenkiella
Brassica
Cakile
Brassica
Cakile
Brassica
Cakile
A
rabidopsis
Eutrema
Schrenkiella
Brassica
Cakile
Brassica
Cakile
Brassica
Cakile
Cakile
5979 SC 2621 WGT1284 WGT+SSD3009 SSD
197 Br-α 352 Shared 576 Cakile
A
B
FIGURE 4 Expanded gene families and orthogroup classification for Cakile maritima. (A) Summary of CAFE results for significantly expanding/
contracting orthogroups. The blue and red numbers respectively indicate the number of significant (P< 0.05) gene family expansions and contractions per
branch as inferred by CAFE. Significantly expanding orthogroups at the Br‐⍺and C. maritima branches were extracted and grouped as Br‐⍺‐specific,
C. maritima‐specific, and shared orthogroups. Counts for these groups are in gold, green, and purple, respectively. (B) Duplication classification workflow of
C. maritima genes. Inferred orthogroups were classified as single copy (SC), small‐scale duplication (SSD), whole‐genome triplication (WGT), or WGT+SSD
based on their duplication status in relation to the C. maritima and Br‐⍺nodes. Phylogenetic tree under each classification show which branches are
enriched for duplications and the numbers next to them represent the number of orthogroups in each class. Green X: SSD duplication event; yellow star:
Br‐⍺WGT.
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genes in other species sharing the polyploidy were lost and
not retained. Further it is possible for duplicates showing a
WGT pattern to be a retained SSD in the ancestral lineage.
For example, an SSD in the ancestor of Cakile and Brassica
would be inferred as being derived from WGT as it would
appear as a duplication at the Br‐⍺branch.
The WGT groups (WGT and WGT+SSD) were
subjected to additional filtering based on the distribution
of the synonymous divergence (Ks) between paralogs in
Cakile. The FASTKs pipeline (McKain et al., 2016) was used
to obtain the Ks distribution for Cakile and assess pairwise
ortholog divergence between Cakile and Brassica to
Arabidopsis, and Cakile to Brassica. In this pipeline, CDS
sequences for each sample were aligned against themselves
with an e‐value cutoffof 1e‐40 using BLAST (Altschul
et al., 1990). Putative pairs were identified as BLAST hits
when they met the following criteria: (1) greater than 40%
identity in alignment, (2) not an exact match (100%
identity), and (3) longer than 300 bp of alignment length.
Amino acid sequences for the putative pairs were aligned
with MUSCLE (Edgar, 2004) and converted to CDS using
PAL2NAL (Suyama et al., 2006). Ks, Ka, and Ka/Ks were
estimated for the aligned pairs using codeml in PAML v.4.8
(Yang, 2007) with the same parameters used by McKain
et al. (2012). The previously identified WGT groups were
filtered to only include orthogroups containing Cakile genes
within the Ks range of 0.082 to 0.538 indicative of the Br‐⍺
WGT (Qi et al., 2021). Despite some inferential limitations,
as stated above, we expect that large‐scale patterns of gene
duplication classes described in this study are accurate.
Functional enrichment of orthogroup classes
The previously identified significantly expanding orthogroups
and duplication classes were assessed for Gene Ontology
(GO) term enrichment. Orthologous Arabidopsis genes in
these classes were used as the input to gain a putative
understanding of functional binning of all biological processes
and specific processes related to salt, metal, amino acid
metabolism, and ROS. For each class, GO enrichment analysis
was performed using ShinyGO 0.77 (Ge et al., 2020).
Arabidopsis was used as the reference database and the
parameters FDR cutoff= 0.05 and Pathway size min‐
max = 20–2000 were used. After enrichment, the resulting
FDR enrichment values and GO Biological Process terms
were extracted for the duplication classes. These were used as
inputs for REVIGO (Supek et al., 2011) to cluster terms based
on semantic similarity.
Associated loci to the GO terms response to salt stress
(GO:0009651) and response to cadmium ion (GO:0071276)
from The Arabidopsis Information Resource (TAIR) data-
base were extracted and mapped to duplication classes to
gain a specific understanding of salt and cadmium functional
binning per duplication class. For each duplication class, GO
enrichment analysis was performed using ShinyGO version
0.77 (Ge et al., 2020). Arabidopsis was used as the reference
database and all other parameters were default (FDR
cutoff=0.05;pathwaysizemin‐max = 2–2000). After enrich-
ment, the resulting FDR enrichment values, GO molecular
function, and KEGG terms were extracted.
RESULTS
Cakile is tolerant to salt and cadmium stress
To quantify the degree of abiotic stress tolerance, we
challenged Cakile along with other Brassicaceae lineages
Arabidopsis and Brassica, both known to be salt and
cadmium sensitive, with salt and cadmium stress. Arabi-
dopsis and Brassica showed significantly shorter roots and
photobleaching on cadmium and salt (Figure 1B; Appen-
dix S2), whereas Cakile was not affected by these treatments
and maintained root growth (Figure 1B; Appendix S2).
Cakile uptakes salt and cadmium
in a tissue‐specific manner
To investigate the ability of Cakile to accumulate elements in a
tissue‐specific manner, we performed elemental profiling
(ionomics) from various tissues of Cakile grown in hydroponic
media during stress treatments. Upon the addition of salt in the
hydroponic media, Na
+
levels increased slightly in both tissues
over the 48 h of salt treatment (Figure 2). K
+
levels were
consistently higher in leaves compared to roots, with a slight
decrease in the roots over the 48‐hperiod(Figure2). Fe
2+
levels
showed a transient spike in the roots after 24 h and then
returned to baseline levels, whereas they did not change in the
leaf tissue (Figure 2). Zn
2+
levels did not change substantially
upon salt stress and as expected, Cd
2+
was not detectable
(Figure 2). When cadmium was applied in the hydroponic
media, Cd
2+
levels increased in the roots, but not the leaves,
indicating that Cakile does not exclude Cd
2+
,butdoes,atleast
in this context, take up Cd
2+
in its roots (Figure 2). Cakile does
not appear to move Cd
2+
into its leaves within 48 h (Figure 2).
Zn
2+
,Fe
2+
,K
+
,andNa
+
levels were unaltered following
cadmium stress (Figure 2). When salt and cadmium were
applied together, the response was similar to the salt‐only
treatment, including a transient spike in Fe
2+
at 24 h in the
roots, but not leaves (Figure 2). Finally, there was a very minor
increase in Cd
2+
in the roots after 48 h, but not as dramatic
a change as observed when Cd
2+
stress was applied alone
(Figure 2).
Cakile metabolic response during salt stresses
To better understand how Cakile metabolically responds to
salt stress, we extracted and quantified total free amino acids
from various tissues in the presence and absence of stress. In
general, roots contained the least amount of total free amino
acids and leaves the most across the species (Appendix S3).
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Cakile had the lowest amount of total free amino acids
compared to Arabidopsis and Brassica (Appendix S3), making
comparisons of absolute levels of amino acids between species
challenging. Additionally, changes in low‐abundance amino
acids can be masked by the most‐abundant amino acids, for
example, glutamine, glutamate, and aspartate (Appendix S4).
Relative amino acid composition can capture important, but
subtle, metabolic changes across the species; thus, percentage
compositions (the amount of an individual amino acid over
the sum of all free amino acids in a single extraction) were
compared across species (Appendix S5). The percentage
compositions of most amino acids were consistent across
species and tissues, for example, serine, valine, and tyrosine
(Appendix S5). One major exception was the relative proline
(Pro) composition. As a percentage of the total free amino
acids, Pro was dramatically higher in Cakile compared with
Arabidopsis and Brassica (Figure 3A). In Cakile, Pro
contributed to 10.8, 32.2, and 23.5% of the amino acid
composition in the roots, stems, and leaves, respectively
(Figure 3A), compared to 5.7, 8.5, and 7.2% Pro in Arabidopsis
roots, stems, and leaves, respectively, and 2.7, 12.9, and 14.2%
Pro in Brassica roots, stems, and leaves (Figure 3A).
Cakile, Arabidopsis, and Brassica were also investigated for
amino acid composition changes during salt treatment. Whole
seedlings were harvested at 0, 2, 24, and 72 h during salt
treatment to analyze amino acids. Amino acid changes could
be classified into three general categories during salt stress: no
change, increase, or decrease. Amino acids such as alanine had
no substantial change during salt treatment (Figure 3B;
Appendix S6). Amino acids such as threonine and Pro
increased during salt treatment; however, Pro increased
substantially more in Arabidopsis and Brassica (13.5‐and
15‐fold, respectively) compared with Cakile (2.6‐fold) during
salt stress (Figure 3B; Appendix S6). Amino acids such as
glutamine tended to decrease during salt treatment (Figure 3B;
Appendix S6). A principal component analysis of the amino
acidcontentduringsaltstressrevealedthatthethreespecies
could be distinctly grouped (Appendix S7A). The major amino
acids contributing to the model were the branched chain
amino acids (leucine, valine, and isoleucine), and amino acids
involved in nitrogen metabolism including aspartate, aspara-
gine, glutamate, and glutamine (Appendix S7B), whereas Pro
was only a minor contributor to the model (Appendix S7B).
Nitrogen metabolism amino acids were inversely correlated
with Pro, suggesting that when Pro levels increased, generally
nitrogen metabolism amino acids decreased (Figure 3B;
Appendices S6,S7B).
Expanding and contracting Cakile gene families
To identify pathways and genes contributing to Cakile abiotic
stress response, we first identified expanding and contracting
gene families at the Br‐⍺WGT branch and the Cakile specific
branch in relation to the other Brassicaceae species used in the
phylogenetic analyses. In total, 24,813 orthogroups were
identified and after filtering served as the input for CAFE5
analysis (Mendes et al., 2021). Testing multiple evolutionary
rate hypotheses identified a model where the Br‐⍺clade had a
separate evolutionary rate from the rest of the tree as the
optimal model with the highest likelihood (final likelihood
–lnL: 234,507). This model identified 549/708 significantly
expanding/contracting orthogroups at the Br‐⍺branch and
928/176 significantly expanding and contracting orthgroups at
the Cakile specific branch (Figure 4A). GO enrichment
of expanding orthogroups identified biological processes
related to specialized metabolism, photosynthesis, response to
amino acid and defense response enriched at Br‐⍺;protein
ubiquitination, proteolysis, RNA dependent DNA biosynthesis
at the Cakile branch; RNA‐dependent DNA biosynthesis,
fungal defense response, pollen recognition, unsaturated fatty
acid metabolism shared at both branches (Appendix S8).
Classification of duplicates in Cakile
We hypothesized that duplication and divergence has
contributed to Cakile abiotic stress adaptation; therefore,
expanded gene families in Cakile could arise from
polyploidy events, including the Br‐⍺WGT and/or
Cakile‐specific SSDs. Orthogroups were classified based on
their duplication status: single copy (SC), small‐scale
duplication (SSD), whole‐genome triplication (WGT), and
whole‐genome triplication plus small‐scale duplication
(WGT+SSD). These classes were inferred based on the
phylogenetic context of orthogroup gene trees and the
distribution of the synonymous divergence (Ks) between
Cakile paralogs (Figure 4B). For example, when a single
copy was phylogenetically distributed across all five species
in a gene tree, these were classified as SC. Gene trees with
duplications localized to a Cakile specific branch were
classified as SSD, and gene trees classified as WGT only
contained duplications at branches shared by Cakile and
Brassica (Br‐⍺). Finally, WGT+SSD represent cases where
gene trees contained duplications at both Cakile‐specific
branches and Br‐⍺branches. Of the 24,813 orthogroups,
17,152 were present across all five species (Cakile, Brassica,
Schrenkiella, Eutrema, and Arabidopsis); 5979 of these were
present as single‐copy genes across all the species analyzed
(Figure 4B); 3009 orthogroups were classified as resulting
from SSD only and showed enrichment only in Cakile
(Figure 4B); 2621 orthogroups were classified as resulting
from WGT only; and 1284 were classified as resulting from
both WGT and SSD (WGT+SSD) (Figure 4B). The
remaining orthogroups were enriched for duplications only
at nodes outside of Br‐⍺and Cakile and were not included
in downstream analyses.
Partitioning of biological process terms across
Cakile duplication classes
Using GO term enrichment of biological processes, we
discerned the partitioning of biological processes across
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Cakile duplication classes and identified where terms related
to salt and cadmium tolerance occurred. The Arabidopsis
orthologs in orthogroups identified as different Cakile
duplication classes and were subject to gene enrichment
analyses. All significantly enriched terms per duplication class
can be found in Appendix S9. In summary, 107, 65, 681, and
304 GO biological process terms were enriched (FDR <0.05)
in the SC, SSD, WGT and WGT+SSD duplication classes,
respectively. The SC category contained GO terms associated
with core cellular functions such as DNA repair, RNA
modification, macromolecule modification, and plastid
organization (Figure 5A). Transport and metabolic processes
such as amide biosynthetic process, peptide biosynthetic
process and cellular amide metabolic process were enriched
in the SSD category (Figure 5B). The WGT category had
many functionally enriched categories associated with
regulation/regulatory elements, abiotic responses, and core
developmental functions, such as regulation of metabolic
A
B
C
D
FIGURE 5 Gene ontology enrichment of Cakile maritima duplication classes. Arabidopsis thaliana genes from orthogroups were used as input for GO
Enrichment using ShinyGO (Ge et al., 2020). Enriched GO biological process terms and their FDR enrichment values were used as inputs for REVIGO
(Supek et al., 2011) to cluster semantically similar terms. The x‐and y‐axes represent semantic space used to cluster semantically similar GO terms (Supek
et al., 2011). Displayed terms include the most significant terms based on EnrichmentFDR value (SC: value <–10; SSD: value <–2; WGT: value <–20; WGT
+SSD: value <–4.59). Salt‐or cadmium‐specific terms are shown in red; terms with an asterisk did not meet the cutofffor the displayed term. The full list of
significantly enriched categories can be found in Appendix S9. Color of circles is based on EnrichmentFDR value; size of circles indicates the number of
terms clustered. (A) Single‐copy (SC) orthogroups. (B) Small‐scale duplication (SSD) orthogroups. (C) Whole‐genome triplication orthogroups. (D) Whole‐
genome triplication + small‐scale duplication (WGT+SSD) orthogroups. Phylogenetic trees show which branches are enriched for duplications. Green X:
SSD duplication event; yellow star: Br‐⍺WGT.
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process, developmental process, and DNA‐templated tran-
scription (Figure 5C). Finally, the WGT+SSD category was
enriched for response to cadmium ion, xenobiotic transport,
amino acid metabolism, and terms related to core cellular
process processes (Figure 5D).
Given the metabolic and ionomic response of Cakile
followingabioticstress,weexpectedtoidentifybiologi-
cal processes related to salt, metal, amino acid metabo-
lism, and ROS. Consistent with our hypothesis, salt‐
specific terms were found exclusively in the WGT
(response to salt stress and regulation of response to
salt stress) and WGT+SSD (response to salt stress and
cellular response to salt stress) categories (Figure 5).
Metal‐related terms were identified across all duplication
classesexceptSC,butcadmium‐specifictermswere
identified only in the WGT and WGT+SSD categories
(Figure 5). In relation to the high potassium abundance
in salt‐treated Cakile (Figure 2), potassium ion transport
was found in the WGT category (Figure 5C). Amino acid
metabolism terms were found in all categories except SC.
Specifically, amide biosynthetic process and peptide
biosynthetic process were among the top enriched terms
in the SSD and WGT+SSD categories (Figure 5). This
term was also enriched in the WGT category; however, it
was the 523rd ranked term by FDR enrichment
(Figure 5). Enrichment of amino‐acid‐related metabolic
processes tend to be retained more commonly following
Cakile‐specificduplicationsandmaybeinvolvedin
metabolic pre‐adaptation to abiotic stress. Finally, ROS‐
specific terms were found in all categories except for SC.
Response to oxidative stress was found in the SSD and
WGT categories, reactive oxygen metabolic process was
found in SSD, and response to oxygen‐containing
compound was found in the WGT and WGT+SSD
categories (Figure 5). Based on the trends observed,
duplicate retention post polyploidy is enriched for
regulation and stress terms, whereas Cakile‐specific
duplications are enriched in metabolic processes, high-
lighting the relevance of duplication type in various
aspects of abiotic stress response.
Salt‐and cadmium‐responsive genes show
evidence of duplication and divergence
Next, we investigated the duplication status and divergence of
known salt‐and cadmium‐responsive genes to understand
what pathways may be contributing to Cakile stress tolerance.
Enriched GO molecular function and KEGG pathways were
identified from known salt‐(Figure 6) and cadmium‐
responsive genes (Appendix S10). There were 501 salt‐
responsive genes identified across the duplication classes and
of those that were classified in SSD, WGT, and SSD+WGT,
terms related to transport, cofactor, amino acid, and sugar
metabolism were highly enriched (Figure 6A, B). Addition-
ally, Pro metabolism was enriched in the SSD only category
(Figure 6B).
To identify whether any of these duplicates show
evidence of divergence, we determined Ka/Ks ratios between
Cakile and Arabidopsis orthologs for salt‐responsive genes
(Figure 6C). Salt‐responsive genes falling within the SC and
WGT categories had low Ka/Ks ratios, indicating sequence
conservation (Figure 6C), whereas salt‐responsive genes
within the SSD and WGT+SSD categories had a bimodal
distribution and higher mean Ka/Ks ratios, which were
significantly different from the distributions observed in the
SC category (Figure 6C; Appendix S11). Ka/Ks ratios between
Brassica and Arabidopsis, and Cakile and Brassica were also
investigated (Appendix S12). Brassica and Arabidopsis had
similar Ka/Ks distribution patterns to those of Cakile and
Arabidopsis (Appendix S12A). However, Ka/Ks distribution
patterns between Cakile and Brassica had higher means
across all categories, and SSD, WGT, and WGT+SSD
distributions of Cakile and Brassica all significantly differed
from the SC distribution (Appendix S12B). Cakile and
Brassica also shared more total ortholog pairs compared to
Arabidopsis, which may influence Ka/Ks ratio distributions.
Overall, higher Ka/Ks ratios between orthologs in the SSD
and WGT+SSD categories indicate that some genes are
diverging in sequence, perhaps indicators of sub‐or
neofunctionalization and abiotic stress response. However,
in the absence of expression data, we cannot determine
whether Cakile genes with high Ka/Ks ratios are expressed,
which would be crucial for functionalization.
A similar analysis was performed using all the cadmium‐
responsive genes, which consists of a much smaller list than
salt‐responsive genes (42 cadmium‐responsive genes). Some
overlapping categories were identified in the SSD, WGT, and
WGT+SSD categories including cofactor, amino acid, sugar,
and specialized metabolism, but protein degradation catego-
ries were unique to cadmium‐responsive genes (Appen-
dix S10A,S10B). Ka/Ks ratios were calculated for all the
cadmium‐responsive genes, and we saw no significant
differences between groups. (Appendix S10C). The low
number of cadmium‐responsive genes limits further inter-
pretation of these data.
DISCUSSION
Duplications are important for stress tolerance
Duplications are a source of genetic material for the evolution
of novel traits. Although gene loss is often the outcome
following duplication, maintenance and functional diver-
gence can lead to new functions (DeGiorgio and Assis, 2021).
Expansions and contractions in gene families post polyploidy
have contributed to the evolution of functional networks
enabling crop domestication and environmental adaptation
(Salman‐Minkov et al., 2016; Zhang et al., 2019;VandePeer
et al., 2021;Xuetal.,2021;Tossietal.,2022;Thomas
et al., 2023). Abiotic stress tolerance is a multifaceted
response encompassing morphological, transport, metabolic,
and regulatory changes (Mickelbart et al., 2015;Slama
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A
B
C
FIGURE 6 Top 10 GO terms/pathways per duplication class enriched with genes associated with response to salt stress. TAIR loci associated with
response to salt stress (GO:0009651) found in duplication classes were used as inputs for functional enrichment using ShinyGO (Ge et al., 2020). The top 10
terms per duplication class for (A) GO molecular function and (B) KEGG. The size of circles indicates the proportion of genes in enrichment/total genes
associated with the term/pathway; circle color indicates –log (Enrichment FDR). (C) Ka/Ks distributions per duplication class for loci associated with salt
stress. Violin plots with nested boxplots of Ka/Ks ratios for loci associated with response to salt stress gene ontology term (GO:0009651). The t‐test
comparisons with P< 0.05 are displayed above distributions.
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et al., 2015; Gul et al., 2022). In mangrove species, polyploid‐
derived genes have enabled environmental adaptations to
fluctuating salinity (Xu et al., 2021; Thomas et al., 2023).
Small‐scale duplications (SSDs) also contribute to novel
phenotypes; for example, genomes of salt‐tolerant Brassica-
ceae species Eutrema and Schrenkiella are enriched for SSDs
linked to salt tolerance (Kazachkova et al., 2018). In the
present study, duplicated Cakile genes were classified as
resulting from SSD, whole‐genome triplication (WGT) or
retained after both (WGT+SSD). The duplicated Cakile genes
associated with putative salt and heavy metal responses are
more likely retained in SSD, WGT, and WGT+SSD,
suggesting that different duplication classes contribute to
various aspects of abiotic stress tolerance in Cakile.
Dosage sensitivity explains patterns
of duplicate retention
The ability of gene(s) and pathways to tolerate changes in
copy number have dramatic impacts on the evolution of
novel traits. The gene balance hypothesis proposes that the
stoichiometric balance of genes whose products act in multi‐
subunit complexes (gene networks) is important for a
complete and functional network (Birchler and Newton, 1981;
Birchler et al., 2001; Edger and Pires, 2009; Freeling, 2009;
Makino and McLysaght, 2010; Birchler and Veitia, 2012;
Conant et al., 2014;Conant,2014). Dosage sensitive genes are
genes in which a change in copy number alters expression
and protein abundance and may interrupt the stoichiometric
balance of a functional gene network (Bird et al., 2023).
Duplications in dosage sensitive genes are tolerated when the
entire network is duplicated (i.e., via polyploidy) and gene
balance is not perturbed; however, duplications such as SSD
that only duplicate one or a few members of a network at a
time are not tolerated because these disrupt network/gene
balance. In contrast, dosage insensitive genes are more
amenable to SSDs because they are not as highly connected
and reliant on the stoichiometry of a network. This pattern of
dosage sensitive genes tending to be over‐retained post
polyploidy and dosage insensitive genes tending to be over‐
retained after SSD is called reciprocal retention and has been
shown in many studies (Edger and Pires, 2009;Freeling,2009;
Makino and McLysaght, 2010;DeSmetetal.,2013; Conant
et al., 2014; Conant, 2014; Tasdighian et al., 2017; Emery
et al., 2018; Gout et al., 2023).
Overall, our results are consistent with patterns of
reciprocal retention in Cakile after gene duplication. GO
terms were classified as dosage sensitive, dosage insensitive,
or neither (Appendix S13A) based on reciprocal retention of
Arabidopsis duplicates (Song et al., 2020). In Cakile, the
WGT class contained a higher proportion of dosage sensitive
GO terms (Appendix S13A), including highly connected
functions such as macromolecular complexes, transcriptional
regulation, and signal transduction (Figure 5). On the other
hand, the SSD class had a higher proportion of dosage
insensitive compared to dosage sensitive genes, which may
indicate that Cakile genes retained after SSD are involved in
metabolism and biosynthetic processes (Figure 5). However,
across all duplication classes, most GO terms were not
classified as dosage sensitive or insensitive. Response to salt
stress (GO:0009651) and response to cadmium ion
(GO:0046686) GO terms were both found in the WGT and
WGT+SSD categories, but were not found to be dosage
sensitive or insensitive because they had a higher than
average retention after both polyploidy and SSD (Appen-
dix S13B). These data suggest that functions associated with
abiotic stress adaptation have either weak or no dosage
sensitivity and can diversify and contribute to adaptation
through combinations of polyploidy and SSD.
Salt‐tolerance functions are partitioned into
various duplication classes
Based on patterns of reciprocal retention, we hypothesized
that Cakile duplicated genes involved in different aspects of
abiotic stress response would be partitioned into the SSD,
WGT, and WGT+SSD classes. Within salt‐response GO
terms, we found enrichment in molecular function and
metabolic pathways generally associated with amino acid
metabolism, cofactor metabolism, lipid metabolism, spe-
cialized metabolism, and sugar metabolism and transport
(Figure 6A, B). Amino acid metabolism terms were found
across all duplication classes, and specifically, Pro metabo-
lism was enriched in the SSD class (Figure 6B). Cofactor and
specialized metabolism were mostly enriched in the SSD
class (Figure 6A, B). Lipid and sugar metabolism terms were
mainly partitioned into the WGT class and transport was
mainly found in the WGT+SSD class (Figure 6). Our results
are consistent with previous studies that show specialized
metabolic genes are over‐retained post SSD but not
polyploidy, and metabolism and transport are often retained
in multiple copies and are seen as drivers of environmental
stress tolerance (Dunham et al., 2002; Gresham et al., 2008;
Selmecki et al., 2006,2015; Sunshine et al., 2015; Moore
et al., 2019). Genes for core metabolic processes are more
conserved compared with evolutionarily agile genes for
specialized metabolism (Schenck and Last, 2020; Schenck
and Busta, 2022), and we find that genes for lipid and sugar
metabolism may be more constrained by dosage balance
than those involved in specialized metabolism. Noncoding
RNAs (ncRNA) play roles in the regulation of gene
expression in response to stress (Di et al., 2014; Wang
et al., 2017). Although ncRNA were not identified in this
study, there are likely many in the Cakile genome that could
contribute to stress tolerance and occur through different
duplication types.
Ionomic response to abiotic stress
Plants that are salt and/or heavy metal tolerant have evolved
diverse adaptive strategies, including exclusion, uptake, and
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sequestration (Mendoza‐Cózatl et al., 2011; Hasanuzzaman
et al., 2014; Kazachkova et al., 2018; Yan et al., 2020). The
elemental profiling suggests that Cakile can tolerate salt by
accumulating Na
+
in leaf tissue and cadmium through Cd
2+
sequestration in the root tissues with limited translocation
to the leaves (Figure 2). The addition of salt in the
hydroponic media tended to show a pattern of higher K
+
in
leaf tissue; however, this result was not statistically
significant (Figure 2). During salt stress, Na
+
ions can
compete with K
+
ions for binding to K
+
transporters due to
ionic similarities (Munns, 2002). Maintenance of high K
+
/
Na
+
ratios can mitigate ionic competition, and the pattern
of high K
+
in Cakile leaf tissue could suggest that Cakile
maintains high K
+
/Na
+
ratios to mitigate salt stress. During
cadmium treatments, there was a significant reduction of K
+
in roots at 24 h (cadmium only) and at 24 and 48 h (both
stressors applied). Cadmium stress has been shown to
negatively impact nutrient uptake (Qin et al., 2020; Naciri
et al., 2021). With Zn
2+
, there were no obvious patterns that
emerged from the data; however, there was a significant
increase of Zn
2+
at 48 h in leaf tissue when salt and
cadmium were applied. Interestingly, salt stress induced a
transient spike in Fe
2+
levels in the roots after 24 h, but Cd
2+
stress alone had little effect on Fe
2+
levels (Figure 2). Salt
stress induces ROS, which can degrade Fe‐S clusters, so
perhaps Cakile increases Fe
2+
uptake to compensate for this
degradation (Zandalinas et al., 2020). We found ROS‐
related terms were enriched across all duplication classes
and may be related to this pattern. When Cakile was grown
on cadmium, an increase in Cd
2+
was observed in the roots,
but not the leaves, suggesting uptake in the roots but not
translocation, at least within the 48‐h window monitored
(Figure 2). However, when salt and cadmium treatments
were applied together, an increase in Cd
2+
was observed in
the roots; however, the increase was substantially lower than
when cadmium was applied alone (Figure 2). Cakile may
respond differently when multiple abiotic stresses are
supplied, which could have led to the decreased Cd
2+
uptake observed (Figure 2). These data provide initial
insights into the ionomic changes during abiotic stress
response in Cakile.
Genes for transporters have been reported as dosage
sensitive (Blanc and Wolfe, 2004; Maere et al., 2005);
therefore, we expected to find transporters enrichment in
the WGT category. However, we found that transporters
tolerated duplications post WGT and SSD because they
were mainly found in the WGT+SSD category (Figure 6).
HKT1 is a sodium transporter in Arabidopsis and other
plants (Kazachkova et al., 2018). Natural variation of
AtHKT1 is associated with salt tolerance in some coastal
Arabidopsis accessions (Busoms et al., 2018). Cakile
possesses four HKT1 orthologs in the SSD category, two
of which have greater than mean Ka/Ks values (Appen-
dix S11A,S14) when compared to Arabidopsis, and further
comparing Ka/Ks ratios of Cakile and Brassica HKT1
orthologs, we see similar levels of divergence. These data
suggest that HKT1 orthologs may have diverged in Cakile.
Multiple HKT1 orthologs are found in other salt‐tolerant
plants. In closely related Brassicaceae species, HKT1
expansion was the result of SSD (Kazachkova et al., 2018),
whereas in mangrove HKT1 expansion is the result of
polyploidy (Xu et al., 2021; Thomas et al., 2023). Another
sodium transporter, AtNHX1, is also associated with salt
tolerance in Arabidopsis (Pehlivan et al., 2016). Cakile has
three AtNHX1 homologs; however, none had high diver-
gence rates (Appendix S11A). It is possible that expansion
of AtNHX1 in Cakile contributes to abiotic stress tolerance;
however, this divergence could be through transcriptional
or subcellular localization changes, which were not captured
in this study. Thus, evolutionary context and environmental
pressures influence the fates of duplicated genes.
Metabolic response to abiotic stress
Metabolically stress‐adapted plants often accumulate osmo-
protectants, such as Pro, before stress or in response to
stress (Verslues and Sharma, 2010; Slama et al., 2015).
Amino acid profiling of Cakile supports that it is
metabolically prepared for stress (Figure 3). Before salt
stress, Pro overaccumulates in Cakile compared to Arabi-
dopsis and Brassica, and only minor changes were observed
in Pro levels following salt stress in Cakile (Figure 3;
Appendices S5,S6). Over 30% of the amino acid composi-
tion in Cakile stems consists of Pro before stress treatment
(Figure 3A). Other Brassicaceae species including Schren-
kiella and Eutrema show metabolic stress adaptation via
increases in Pro content (Tran et al., 2022). Downregulation
of Pro catabolic genes appear to enable Pro overaccumula-
tion in these species and to lead to stress tolerance (Gong
et al., 2005; Tran et al., 2022). Amino‐acid‐related processes,
including Pro metabolism, were enriched in SSD categories
and show evidence of sequence divergence (Figure 6),
suggesting that Pro metabolism in Cakile may have diverged
to support Pro accumulation. However, not all Pro
biosynthetic or catabolic genes have greater than mean
Ka/Ks (Appendices S11,S14), indicating that Cakile Pro
metabolism may have diverged at a transcriptional level, like
in other salt‐tolerant species (Tran et al., 2022). Although
Pro levels increased in Brassica and Arabidopsis following
stress treatment (Figure 3B; Appendix S6), this increase did
not translate into salt tolerance (Figure 1B), suggesting that
Pro accumulation by itself is insufficient for some abiotic
stresses and additional metabolic processes likely contribute
to Cakile stress tolerance. Other metabolic processes
including sugar and lipid metabolism were mostly enriched
in WGT (Figure 6). Cytochrome P450 (CYP) genes play key
roles in metabolic pathways related to plant development
and stress response (Xu et al., 2015). Many CYPs were
enriched in both the SSD (CYP71B families) and WGT
+SSD (CYP72A and CYP709B families) classes, and several
showed higher than mean Ka/Ks ratios (Appendi-
ces S11,S12,S14), which may suggest functional divergence.
In particular, Cakile possesses two CYP709B3 orthologs
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with high sequence divergence, and this gene has been
characterized in Arabidopsis to play a role in the regulation
of salt stress response (Mao et al., 2013). These CYP
duplicate genes may contribute to novel specialized
metabolites in Cakile. Although we did not capture sugars,
lipids, or specialized metabolites in our metabolic profiling,
alteration in these pathways may also contribute to abiotic
stress tolerance in Cakile. Large‐scale metabolomic charac-
terization could give further insights into patterns seen by
the comparative genomics analyses.
CONCLUSIONS
This study identifies duplicated genes in Cakile, a mustard
crop wild relative, which may mediate tolerance to salt and
cadmium stress. Abiotic‐stress‐related genes in Cakile
grouped into different duplication classes, and sequence
divergence of some of these genes suggest potential sub‐/
neofunctionalization. Validation of these putative stress‐
related Cakile genes can provide experimental evidence into
their specific functions in abiotic stress tolerance. This study
demonstrates the power of using crop wild relatives because
they can provide insights into the evolution of environ-
mental adaptation. Further they provide novel genetic
resources that can aid in the development of stress‐
resilient crops to meet global agricultural challenges.
AUTHOR CONTRIBUTIONS
The conception and design of the experiments were
performed by S.K.T., C.A.S., D.M.C., J.C.P., R.A., and J.W.
Data acquisition and analyses were performed by S.K.T.,
C.A.S., T.O., K.V.H., H.N.D., and D.M.C. The first draft of
the manuscript was written by S.K.T., K.V.H., and C.A.S. All
authors read, revised, and approved the final version of the
manuscript.
ACKNOWLEDGMENTS
The computational aspect of this work was done using the
high‐performance computing infrastructure provided by
Research Computing Support Services and funded in part
by the National Science Foundation under grant number
CNS‐1429294 at the University of Missouri, Columbia MO
(MU). We thank Armando Moreno Geraldes and Kathryn
Hodgins for providing Cakile seeds. We thank Emily Walter
for help with hydroponics and Norma Castro‐Guerrero for
help with processing samples for elemental profiling. We
thank Brian Mooney of the Gehrke Proteomics Center at
MU for help with amino acid profiling. We thank Jim
Birchler, Kevin Bird, and Makenzie Mabry for providing
useful comments. We thank the Bioinformatics in Plant
Science (BIPS) program at MU for providing funding for
K.V.H. and T.O. (NSF MCB −2224839). We thank the
Bioinformatics and Analytics Core at MU for providing
funding for SKT. We thank the Department of Energy Joint
Genome Institute and collaborators for prepublication
access to the Cakile maritima genome sequence. The work
(proposal: 10.46936/10.25585/60000980) conducted by the
U.S. Department of Energy Joint Genome Institute (https://
ror.org/04xm1d337), a DOE Office of Science User Facility,
is supported by the Office of Science of the U.S. Department
of Energy operated under Contract No. DE‐AC02‐
05CH11231. We thank the reviewers for careful reading
and comments that have improved the manuscript.
DATA AVAILABILITY STATEMENT
Genome data used in this study were obtained from DOE‐
JGI Phytozome (https://phytozome-next.jgi.doe.gov/). Spe-
cific data sources can be found in Appendix S1. Scripts, raw
and intermediate data available at https://github.com/
shawnkt/HalophytesAndHeavyMetals.
ORCID
Shawn K. Thomas http://orcid.org/0000-0002-6198-8847
Kathryn Vanden Hoek http://orcid.org/0000-0001-
6419-3364
Tasha Ogoti http://orcid.org/0000-0002-7813-4282
Ha Duong http://orcid.org/0000-0001-6087-746X
Ruthie Angelovici http://orcid.org/0000-0001-5150-0695
J. Chris Pires http://orcid.org/0000-0001-9682-2639
David Mendoza‐Cozatl http://orcid.org/0000-0002-
9616-0791
Jacob Washburn http://orcid.org/0000-0003-0185-7105
Craig A. Schenck http://orcid.org/0000-0002-5711-7213
REFERENCES
Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990.
Basic local alignment search tool. Journal of Molecular Biology 215:
403–410.
Angelovici, R., A. E. Lipka, N. Deason, S. Gonzalez‐Jorge, H. Lin, J. Cepela,
R. Buell, et al. 2013. Genome‐wide analysis of branched‐chain amino
acid levels in Arabidopsis seeds. Plant Cell 25: 4827–4843.
Arbelet‐Bonnin, D., I. Ben Hamed‐Laouti,P.Laurenti,C.Abdelly,
K. Ben Hamed, and F. Bouteau. 2018. Cellular mechanisms to survive
salt in the halophyte Cakile maritima.Plant Science 272: 173–178.
Arbelet‐Bonnin, D., I. Ben‐Hamed‐Louati, P. Laurenti, C. Abdelly, K. Ben‐
Hamed, and F. Bouteau. 2019. Cakile maritima, a promising model
for halophyte studies and a putative cash crop for saline agriculture.
Advances in Agronomy, 155: 45–78.
Arias, T., and J. C. Pires. 2012. A fully resolved chloroplast phylogeny
of the brassica crops and wild relatives (Brassicaceae: Brassiceae):
novel clades and potential taxonomic implications. Taxon 61:
980–988.
Birchler, J. A., U. Bhadra, M. P. Bhadra, and D. L. Auger. 2001. Dosage‐
dependent gene regulation in multicellular eukaryotes: implications
for dosage compensation, aneuploid syndromes, and quantitative
traits. Developmental Biology 234: 275–288.
Birchler, J. A., and K. J. Newton. 1981. Modulation of protein levels in
chromosomal dosage series of maize: the biochemical basis of
aneuploid syndromes. Genetics 99: 247–266.
Birchler, J. A., and R. A. Veitia. 2012. Gene balance hypothesis: connecting
issues of dosage sensitivity across biological disciplines. Proceedings of
the National Academy of Sciences, USA 109: 14746–14753.
Birchler, J. A., and H. Yang. 2022. The multiple fates of gene duplications:
deletion, hypofunctionalization, subfunctionalization, neofunctiona-
lization, dosage balance constraints, and neutral variation. Plant Cell
34: 2466–2474.
Bird, K. A., J. C. Pires, R. VanBuren, Z. Xiong, and P. P. Edger. 2023.
Dosage‐sensitivity shapes how genes transcriptionally respond to
14 of 18
|
HALOPHYTES AND HEAVY METALS
15372197, 0, Downloaded from https://bsapubs.onlinelibrary.wiley.com/doi/10.1002/ajb2.16310 by University Of Missouri Columbia, Wiley Online Library on [12/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
allopolyploidy and homoeologous exchange in resynthesized Brassica
napus.Genetics 225: iyad114.
Blanc, G., and K. H. Wolfe. 2004. Functional divergence of duplicated
genes formed by polyploidy during Arabidopsis evolution. Plant Cell
16: 1679–1691.
Bromham, L. 2015. Macroevolutionary patterns of salt tolerance in
angiosperms. Annals of Botany 115: 333–341.
Busoms, S., P. Paajanen, S. Marburger, S. Bray, X.‐Y. Huang,
C. Poschenrieder, L. Yant, and D. E. Salt. 2018. Fluctuating selection
on migrant adaptive sodium transporter alleles in coastal Arabidopsis
thaliana.Proceedings of the National Academy of Sciences, USA 115:
E12443–E12452.
Chen, A., E. A. Komives, and J. I. Schroeder. 2006. An improved grafting
technique for mature Arabidopsis plants demonstrates long‐distance
shoot‐to‐root transport of phytochelatins in Arabidopsis. Plant
Physiology 141: 108–120.
Conant, G. C. 2014. Comparative genomics as a time machine: How
relative gene dosage and metabolic requirements shaped the time‐
dependent resolution of yeast polyploidy. Molecular Biology and
Evolution 31: 3184–3193.
Conant, G. C., J. A. Birchler, and J.C. Pires. 2014. Dosage, duplication, and
diploidization: clarifying the interplay of multiple models for
duplicate gene evolution over time. Current Opinion in Plant
Biology 19: 91–98.
Dassanayake, M., D.‐H. Oh, J. S. Haas, A. Hernandez, H. Hong, S. Ali, D.‐J.
Yun, et al. 2011. The genome of the extremophile crucifer
Thellungiella parvula.Nature Genetics 43: 913–918.
DeGiorgio, M., and R. Assis. 2021. Learning retention mechanisms and
evolutionary parameters of duplicate genes from their expression
data. Molecular Biology and Evolution 38: 1209–1224.
DeSmet,R.,K.L.Adams,K.Vandepoele,M.C.E.VanMontagu,S.Maere,
andY.VandePeer.2013.Convergentgenelossfollowinggeneand
genome duplications creates single‐copy families in flowering plants.
Proceedings of the National Academy of Sciences, USA 110: 2898–2903.
Di, C., J. Yuan, Y. Wu, J. Li, H. Lin, L. Hu, T. Zhang, et al. 2014.
Characterization of stress‐responsive lncRNAs in Arabidopsis thali-
ana by integrating expression, epigenetic and structural features.
Plant Journal 80: 848–861.
Dunham, M. J., H. Badrane, T. Ferea, J. Adams, P. O. Brown,
F. Rosenzweig, and D. Botstein. 2002. Characteristic genome
rearrangements in experimental evolution of Saccharomyces cerevi-
siae.Proceedings of the National Academy of Sciences, USA 99:
16144–16149.
Edgar, R. C. 2004. MUSCLE: multiple sequence alignment with high
accuracy and high throughput. Nucleic Acids Research 32: 1792–1797.
Edger, P. P., and J. C. Pires. 2009. Gene and genome duplications: the
impact of dosage‐sensitivity on the fate of nuclear genes. Chromosome
Research 17: 699–717.
Emery, M., M. M. S. Willis, Y. Hao, K. Barry, K. Oakgrove, Y. Peng,
J. Schmutz, et al. 2018. Preferential retention of genes from one
parental genome after polyploidy illustrates the nature and scope of
the genomic conflicts induced by hybridization. PLoS Genetics 14:
e1007267.
Emms, D. M., and S. Kelly. 2019. OrthoFinder: phylogenetic orthology
inference for comparative genomics. Genome Biology 20: 238.
Flowers, T. J., and T. D. Colmer. 2008. Salinity tolerance in halophytes.
New Phytologist 179: 945–963.
Flowers, T. J., H. K. Galal, and L. Bromham. 2010. Evolution of halophytes:
multiple origins of salt tolerance in land plants. Functional Plant
Biology 37: 604–612.
Freeling, M. 2009. Bias in plant gene content following different sorts of
duplication: tandem, whole‐genome, segmental, or by transposition.
Annual Review of Plant Biology 60: 433–453.
Ge, S. X., D. Jung, and R. Yao. 2020. ShinyGO: a graphical gene‐set
enrichment tool for animals and plants. Bioinformatics 36:
2628–2629.
Gong, Q., P. Li, S. Ma, S. Indu Rupassara, and H. J. Bohnert. 2005. Salinity
stress adaptation competence in the extremophile Thellungiella
halophila in comparison with its relative Arabidopsis thaliana.Plant
Journal 44: 826–839.
Gout, J.‐F., Y. Hao, P. Johri, O. Arnaiz, T. G. Doak, S. Bhullar, A. Couloux,
et al. 2023. Dynamics of gene loss following ancient whole‐genome
duplication in the cryptic Paramecium complex. Molecular Biology
and Evolution 40: msad107.
Gresham, D., M. M. Desai, C. M. Tucker, H. T. Jenq, D. A. Pai, A. Ward,
C. G. DeSevo, et al. 2008. The repertoire and dynamics of
evolutionary adaptations to controlled nutrient‐limited environments
in yeast. PLoS Genetics 4: e1000303.
Gul, Z., Z.‐H. Tang, M. Arif, and Z. Ye. 2022. An insight into abiotic stress
and influx tolerance mechanisms in plants to cope in saline
environments. Biology 11: 597.
Hamed‐Louati, I. B., F. Bouteau, C. Abdelly, and K. B. Hamed. 2016.
Impact of repetitive salt shocks on seedlings of the halophyte Cakile
maritima.Environmental Control in Biology 54: 23–30.
Hasanuzzaman, M., K. Nahar, M. M. Alam, P. C. Bhowmik, M. A. Hossain,
M. M. Rahman, M. N. V. Prasad, et al. 2014. Potential use of
halophytes to remediate saline soils. BioMed Research International
2014: 589341.
Hauser, F., and T. Horie. 2010. A conserved primary salt tolerance
mechanism mediated by HKT transporters: a mechanism for sodium
exclusion and maintenance of high K
+
/Na
+
ratio in leaves during
salinity stress. Plant, Cell & Environment 33: 552–565.
Hendriks, K. P., C. Kiefer, I. A. Al‐Shehbaz, C. D. Bailey, A. Hooft van
Huysduynen, L. A. Nikolov, L. Nauheimer, et al. 2023. Global
Brassicaceae phylogeny based on filtering of 1,000‐gene dataset.
Current Biology 33: 4052–4068.e6.
Kazachkova, Y., G. Eshel, P. Pantha, J. M. Cheeseman, M. Dassanayake,
and S. Barak. 2018. Halophytism: What have we learnt from
Arabidopsis thaliana relative model systems? Plant Physiology 178:
972–988.
Lamesch, P., T. Z. Berardini, D. Li, D. Swarbreck, C. Wilks, R. Sasidharan,
R. Muller, et al. 2012. The Arabidopsis Information Resource (TAIR):
improved gene annotation and new tools. Nucleic Acids Research 40:
D1202–D1210.
Lopes, M. S., I. El‐Basyoni, P. S. Baenziger, S. Singh, C. Royo, K. Ozbek,
H. Aktas, et al. 2015. Exploiting genetic diversity from landraces in
wheat breeding for adaptation to climate change. Journal of
Experimental Botany 66: 3477–3486.
Lou, P., S. Woody, K. Greenham, R. VanBuren, M. Colle, P. P. Edger,
R. Sartor, et al. 2020. Genetic and genomic resources to study natural
variation in Brassica rapa.Plant Direct 4: e00285.
Lysak,M.A.,K.Cheung,M.Kitschke,andP.Bures.2007.Ancestral
chromosomal blocks are triplicated in Brassiceae species with varying
chromosome number and genome size. Plant Physiology 145: 402–410.
Lysak, M. A., M. A. Koch, A. Pecinka, and I. Schubert. 2005. Chromosome
triplication found across the tribe Brassiceae.Genome Research 15:
516–525.
Maere, S., S. De Bodt, J. Raes, T. Casneuf, M. Van Montagu, M. Kuiper,
and Y. Van de Peer. 2005. Modeling gene and genome duplications in
eukaryotes. Proceedings of the National Academy of Sciences, USA 102:
5454–5459.
Makino, T., and A. McLysaght. 2010. Ohnologs in the human genome are
dosage balanced and frequently associated with disease. Proceedings of
the National Academy of Sciences, USA 107: 9270–9274.
Mao, G., T. Seebeck, D. Schrenker, and O. Yu. 2013. CYP709B3, a
cytochrome P450 monooxygenase gene involved in salt tolerance in
Arabidopsis thaliana.BMC Plant Biology 13: 169.
McKain, M. R., H. Tang, J. R. McNeal, S. Ayyampalayam, J. I. Davis,
C. W. dePamphilis, T. J. Givnish, et al. 2016. A phylogenomic
assessment of ancient polyploidy and genome evolution across the
Poales. Genome Biology and Evolution 8: 1150–1164.
McKain, M. R., N. Wickett, Y. Zhang, S. Ayyampalayam, W. R. McCombie,
M. W. Chase, J. C. Pires, et al. 2012. Phylogenomic analysis of
transcriptome data elucidates co‐occurrence of a paleopolyploid
event and the origin of bimodal karyotypes in Agavoideae
(Asparagaceae). American Journal of Botany 99: 397–406.
HALOPHYTES AND HEAVY METALS
|
15 of 18
15372197, 0, Downloaded from https://bsapubs.onlinelibrary.wiley.com/doi/10.1002/ajb2.16310 by University Of Missouri Columbia, Wiley Online Library on [12/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Melino, V., and M. Tester. 2023. Salt‐tolerant crops: time to deliver.
Annual Review of Plant Biology 74: 671–696.
Mendes, F. K., D. Vanderpool, B. Fulton, and M. W. Hahn. 2021. CAFE 5
models variation in evolutionary rates among gene families.
Bioinformatics 36: 5516–5518.
Mendoza‐Cózatl, D. G., T. O. Jobe, F. Hauser, and J. I. Schroeder. 2011.
Long‐distance transport, vacuolar sequestration, tolerance, and
transcriptional responses induced by cadmium and arsenic. Current
Opinion in Plant Biology 14: 554–562.
Mickelbart, M. V., P. M. Hasegawa, and J. Bailey‐Serres. 2015. Genetic
mechanisms of abiotic stress tolerance that translate to crop yield
stability. Nature Reviews Genetics 16: 237–251.
Milla, R., J. M. Bastida, M. M. Turcotte, G. Jones, C. Violle, C. P. Osborne,
J. Chacón‐Labella, et al. 2018. Phylogenetic patterns and phenotypic
profiles of the species of plants and mammals farmed for food. Nature
Ecology & Evolution 2: 1808–1817.
Minhas, P. S., J. Rane, and R. K. Pasala. 2017. Abiotic stress management
for resilient agriculture. Springer, Singapore.
Moore, B. M., P. Wang, P. Fan, B. Leong, C. A. Schenck, J. P. Lloyd,
M. D. Lehti‐Shiu, et al. 2019. Robust predictions of specialized
metabolism genes through machine learning. Proceedings of the
National Academy of Sciences, USA 116: 2344–2353.
Munns, R., 2002. Comparative physiology of salt and water stress. Plant,
Cell & Environment. 25: 239–250. https://doi.org/10.1046/j.0016-
8025.2001.00808.x
Naciri, R., M. Lahrir, C. Benadis, M. Chtouki, and A. Oukarroum. 2021.
Interactive effect of potassium and cadmium on growth, root
morphology and chlorophyll a fluorescence in tomato plant.
Scientific Reports 11: 5384.
Nguyen, N. T., S. A. McInturf, and D. G. Mendoza‐Cózatl. 2016.
Hydroponics: a versatile system to study nutrient allocation and
plant responses to nutrient availability and exposure to toxic
elements. Journal of Visualized Experiments 113: e54317.
Oh, D.‐H., B. J. Barkla, R. Vera‐Estrella, O. Pantoja, S.‐Y. Lee,
H. J. Bohnert, and M. Dassanayake. 2015. Cell type‐specific responses
to salinity –the epidermal bladder cell transcriptome of Mesembry-
anthemum crystallinum.New Phytologist 207: 627–644.
Oh, D.‐H., H. Hong, S. Y. Lee, D.‐J. Yun, H. J. Bohnert, and
M. Dassanayake. 2014. Genome structures and transcriptomes signify
niche adaptation for the multiple‐ion‐tolerant extremophyte Schren-
kiella parvula.Plant Physiology 164: 2123–2138.
Panchy, N., M. Lehti‐Shiu, and S.‐H. Shiu. 2016. Evolution of gene
duplication in plants. Plant Physiology 171: 2294–2316.
Pehlivan, N., L. Sun, P. Jarrett, X. Yang, N. Mishra, L. Chen, A. Kadioglu,
et al. 2016. Co‐overexpressing a plasma membrane and a vacuolar
membrane sodium/proton antiporter significantly improves salt
tolerance in transgenic Arabidopsis plants. Plant & Cell Physiology 57:
1069–1084.
Qi, X., H. An, T. E. Hall, C. Di, P. D. Blischak, M. T. W. McKibben, Y. Hao,
et al. 2021. Genes derived from ancient polyploidy have higher
genetic diversity and are associated with domestication in Brassica
rapa.New Phytologist 230: 372–386.
Qin, S., H. Liu, Z. Nie, Z. Rengel, W. Gao, C. Li, and P. Zhao. 2020.
Toxicity of cadmium and its competition with mineral nutrients for
uptake by plants: a review. Pedosphere 30: 168–180.
Rody, H. V. S., G. J. Baute, L. H. Rieseberg, and L. O. Oliveira. 2017. Both
mechanism and age of duplications contribute to biased gene
retention patterns in plants. BMC Genomics 18: 46.
Salman‐Minkov, A., N. Sabath, and I. Mayrose. 2016. Whole‐genome
duplication as a key factor in crop domestication. Nature Plants 2:
16115.
Schenck, C. A., and R. L. Last. 2020. Location, location! Cellular
relocalization primes specialized metabolic diversification. FEBS
Journal 287: 1359–1368.
Schenck, C. A., and L. Busta. 2022. Using interdisciplinary, phylogeny‐
guided approaches to understand the evolution of metabolism. Plant
Molecular Biology 109: 355–357.
Schranz, M. E., S. Mohammadin, and P. P. Edger. 2012. Ancient whole
genome duplications, novelty and diversification: the WGD Radiation
Lag‐Time Model. Current Opinion in Plant Biology 15: 147–153.
Selmecki, A., A. Forche, and J. Berman. 2006. Aneuploidy and isochromosome
formation in drug‐resistant Candida albicans.Science 313: 367–370.
Selmecki, A. M., Y. E. Maruvka, P. A. Richmond, M. Guillet, N. Shoresh,
A. L. Sorenson, S. De, et al. 2015. Polyploidy can drive rapid
adaptation in yeast. Nature 519: 349–352.
Slama, I., C. Abdelly, A. Bouchereau, T. Flowers, and A. Savouré. 2015.
Diversity, distribution and roles of osmoprotective compounds
accumulated in halophytes under abiotic stress. Annals of Botany 115:
433–447.
Song, M. J., B. I. Potter, J. J. Doyle, and J. E. Coate. 2020. Gene balance
predicts transcriptional responses immediately following ploidy
change in Arabidopsis thaliana.Plant Cell 32: 1434–1448.
Sunshine, A. B., C. Payen, G. T. Ong, I. Liachko, K. M. Tan, and
M. J. Dunham. 2015. The fitness consequences of aneuploidy are driven
by condition‐dependent gene effects. PLoS Biology 13: e1002155.
Supek, F., M. Bošnjak, N. Škunca, and T. Šmuc. 2011. REVIGO
summarizes and visualizes long lists of gene ontology terms. PLoS
One 6: e21800.
Suyama, M., D. Torrents, and P. Bork. 2006. PAL2NAL: robust conversion
of protein sequence alignments into the corresponding codon
alignments. Nucleic Acids Research 34: W609–W612.
Taamalli, M., R. Ghabriche, T. Amari, M. Mnasri, L. Zolla, S. Lutts,
C. Abdely, and T. Ghnaya. 2014. Comparative study of Cd tolerance
and accumulation potential between Cakile maritima L. (halophyte)
and Brassica juncea L. Ecological Engineering 71: 623–627.
Tang, H., M. R. Woodhouse, F. Cheng, J. C. Schnable, B. S. Pedersen,
G. Conant, X. Wang, et al. 2012. Altered patterns of fractionation and
exon deletions in Brassica rapa support a two‐step model of
paleohexaploidy. Genetics 190: 1563–1574.
Tasdighian, S., M. Van Bel, Z. Li, Y. Van de Peer, L. Carretero‐Paulet, and
S. Maere. 2017. Reciprocally retained genes in the angiosperm lineage
show the hallmarks of dosage balance sensitivity. Plant Cell 29:
2766–2785.
Thomas, S. K., H. An, and J. C. Pires. 2023. Mangroves and multiplications:
influence of genome duplications on salt tolerance. Molecular Ecology
32: 275–277.
Tossi, V. E., L. J. Martínez Tosar, L. E. Laino, J. Iannicelli, J. J. Regalado,
A. S. Escandón, I. Baroli, et al. 2022. Impact of polyploidy on plant
tolerance to abiotic and biotic stresses. Frontiers in Plant Science 13:
869423.
Tran, K.‐N., G. Wang, D.‐H. Oh, J. C. Larkin, A. P. Smith, and
M. Dassanayake. 2022. Multiple paths lead to salt tolerance ‐pre‐
adaptation vs dynamic responses from two closely related extremo-
phytes. bioRxiv https://doi.org/10.1101/2021.10.23.465591 [preprint].
Van de Peer, Y., T.‐L. Ashman, P. S. Soltis, and D. E. Soltis. 2021.
Polyploidy: an evolutionary and ecological force in stressful times.
Plant Cell 33: 11‐26.
Vance, Z., and A. McLysaght. 2023. Ohnologs and SSD paralogs differ in
genomic and expression features related to dosage constraints.
Genome Biology and Evolution 15: evad174.
Vanneste, K., G. Baele, S. Maere, and Y. Van de Peer. 2014. Analysis of 41
plant genomes supports a wave of successful genome duplications in
association with the Cretaceous–Paleogene boundary. Genome
Research 24: 1334–1347.
Verslues, P. E., and S. Sharma. 2010. Proline metabolism and its
implications for plant‐environment interaction. Arabidopsis Book 8:
e0140.
Walden, N., D. A. German, E. M. Wolf, M. Kiefer, P. Rigault, X.‐C. Huang,
C. Kiefer, et al. 2020. Nested whole‐genome duplications coincide
with diversification and high morphological disparity in Brassicaceae.
Nature Communications 11: 3795.
Wang, J., X. Meng, O. B. Dobrovolskaya, Y. L. Orlov, and M. Chen. 2017.
Non‐coding RNAs and their roles in stress response in plants.
Genomics, Proteomics & Bioinformatics 15: 301–312.
16 of 18
|
HALOPHYTES AND HEAVY METALS
15372197, 0, Downloaded from https://bsapubs.onlinelibrary.wiley.com/doi/10.1002/ajb2.16310 by University Of Missouri Columbia, Wiley Online Library on [12/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Xu, J., X. Y. Wang, and W. Z. Guo. 2015. The cytochrome P450
superfamily: key players in plant development and defense. Journal of
Integrative Agriculture 14: 1673–1686.
Xu, S., Z. Guo, X. Feng, S. Shao, Y. Yang, J. Li, C. Zhong, et al. 2021. Where
whole‐genome duplication is most beneficial: adaptation of man-
groves to a wide salinity range between land and sea. Molecular
Ecology 32: 460–475.
Yan, A., Y. Wang, S. N. Tan, M. L. Mohd Yusof, S. Ghosh, and Z. Chen.
2020. Phytoremediation: a promising approach for revegetation of
heavy metal‐polluted land. Frontiers in Plant Science 11: 359.
Yang, R., D. E. Jarvis, H. Chen, M. A. Beilstein, J. Grimwood, J. Jenkins,
S. Shu, et al. 2013. The reference genome of the halophytic plant
Eutrema salsugineum.Frontiers in Plant Science 4: 46.
Yang, Z. 2007. PAML 4: phylogenetic analysis by maximum likelihood.
Molecular Biology and Evolution 24: 1586–1591.
Yobi, A., C. Bagaza, A. Batushansky, V. Shrestha, M. L. Emery, S. Holden,
S. Turner‐Hissong, et al. 2020. The complex response of free and
bound amino acids to water stress during the seed setting stage in
Arabidopsis.Plant Journal 102: 838–855.
Zandalinas, S. I., L. Song, S. Sengupta, S. A. McInturf, D. G. Grant, H.‐B.
Marjault, N. A. Castro‐Guerrero, et al. 2020. Expression of a
dominant‐negative AtNEET‐H89C protein disrupts iron‐sulfur
metabolism and iron homeostasis in Arabidopsis.Plant Journal 101:
1152–1169.
Zhang, K., X. Wang, and F. Cheng. 2019. Plant polyploidy: origin,
evolution, and its influence on crop domestication. Horticultural
Plant Journal 5: 231–239.
SUPPORTING INFORMATION
Additional supporting information can be found online in
the Supporting Information section at the end of this article.
Appendix S1. Taxon sampling and genome data sources.
Appendix S2. Representative images following salt and
cadmium stress. Arabidopsis, Brassica, and Cakile were grown
for 6 days on ½ MS plates and moved to plates containing
150 mM NaCl (salt stress), 20 μMcadmium(Cd
2+
heavy metal
stress) or control plates and grown for an additional 5 days.
Appendix S3. Total absolute free amino acid levels in
Arabidopsis, Brassica, and Cakile tissues grown under
standard conditions. Amino acids were extracted from root,
stem, and leaf tissue under standard growth conditions in
the absence of exogenous abiotic stress. Amino acids are
shown as the sum of the absolute amount of each individual
amino acid. Data are the mean of N≥4 biological replicates
± standard deviation.
Appendix S4. Absolute levels of individual amino acids in
root, stem, and leaf tissue of Arabidopsis, Brassica, and
Cakile grown under standard conditions in the absence of
exogenous abiotic stress. The three‐letter abbreviations are
used for each amino acid. Data are the means of N≥4
biological replicates ± standard deviation.
Appendix S5. Free amino acid composition in leaf, root,
and stems of Arabidopsis, Brassica, and Cakile grown under
standard conditions in the absence of exogenous abiotic
stress. Amino acids are expressed as a percentage of the total
amino acid complement in different tissues. The three‐letter
abbreviations are used for each amino acid. Data are the
mean of N≥4 biological replicates.
Appendix S6. Free amino acid composition of Cakile,
Brassica, and Arabidopsis during salt stress. Amino acids
were extracted from whole seedlings of Arabidopsis,
Brassica, and Cakile under salt stress for different times.
Plants were grown for 6 days on ½ MS plates and moved to
plates containing 150 mM NaCl. Whole seedlings were
collected and analyzed for amino acids after 0, 2, 24, and
72 h of salt stress. Amino acids are expressed as a percentage
of the total amino acid complement at each timepoint. The
three‐letter abbreviations are used for each amino acid. Data
are the mean of N≥4 biological replicates.
Appendix S7. PCA of amino acid changes during salt stress.
(A) PCA of amino acid content from whole seedlings of
Arabidopsis, Brassica, and Cakile during salt stress for different
times. Amino acid content was normalized by subtracting
means and dividing by the standard deviations. PC1 and PC2
accounted for 79.59% of variance. Symbol size is based on the
squared cosine values (cos2). (B) Loadings plot shows relative
contributions and correlations of each amino acid to changes
observed during salt stress. The standard three‐letter code is
used to abbreviate each amino acid.
Appendix S8. GO biological processes enriched at significantly
expanding gene families. TAIR loci found in significantly
expanding gene families that are (A) Br‐⍺branch specific, (B)
shared at the Br‐⍺and Cakile branches, and (C) Cakile branch
specificwereusedasinputsforfunctionalenrichmentusing
ShinyGO (Ge; Jung and Yao, 2020). The top enriched GO
biological process terms per group are shown. The size of
circles indicates the number of genes, circle color indicates –log
(EnrichmentFDR),andfoldenrichment is indicated by the
length of the lines in the chart.
Appendix S9. Partitioning GO biological process terms
across Cakile duplication classes. (A) Single‐copy (SC)
global enrichment. (B) Small‐scale duplication (SSD) global
enrichment. (C) Whole‐genome triplication (WGT) global
enrichment. (D) Whole‐genome triplication+small‐scale
duplication (WGT+SSD) global enrichment.
Appendix S10. Top 10 GO terms/pathways enriched with
loci associated with response to cadmium ion. TAIR loci
associated with response to cadmium ion (GO:0046686) and
found in duplication classes were used as inputs for
functional enrichment using ShinyGO (Ge et al., 2020).
The top 10 terms per duplication class for (A) GO
molecular function and (B) KEGG per duplication class.
The size of circles indicates the proportion of genes in
enrichment/total genes associated with the term/pathway,
and circle color indicates –log (Enrichment FDR). (C)
Ka/Ks distributions per duplication class for response to
cadmium ion loci. Violin plots with nested boxplots of Ka/
Ks ratios for loci associated with response to cadmium ion
(GO:0046686). The t‐test comparisons with P< 0.05 are
displayed above distributions.
Appendix S11. Ka, Ks, Ka/Ks values between (A) Cakile
and Arabidopsis genes in the salt‐specific enrichment and
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their duplication class; (B) Cakile and Arabidopsis genes in
the cadmium‐specific enrichment and their duplication
class; (C) Brassica and Arabidopsis genes in the salt‐specific
enrichment and their duplication class; (D) Cakile and
Brassica genes in the salt‐specific enrichment and their
duplication class.
Appendix S12. Ka/Ks distributions per duplication class for
response to salt stress loci. Violin plots with nested boxplots
of Ka/Ks ratios for loci associated with response to salt
stress (GO:0009651). The t‐test comparisons with P< 0.05
are displayed above distributions. (A) Brassica vs. Arabi-
dopsis. (B) Cakile vs. Brassica.
Appendix S13. Response to salt stress and response to
cadmium ion GO Terms were not classifiable as dosage
sensitive or insensitive. (A) Log10‐transformed counts of
enriched GO biological process terms with FDR < 0.05 per
duplication class classified as dosage sensitive, insensitive,
and NA per Song et al. (2020) assignments of GO terms. (B)
Adaptation of Figure 1a from Song et al. (2020). Yellow data
points indicate class I GO terms (putatively dosage balance
insensitive; unbalanced > 0.38 and α< 0.3), and blue data
points indicate class II GO terms (putatively dosage balance
sensitive; unbalanced < 0.38 and α> 0.3). Response to salt
stress (GO:0009651) and response to cadmium ion
(GO:0046686) did not fall into either group and had
unbalanced = 0.44, α= 0.35 and unbalanced = 0.41, α= 0.37,
respectively.
Appendix S14. Arabidopsis gene annotations of Ka/Ks
values above means between Cakile and Arabidopsis genes.
(A) small‐scale duplication (SSD) class. (B) whole‐genome
triplication+SSD (WGT+SSD) class.
How to cite this article: Thomas, S. K., K. Vanden
Hoek, T. Ogoti, H. Duong, R. Angelovici, J. C. Pires,
D. Mendoza‐Cozatl, J. Washburn, and C. A. Schenck.
2024. Halophytes and heavy metals: A multi‐omics
approach to understand the role of gene and genome
duplication in the abiotic stress tolerance of Cakile
maritima.American Journal of Botany 111: e16310.
https://doi.org/10.1002/ajb2.16310
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