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ORIGINAL ARTICLE
A transcriptomic, metabolomic and cellular approach to the
physiological adaptation of tomato fruit to high temperature
Juliana Almeida | Laura Perez-Fons | Paul D. Fraser
Department of Biological Sciences, Royal
Holloway University of London, Egham, UK
Correspondence
Paul D. Fraser, Department of Biological
Sciences, Royal Holloway University of
London, Egham, Surrey TW20 0EX, UK.
Email: p.fraser@rhul.ac.uk
Funding information
H2020 Food, Grant/Award Number: H2020
programme TomGEM / 679766
Abstract
High temperatures can negatively influence plant growth and development. Besides
yield, the effects of heat stress on fruit quality traits remain poorly characterised. In
tomato, insights into how fruits regulate cellular metabolism in response to heat
stress could contribute to the development of heat-tolerant varieties, without detri-
mental effects on quality. In the present study, the changes occurring in wild type
tomato fruits after exposure to transient heat stress have been elucidated at the
transcriptome, cellular and metabolite level. An impact on fruit quality was evident as
nutritional attributes changed in response to heat stress. Fruit carotenogenesis was
affected, predominantly at the stage of phytoene formation, although altered
desaturation/isomerisation arose during the transient exposure to high temperatures.
Plastidial isoprenoid compounds showed subtle alterations in their distribution within
chromoplast sub-compartments. Metabolite profiling suggests limited effects on pri-
mary/intermediary metabolism but lipid remodelling was evident. The heat-induced
molecular signatures included the accumulation of sucrose and triacylglycerols, and a
decrease in the degree of membrane lipid unsaturation, which influenced the volatile
profile. Collectively, these data provide valuable insights into the underlying bio-
chemical and molecular adaptation of fruit to heat stress and will impact on our abil-
ity to develop future climate resilient tomato varieties.
KEYWORDS
carotenoids, fruit quality, fruit ripening, heat stress, isoprenoids, metabolomics, plastoglobuli,
tomato, transcriptomics
1|INTRODUCTION
Plant growth and development are vulnerable to abiotic stresses such
as higher temperatures, leading to detrimental effects on agricultural
yield (Battisti & Naylor, 2009; Bita & Gerats, 2013). Increased global
mean temperatures and extreme climate-related events are now
occurring with increased frequency and intensity (Horton et al., 2015;
IPCC, 2013). The consequences arising from the warmer temperatures
have already been observed and hold potential to destabilise food
systems and to threaten local to global food security (Lesk
et al., 2016; Zhao et al., 2017). Higher temperatures can trigger devel-
opmental, physiological, cellular stress responses in plants which is
highly dependent on duration and severity of stress as well as sensi-
tivity of plant cell type and developmental stage (Larkindale
et al., 2007; Ohama et al., 2017). When heat stress is moderate, the
changes occurring to crops may be rapidly reversible, but severe epi-
sodes of elevated temperatures are irreversible and can lead to crop
failure (Zhang et al., 2010).
Received: 24 February 2020 Revised: 2 July 2020 Accepted: 12 July 2020
DOI: 10.1111/pce.13854
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.
© 2020 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.
Plant Cell Environ. 2020;1–19. wileyonlinelibrary.com/journal/pce 1
Tomato (Solanum lycopersicum) is among the crops for which yield
losses have been well documented when different high-temperature
regimes occur during reproductive phase. Yield (e.g., fruit number and
weight) is adversely affected by daily mean temperatures above 29C,
ranging from a few days—when pollen development or fruit set is
disturbed—to a whole developmental period (Peet et al., 1998; Press-
man et al., 2002; Sato et al., 2000, 2006). Notably, the tomato vegeta-
tive development is less sensitive to episodic temperature increases
as structural damages of photosystem II were not detected for tem-
peratures reaching 38C (Lu et al., 2017; Spicher et al., 2017). Never-
theless, the influence of heat stress on tomato nutritional composition
and quality has received less attention. This is surprising considering
tomato is one of the most widely consumed fruits globally
(Bergougnoux, 2014), it is grown worldwide and is an important
source of vitamins and bioactives in the human diet (Viuda-Martos
et al., 2014).
Metabolite composition of tomato fruit is affected by adverse
environmental conditions (Quinet et al., 2019). While some abiotic
stresses as water deficit lead to an increase in sugars, organic acids,
vitamin C and carotenoids (Albert et al., 2016), high-temperature con-
ditions seem to have a diverse impact on fruit quality. Most informa-
tion relies on studies performed with differing post-harvest
conditions. Early studies have demonstrated that the long-known fail-
ure to achieve normal pigmentation of excised tomatoes ripening at
high temperatures (Tomes, 1963) is associated with changes in ethyl-
ene production, fruit softening and colour development, a phenotype
that could be reversed when fruits were transferred to optimal tem-
perature (Lurie et al., 1996; Picton & Grierson, 1988). More recent
evaluations corroborated these findings highlighting the heat-
sensitivity of antioxidant accumulation as carotenoids and vitamin C
(Gautier et al., 2008; Massot et al., 2013). For vine-attached fruits, the
detrimental effects of high ambient temperature on bioactive com-
pounds have also been observed (Hernández et al., 2015; Mulholland
et al., 2003). Vitamin C (ascorbate) and carotenoids (provitamin A) are
significantly lower when a heat-stress treatment is imposed during
the advanced stages of fruit development. However, when the tem-
perature is raised at earlier stages, the lack of effects indicates differ-
ential thermo-sensitivity of fruit developmental stages (Hernández
et al., 2015). While these changes in tomato metabolism have been
reported, a comprehensive evaluation exploring the cellular and
molecular modifications associated with heat response awaits
elucidation.
Alteration in metabolite composition can be direct consequence
of several molecular mechanisms underlying the heat stress
responses. A typical signature response is a wide-scale transient
reprogramming of gene expression, including the expression of heat
shock proteins (HSPs, Kotak et al., 2007; Ohama et al., 2017). The
majority of HSPs function as molecular chaperones which act not only
in protection against stress damage but also in folding, intracellular
distribution and degradation of proteins (Mishra et al., 2002). Interest-
ingly, HSPs seem to be important for tomato fruit ripening
(Fragkostefanakis et al., 2015; Neta-Sharir et al., 2005). To maintain
membrane stability and deal with oxidative stress generated in
response to heat, plants induce synthesis of hormones, and other pro-
tective molecules including osmoprotectants and antioxidants (Gray &
Brady, 2016; Wahid et al., 2007). The antioxidant network, in part, is
based on the action of several molecules including carotenoids,
tocopherols (vitamin E), ascorbate and phenolic compounds, all with
potential to contribute to fruit nutritional proprieties (Li et al., 2018b).
Furthermore, as heat can lead to membrane damage caused by lipid
hyper-fluidity and lipid peroxidation, modifying membrane lipid com-
position, particularly the acyl moieties of glycerolipids, is another criti-
cal aspect of plant thermotolerance (Falcone et al., 2004; Murakami
et al., 2000; Higashi & Saito, 2019) that is linked to fruit quality.
Changes in 18-carbon (C18) polyunsaturated fatty acids can affect the
enzymatic/nonenzymatic formation of oxylipins derived thereof
(Feussner & Wasternack, 2002). Lipoxygenase (LOX) pathway pro-
duces hydroperoxide intermediates for the synthesis of different com-
pounds, including jasmonic acid and volatiles, the latter an essential
aspect of fruit quality (Tieman et al., 2017). Lipid-derived signaling
molecules as oxidised derivatives constitute important components of
heat stress response, acting, for example, in the control of gene
expression related to protective responses (Balogh et al., 2013;
Farmer & Mueller, 2013; Hou et al., 2016).
In this present study, transcriptomic, metabolomic and cellular
analysis has been applied to tomato fruits at different ripening transi-
tions, following exposure to transient high-temperature treatment
(40C day/ 30C night). These conditions may replicate events of heat
stress experienced during commercial production. Collectively, the
data provide new insights into the metabolic plasticity of tomato fruit
to heat stress episodes and may contribute generically to the develop-
ment of climatic resilient crops.
2|MATERIAL AND METHODS
2.1 |Plant material, growth conditions and
transient high-temperature treatment
Tomato plants (Solanum lycopersicum, cv. Ailsa Craig) were grown
under greenhouse conditions with a 16/8-hr day night photoperiod at
25C /19C, respectively.
For transient heat stress treatment (H), the greenhouse chamber
temperatures were set at 40C/30C (day/night) over a 48 hr dura-
tion. Photoperiod and lighting were the same as the control plant
chamber, and watering adjusted to keep soil water content near field
capacity. Non-stressed plants were kept at control conditions (C) in a
parallel chamber. Flowers were tagged at anthesis. Treatment H was
imposed on three different groups formed by at least five tomato
plants (between 12- and 13-week-old), each one used to harvest a
specific fruit ripening stage, that was heat-stressed only once.
After H, five biological replicates of fruits, that is, fruits from five dif-
ferent plants, were harvested as follows: mature green (H
MG
,
37–39 days after anthesis, DAF), breaker (H
B
,39–41 DAF) and 3 days
post breaker (H
B3
,42–44 DAF). Leaves from heat-stressed plants (H
L
)
were also collected. Plants dedicated to H
B
and H
B3
treatment could
2ALMEIDA ET AL.
recover (R) under control conditions, and fruits were harvested at red
ripe stage (7 days post breaker, B7); for H
B
, after 7-days recovery
period (H
B
R7); for H
B3
, after 4-days recovery period (H
B3
R4). Overall
fruit from five plants, representing five biological replicates, were col-
lected for each treatment described above and each biological repli-
cate was analysed independently. Samples were frozen immediately in
liquid N
2
upon collection and stored at −80C until metabolic and
molecular analysis.
2.2 |Isoprenoid determination and quantification
Isoprenoids (carotenoids, tocochromanols and chlorophylls) were
extracted from lyophilised tissue powder (15 mg) as described by
Enfissi et al. (2010). Compounds were analysed by reverse-phase
chromatography using an ultra-performance liquid chromatography
(UPLC) system (Acquity, Waters) equipped with a Photo Diode Array
(PDA) detector (Acquity, Waters). A UPLC BEH-C18 column
(100 mm ×2.1 mm; 1.7 μm, Acquity, Waters) was used for separation
as described by Nogueira et al. (2013). Peak identification was
achieved by comparison of characteristic UV/Vis spectrum with
authentic standards, reference spectra and retention times (Fraser
et al., 2007). Quantification was performed using dose–response cur-
ves obtained from authentic standards.
2.3 |Metabolite profiling by gas chromatography
(GC)-MS
Polar extracts were prepared from freeze-dried fruit powder (10 mg),
extracted with 1 ml of solution containing methanol and water acidified
with 0.1% formic acid [80:29.9:0.1, (v/v/v)] and agitated for 1 hr. After
centrifugation, the polar extract was spiked with ribitol (1 mg/ml in
MeOH; 10 μg final concentration) as the internal standard. For nonpolar
extracts, alkaline hydrolysis with KOH was performed with a fruit pow-
der aliquot (10 mg) during 1 hr at 40C followed by extraction as
described for isoprenoids. Nonpolar extracts were spiked with deuter-
ated myristic acid-
d27
as the internal standard. The dried residues were
derivatised in methoxyamine hydrochloride (in pyridine) followed by
silylation with N-methyl trimethylsilyl trifluoroacetamide. The GC–MS
analysis was achieved on Agilent 7890A GC system interfaced with a
5975C mass-selective detector as described in Uluisik et al. (2016).
For lipid and fatty acid compositional analysis, extraction was per-
formed as described for isoprenoids and resolved on high-
performance thin-layer chromatography (HTLC) silica gel 60 F254
plates (Merck) developed in a solvent mixture of acetone, toluene,
and water [91:30:7, (v/v/v)]. Regions containing the lipid classes were
identified based on the comparison with authentic standards vis-
ualised with iodine vapour and scraped from the HTLC plate. Elution
and conversion to fatty acid methyl esters (FAMEs) by acid-catalysed
transmethylation, followed by quantification using GC–MS were per-
formed as previously described by Nogueira et al. (2013). FAMEs
were quantified using myristic-
d27
acid as an internal standard.
Components were identified using a mass spectral library built
from in-house standards and NIST11 database. Each analytical batch
was validated with quality control samples.
2.4 |Profiling of volatile compounds by GC–MS
Frozen fruit samples were ground in liquid N
2
and aliquots (0.5 g) used
for the analysis of volatile compounds. Homogenates were weighed
out into screw-top headspace amber glass vials (20 ml) and spiked in
with deuterated acetophenone-
d3
as internal standard (20 ppb). Capped
vials were incubated at 40C and shaken for 30 min. Volatile compounds
were then adsorbed onto a SMPE fibre (Car/DVB/PDSM) for 20 min,
followed by desorption into the injection port for 5 min. Chromatographic
separation was conducted in a DB-5MS 30 m ×250 μm×0.25 μmcol-
umn (J&W Scientific, Folsom, CA), equipped with a 10 m guard column
and using a step-temperature gradient from 40 to 300Cat5
C/min. The
linear temperature gradient included a 2 min hold-temperature and then
steps at 40, 120, 250Cand5minat300
C. Helium was employed as
thecarriergasandtheflowratewas1ml/min.Theinletandthemass
spectrometer transfer line were heated to 250C. A 7890B-5977B GC-
MS system (Agilent Technologies, Palo Alto, CA) was used in splitless
mode, and data processing and analysis proceeded using AMDIS (version
2.73) software.
2.5 |Subchromoplast fractionation
Chromoplasts were isolated from fruits (90 g) at B3 to B4 stage, and
sub-compartments were fractionated using a discontinuous gradient
of sucrose, according to Nogueira et al. (2013).
2.6 |Transmission electron microscopy
Pericarp fruit segments were fixed at room temperature in solution
[3% (v/v) glutaraldehyde, 4% (v/v) formaldehyde buffered with 0.1 M
PIPES buffer pH 7.2] and then stored at 4C for at least 24 hr until
processing. Samples were post-fixed in buffered 1% (w/v) osmium
tetroxide and uranyl acetate, washed, dehydrated in a graded series of
acetone, and embedded in resin. Ultrathin sections were stained with
Reynolds lead citrate and imaged on a Tecnai T12 Transmission Elec-
tron Microscope (Field Electron and Ion Company).
2.7 |qPCR expression analyses
Total RNA was extracted from frozen leaves and fruit pericarps using
the RNeasy kit (Qiagen) according to manufacturer's instructions. RNA
from at least four biological replicates was prepared from each tissue
and ripening stage. RNA quality was assessed by agarose gel electro-
phoresis. Total RNA (1 μg) was treated with DNase and converted into
cDNA using the QuantiTect Reverse Transcription kit (Qiagen),
ALMEIDA ET AL.3
according to the manufacturer's protocols. Real-time quantitative PCR
(qPCR) assays were performed in technical duplicates using RotorGene
SYBR green PCR kit (Qiagen) on Rotor-Gene Q, with approximately
10 ng of reverse-transcribed RNA. Primer sequences are listed in
Table S1. Relative expression was calculated as described by Quadrana
et al. (2013). For reference gene selection, expression stability of five
known reference genes (CAC,EXP,GAGA,ACT1 and ACT2)(Cheng
et al., 2017; Exposito-Rodriguez et al., 2008) was evaluated on control
and heat-stressed samples using GeNorm (Vandesompele et al., 2002).
ACT2 and CAC were selected based on lowest expression stability
values (M) of 0.362 and 0.438, respectively.
2.8 |RNA sequencing
Total RNA from three biological replicates samples was isolated using
Trizol RNA Purification kit (Thermo Fisher Scientific). cDNA libraries
were prepared and sequenced by IGA Technology Services facility
(Udine, Italy). Single-end sequence reads (75 nt) at a read depth of 31.3
million reads on the average per sample (24.1 to 39.9 M reads) were
obtained from the NextSeq500 platform (Illumina). Raw reads were
processed using ERNE (Del Fabbro et al., 2013) and Cutadapt
(Martin, 2011) software. The reads were mapped onto tomato genome
(S. lycopersicum, cv. Heinz) reference SL3.0, with gene models ITAG3.10,
using STAR (Dobin et al., 2013) applying default parameters. Assembling
and quantification of full-length transcripts were accomplished by
Stringtie (Pertea et al., 2015). The counting was achieved by HTseq-
count (Anders et al., 2015). Gene ontology (GO) term annotation was
performed using Blast2GO Pro (version 5.2.5) (Conesa et al., 2005).
All raw RNA sequencing data are available on NCBI, under the
Bioproject accession number PRJNA603594.
2.9 |Data analyses
Significant differences between the control and heat-stressed condi-
tions were determined by Student's ttest or ANOVA followed by a
Dunnett's multiple comparison with the level of significance set to 0.05,
using GraphPad Prism software. For pair-wise differential expression
analysis of transcriptome data, statistical analyses were performed by
DeSeq2 (Love et al., 2014). Differentially expressed genes were deter-
mined using false discovery rate (FDR) ≤0.01 (adjusted p-value) and
jfold-changej≥1.5 (or jlog
2
FCj≥0.58). GO enrichment analysis with
Fisher's Exact Test was conducted using Blast2GO.
3|RESULTS
3.1 |Heat stress at advanced ripening stages
negatively affects carotenoid accumulation
Plants grown under control conditions (C) were exposed to 48 hr
high-temperature treatment (H). The H was imposed when plants
possessed fruits undergoing specific ripening transitions, from
mature green to breaker (H
B
) and yellow to light-red transition
(H
B3
) (Figure 1). For comparison, an additional H treatment at early
to late mature green (H
MG
) was included. After H, plants were ret-
urned to control conditions (recovery, R) until fruits ripened
(B7 stage).
The levels of plastidial isoprenoids were determined by UPLC-
PDA. H negatively influenced the carotenoid levels in tomato fruit,
and changes from the heat stress were highly dependent on the fruit
stage used (Table 1). Remarkably, phytoene, the first carotene prod-
uct of the pathway, was reduced both in H
B
(10-fold) and H
B3
(4.6-fold) compared to their corresponding non-stressed fruits. The
other carotenes in the pathway, phytofluene and ζ-carotene, when
detected, responded similarly to phytoene when H was applied.
Lycopene, the predominant carotenoid found in red ripe tomato,
was significantly lower, below detection in H
B
fruits and reduced by
50% in H
B3
fruits, compared to corresponding non-stressed controls.
Interestingly, lycopene levels only partially recovered in H
B3
R4
fruits, to about 60% of the content found in ripe C
B7
fruit, despite
the levels of phytoene and phytofluene precursors being fully
restored at this stage (Table 1).
The total fruit carotenoid content was consistent with lycopene
levels at later stages of ripening. While lycopene decreased in H
B3
and
H
B3
R4 fruits, total carotenoids remained unchanged both in H
B
fruits
and in those fruits allowed to recover from heat stress (H
B
R7). By
contrast, H
MG
carotenoid levels were mostly similar to the control,
except for a modest increase in β-carotene levels. The same response
was also observed in heat-stressed leaves (H
L
). Additionally, lutein
levels as well as total carotenoid showed an increase in H
L
compared
to control conditions.
FIGURE 1 Outline of heat stress experiment. Tomato plants were
kept at 25Cday/20
C night (control, C) or exposed to transient high-
temperature treatment (H) at 40C/30C (day/night) for 48 hr. After H,
fruits at the following stages were harvested: mature green (H
MG
),
breaker (H
B
) and 3 days post-breaker (H
B3
). Fruits allowed to recover
(R) under normal conditions were harvested at ripe stage (H
B
R7 and
H
B3
R4) [Colour figure can be viewed at wileyonlinelibrary.com]
4ALMEIDA ET AL.
TABLE 1 Transient changes in isoprenoid profile of tomato fruits and leaves exposed to high-temperature treatment
Fruit Leaf
μg/g DW C
MGa
H
MGa
C
Ba
H
Ba
C
B3a
H
B3a
C
B7a
H
B
R7
a
H
B3
R4
a
C
La
H
La
Phytoene nd nd 2.7 ± 1.5 0.3 ± 0.2* 103.5 ± 15.7 21.9 ± 17** 207.8 ± 39.1 201.8 ± 44.9 147.9 ± 32.3 14 ± 3.3 13.1 ± 5.2
Phytofluene nd nd 1.8 ± 1.5 nd 78.2 ± 13.3 15.7 ± 13.4** 172.9 ± 28.7 165.7 ± 32.8 125.8 ± 25.5 nd nd
ζ-Carotene nd nd nd nd 3.1 ± 1.3 0.7 ± 0.4* 6.7 ± 1.1 7.7 ± 1.6 8.2 ± 1 nd nd
Lycopene nd nd 4.4 ± 5.4 nd 616.3 ± 93.6 227.9 ± 188.3** 1724.7 ± 258.6 1,670.3 ± 279.5 1,066.2 ± 117.3** nd nd
γ-Carotene nd nd nd nd 30.6 ± 6.6 19.9 ± 8.5 46.4 ± 6 38.6 ± 4.3 29.9 ± 10.2 nd nd
β-Carotene 48.9 ± 1.1 54.1 ± 2.5** 64.8 ± 2.4 54.7 ± 6.1* 136.9 ± 7.8 145.5 ± 8.5 242.6 ± 8.4 217.3 ± 12.6 231.7 ± 38.4 499.3 ± 58.6 614.9 ± 61.2*
δ-Carotene nd nd nd nd 3.8 ± 1.5 2.5 ± 1.3 3.4 ± 0.7 7.8 ± 2.6** 2.8 ± 0.6 nd nd
Lutein 57.8 ± 4.5 64.4 ± 6.6 63.2 ± 2.5 61.6 ± 4.6 71.8 ± 11.3 70 ± 3.9 146.3 ± 1.5 158.3 ± 3.1** 145.8 ± 2.2 697.9 ± 84.4 943.2 ± 87.3**
Total carotenoid 106.7 ± 5.5 118.5 ± 8.8 136.9 ± 11.6 116.7 ± 10.5 1,044.2 ± 130.8 504 ± 229.2** 2,550.7 ± 329.5 2,467.5 ± 308.4 1758.1 ± 170.6** 1,211.2 ± 142.5 1,571.2 ± 150.4**
α-Tocopherol 210.7 ± 9.2 284.3 ± 7.8** 291.9 ± 46.6 271.3 ± 28.9 341.8 ± 45.7 421.7 ± 13.5* 425.2 ± 36.2 507.8 ± 33.3** 523.8 ± 35.8** 1,583.3 ± 213.6 1913.4 ± 431.3
β/γ-tocopherol nd nd nd nd 15.2 ± 3.1 10.1 ± 5.0 25.1 ± 3.9 17.1 ± 5.6* 7.7 ± 3.7** nd nd
PC-8 36.7 ± 5.1 48.6 ± 8.5* 38.1 ± 2.9 43.3 ± 4.7 38.9 ± 7.5 44.2 ± 14.2 38.8 ± 5.2 43.3 ± 3.6 44.8 ± 11.6 67.1 ± 13.1 102.1 ± 36.3
Total Chl 311.5 ± 66.2 281.7 ± 39.3 376.5 ± 48.3 430.1 ± 101.9 18.4 ± 7.8 16.1 ± 11.7 nd nd nd 6,168.4 ± 824.3 7,970.6 ± 868.7*
Note: Values are mean ± SD (n= 5). Significant differences compared to corresponding control are indicated in bold (Student's ttests or ANOVA/Dunnett's test, * p< .05; ** p< .01). nd, not detected; DW, dry
weight; C
B3
, nonstressed fruit at 3 days post-breaker; H
B3
, heat-stressed fruit at 3 days post-breaker. C
B7
, non-stressed fruit at 7 days post-breaker; H
B
R7 and H
B3
R4, fruits H
B
and H
B3
, respectively, allowed to
recover under normal conditions and harvested at ripe stage. C
L
, nonstressed leaf; H
L
, heat-stressed leaf.
a
C
MG
, nonstressed fruit at mature green stage; H
MG
, heat-stressed fruit at mature green stage. C
B
, nonstressed fruit at breaker stage; H
B
, heat-stressed fruit at breaker stage.
ALMEIDA ET AL.5
Levels of other lipid-soluble antioxidants were also influenced by
H. α-tocopherol increased 1.2-fold in H
MG
and H
B3
fruits. Interest-
ingly, its levels were still higher even after a recovery period in H
B
R7
and H
B3
R4 fruits compared to control conditions (Table 1).
3.2 |Metabolite profiling of tomato fruit exposed
to transient heat stress
Metabolite profiling using GC–MS was carried out on fruits exposed
to heat stress and concurrently samples were collected for trans-
criptome analysis. Principal component analysis (PCA) was used to
compare primary metabolism of fruits under C, H and R conditions.
The score plot obtained from polar and nonpolar extracts could not
discriminate non-stressed and heat-stressed conditions among the
fruit stages evaluated, though a clear separation between early- and
late-ripening stages was achieved (Figure. 2a, b). Overall primary
metabolism remained unchanged after H and followed by R
(Table S2).
Our high-temperature treatment did not appreciably affect the
levels of known osmoregulators such as proline and GABA, and only a
few metabolites responded significantly to H mostly in a fruit stage-
dependent manner (Figure 2c, d). Sucrose was very responsive to
heat, consistently accumulating in fruits (H
MG
and H
B
) at early ripe
stages (Figure 2c). Some amino acids changed in content, particularly
threonine was increased in H
B
and H
B3
. The tricarboxylic acid (TCA)
FIGURE 2 Effect of heat stress on tomato fruit metabolite profiling. Scores plot obtained by Principal Component Analysis (PCA) for
metabolite levels measured in polar (a) and nonpolar (b) extracts. Metabolites at mature green, MG (c), breaker, B (d), breaker+3, B3 (e), red ripe,
B7 (f) stage of fruits kept at C, exposed to H or followed R. Quantification was determined relative to the internal standard and values are
presented as mean ± SD from five biological replicates. Nonpolar compounds are shown as left insets in the graphs. Only significant changes
compared to respective control are shown (pair-wise ttest corrected for multiple comparison using Holm-Sidak's post-test; * Adjusted p< .05,
** p< .01, *** p< .001). Full data set available in Table S2. C22:0, Docosanoic acid; C24-ol, tetracosanol; Ribose/xylose (f), Ribose/xylose in
furanose ring form [Colour figure can be viewed at wileyonlinelibrary.com]
6ALMEIDA ET AL.
intermediates showed a variable response, with aconitic acid accumu-
lating upon H. From non-polar fraction, β-sitosterol levels significantly
accumulated in H
B
and H
B3
.
After R, primary metabolism of ripe fruits previously heat-
stressed at B transition (H
B
R7) was largely the same than their non-
stressed counterparts, except for high levels of α-tocopherol. This
quantitative tocopherol response to heat was observed both within
the GC–MS and UPLC-PDA derived datasets confirming the utility of
the approaches (Table S2).
3.3 |Fruit lipid metabolism is highly responsive
to heat
Lipid remodelling while-decreasing the level of lipid unsaturation is a
crucial aspect of plant thermotolerance under suboptimal temperature
conditions (Falcone et al., 2004). Nevertheless, the analysis of the
total lipid fraction by GC–MS showed no significant differences
between C and H in fatty acid composition. To increase the sensitivity
and address the potential changes in complex lipid moieties, lipid spe-
cies were first separated by TLC and then analysed by GC–MS.
Analysis of lipid classes revealed a relative increase in the stor-
age lipid triacylglycerol (TAG) under H at both stages B and B3 com-
pared to control (Figure 3a, c). Extraplastidic classes of
phospholipids, phosphatidylcholine (PC), phosphatidylserine (PS) and
phosphatidylethanolamine (PE) showed variable response to heat.
For the fatty acid composition of the lipid classes (Figure 3), the gen-
eral trend was the lower levels of the trienoic fatty acid C18:3
under H, particularly in the plastidial membrane lipids mono-
galactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol
(DGDG) in comparison to control conditions. This decline was pre-
dominantly mirrored by an increase in the corresponding dienoic
fatty acid precursor, linoleic acid (C18:2), and other less unsaturated
and saturated acyl moieties (Figure 3b, d). Consistently, a greater
reduction of the C18:3/C18:2 ratio was observed in MGDG and
DGDG (Table S3). In contrast, H
B
fatty acid composition of the TAG
fraction showed an opposite response with higher levels of the tri-
enoicacidC18:3proportioncomparedtocontrolC
B
(Figure 3).
Overall, tomato fruit response to high-temperature conditions
includes lipid remodelling, which leads to TAG accumulation, and
decreasing the level of membrane lipid unsaturation, particularly tri-
enoic acid composition.
FIGURE 3 Lipid profile of tomato fruits after heat stress. Lipids were separated by TLC and quantified by GC–MS. (a, c) Relative abundance
of lipid content (mol % of total) based on detector response calculated from internal standard. The values are shown as mean ± SD of three
measurements from a pool of four biological replicates. (b, d) Heat map representation of changes in fatty acid composition (mol% of total) of
each lipid class under heat. The ratio % in H versus % in C were obtained and showed as log
2
fold change. Dark red or dark blue indicates that the
acyl moiety is relatively increased or decreased, respectively, under high-temperature conditions. Grey colour indicates acyl moieties not
detected. MGDG, monogalactosyldiacylglycerol; DGDG, digalactosyldiacylglycerol; PC/PS, phosphatidylcholine/phosphatidylserine; PE,
phosphatidylethanolamine; TAG, triacylglycerol [Colour figure can be viewed at wileyonlinelibrary.com]
ALMEIDA ET AL.7
The volatile organic compounds (VOC) analysis revealed C18 fatty
acid-derived volatiles were altered in heat-stressed fruits (Table S4). For
the C18:3-derived flavour molecules produced via LOX action, a nota-
ble decrease in C6 volatiles was observed. Interestingly, levels of the
C18:2-decomposition product hexanal were significantly lower only at
H
B
fruits, whilst H
B3
exhibited hexanal at the same levels of non-
stressed fruits. Contrastingly, C5 volatiles as pentanal and 2-pentanone
accumulated in H
B3
fruits. Carotenoid-derived volatiles detected were
unaffected except for the lycopene derived 6-methyl-5-hepten-2-one;
which was found to be lower in H
B3
compared to C
B3
. These data com-
plement the lower levels of lycopene found in these samples following
H. Finally, the accumulation of different VOCs belonging to terpenoid
parent molecules, such as α-pinene, δ-4-carene, cymene, β-phellandrene
was detected in at least one ripening stage either immediately after
stress and/or after recovering. A similar trend was found for the pheno-
lic derived molecule o-guaiacol (Table S4).
3.4 |Chromoplast structure of fruits exposed to
high temperatures
Heat stress-induced changes in carotenoids, tocopherols and neutral
lipid levels may be associated with perturbations not only in the
metabolic pathways but also in compound sequestration (Spicher
et al., 2017; Zhang et al., 2010). To ascertain whether high tempera-
ture alters the distribution of liposoluble antioxidants into plastidial
sub-compartments, analysis of fractionated chromoplast from heat-
stressed and non-stressed fruits was carried out (Figure 4).
First, the total amounts obtained from the sum of all fractions
(i.e., plastoglobuli, envelope membranes, stroma, thylakoids) corrobo-
rated the lower levels of phytoene, phytofluene and lycopene in H
B3
fruits (Table S5). Overall, the proportions of all carotenoids, tocoph-
erols and plastochromanol (PC-8), this latter also an antioxidant typi-
cally accumulating at plastoglobuli (Kruk et al., 2014) followed the
same distribution pattern immediately after H, suggesting that sub-
compartmentation was not largely altered by heat stress. Yet, the
lower phytofluene levels found in H
B3
fruits preferentially accumu-
lated into plastoglobular fraction (Figure 4b). No changes in the pro-
portion of carotenoids and tocopherols arose in the plastoglobular
fraction in the fruits that undergone heat stress at B stage following a
short recovery (H
B
R4). However, the presence of the fractions termed
membrane II was more pronounced in H
B
R4, which indicates the per-
sistence of thylakoids remnants (Figure 4d).
Analysis of chromoplast ultrastructure by transmission electron
microscopy suggested that non-stressed fruit chromoplasts displayed
more mature lycopene crystals (Figure S1) while fruits experiencing
FIGURE 4 Profile of carotenoid and tocopherol across the chromoplast subcompartments after heat stress. (a, c) Fruit harvest time point and
separation of membranes from isolated tomato chromoplasts (H
B3
and H
B
R4) by flotation on a discontinuous sucrose gradient. (b, d) Plastidial
isoprenoid profile of fractions. Values represent % of the total amount found in each fraction [Colour figure can be viewed at
wileyonlinelibrary.com]
8ALMEIDA ET AL.
stress at B stage (H
B
R4) had smaller lycopene crystals and a notable
persistence of thylakoid membrane which could reflect delays in fruit
ripening.
3.5 |Transcriptome analysis
To address the molecular mechanisms underlying metabolic responses
to heat stress, transcriptome analysis of B and B3 fruits under
stressed and non-stressed conditions was carried out. Numerous
differentially expressed (DE) transcripts were detected as a result of H
imposed (Table S6). In total, 8,141 and 7,006 genes were found DE in
the comparison C
B
versus H
B
and C
B3
versus H
B3
, respectively.
Among the DE, 2067 genes were up-regulated, and 2,368 were
down-regulated under heat, irrespectively of the fruit stage when the
stress was imposed. These subsets of commonly heat-stressed regu-
lated genes were used in the gene ontology (GO) enrichment analysis
(Figure 5a).
Considering the most specific GO terms, up-regulated genes were
significantly enriched for only a few biological process GOs
FIGURE 5 Gene expression changes associated with heat stress in tomato fruit. (a) Venn-diagrams of the up-regulated and down-regulated
differentially expressed (DE) genes following H at B and B3 fruit stages. (b) GO terms enriched in the common set of DE genes observed at B and
B3 stages according to Fisher's exact test (FDR < 0.05). Only the most specific GO terms for biological process category were shown. (c) Relative
expression of HsfA2 and PSY1 by qPCR. Abbreviations and colour codes for fruit treatments are the same as in Figure 1. Nonstressed leaves (C
L
)
and heat-stressed (H
L
, brown bars) leaf samples were included for comparison. Values are expression levels normalized to CAC and ACT2
reference genes (mean ± SEM of at least four biological replicates) from samples kept at C, exposed to H or followed R. Significant differences
(Student's ttest, * p< .05, ** p< .01, *** p< .001) between heat and control conditions at corresponding organ/developmental stage are shown
[Colour figure can be viewed at wileyonlinelibrary.com]
ALMEIDA ET AL.9
(10) associated with general terms as RNA processing, mitotic cell
cycle and chromatin remodelling (Figure 5b). When a more relaxed
significance threshold (p-value <.01) was applied, GO terms as “mRNA
splicing, via spliceosome”(GO:0000398; p-value 2.15 E
−03
), and
molecular function “SWI/SNF superfamily-type complex”
(GO:0070603, p-value 4.68 E
−04
), the latter acting in chromatin
remodelling processes (Table S7) were found. DNA de novo methyla-
tion results in part from the activity of DOMAIN REARRANGED
METHYLTRANSFERASE (DRM) and chromatin remodelers as DEFEC-
TIVE IN RNA-DIRECTED DNA METHYLATION1 (DRD1) (Zhang
et al., 2018). As a response to heat, for example, gene encoding
tomato homologs of DRM (Solyc10g078190, Solyc05g053260) and
DRD1 (Solyc01g109970) were found up-regulated upon H (Table S6).
The epigenetic mechanisms also featured when the heat-induced
genes were queried for each comparison separately; “histone modifi-
cation”and “RNA processing”processes were overrepresented among
up-regulated H
B3
genes (Table S7).
By contrast, genes down-regulated by H were enriched for GOs
mainly associated with defence response to biotic stress, hormone
synthesis and signaling pathway, metabolic processes related to lipids,
carbohydrate, amino acids, and redox-related compound glutathione
(Figure 5b). GO terms found overrepresented such as “carotenoid bio-
synthetic process,”“carbohydrate metabolic process,”“alpha-amino
acid biosynthetic process”are closely related to the metabolic repro-
gramming triggered by heat in fruits.
Extreme temperature is known to induce the expression of HEAT
SHOCK TRANSCRIPTION FACTORS (Hsfs). In tomato, HsfA1, which is
constitutively expressed and post-translationally regulated, is respon-
sible for the initial heat stress response controlling the HS-induced
expression of HsfA2 and HsfA3 (Fragkostefanakis et al., 2015; von
Koskull-Döring et al., 2007). Both HsfA2 and HsfA3 were found up-
regulated under H in RNA-seq dataset compared to control conditions
(Table S6). qPCR assays confirmed higher HsfA2 transcript levels not
only for H
B
and H
B3
but also in heat-stressed leaves (H
L
), though sig-
nificant differences for H
MG
were not detected. Importantly, higher
HsfA2 transcripts were not sustained after R (Figure 5c).
3.6 |Ripening regulators and targeted pathways
Given that processes related to fruit ripening were significantly
enriched among the genes repressed by heat (Figure 5b), we explored
the heat-induced transcriptional regulation associated with ripening
as well as downstream targeted pathways such as isoprenoid metabo-
lism (Karlova et al., 2014; Li et al., 2019; Quadrana et al., 2013)
(Figure 6a, Table S8).
FIGURE 6 Transcriptional regulation of genes involved in fruit ripening and lipid metabolism under heat stress. Bars represent the log
2
-fold
change based on transcriptome comparison of H versus C at B (dark grey) and at B3 (light grey) for DEG. (a) Ripening-related genes. (b) Lipid-
related genes. Full data set available in Tables S8 and S9. Gene abbreviatures according to Tables S8 and S9
10 ALMEIDA ET AL.
In tomato, climacteric ripening is controlled by several transcrip-
tion factors in conjunction with different phytohormones, such as eth-
ylene (Karlova et al., 2014). Genes encoding key ripening-associated
transcription factors such as COLORLESS NON-RIPENING (CNR), NON-
RIPENING (NOR), FRUITFULL1 (FUL1), TOMATO AGAMOUS-LIKE1
(TAGL1) and AUXIN RESPONSE FACTOR2A (ARF2A) were strongly
suppressed by heat. Interestingly, the expression of RIPENING-INHIBI-
TOR (RIN) was not found to be heat-sensitive (Figure 6a). Transcripts
encoding key repressors of photomorphogenesis as PHYTOCHROME
INTERACTING FACTORS (PIFs), which are degraded in the light upon
interaction with photoactivated phytochromes (Leivar &
Monte, 2014), specifically PIF1a and PIF3, as well as DEETIOLATED1
(DET1/HP2), CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1)
responded positively to heat at both ripening stages analysed.
Ripening-inducible genes related to ethylene biosynthesis and sig-
naling, namely 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase
and ACC synthase (ACO1,ACS2,ACS4), cell wall metabolism
(e.g., polygalacturonase, PG2A), were significantly down-regulated as a
result of heat stress exposure (Figure 6a). Moreover, expression of the
gene encoding the fruit specific phytoene synthase (PSY1), the first
enzyme of carotenoid pathway, was significantly repressed under
H. This result was also confirmed by qPCR analysis in H
B
and H
B3
samples (Figure 5c). Indeed, the expression profile obtained from all
samples suggests that PSY1 suppression under heat seemed to be
linked to later ripening stages when chromoplast differentiates,
because in H
MG
fruits the PSY1 expression was yet slightly up-
regulated.
Besides PSY1, other carotenogenic-related genes were DE
upon heat exposure. Genes encoding 4-diphosphocytidyl-2-C-
methyl-D-erythritol kinase (ISPE), geranylgeranyl pyrophosphate
synthase (GGPPS2,GGPPS3), plastidial terminal oxidase (PTOX),
carotenoid isomerase (CrtISO) were significantly down-regulated.
Genes associated with vitamin E metabolism showed also lower
expression (VTE2,VTE3.1,HPPD2) under H. Regarding the plas-
toglobuli-related proteins involved in the isoprenoid sequestration,
tomato genes encoding FIBRILLIN (FBN)homologous to those
described in Arabidopsis thaliana (van Wijk & Kessler, 2017)
responded differently to heat stress. While some highly expressed
members were down-regulated as FBN4, other members with less
abundant transcripts in fruits were consistently up-regulated under
H(TableS8).
The sucrose accumulation in heat-stressed fruit may be related to
differences in sucrose turnover enzyme abundance (Qin et al., 2016).
Although sucrose accumulated only in H
B
fruits (Figure 2c), perturba-
tions in the expression of genes related to sugar metabolism were
detected at both B and B3 stages, with notable repression of the tran-
scripts of vascular invertase (VI), cell wall invertase (LIN5) and sucrose
synthase (Susy6) (Figure 6a). Sucrose turnover enzymes coding genes
were higher repressed in H
B
than H
B3
(e.g., for VI, fold change ratio
was about four-fold and two-fold in C
B
vs H
B
and C
B3
vs H
B3
, respec-
tively), though the transcripts encoding a vacuolar invertase inhibitor
(VIF) were found slightly up-regulated in H
B3
, which suggests further
capping to invertase activity.
3.7 |Lipid metabolism
Upon heat stress, lipid metabolism-related transcripts were found
to be overrepresented among downregulated genes (Figure 5(b)).
From a manually curated list derived from tomato loci showing
homology to Arabidopsis genes associated with acyl-lipid metabo-
lism (Higashi et al., 2015), a subset of genes putatively involved in
the plastidial de novo fatty acid biosynthesis was suppressed
under H in both B and B3 stages (Figure 6b, Table S9). First, the
tomato homologs encoding the plastidial pyruvate dehydrogenase
complex (PDH-E1 α,E1 β) producing acetyl-CoA precursors that
feed fatty acid synthesis; then the subunits of heteromeric acetyl-
CoA carboxylase (α-CT/CAC3,BCCP2), the acyl carrier protein
(ACP), the malonyl-CoA:ACP malonyltransferase (MCMT), the
3-ketoacyl-ACP synthase 3 and 1 (KASIII,KASI) and the reductase
(KAR), the hydroxyacyl ACP dehydratase (HAD), the enoyl-ACP
reductase (ENR) were all strongly down-regulated under heat. The
same trend was observed for the genes related to the acyl-ACP
hydrolysis (FATB) involved in the acyl moieties releasing for the
trafficking between plastid and endoplasmic reticulum (ER, Hölzl &
Dörmann, 2019).
For the plastidial galactolipid synthesis, heat-inducible tran-
scripts included those involved in the remodelling of galactolipids;
for example, the galactolipid galactosyltransferase (SFR2/GGGT),
which converts MGDG to oligogalactolipids and diacylglycerol
(DAG, Higashi & Saito, 2019), and digalactosyl-DAG synthase (DGD).
In contrast, genes encoding members of the plastid-localized lipid
phosphatidate phosphatases (LPP), promoting dephosphorylation of
phosphatidic acid (PA) that yields DAG, were suppressed by heat
(Figure 6b).
In Arabidopsis, fatty acid desaturation, a key aspect of
thermotolerance (Falcone et al., 2004), is regulated by genes encoding
for the plastid localised fatty acid desaturase (FAB2/SAD, FAD4,
FAD5, FAD6, FAD7, FAD8) and the ER-localized fatty acid
desaturases (FAD2 and FAD3). For the plastid-residing FADs, genes
encoding homologs for the 18:0-ACP desaturase (FAB2) and the
16:2/18:2 galactolipid ω3 desaturase (FAD7/FAD8/LeFAD7, Nakamura
et al., 2016) were repressed. The latter is in line with the decrease in
the plastid membrane trienoic fatty acids (Figure 3). In the present
study, a specific 16:0-MGDG Δ7-desaturase (FAD5) coding gene was
found to be highly up-regulated after H. In addition, major differences
were detected in ER lipid biosynthetic gene transcripts in the H
B3
samples, where the highly expressed gene coding for
18:1-phosphatidylcholine ω6 desaturase (FAD2-1, Lee et al., 2020)
isoform and 16:2/18:2 galactolipid ω3 desaturase (FAD3,Yu
et al., 2009) were repressed.
For TAG biosynthesis, higher levels of expression were associated
with biosynthetic pathway genes. Different types of enzymes can syn-
thesise TAG from DAG, including acyl-CoA dependent enzymes, acyl-
CoA:DAG acyltransferases (DGATs), and diacylglycerol acyltransferase
(PDAT) which uses PL as acyl donor (Fan et al., 2017). Genes encoding
DGAT and PDAT were upregulated under heat, following the higher
levels of TAG observed in H
B
and H
B3
(Figure 3). A similar trend was
ALMEIDA ET AL.11
found for the transcripts encoding proteins associated with TAG
hydrolysis, which were predominantly upregulated at both stages
analysed (Figure 6b).
Fatty acid β-oxidation pathway was overrepresented among the
genes down-regulated by heat (Figure 5b). In accordance, the genes
encoding enzymes were found actively repressed under heat, such as
acyl-CoA oxidase (ACX1a, Li et al., 2005) and members of the
multifunctional protein (MFP), as well as the peroxisomal isoform of
long-chain acyl-CoA synthetase (LACS), which activates free fatty
acids to acyl-CoA thioesters to generate acyl-CoA derivatives. Indeed,
genes associated with oxylipin biosynthesis were repressed after H,
including allene oxide cyclase (AOC3), allene oxide synthase (AOS2)
and acyl-hydrolase patatin-like, involved in the production of
jasmonate from polyunsaturated fatty acids.
For the volatiles production, 13-LOXs and hydroperoxide lyase
(HPL) are the main enzymes catalysing the conversion of C18 polyun-
saturated fatty acids to C5 and C6 volatiles in tomato fruit (Shen
et al., 2014). Among the tomato 13-LOXs, LoxC is essential for the
generation of fruit C5- (1-penten-3-ol, 1-penten-3-one, pentanal, (Z)-
2-penten-1-ol, and 1-pentanol) and C6-flavour volatiles (Shen
et al., 2014), while LoxB and LoxA possibly support C5 synthesis
(Griffiths et al., 1999). The heat-induced response of genes encoding
tomato 13-LOX varied between H
B
and H
B3
, except for LoxB, which
was downregulated at both ripening stages addressed. Importantly,
ripening-inducible LoxC was found only repressed in H
B
.HPL tran-
scripts were concordantly repressed under H (Figure 6b, Table S9).
As the levels of β-sitosterol increased after heat stress (Figure 2e),
the transcripts encoding sterol 22-desaturase (CYP710A11), involved
in the conversion of β-sitosterol to stigmasterol, were checked, reveal-
ing a heat-sensitive expression pattern.
4|DISCUSSION
Our study has undertaken an integrative approach to address the
metabolic, cellular and molecular changes associated with transient
heat stress imposed on tomato fruits, elucidating several key features
that impact on fruit quality traits.
4.1 |Nutritional attributes were altered by heat
stress
Our findings revealed that transient heat stress can alter carotenoid
accumulation in tomato fruits, with sensitivity to temperature increas-
ing as ripening advanced. The negative impact of heat on carotenoid
levels was associated with changes in the initial steps in carotene
formation.
Our study showed that ripe fruits have tremendous plasticity to
restore carotenoid levels following a heat wave, suggesting no perma-
nent damage was achieved. The temperature of 40C caused moder-
ate stress in tomato as reported earlier (Spicher et al., 2017).
Reversible effects of heat treatment have been observed on vine
detached fruits previously (Lurie et al., 1996). Nevertheless, the lower
carotenoids levels in ripe fruits experiencing heat at B3 transition
implies that the length of the recovery period may be critical. Boosting
in carotenoid synthesis during fruit ripening is achieved predominantly
by the up-regulation of genes encoding key biosynthetic enzymes
(Hirschberg, 2001; Enfissi et al., 2017). Thus, the heat-induced tran-
scriptional misregulation at advanced ripening stages (Figure 6a) may
explain the decrease in fruit carotenoid levels. Firstly, both phytoene
formation and subsequent isomerisation are potentially compromised
as the expression of fruit-specific PSY1 (Figure 5), encoding the major
flux-controlling enzyme of carotenogenic pathway (Fraser et al., 2002,
2007), and CrtISO (Isaacson et al., 2002) were repressed by heat. Sec-
ondly, efficient carotenoid desaturation conducted by phytoene desa-
turase (PDS) and ζ-carotene desaturase (ZDS) depends on the redox
status of plastoquinone/plastoquinol pool dependent on the activity
of PTOX (Shahbazi et al., 2007) whose transcripts were found heat-
sensitive. Finally, methyl-erythritol phosphate (MEP)-derived precur-
sors for carotenogenesis (Almeida et al., 2015; Nogueira et al., 2018)
might be altered as the expression of ISPE,GGPPS3 and the ripening-
induced GGPPS2 were suppressed by heat. Together, these perturba-
tions emphasise the transcriptional heat-sensitivity of the early
carotenogenesis in fruits.
Carotenogenic enzymes are also post-translationally regulated by
protease complexes which are highly active under heat (D'Andrea
et al., 2018). The MEP enzyme DXS and carotenogenic PSY are direct
substrates of Clp protease complex whose activity, in turn, is
counterbalanced by other chaperones, for example, orange (OR;
Welsch et al., 2018). Their interaction adjusts DXS and PSY functional
forms controlling enzyme level and activity. Interestingly, Clp-
defective tomato fruits have improved carotenoid accumulation under
higher temperature post-harvest treatment compared to control
(D'Andrea et al., 2018). Therefore, it is expected some contribution of
post-translational mechanisms curbing the activity of carotenoid-
related enzymes under heat stress. It is worth noting that this
response is likely fruit-specific as heat-stressed leaves showed an
opposite effect on carotenoids; indeed, the increased levels of lutein
observed in H
L
were consistent with a previous report using tomato
(Spicher et al., 2017).
Higher vitamin E levels found in heat-stressed fruits and their
recovered counterparts may serve as a molecular signature of fruits
which have experienced stress previously irrespective of their devel-
opmental stage. The production of tocopherols has been linked to
high-temperature response in tomato leaves supported by transcrip-
tional regulation (Spicher et al. 2016, 2017). In our study, the lack of
correlation between fruit tocopherol content (Table 1) and the
expression of genes involved in tocopherol biosynthesis (Figure 6a),
could be associated with the redirection of isoprenoid precursors
from carotenoid formation into tocopherol formation instead
(Almeida et al., 2015; Fraser et al., 2007). Importantly, carotenoid
and tocopherol heat-responsive genes typically exhibit ripening-
associated expression pattern (Quadrana et al., 2013; Sato
et al., 2012;) and the changes observed may be due to inhibition of
fruit ripening.
12 ALMEIDA ET AL.
4.2 |Ripening related processes are misregulated
in heat-stressed fruits
In tomato, fruit ripening encompasses highly coordinated processes
orchestrated by a network of interacting genes and signaling path-
ways, which involves differentiation of chloroplasts into chromoplasts
(Liu et al., 2015; Seymour et al., 2013). A peak in ethylene production
and burst in cellular respiration are associated with profound meta-
bolic transitions, leading to alterations not only in pigmentation but
also in sugar accumulation, tissue softening and volatile production
(Klee & Giovannoni, 2011; Rambla et al., 2015).
Several transcription factors acting as regulators of tomato fruit
ripening including CNR,NOR,FUL1,TAGL1 and ARF2a (Bemer
et al., 2012; Giovannoni, 2007; Hao et al., 2015; Manning et al., 2006;
Vrebalov et al., 2009) were transcriptionally suppressed by heat.
Besides, repressors of carotenogenesis PIF1a,DET1 and COP1, which
transduce phytochrome-sensed changes in the environmental light,
hence affecting carotenoid biosynthesis and plastid development
(Enfissi et al., 2010; Liu et al., 2004; Llorente et al., 2015), were found
to be up-regulated under heat conditions. As PIF1a binds to PSY1 pro-
moter region and represses PSY1 transcription in tomato fruits
(Llorente et al., 2015), the higher PIF1a expression may cause
temperature-induced PSY1 repression.
Expression profile of the ripening regulators mined from public
database (TomExpress; Zouine et al., 2017) at non-stressed conditions
supports the view that heat-induced repression of CNR and NOR are
not likely due to ripening delay (as it cannot be ruled out for FUL1 and
TAGL1) but rather a bona fide feedback transcriptional regulation of
fruits in response to higher temperatures. Similarly, for the known
carotenogenic repressors PIF1a, COP1 and DET1 and possibly PIF3,
all plausibly act as core components of heat-triggered transcriptional
reprogramming in tomato fruits.
Importantly, PIF-dependent signaling is a central pathway for
thermoresponsiveness under warmer but non-stressful ambient tem-
peratures (Rosado et al., 2019; Vu et al., 2019;). Our findings thereby
extend the role of the light signaling components in response to dif-
ferent temperature cues. The prevalence of phytochrome-signaling
repressors, together with the suppression of CNR and NOR, may
inhibit the ripening program in fruits experiencing heat stress at
advanced ripening stages, which is in line with the down-regulation of
genes involved in ethylene biosynthesis/signalling (ACO1,ACS2,
ACS4,E8), cell wall degradation (PG2a,EXP1) (Li et al., 2019), and also
with the persistence of thylakoid-like membranes (Figure 4,
Figure S1). Moreover, consistent with previous studies in tomato
(Mishra et al., 2002), fruit heat transcriptional response seems to be
mediated by Hsfs (Figure 5). Finally, our transcriptome analysis
suggested a role of epigenetic mechanisms mediating heat-induced
transcriptional changes as chromatin remodelling and histone methyl-
ation were enriched among up-regulated genes in response to heat
(Figure 5, Table S7). Epigenetic mechanisms as DNA methylation add
another layer of regulation for the tomato ripening program. DNA
methylation relies, in part, on the RNA directed DNA methylation
(RdDM) pathway, dependent on small RNAs and the activity of DRM
and DRD1 (Gallusci et al., 2016). The up-regulation of tomato homo-
logs DRM1 and DRD1 (Table S6) might contribute to rearrangements
of epigenome landscape under high-temperature treatment and raises
the possibility that plant adaptive responses to heat mediated by epi-
genetic mechanisms (Li et al., 2018a; Quadrana et al., 2019; Ohama
et al., 2017) also operate in tomato fruit.
4.3 |Fruit primary metabolism changes in
response to heat stress
The absence of signatures commonly shared through all stressed sam-
ples (Figure 2, Table S2) further supports the idea the thermo-
responsive is highly dependent on fruit stage. Among the known
osmoregulators, higher threonine levels at advanced ripening stages
are in line with its conserved biomarker for abiotic stress, accumulat-
ing in Arabidopsis leaves under heat stress (Obata & Fernie, 2012).
Notably, citric and malic acids that contribute most to the typical acid-
ity of tomato fruit (Baldwin et al., 2008) remained unaffected.
Sucrose accumulation in heat-stressed fruit at early ripening stages
(Figure 2) correlated with the sensitivity of sucrose metabolism to high
temperatures previously reported in tomato male reproductive system
and in fruits after pollination (Li et al., 2012; Liu et al., 2016; Sato
et al., 2006). Indeed, enhanced LIN and VI activity in tomato has been
associated with fruit thermotolerance at early developmental stages
(Li et al., 2012; Liu et al., 2016). It is known that, at late-ripening stages,
sucrose accumulation is limited since invertase activities intensify as ripen-
ing progresses, with VI controlling sucrose/hexose ratio (Biais et al., 2014;
Klann et al., 1996; Qin et al., 2016; Yelle et al., 1991). In H
B
fruits, lower VI
transcripts may explain why sucrose increased. However, control sucrose
levels found at H
B3
did not correlate with down-regulation of VI,LIN5 and
Susy6. In this case, sugar metabolic fluxes at B3 stage may prevent sucrose
from accumulating under heat. Lack of correlation between invertase
activity and sucrose/hexose levels has been reported in tomato fruits of
lines with increased LIN activity (Liu et al., 2016).
Effects on fruits seem to be minor compared to the vegetative
system or pollen development, where altered carbon metabolism
upon exposure to high temperatures can promote yield losses (Ruan
et al., 2010; Rieu et al., 2017). Moreover, fluctuations in sugar levels
are important, as cell signals, since they can act in crosstalk with hor-
mones (e.g., auxin) and reactive oxygen species (ROS) signaling path-
ways during stress responses; sugars can also contribute to stress
alleviation by facilitating production of HSP even in reproductive sys-
tems, property that correlates to high fruit sensitivity under stress (Liu
et al., 2013). Molecule signaling and protective roles are, therefore,
possible to intersect under heat stress in fruits.
4.4 |Lipid remodelling triggered by heat stress in
fruits
Lipid remodelling was a pronounced feature of heat-stressed fruits,
leading to TAG accumulation, and decreasing the level of membrane
ALMEIDA ET AL.13
lipid unsaturation (Figure 3, Table S3). Polyunsaturated acyl chains
contribute to membrane fluidity and stability and, in response to
higher temperatures, the degree of unsaturation is decreased to main-
tain optimal fluidity and integrity of membranes (Nishida &
Murata, 1996; Murakami et al., 2000; Falcone et al., 2004; Zheng
et al., 2011). Moreover, the observed changes in sterols might also be
linked to membrane stability. Heat-induced β-sitosterol accumulation
may contribute to control membrane permeability and membrane pro-
tein activity (Guo et al., 2019).
TAG accumulation associated with heat stress-induced lipid
remodelling has been reported in photosynthetic organisms (Légeret
et al. 2016; Mueller et al., 2015; Narayanan et al., 2016). The
FIGURE 7 Impact of heat stress on tomato fruit. (a) Fruit metabolic processes affected by heat. Compounds targeted on metabolite profiling
are shown in bold. Amino acid (aa) metabolism was omitted for simplicity. Dotted lines indicate multiple metabolic steps; (b) Summary of heat-
induced metabolic changes and associated transcriptional regulation found in B and B3 fruits exposed to high-temperature treatment. Arrows
indicate metabolite changes. Up-regulated and down-regulated genes (or pathways) are indicated in blue and red, respectively. ACP, acyl carrier
protein; CHL, chlorophyll; DAG, diacylglycerol; FA, fatty acid; MVA, mevalonate; MEP, methyl-erythritol phosphate; MGDG,
monogalactosyldiacylglycerol; DGDG, digalactosyldiacylglycerol; PC/PS, phosphatidylcholine/phosphatidylserine; PE, phosphatidylethanolamine;
TAG, triacylglycerol; Suc, sucrose; α-toc, α-tocopherol; TCA, tricarboxylic acid; ox-lipids, oxidised phospholipids; VOC, volatile organic compound
[Colour figure can be viewed at wileyonlinelibrary.com]
14 ALMEIDA ET AL.
substantial heat-induced decrease in C18:3/C18:2 ratio of plastid
membrane lipids in fruits is similar to previous reports in heat-stressed
tomato leaves (Spicher et al., 2016). Besides the prevention of
physical–chemical damages of membranes, it has been proposed that
selective decline of trienoic acid acyl moieties might confer survival
advantage imposed by cellular oxidative stress associated to excessive
ROS generated under heat (Larkindale et al., 2007; Légeret
et al., 2016). As galactolipids are highly enriched in polyunsaturated
fatty acids, and thus easily prone to lipid peroxidation, photosynthetic
organisms may transfer trienoic fatty acids from membrane lipids to
TAG sequestered in lipid droplets as a strategy to control the exten-
sion of lipid oxidation (Du et al., 2018; Légeret et al., 2016).
Our transcriptome data provided insights into molecular mecha-
nisms supporting lipid remodelling (Figure 6b, Table S9). The heat-
induced decrease in the level of plastid lipid unsaturation coincided
with the down-regulation of FADs, mainly the FAD7/FAD8 encoding
the plastidial ω3 desaturase. Transcripts of the ER-counterpart desa-
turase (FAD3) were only significantly repressed in H
B3
, following the
temperature-induced decrease of extraplastidial 18:3-acyl-containing
lipids specific to this stage (Figure 3). Lipid remodelling may also be
supported for the up-regulation of (a) SFR2/GGGT, whose
corresponding enzyme contributes to diminish the MGDG/
oligogalactolipids ratio and to release DAG further used for TAG bio-
synthesis (Higashi & Saito, 2019; Moellering & Benning, 2011), and
(b) DGAT and PDAT, which encode TAG biosynthetic-related
enzymes (Fan et al., 2017). Together, these data suggest that the
higher amounts of 18:3-acyl-containing TAGs upon heat, at B stage,
may have been derived from C18:3 released from membrane lipids
than from de novo synthesised fatty acids (Légeret et al., 2016),
therefore reflecting the changes in the proportion of membrane
glycerolipid composition (Higashi & Saito, 2019). By contrast, at B3
stage, 18:3 acyl moieties are likely redirected to lipid oxidation path-
ways(Schilmilleretal.,2007),forexample, volatile production. In
tomato, levels of C18:2 and C18:3 positively correlate to volatile
derivatives hexanal and hexenal, respectively (Domínguez
et al., 2010; Ties & Barringer, 2012). The lipid remodelling triggered
by heat under our experimental conditions, affecting plastidial C18:3
availability, may explain the decrease in unsaturated C6 volatiles as
hexenal. The increased C18:2 precursors may sustain non-stressed
levels of C18:2-derived hexanal at H
B3
, as opposed to lower levels
foundinH
B
, even under a possible constraint in LOX/HPL-
dependent pathway as suggested by heat-induced repression of HPL
transcripts. C6-volatiles are synthesised via the action of 13-LOX
and HPL enzymes, whilst C5-volatiles are LOX-dependent though
HPL-independent (Shen et al., 2014). C5-volatiles accumulated only
in H
B3
as, in this case, 18:3-acyl-containing TAGs were unaffected.
A decrease in HPL activity by heat might enhance the hydroperoxide
pool, which can be redirected towards the C5 branch of LOX-
pathway as proposed previously based on tomato HPL-deficient
lines (Shen et al., 2014). Notably, the tomato fruit volatile profile is
highly sensitive to heat exhibiting alterations even when the stress
ceased.
In conclusion, our findings illustrated the impact of brief exposure
to high-temperature events on tomato fruit quality and revealed
potential molecular mechanisms associated with heat response
(Figure 7). Depending on the ripening stage, heat may have under-
estimated yet significant effects on nutritionally value and other
quality-related attributes in tomato, with sensitivity to high tempera-
ture increasing in more advanced ripening stages. Several heat-stress
responsive genes, including fruit ripening regulators, have been identi-
fied from transcriptome analysis correlating with the metabolite
changes. Collectively the data acquired provides a significant advance-
ment to our understanding of fruit metabolic reprogramming associ-
ated with heat stress. It is now clear that cold storage is not the only
stress affecting fruit quality but perturbations in heat will also alter
quality attributes. These data provide an exploitable resource for the
development of climate resilient crop varieties.
ACKNOWLEDGMENTS
This work is supported by the H2020 programme No. 679766.
TomGEM; A holistic multi-actor approach towards the design of new
tomato varieties and management practices to improve yield and qual-
ity in the face of climate change. The authors thank Mr Chris Gerrish
for the technical assistant with fractionation experiments. IGA Tech-
nology Services facility for assistance in the utilisation of RNA-seq
data and Dr Genny Enfissi for advice and input in the experimental
approach.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
AUTHOR CONTRIBUTIONS
Juliana Almeida and Paul D. Fraser conceived the original research
plan. Juliana Almeida performed the experimental programme, data
analysis and statistics. Laura Perez-Fons carried out the volatile analy-
sis and components of the metabolite analysis. Juliana Almeida and
Paul D. Fraser wrote the manuscript. All authors contributed to data
discussion and conclusions. Paul D. Fraser acquired the funding and
accepts to serve as the contact point author for communication. All
authors have read and approved the final manuscript.
ORCID
Paul D. Fraser https://orcid.org/0000-0002-5953-8900
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SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section at the end of this article.
How to cite this article: Almeida J, Perez-Fons L, Fraser PD. A
transcriptomic, metabolomic and cellular approach to the
physiological adaptation of tomato fruit to high temperature.
Plant Cell Environ. 2020;1–19. https://doi.org/10.1111/pce.
13854
ALMEIDA ET AL.19
