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Grain Transcriptome Dynamics Induced by Heat in Commercial and Traditional Bread Wheat Genotypes

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High temperature (HT) events have negative impact on wheat grains yield and quality. Transcriptome profiles of wheat developing grains of commercial genotypes (Antequera and Bancal) and landraces (Ardito and Magueija) submitted to heatwave-like treatments during grain filling were evaluated. Landraces showed significantly more differentially expressed genes (DEGs) and presented more similar responses than commercial genotypes. DEGs were more associated with transcription and RNA and protein synthesis in Antequera and with metabolism alterations in Bancal and landraces. Landraces upregulated genes encoding proteins already described as HT responsive, like heat shock proteins and cupins. Apart from the genes encoding HSP, two other genes were upregulated in all genotypes, one encoding for Adenylate kinase, essential for the cellular homeostasis, and the other for ferritin, recently related with increased tolerance to several abiotic stress in Arabidopsis. Moreover, a NAC transcription factor involved in plant development, known to be a negative regulator of starch synthesis and grain yield, was found to be upregulated in both commercial varieties and downregulated in Magueija landrace. The detected diversity of molecular processes involved in heat response of commercial and traditional genotypes contribute to understand the importance of genetic diversity and relevant pathways to cope with these extreme events.
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ORIGINAL RESEARCH
published: 17 June 2022
doi: 10.3389/fpls.2022.842599
Edited by:
Felipe Klein Ricachenevsky,
Federal University of Rio Grande do
Sul, Brazil
Reviewed by:
Klára Kosová,
Crop Research Institute (CRI),
Czechia
Manosh Kumar Biswas,
University of Leicester,
United Kingdom
*Correspondence:
Manuela Silva
manuelasilva@isa.ulisboa.pt
Specialty section:
This article was submitted to
Plant Abiotic Stress,
a section of the journal
Frontiers in Plant Science
Received: 23 December 2021
Accepted: 16 May 2022
Published: 17 June 2022
Citation:
Tomás D, Viegas W and Silva M
(2022) Grain Transcriptome Dynamics
Induced by Heat in Commercial and
Traditional Bread Wheat Genotypes.
Front. Plant Sci. 13:842599.
doi: 10.3389/fpls.2022.842599
Grain Transcriptome Dynamics
Induced by Heat in Commercial and
Traditional Bread Wheat Genotypes
Diana Tomás, Wanda Viegas and Manuela Silva*
LEAF Linking Landscape, Environment, Agriculture and Food, TERRA Laboratory for Sustainable Land Use and
Ecosystem Services, Instituto Superior de Agronomia, Universidade de Lisboa, Lisbon, Portugal
High temperature (HT) events have negative impact on wheat grains yield and
quality. Transcriptome profiles of wheat developing grains of commercial genotypes
(Antequera and Bancal) and landraces (Ardito and Magueija) submitted to heatwave-
like treatments during grain filling were evaluated. Landraces showed significantly
more differentially expressed genes (DEGs) and presented more similar responses
than commercial genotypes. DEGs were more associated with transcription and RNA
and protein synthesis in Antequera and with metabolism alterations in Bancal and
landraces. Landraces upregulated genes encoding proteins already described as HT
responsive, like heat shock proteins and cupins. Apart from the genes encoding
HSP, two other genes were upregulated in all genotypes, one encoding for Adenylate
kinase, essential for the cellular homeostasis, and the other for ferritin, recently related
with increased tolerance to several abiotic stress in Arabidopsis. Moreover, a NAC
transcription factor involved in plant development, known to be a negative regulator
of starch synthesis and grain yield, was found to be upregulated in both commercial
varieties and downregulated in Magueija landrace. The detected diversity of molecular
processes involved in heat response of commercial and traditional genotypes contribute
to understand the importance of genetic diversity and relevant pathways to cope with
these extreme events.
Keywords: bread wheat, commercial varieties, landraces, heatwave, grain transcriptome, RNA sequencing
INTRODUCTION
Wheat is the second most produced and consumed cereal worldwide on a daily basis (FAO©, 2019)
and hexaploid bread wheat (Triticum aestivum L, 2n = 42) represents 90–95% of this production.
However, the current growth rate of wheat production is not sufficient to cover the predicted global
demand in 2050. Specifically in European countries, the stagnation of wheat yield increase is related
with the progressive global warming (Brisson et al., 2010;Ray et al., 2013;Gaupp et al., 2019).
The increase of mean temperature during wheat development affects grain yield and quality, due
to reduction in lifecycle, pollen abortion, kernel shrinkage, and decrease in seed reserves (Asseng
et al., 2014;Nuttall et al., 2018;Wang et al., 2019). The required optimum temperature for wheat
anthesis and grain filling ranges from 12 to 22C (Tewolde et al., 2006) and the overall acceleration
of grain development observed under high temperature regimes is associated with the speed up of
transcriptomic events (Altenbach and Kothari, 2004;Wan et al., 2008).
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Tomás et al. Wheat Grain Transcriptome Under Heat
Transcription modulation of genes encoding heat shock
proteins (HSPs) is the most studied molecular response under
heat stress (Wahid et al., 2007). A recent study identified
and characterized 753 HSP genes expressed in bread wheat,
revealing the developmental stage and stress situation at which
they are responsive (Kumar et al., 2020). HSPs transcripts were
also differentially detected after 1 h and 1 d at 40C using
Wheat Genome Array profiles in seedlings of two genotypes
with contrasting thermotolerances (Qin et al., 2008). The same
work also detected transcription factors and genes involved in
phytohormone biosynthesis/signaling, calcium and sugar signal
pathways, RNA metabolism, ribosomal proteins, primary and
secondary metabolisms synthesis, and biotic and abiotic stress
responses. Chauhan et al. (2011) identified heat responsive genes,
after 2 h of heat stress treatments (34 and 40C), implicated in
metabolites and protein synthesis in seedling shoot, flower tissues
and developing grain through subtractive hybridization.
Whole transcriptome sequencing of wheat seedlings reported
similar transcripts profiles after heat, drought and their
combination treatments of 1 and 6 h (Liu et al., 2015).
The main biological groups associated with upregulated genes
were stress response, hormone stimulus response and nutrient
metabolic processes, while downregulated genes were mainly
enriched in photosynthesis and nutrient biosynthesis pathway.
A more recent study used RNA Sequencing data obtained from
developing grains of genotypes with distinct thermotolerances
that underwent post anthesis heat stress for 3 days, identified
different clusters of genes unique to tolerant and susceptible
genotypes (Rangan et al., 2019). This work also refers that most
genes uniquely expressed in tolerant genotype during heat stress
are detected in both early and late grain filling reinforcing their
role in heat stress response. Other work from Kino et al. (2020)
compared RNA Sequencing data obtained from whole grains
after post anthesis high temperature treatment (35C during 2–
12 days) against existing sequence data from individual pericarp
and endosperm tissue. A significant down-regulation of pericarp
genes with a known role in regulation of cell wall expansion
was observed. For that reason, the authors suggested that heat
treatment induces reduced expansion capability of the pericarp,
which may result in a physical constraint of endosperm growth.
Several studies have shown increasing genetic erosion caused
by the replacement of diverse old landraces by comparatively
few and homozygous modern cultivars (Gregová et al., 1999;
Caballero et al., 2001;Srinivasan et al., 2003). Landraces are
dynamic populations of cultivated species lacking formal crop
improvement, locally adapted and often genetically diverse
[reviewed in Villa et al. (2005)]. Thus, landraces provide notable
successes in crop improvement [reviewed in Dwivedi et al.
(2016)] as sources of nutritional and technological quality traits
and marginal environment tolerance [reviewed in Newton et al.
(2010)]. They are considered extremely valuable agrobiodiversity
pools in changing environmental conditions (Trethowan and
Mujeeb-Kazi, 2008;Lopes et al., 2015) that may constitute a key
resource facing extreme heat events like heatwaves. Heatwaves
are defined by World Meteorological Organization [WMO]
(2015) as five or more consecutive days of heat in which the
daily maximum temperature is at least 5C higher than the
average maximum temperature. These adverse environmental
events are foreseen to be increasingly frequent (Cardoso et al.,
2019). The main goal of this work was to evaluate whole
transcriptomic alterations induced by heatwave-like treatment
during grain filling. This study was comparatively performed
in two commercial varieties and two Portuguese landraces,
chosen based on previous evaluations of high temperature (HT)
responses regarding yield and grain composition (Tomás et al.,
2020a,b,c).
MATERIALS AND METHODS
Plant Material and High Temperature
Treatment
The genotypes studied in this work comprehend two bread wheat
(Triticum aestivum L., 2n = 6×= 42, AABBDD) commercial
varieties recommended to be used in Portugal (ANPOC et al.,
2014), Antequera and Bancal, and two old Portuguese landraces
from Vasconcellos collection, established in the 30s of the
last century (Vasconcellos, 1933), Ardito and Magueija. Seeds
of commercial varieties were gently supplied by ANSEME
(Portugal) and seeds of traditional landraces by EAN Germplasm
Bank (Oeiras, Portugal, PRT005). Twenty seeds from each
genotype obtained after 2 years of controlled propagation were
germinated and grown in controlled conditions–8 h of dark at
20C and a 16 h light period divided in 6 h increasing to 25C, 4 h
at 25C, and 6 h decreasing to 20C. Three-week old plants were
transferred individually to seven liters soil pots and maintained
in greenhouse conditions.
When the first anther was observed in the first spike (anthesis),
plants were transferred to growth chambers with the previously
described control conditions. Ten days after anthesis (daa)
subsets of ten plants (biological replicates) of each genotype
were submitted to two different growth conditions for 7 days:
control conditions above described or high temperature (HT)
regime with a daily plateau of 40C maximum temperature
(Supplementary Figure 1). Immediately after the period of
4 h at maximum temperature in the last day of the treatment,
two immature grains from the middle of each first spike of
each plant were collected (17 daa) and stored at –80C for
posterior RNA extraction.
RNA Extraction, Library Preparation, and
Sequencing
Total RNA was individually extracted from control and heat-
treated immature grains using the SpectrumTM Plant Total RNA
Kit (Sigma-Aldrich, Inc., Spain) and following manufacturer’s
instructions. For the RNA sequencing, three biological replicates
were analyzed per condition and genotype, each composed
of a pool of three immature grains (total 100 ng of RNA).
Both library preparation and sequencing were performed and
optimized by the Genomics Unit of the Instituto Gulbenkian
Ciência, Oeiras. mRNA-libraries were prepared using the
SMART-seq2 protocol adapted from Macaulay et al. (2016)
Illumina R
libraries performed used the Nextera protocol adapted
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Tomás et al. Wheat Grain Transcriptome Under Heat
from Baym et al. (2015). The libraries quantification and quality
verification were done using the Agilent Fragment Analyzer in
combination with HS NGS Kit (Agilent Technologies, Santa
Clara, California). Libraries were sequenced in the NextSeq500
Illumina R
Sequencer using 75 SE high throughput kit (Illumina,
San Diego, California) and 937302653 reads were obtained
from the 24 samples.
RNA Sequencing Data Processing and
Differential Gene Expression Analysis
Bioinformatic analysis from quality assessment to differential
expression analysis were performed by BioData.pt. Quality
control was evaluated on raw reads using FastQC (Andrews
et al., 2010). Raw reads were then trimmed using fastp (Chen
S. et al., 2018) to the longest continuous segment of Phred-
quality (threshold of 30 or above) in order to improve overall base
quality, and remove the Illumina R
Smart-Seq2 adaptors from
sequencing. A new quality control with FastQC was performed.
The trimmed reads were mapped to Triticum aestivum genome1
using hisat2 with default parameters (Kim et al., 2015). Quality
control of the mapping procedure was accessed with Qualimap
(Okonechnikov et al., 2016).
Read assignment to genomic features and gene expression
quantification were made using featureCounts (Liao et al., 2014).
Differential gene expression was tested using DESeq2 (Love et al.,
2014) between transcript sets of control and HT treated samples.
Manual search of gene ID and encoding products was made in
Ensemble Plants BioMart (Kinsella et al., 2011).
R software (R Core Team, 2018) was used to integrate all the
analysis and obtain multi-dimensional scaling analysis (MDS)
plot (to show the general relationship between the samples) and
hierarchical clustering of samples for all varieties and conditions
represented as an heatmap and Venn diagrams (showing the
relationships between the differentially expressed genes lists of all
varieties and conditions).
Gene Ontology Enrichment Analysis
Gene enrichment (GO) analysis was done in AgriGOv22
web-based tool (Tian et al., 2017). AgriGO SEA parameter
settings were as follows: Fisher test, with Bonferroni multi-
test adjustment method, 0.05 significance level, five minimum
mapping entries, and complete gene ontology. The GO database3
was used to analyze GO terms enrichment of DEGs, and the
Kyoto Encyclopedia of Genes and Genomes (KEGG) database4
was used to identify the enriched metabolic pathways, as well as
the enzymes involved.
RESULTS AND DISCUSSION
In this work, plants of four wheat genotypes, Antequera,
Ardito, Bancal, and Magueija were submitted to HT treatment
1ftp://ftp.ensemblgenomes.org/pub/plants/release-48/fasta/triticum_aestivum/
dna/Triticum_aestivum.IWGSC.dna.toplevel.fa.gz
2http://systemsbiology.cau.edu.cn/agriGOv2/index.php
3http://geneontology.org
4http://www.kegg.jp/kegg
simulating a heatwave, for 1 week during grain filling stage.
Transcriptome profiles of immature grains collected immediately
after treatment period (17 days after anthesis) from control and
treated plants were analyzed.
Traditional Genotypes Presented a More
Similar High Temperature Response
Than Commercial Varieties
The reference genome used to map transcripts was the IWGSC
RefSeq v1.0 assembly (the first version of the reference sequence
obtained from the bread wheat variety Chinese Spring). Overall,
about 90% of the transcripts were mapped against the reference
genome, and from these 37% mapped to multiple sites and the
other 53% mapped specifically to one site in the genome. From
the mapped transcripts, an average of 68% of the reads aligned
to exonic regions, 29% to the intergenic regions and only 3% to
intronic regions. The great percentage of transcripts mapped to
intergenic domains is probably due to the incomplete genome
annotation. Interestingly, commercial varieties Antequera and
Bancal presented a significantly (p<0.05) higher percentage
(71.5%) of transcripts mapped to the exonic regions than the
traditional ones Ardito and Magueija (61%). Concomitantly, the
number of transcripts mapped to both intronic and intergenic
regions is higher in the landraces. This may be explained by
the fact that old traditional genotypes, collected in the 1930s,
are more distinct from the reference genome than commercial
varieties. Lastly, the results summarized in Figure 1A indicate
that most reads (between 73 and 82%) were assigned as protein
coding regions, the next most found class was non-translating–
coding sequence (between 18 and 27%), and, in a very small
amount ribosomal DNA (less than 3%).
Also a hierarchical clustering (Figure 1B) of sample-to-sample
correlations revealed great intravarietal similarity between
Antequera and Bancal samples, independently of the treatment,
while for Ardito and Magueija, the similarities were greater
between samples of the same condition (control/HT). In fact, the
MDS (multi-dimensional scaling) plot grouping (Figure 1C) has
shown that five of the six samples of each commercial varieties are
closer to each other, while in landraces a clear separation between
control and treatment samples was observed.
Differentially expressed genes (DEGs) between transcriptomes
of immature grains from plants kept in control conditions
and submitted to high temperature were considered significant
with and adjusted p-value (padj) <0.05 for all genotypes.
Up and downregulated genes were obtained filtering the log2
foldchange absolute value higher than 1. For the four genotypes
analyzed, a total of 10,366 DEGs were identified, 86% of
them referent to Ardito and Magueija traditional genotypes,
showing that they have a greater response to high temperature
treatment. In a similar study done recently (Rangan et al.,
2019), grain transcriptomes of three genotypes showed a
higher number (more than 80%) of downregulated genes in
susceptible genotypes, comparing with tolerant ones (2% of
the DEGs were downregulated). Thus, the HT response of our
landraces was similar to susceptible genotypes, as they present a
higher number of DEGs.
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FIGURE 1 | Reads assignments by gene type and relationship between samples. (A) Read assignments and relative abundance of reads per type of gene.
(B) Hierarchical clustering of sample-to-sample correlations based on Pearson correlations (right). (C) Multi-dimensional scaling (MDS) plot showing similarity
between all samples. The comparisons were made between control (C) and high temperature (HT) reads sets of commercial varieties Antequera and Bancal and
landraces Ardito and Magueija.
The number of DEGs is significantly different between
all genotypes (p<0.05), although these differences were
less accentuated between Ardito and Magueija (Figure 2A).
Particularizing to each genotype, the commercial variety Bancal
presented the lower number of DEGs of all varieties studied,
129 in total, 69 upregulated and 60 downregulated. A ten times
higher number of DEGs were identified in Antequera (1298),
339 upregulated and 959 downregulated. Considering the work
above referred (Rangan et al., 2019), the higher number of
DEGs detected in Antequera is in accordance with the previously
reported worse heat response of this genotype in comparison
to Bancal regarding grain protein content and grain yield
(Tomás et al., 2020b). In Ardito, 4,375 DEGs were identified,
2,397 upregulated and 1,978 were downregulated. The genotype
with greater number of DEGs (4,564) in response to high
temperature treatment was Magueija, with 2,661 downregulated
and 1,903 upregulated.
Our first approach was to investigate if any of A, B, and
D genomes or distinct chromosomes were particularly affected
by high temperature treatment, since is already documented
that chromosomes 3A and 3B harbor genes involved in high
temperature response [reviewed in Ni et al. (2018)]. Although,
no significant differences were detected between genomes neither
between chromosomes (Supplementary Figure 2).
The results presented in Figure 2B have shown that from the
1,199 upregulated genes common to more than one genotype,
only six genes were common to Antequera, Bancal, Ardito,
and Magueija. These six upregulated genes common to all
genotypes (Table 1) encompassed annotated genes encoding
three small heat shock proteins HSP20, one adenylate kinase, a
BAG domain proteins and a ferritin. Adenylate kinase catalyzes
a reversible transphosphorylation reaction that converts adenine
nucleotides (ADP to ATP and AMP), and is critical for many
processes in living cells (Pradet and Raymond, 1983), as, for
example cellular activities maintenance during abiotic stress
situations (Komatsu et al., 2014). BAG domain proteins are
responsible for the modulation of chaperones activity as they
bind to HSP70 proteins and promote the substrate release.
Lastly, ferritin is a protein that functions in the iron storage
in a soluble, non-toxic, readily available form. A recent study
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Tomás et al. Wheat Grain Transcriptome Under Heat
FIGURE 2 | Differentially expressed genes (DEGs). (A) Number of DEGs between control and high temperature treated samples of commercial varieties Antequera
and Bancal and landraces Ardito and Magueija. Red and blue indicate down and upregulated genes, respectively. (B–E) Venn diagrams of differentially expressed
genes in commercial varieties Antequera and Bancal and landraces Ardito and Magueija: (B) upregulated in all genotypes; (C) downregulated in all genotypes; (D)
downregulated in commercial varieties and upregulated in landraces; (E) upregulated in commercial varieties and downregulated in landraces. Non-overlapping
regions represent the number of genes exclusive to one genotype. Overlapping regions indicate the number of genes common to two, three or four genotypes.
(Zang et al., 2017) showed that the overexpression of a gene
encoding a ferritin (TaFER-5B) functionally complemented the
heat stress-sensitive phenotype of a ferritin-lacking mutant of
Arabidopsis enhancing heat, drought, oxidative, and excess iron
stress tolerance associated with the ROS scavenging, as well as
leaf iron content. Thus, the present work not only identified
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Tomás et al. Wheat Grain Transcriptome Under Heat
TABLE 1 | Upregulated genes common to all genotypes analyzed.
Gene ID Encoded protein
TraesCS3B02G155300 Adenylate kinase
TraesCS4D02G086200 Small heat shock protein (Hsp20 family)
TraesCS4A02G092900 Small heat shock protein (Hsp20 family)
TraesCS5A02G548000 BAG domain
TraesCS4B02G089800 Small heat shock protein (Hsp20 family)
TraesCS7D02G428200 Ferritin
genes commonly modulated by HT in distant related hexaploidy
wheats, but also pointed out an upregulated one that seem to be
involved in HT response not only of wheat genotypes but also of
dicot plants like Arabidopsis.
There is a great difference between the number of upregulated
genes common to both commercial varieties and the number
of these genes common to both traditional ones, as can be
seen in Figure 2B. Only 2 of the 418 (0.48%) HT upregulated
genes were common in both commercial varieties. These genes
encode for a protein induced by water deficit or abscisic acid
stress and ripening, and a NAC transcription factor involved in
plant development (NAC019-A1). A recent work revealed that
this transcription factor is known to be a negative regulator
of starch synthesis, kernel weight, and kernel width in wheat
developing grains (Liu et al., 2020). In fact, our previous analyses
of mature grains of these genotypes subjected to HT during
grain filling revealed a reduction of starch amount in both
commercial varieties and an increase in both landraces (Tomás
et al., 2020a,b). On the other hand, a much higher proportion
of upregulated genes, 1,097 of the 5,058 (21.7%) are shared by
the landrace genotypes. These genes are associated with 1,747
biological processes gene ontologies, being the most represented
terms related with protein folding and metabolic process.
Concerning downregulated genes, none was commonly
detected in all genotypes, and it was also observed a much
higher number of genes common to both traditional landraces
(572–87.6%) than between commercial varieties (6–0.92%)
(Figure 2C). These results reinforce the already referred
suggestion that traditional genotypes have a more similar
response to the HT treatment than commercial ones.
Among the 110 genes downregulated in commercial
genotypes and upregulated in landraces (Figure 2D), there
were genes encoding for several HSP of different classes, related
with HT response proteins involved in nitrogen metabolism
and seed storage proteins, that are mainly involved in the seed
quality. On the other hand, only 34 genes were upregulated
in commercial varieties and downregulated in traditional
genotypes (Figure 2E), and the gene products are very diverse,
encompassing proteins involved in DNA binding, zinc finger
domains, transport proteins, and several No Apical Meristem
(NAM) proteins, referred before as a negative regulator of starch
synthesis (Liu et al., 2020).
Looking forward to unravel if there was any HT common
response related with the more affected genes, we analyzed
the ten most up and downregulated genes of each genotype
(Supplementary Table 1). It was possible to note that
in the commercial varieties, upregulated genes encode for
diverse products, several involved in the RNA processing. For
example, pentatricopeptide-repeat-containing proteins (PPR)
were encoded by these upregulated genes in both commercial
genotypes. They are known to influence the expression of
several organellar genes by altering RNA sequence, turnover,
processing, or translation (Barkan and Small, 2014). Also PPR
proteins have crucial roles in response to different abiotic
stresses in rice and were found as miRNAs target genes
associated with thermotolerance in wheat (Tan et al., 2014;
Chen G. et al., 2018;Ravichandran et al., 2019). On the
other hand, 60% of landraces upregulated genes encode for
products involved in heat shock response, as heat shock proteins
or heat shock factors, well-documented as high temperature
responsive genes [reviewed in Kaur et al. (2019)]. One of the
Magueija up regulated genes is the already identified in leaves
and roots TaHsfA6f, associated with increased thermotolerance
(Xue et al., 2015;Bi et al., 2020) and, to our knowledge, it
is for the first time identified in developing grains. As for
the downregulated genes, the only characteristic that stood
out was that in Antequera 7 out of the 10 downregulated
genes encode for products related with protein synthesis and
regulation, which can be related with the reduction in grain
protein content observed in this variety after HT treatment
(Tomás et al., 2020b).
Functional Annotation and Gene
Ontology Mapping of High Temperature
Differentially Expressed Genes
In a more global perspective regarding each genotype response
to high temperature treatment, functional annotation of DEGs
of each genotype was made through the assignment of gene
ontologies (GO) for biological processes, molecular functions
and cellular components (Figure 3 and Supplementary Table 2).
Figure 3 indicates the percentage of up and downregulated
genes of each genotype, assigned to second and third levels of
categories associated with each ontology. For all categories of
the three ontologies the proportions of up and downregulated
genes associated are very similar, being the classes with
higher and lower number of genes the same in both cases
for the four genotypes. This may indicate that, although
the number of altered genes may be different in distinct
genotypes (Figure 3), the functional roles in which the DEGs
are involved constitute a common feature in wheat heat
stress response.
In biological processes for both up and downregulated
genes, the most represented categories are biological regulation
(GO:0065007), cellular process (GO:0009987), metabolic process
(GO:0008152), and response to stimulus (GO:0050896). It
was also notorious that Bancal had a great percentage
of downregulated genes assigned to several categories. For
molecular functions ontology, catalytic activity and binding,
mainly protein (GO:0005515) and organic cyclic compound
binding (GO: 0097159) were clearly highlighted as compared
with the other classes. Regarding cellular component GO, the
most represented class was organelle (GO:0043226), with more
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FIGURE 3 | Gene ontology percentage of up and downregulated genes in commercial varieties Antequera and Bancal and landraces Ardito and Magueija assigned
to second and third levels of biological processes, molecular functions and cellular component gene ontologies. Red and blue indicate down and upregulated genes,
respectively.
than 50% of the DEGs in almost all the genotypes, and the other
was membrane (GO:0016020) with half of this amount.
Several GO terms were significantly represented in each
genotype (Supplementary Table 2), except for Bancal up
regulated genes, that were only significantly enriched in 16
molecular function ontologies. In total 395, 129, and 154 distinct
ontologies were identified for biological processes, molecular
functions and cellular components, respectively. Ardito and
Magueija present a closer response as several common ontologies
were significatively represented, namely some categories of
response to stress, establishment of cell polarity, protein
complex biogenesis, de novo protein folding and carbohydrate
catabolism for upregulated genes and DNA metabolism,
regulation of gene expression and protein complex biogenesis
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FIGURE 4 | KEGG pathways enrichment percentage of up and downregulated genes in commercial varieties Antequera and Bancal and landraces Ardito and
Magueija associated with metabolic, environmental and genetic information processing pathways. Red and blue indicate down and upregulated genes, respectively.
for downregulated genes. We also found common categories
in which Antequera and both landraces upregulated genes
were enriched, such as protein folding, response to light and
reactive oxygen species and heat acclimation biological processes
and peroxisome and microbody cellular components. On the
other hand, categories significantly enriched by downregulated
genes common between commercial and landraces were only
identified in cellular components, for instance related to nuclear
lumen and thylakoid.
Particularly, from all the DEGs engaged in high temperature
treatment response, 512 were assigned to response to heat
category (GO:0009408), most of them presenting increased
expression levels in the traditional varieties while in the
commercial ones, only a small part was affected (Supplementary
Table 3). These genes are related with other 106 biological
processes, being the most represented protein folding (16%)
and transcription regulation (8%), 41 cellular components with
nucleus and integral component membrane being associated
with a greater number of genes (14% each), and 105
molecular functions, being the most represented ATP binding
(12.6%), protein binding (8.7%), and unfolded protein binding
(7.3%). These classes of DEGs where also recently associated
with wheat tolerance to drought during grain filling stage
(Nouraei et al., 2022). Regarding unfolded protein binding, as
expected, a great number of genes encode heat shock proteins
(Hsp) and heat shock factors (Hsf). About 30% of the genes
encode for the different Hsp20, Hsp40 (DNAJ domain), Hsp70
and Hsp90, and the great majority were upregulated in the
traditional genotypes and remain unaltered or downregulated
in the commercial ones. Also in the traditional genotypes, 12
genes encoding Apetala 2 proteins were identified as upregulated.
Several proteins of this class were involved in grain and
spike morphology, plant height, and spike emergence time
determination, and play a key role in growth and development,
including regulation of plant architecture and yield-related traits
(Li et al., 2016;Zhao et al., 2019).
To further disclose biological functions of DEGs and
determine if any pathway have a significant involvement in heat
tolerance, we investigated DEGs involved in Kyoto Encyclopedia
of genes and Genomes (KEGG) pathways and 749 DEGs were
assigned related with 107 KEGG pathways (Figure 4 and
Supplementary Table 4). Antequera was the genotype with less
DEGs associated with these pathways (0.5%), the traditional
genotypes revealed 9% each, and Bancal was the genotype that
presented the higher percentage (57%).
Analyzing the pathways associated with products encoded
by downregulated genes, the ones related with carbohydrate
metabolism were the most influenced in Bancal and both
traditional genotypes. The only carbohydrate pathway associated
with downregulated genes of all four genotypes were the
Glycolysis/Gluconeogenesis pathway, although neither the genes
nor the encoded enzymes were common. Although, inside
this category, the majority of Bancal downregulated genes
encoded for enzymes involved in pentose and glucuronate
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Tomás et al. Wheat Grain Transcriptome Under Heat
FIGURE 5 | Differentially expressed genes involved in nutrient reservoir activity ontology in commercial varieties Antequera and Bancal and landraces Ardito and
Magueija. Red and blue indicate down and upregulated genes, respectively, and color intensity are related with the degree of gene expression alteration; gray
represents unaltered genes.
interconversions pathway and in landraces encoded for starch
and sucrose metabolism, with the majority of encoded enzymes
associated with glucose synthesis. Some of the enzymes
categorized in the pentose and glucuronate interconversions were
pectinesterases known to be involved in cell wall remodeling
that occurs during high temperature response (Wu et al.,
2018). Kino et al. (2020) reported also a downregulation of
genes involved in pericarp cell wall expansion due to high
temperatures exposure during post anthesis, and speculate
that this can be related with the reduction in grain weight
observed after this stress. Our work also corroborated this
suggestion since the majority of DEGs encoding pectinesterases
were downregulated in landraces in which a reduction in
grain weight was observed (Tomás et al., 2020a). The second
most affected pathways were the ones involved in amino acid
metabolism, with the majority of DEGs assigned to cysteine and
methionine metabolic pathways for Bancal and both landraces.
This was an unexpected result as the accumulation of this
amino acid was reported in high temperature conditions (Tao
et al., 2018). Again, only one pathway, the glycine, serine
and threonine metabolism, was identified as being associated
with downregulated genes in all the genotypes, but also again
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Tomás et al. Wheat Grain Transcriptome Under Heat
FIGURE 6 | Integrative analysis of grain yield, composition and transcriptomic analysis of the commercial varieties and the old landrace responses to heatwave at
the grain-filling stage.
none of these genes was common to all the genotypes. An
interesting result was the percentage of Bancal downregulated
genes encoding for Aminoacyl-tRNA synthetases (nine different
genes encode for six different synthases), classified in the
translation pathways. This was not an expected result as
several works in distinct species report an increase in different
enzymes of this family in abiotic stress situations (Giritch
et al., 1997;Thimm et al., 2001;Kobayashi et al., 2005;
Baranaši´
c et al., 2021). Lastly, several downregulated genes
in Bancal were associated with nucleotide metabolism, more
specifically with purine metabolism, and encoded for Adenosine
triphosphatase (ATPase).
Upregulated genes were also associated with most of the
mentioned pathways for downregulated genes. In fact, the
encoded products were in some cases the same as for
downregulated genes, suggesting that they include several
cases of different enzyme isoforms or homologous genes with
different functions, as already reported (Liu et al., 2015;
Kaushik et al., 2020). Carbohydrate pathways include the
greater number of associated upregulated genes for Bancal
and both landraces. Particularizing, starch and sugar pathway
was the most common, and glycolysis was the second. For
Bancal, nucleotide metabolism was again the pathway with
the higher percentage, although the encoded enzymes were
involved in the dephosphorylation of ATP molecules, as well
as translation pathways, in which were detected transcripts
for enzymes involved in glutamate and tryptophan tRNA
synthesis. Upregulated genes of Bancal and both landraces
encoded also for enzymes in lipid metabolism. Specifically
involved in cutin, suberine and wax biosynthesis, glycerolipid
metabolism, fatty acid elongation, fatty acid biosynthesis,
the latter two being related only with upregulated genes in
landraces. This may indicate an alteration in lipids proportions
in response to high temperature as previously reported
[reviewed in Abdelrahman et al. (2020)].
High Temperature Effects in Storage
Proteins Encoding Genes
Gluten is determinant for the wheat suitability to produce bread
as it is a protein network that entrains air bubbles during dough
fermentation. It is composed from two classes of storage proteins,
glutenins, responsible for the dough strength and elasticity,
and gliadins which confer extensibility and viscous properties
to gluten required for dough development. Gliadin/glutenin
ratio is determinant for rheological characteristics (Dhaka and
Khatkar, 2015), being for that reason important to access if
these proteins encoding genes’ are affected by high temperature
treatment. Storage proteins encoding genes are classified in the
nutrient reservoir activity ontology and the expression levels of
DEGs associated with this category are presented as heatmap
in Figure 5. None of the genes presented altered expression in
Bancal genotype. Additionally, about 60% of the DEGs were
related with two protein families, Cupins and Gliadins.
The results obtained revealed 12 gliadin encoding genes
differentially expressed, mostly upregulated in Magueija and
Antequera, that may have implications in grain quality. In fact,
several studies showed that an increase in gliadin fraction has a
detrimental effect on the technological characteristics of wheat.
Flours with higher gliadin content present weaker gluten quality
and dough, with increased viscosity and stickiness (Barak et al.,
2015). Additionally, Antequera and Ardito presented 6 and 2
downregulated genes encoding for Cupins, respectively, while
three genes were upregulated, 1 in Ardito and 2 in Magueija.
Cupins were already described as heat responsive proteins
with an unusual thermostable character which facilitates their
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Tomás et al. Wheat Grain Transcriptome Under Heat
accumulation in a number of heat-stressed organisms (Dunwell
et al., 2001). A more recent work shows that these proteins are
preferentially accumulated when protein synthesis components
are generally decreased during heat stress, suggesting that they
may provide valuable insights into improving the protein content
of wheat (Wang et al., 2018). A significative reduction of protein
content was previously observed in Antequera mature grains
after heat stress treatment (Tomás et al., 2020b). Altogether, these
results show that gliadins are more affected by high temperature
treatment than glutenins in both wheat commercial varieties and
landraces and reinforce the need to investigate the cupins role in
heat stress response.
It must be emphasized that the expression levels of genes
related with flour quality, like the ones encoding glutenins
and enzymes involved in starch and puroindolines synthesis,
were already evaluated through qRT-PCR (Tomás et al.,
2020c), and the results obtained confirm the RNA Sequencing
analysis here presented.
Overall, the results obtained in this and past works (Tomás
et al., 2020a,b,c) show that high temperature treatment tend
to reduce yield and quality differences observed between
commercial varieties in control conditions. Conversely,
differences observed between old landraces were enhanced in
plants submitted to HT treatments (Figure 6). Characteristics like
grain weight, protein content and transcription profiles of heat
responsive genes on the traditional genotypes studied encourages
a deeper analysis of those genotypes enclosed in Vasconcellos
collection (Vasconcellos, 1933). Nevertheless, accordingly to
all our analysis, the commercial variety Bancal seems to be a
promising genotype to cope with high temperatures.
Several questions arise from this work, confirming that high
temperature response results from a complex of physiological,
cellular and molecular processes, as previously proposed (Jacott
and Boden, 2020;Schaarschmidt et al., 2021). Though, several
pieces are missing to compose the intricate puzzle of plant
response to this abiotic stress (Jagadish et al., 2021;Khan
et al., 2021). A deeper exploitation of RNA sequencing data,
focusing on particular pathways, will be needed to enrich
correlations between specific changes in genes expression
profiles and phenotypic alterations induced by heat wave
like treatments.
DATA AVAILABILITY STATEMENT
The original contributions presented in this study are publicly
available and sequences data can be found here: https://www.
ncbi.nlm.nih.gov/search/all/?term=PRJNA750265.
AUTHOR CONTRIBUTIONS
DT and MS: conceptualization, methodology, and validation.
DT: formal analysis, investigation, visualization, and writing—
original draft preparation. MS: funding acquisition and project
administration. MS and WV: supervision and writing—review
and editing. All authors contributed to the article and approved
the submitted version.
FUNDING
DT was funded by a Fundação para a Ciência e a Tecnologia,
Portugal (FCT) doctoral scholarship (SFRH/BD/93156/2013),
MS by the FCT Investigator Program (IF/00834/2014), and
the research work was financed by FCT research project
IF/00834/2014/CP1219/CT0003, LEAF Unit (Linking Landscape,
Environment, Agriculture and Food) (UID/AGR/04129/2020),
and CEF Unit (Forest Research Centre, UIDB/00239/2020).
ACKNOWLEDGMENTS
We would like to thank Eng. Manuela Veloso (PRT005: EAN
Germplasm Bank, Oeiras) for the landrace accessions studied in
the present study. Also we would like to acknowledge the support
of Ricardo Leite from Genomics Facility in Instituto Gulbenkian
Ciência, on RNA sequencing design and analysis.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fpls.2022.
842599/full#supplementary-material
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Frontiers in Plant Science | www.frontiersin.org 13 June 2022 | Volume 13 | Article 842599
... Hexaploid bread wheat (Triticum aestivum L.) belongs to the Poaceae family and is the second-most grown, regularly fed crop in the world. However, due to the elevatedglobal temperature, the estimated worldwide demand for wheat in coming years cannot be met with the current declining production output [1,2]. According to controlled environment research, wheat grain yield is more influenced by heat stress as compared to drought stress as it decreased by 4% or a yield loss of 190 kg/ha for every degree rise in temperature over 15 • C [3][4][5]. ...
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Temperature is an essential physical factor that affects the plant life cycle. Almost all plant species have evolved a robust signal transduction system that enables them to sense changes in the surrounding temperature, transduce, and accordingly adjust their metabolism and cellular functions to avoid heat stress-related damage. Wheat (Triticum aestivum), as a cool-season crop, is very sensitive to heat stress. Any increase in the ambient temperature, especially at reproductive and grain-filling stages, can cause a drastic wheat yield loss. Heat stress causes lipid peroxidation due to oxidative stress, resulting in damage of thylakoid membranes and disruption of their function, and ultimately decreases photosynthesis and crop yield. The cell membrane/plasma membrane plays prominent roles as an interference system that perceives and translates the changes in environmental signals into intracellular responses. Thus, membrane lipid composition is a critical leap for heat stress tolerance or susceptibility in wheat. In this review, we elucidate the possible involvement of calcium influx as an early heat stress-responsive mechanism in wheat plants. In addition, the physiological implications underlying the changes in lipid metabolism under high-temperature stress in wheat and other plants species will be discussed. In-depth knowledge about wheat lipid reprogramming can help in developing heat-tolerant wheat varieties, and provide approaches to solve the consequences of global climate change.