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A transcriptomic, metabolomic and cellular approach to the physiological adaptation of tomato fruit to high temperature


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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 detrimental 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 primary/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 biochemical and molecular adaptation of fruit to heat stress and will impact on our ability to develop future climate resilient tomato varieties. This article is protected by copyright. All rights reserved.
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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
Paul D. Fraser, Department of Biological
Sciences, Royal Holloway University of
London, Egham, Surrey TW20 0EX, UK.
Funding information
H2020 Food, Grant/Award Number: H2020
programme TomGEM / 679766
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.
carotenoids, fruit quality, fruit ripening, heat stress, isoprenoids, metabolomics, plastoglobuli,
tomato, transcriptomics
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;119. 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 dayswhen pollen development or fruit set is
disturbedto 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
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.
2.1 |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
3739 days after anthesis, DAF), breaker (H
,3941 DAF) and 3 days
post breaker (H
,4244 DAF). Leaves from heat-stressed plants (H
were also collected. Plants dedicated to H
and H
treatment could
recover (R) under control conditions, and fruits were harvested at red
ripe stage (7 days post breaker, B7); for H
, after 7-days recovery
period (H
R7); for H
, after 4-days recovery period (H
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
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 doseresponse cur-
ves obtained from authentic standards.
2.3 |Metabolite profiling by gas chromatography
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-
as the internal standard. The dried residues were
derivatised in methoxyamine hydrochloride (in pyridine) followed by
silylation with N-methyl trimethylsilyl trifluoroacetamide. The GCMS
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 GCMS were per-
formed as previously described by Nogueira et al. (2013). FAMEs
were quantified using myristic-
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 GCMS
Frozen fruit samples were ground in liquid N
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-
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
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),
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-changej1.5 (or jlog
FCj0.58). GO enrichment analysis with
Fisher's Exact Test was conducted using Blast2GO.
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
) and yellow to light-red transition
) (Figure 1). For comparison, an additional H treatment at early
to late mature green (H
) 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
(10-fold) and H
(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
fruits and reduced by
50% in H
fruits, compared to corresponding non-stressed controls.
Interestingly, lycopene levels only partially recovered in H
fruits, to about 60% of the content found in ripe C
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
R4 fruits, total carotenoids remained unchanged both in H
and in those fruits allowed to recover from heat stress (H
R7). By
contrast, H
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
). Additionally, lutein
levels as well as total carotenoid showed an increase in H
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
breaker (H
) and 3 days post-breaker (H
). Fruits allowed to recover
(R) under normal conditions were harvested at ripe stage (H
R7 and
R4) [Colour figure can be viewed at]
TABLE 1 Transient changes in isoprenoid profile of tomato fruits and leaves exposed to high-temperature treatment
Fruit Leaf
μg/g DW C
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
, nonstressed fruit at 3 days post-breaker; H
, heat-stressed fruit at 3 days post-breaker. C
, non-stressed fruit at 7 days post-breaker; H
R7 and H
R4, fruits H
and H
, respectively, allowed to
recover under normal conditions and harvested at ripe stage. C
, nonstressed leaf; H
, heat-stressed leaf.
, nonstressed fruit at mature green stage; H
, heat-stressed fruit at mature green stage. C
, nonstressed fruit at breaker stage; H
, heat-stressed fruit at breaker stage.
Levels of other lipid-soluble antioxidants were also influenced by
H. α-tocopherol increased 1.2-fold in H
and H
fruits. Interest-
ingly, its levels were still higher even after a recovery period in H
and H
R4 fruits compared to control conditions (Table 1).
3.2 |Metabolite profiling of tomato fruit exposed
to transient heat stress
Metabolite profiling using GCMS 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
and H
) at early ripe
stages (Figure 2c). Some amino acids changed in content, particularly
threonine was increased in H
and H
. 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]
intermediates showed a variable response, with aconitic acid accumu-
lating upon H. From non-polar fraction, β-sitosterol levels significantly
accumulated in H
and H
After R, primary metabolism of ripe fruits previously heat-
stressed at B transition (H
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 GCMS 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 GCMS 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 GCMS.
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
fatty acid composition of the TAG
fraction showed an opposite response with higher levels of the tri-
(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 GCMS. (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
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]
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
fruits, whilst H
exhibited hexanal at the same levels of non-
stressed fruits. Contrastingly, C5 volatiles as pentanal and 2-pentanone
accumulated in H
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
compared to C
. 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
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
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
R4). However, the presence of the fractions termed
membrane II was more pronounced in H
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
and H
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]
stress at B stage (H
R4) had smaller lycopene crystals and a notable
persistence of thylakoid membrane which could reflect delays in fruit
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
versus H
and C
versus H
, 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
and heat-stressed (H
, 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]
(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
), and
molecular function SWI/SNF superfamily-type complex
(GO:0070603, p-value 4.68 E
), 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-
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-
cationand RNA processingprocesses were overrepresented among
up-regulated H
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 processare 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
and H
but also in heat-stressed leaves (H
), though sig-
nificant differences for H
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
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
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-
(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
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
and H
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
fruits the PSY1 expression was yet slightly up-
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
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
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
than H
(e.g., for VI, fold change ratio
was about four-fold and two-fold in C
vs H
and C
vs H
, respec-
tively), though the transcripts encoding a vacuolar invertase inhibitor
(VIF) were found slightly up-regulated in H
, 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
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
and H
(Figure 3). A similar trend was
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
and H
, except for LoxB, which
was downregulated at both ripening stages addressed. Importantly,
ripening-inducible LoxC was found only repressed in H
.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.
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
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
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
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.
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
fruits, lower VI
transcripts may explain why sucrose increased. However, control sucrose
levels found at H
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
Lipid remodelling was a pronounced feature of heat-stressed fruits,
leading to TAG accumulation, and decreasing the level of membrane
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]
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
physicalchemical 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
, 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
, as opposed to lower levels
, 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
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
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.
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
The authors declare no conflict of interest.
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.
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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;119.
... The highest level of sucrose agreed with the results of Iwahashi and Hosoda (2000) who reported a lower accumulation of invertase protein in tomato fruit after the application of thermal treatment. Furthermore, sucrose availability has been related to the heat stress tolerance of tomato fruit (Almeida et al., 2020;Alsamir et al., 2021). ...
... In this regard, transcriptomic analysis of tomato fruit (cv. Alisa Craig) showed that heat treatment induced the downregulation of carbohydrate and Krebs cycle metabolic genes (Almeida et al., 2020). Our data and published information support an important role of glutamine for the cold tolerance induced by HWT in tomato cv. ...
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Hot water treatment (HWT) of tomato (Solanum lycopersicum L.) fruit reduces the symptoms of chilling injury (CI). The aim of this study was to identify metabolites associated with HWT-induced CI tolerance in tomato fruit cv. Imperial. Mature green tomatoes with HWT (42°C/5 min) and control were stored under chilling conditions (5°C/20 days) and then ripened (21°C/7 days). Methanol extracts from pericarp were analyzed for total phenolics (TP), antioxidant activity (AoxA), and metabolic profiling by UPLC-DAD-MS and GC-MS. After cold storage and ripening, HWT fruit showed less CI, higher TP, and AoxA than control. It also showed an increased accumulation of phenolics, sugars, and some alkaloids that may be mediated by azelaic acid, glutamine, and tryptophan. The levels of N-feruloyl putrescine, esculeoside AII, and hydroxy-α-tomatine II were reduced. The better metabolic performance of HWT fruit under cold storage was associated with a higher accumulation of several metabolites (e.g., antioxidants and osmolytes) in ripening fruit. Practical application The identification of metabolites associated with the reduction of chilling injury (CI) symptoms in HWT tomato fruit extends the understanding of the mechanisms involved in CI tolerance. This information provides targets that could be used to develop strategies for preventing CI (e.g., genetic improvement of tomato, direct application of key metabolites). The application of such strategies will increase the economic value and decrease postharvest losses.
... Wang et al. [18] documented that microclimatic factors, remarkably, light and temperature, affect the phytochemical profile of fresh horticultural products, resulting in an ongoing modification of their nutritional quality. However, many authors agree that environmental factors which are most likely to affect the nutritional value of tomatoes are temperature and light [3,15,19,20]. Pressman et al. [21] and Sato et al. [22] showed that yield parameters (number and weight of fruits) are negatively affected by average temperatures above 29 • C due to pollination or fruit set defects. ...
... In the literature, it is known that fruit sweetness is strongly influenced by genetic material [26]. Almeida et al. [20] have studied the effects on the accumulation of total soluble solids (TSS) of five genotypes of tomatoes under different environmental conditions. The authors found that the TSS content ranged among the genotypes from 5.6 to 7.2 • Brix, and according to the environmental conditions from 3.8 to 8.9 • Brix. ...
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Tomato (Solanum lycopersicum L.) is one of the most consumed vegetables worldwide due to its low caloric intake and high fiber, minerals, and phenolic compounds, making it a high-quality functional food. However, fruit quality attributes can be affected by pre-harvest factors, especially environmental stresses. This research aimed to evaluate the influence of two shading nets (white net −30% and pearl grey net −40% shading degree) on the yield and phytochemical profile of tomato fruits grown in summer under the Mediterranean climate. Mineral and organic acid content (by ion chromatography-IC), phenolic profile (by ultra-high performance liquid chromatography-UHPLC coupled with an Orbitrap high-resolution mass spectrometry-HRMS), carotenoid content (by high-performance liquid chromatography with diode array detection-HPLC-DAD), and antioxidant activities DPPH, ABTS, and FRAP (by UV-VIS spectrophotometry) were determined. Tomato fruits grown under the pearl grey net recorded the highest values of total phenolic compounds (14,997 µg 100 g −1 of fresh weight) and antioxidant activities DPPH, ABTS, and FRAP, without affecting either fruit color or marketable yield. The reduction of solar radiation through pearl grey nets proved to be an excellent tool to increase the phytochemical quality of tomato fruits during summer cultivation in a Mediterranean environment.
... In Arabidopsis, AP1 has been proposed to be a pioneer TF that can precede increase in DNA accessibility (Pajoro et al. 2014). In tomato fruit, HSFs have been suggested as a modulator of heat stress response, including enrichment of chromatin remodeling and histone methylation (Almeida et al. 2021). HSFs were also found to be involved in the opening of the chromatin structure in mammalian cells and yeast (Fujimoto et al. 2012;Pincus et al. 2018). ...
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Key message In SlHDC-A promoter, SlHDC-A core-ES is an essential region for fruit-specific expression and interacts with GATA, HSF and AP1. Triplication of essential region was proposed as a minimal fruit-specific promoter. In plant biotechnology, fruit-specific promoter is an important tool for the improvement and utilization of tomato fruit. To expand our understanding on fruit-specific expression, it is necessary to determine the promoter region involved in fruit-specific transcriptional activity and transcriptional regulations of the promoter. In previous study, we isolated a fruit-specific SlHDC-A core promoter specifically expressed during tomato ripening stages. In this study, we identified SlHDC-A promoter region (SlHDC-A core-ES) that is essential for fruit-specific expression of the SlHDC-A. To understand the molecular mechanisms of fruit-specific expression of the SlHDC-A promoter, we first identified the putative transcription factor binding elements in the SlHDC-A core promoter region and corresponding putative transcription factors which are highly expressed during fruit maturation. Yeast one hybrid analysis confirmed that GATA, HSF, and AP1 interact with the SlHDC-A core-ES promoter region. Further transactivation analysis revealed that expression of the three transcription factors significantly activated expression of a reporter gene driven by SlHDC-A core-ES promoter. These results suggest that GATA, HSF, and AP1 are involved in the fruit-specific expression of SlHDC-A promoter. Furthermore, the synthetic promoter composed of three tandem repeats of SlHDC-A core-ES showed relatively higher activity than the constitutive 35S promoter in the transgenic tomato fruits at the orange stage. Taken together, we propose a new synthetic promoter that is specifically expressed during fruit ripening stage.
... Transcriptome and metabolome analyses revealed the crucial biological pathways involved in the fast-adaptive response to salt stress, including carotenoid biosynthesis and the metabolism of porphyrin and chlorophyll [131][132][133][134]. An additional multi-omics analysis was used to unveil thermal adaptation strategies of extremophile bacteria [135] and plants [136,137], where the lipid or carotenoid metabolism seems to be implicated. The main effort that requires multi-omics analysis is to select complementary signals in the experimental design, so that the studied signals allow for a deeper understanding of the molecular adaptation of the organism to stress. ...
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Chlorophylls and carotenoids are two families of antioxidants present in daily ingested foods, whose recognition as added-value ingredients runs in parallel with the increasing number of demonstrated functional properties. Both groups include a complex and vast number of compounds, and extraction and analysis methods evolved recently to a modern protocol. New methodologies are more potent, precise, and accurate, but their application requires a better understanding of the technical and biological context. Therefore, the present review compiles the basic knowledge and recent advances of the metabolomics of chlorophylls and carotenoids, including the interrelation with the primary metabolism. The study includes material preparation and extraction protocols, the instrumental techniques for the acquisition of spectroscopic and spectrometric properties, the workflows and software tools for data pre-processing and analysis, and the application of mass spectrometry to pigment metabolomics. In addition, the review encompasses a critical description of studies where metabolomics analyses of chlorophylls and carotenoids were developed as an approach to analyzing the effects of biotic and abiotic stressors on living organisms.
... species, such as tomato (Almeida et al., 2011(Almeida et al., , 2016Quadrana et al., 2013;Gramegna et al., 2019;Burgos et al., 2021), pepper (Arango and Heise, 1998;Koch et al., 2002), olive (Georgiadou et al., 2015(Georgiadou et al., , 2016(Georgiadou et al., , 2019, and mango (Singh et al., 2017). These studies concluded that tocopherol accumulation in fruit is mainly transcriptionally regulated, and that tocopherol biosynthetic genes are modulated in a temporal manner, and also influenced by environmental factors (Almeida et al., 2011(Almeida et al., , 2020Quadrana et al., 2013;Georgiadou et al., 2016Georgiadou et al., , 2019Singh et al., 2017;Gramegna et al., 2019). ...
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Tocopherols are plant-derived isoprenoids with vitamin E activity, which are involved in diverse physiological processes in plants. Although their biosynthesis has been extensively investigated in model plants, their synthesis in important fruit crops as Citrus has scarcely been studied. Therefore, the aim of this work was to initiate a physiological and molecular characterization of tocopherol synthesis and accumulation in Citrus fruits during maturation. For that purpose, we selected fruit of the four main commercial species: grapefruit ( Citrus paradisi ), lemon ( Citrus limon ), sweet orange ( Citrus sinensis ), and mandarin ( Citrus clementina ), and analyzed tocopherol content and the expression profile of 14 genes involved in tocopherol synthesis during fruit maturation in both the flavedo and pulp. The selected genes covered the pathways supplying the tocopherol precursors homogentisate (HGA) ( TAT1 and HPPD ) and phytyl pyrophosphate (PPP) ( VTE5 , VTE6 , DXS1 and 2 , GGPPS1 and 6 , and GGDR ) and the tocopherol-core pathway ( VTE2 , VTE3a , VTE3b , VTE1 , and VTE4 ). Tocopherols accumulated mainly as α- and γ-tocopherol, and α-tocopherol was the predominant form in both tissues. Moreover, differences were detected between tissues, among maturation stages and genotypes. Contents were higher in the flavedo than in the pulp during maturation, and while they increased in the flavedo they decreased or were maintained in the pulp. Among genotypes, mature fruit of lemon accumulated the highest tocopherol content in both the flavedo and the pulp, whereas mandarin fruit accumulated the lowest concentrations, and grapefruit and orange had intermediate levels. Higher concentrations in the flavedo were associated with a higher expression of all the genes evaluated, and different genes are suitable candidates to explain the temporal changes in each tissue: (1) in the flavedo, the increase in tocopherols was concomitant with the up-regulation of TAT1 and VTE4 , involved in the supply of HGA and the shift of γ- into α-tocopherol, respectively; and (2) in the pulp, changes paralleled the expression of VTE6 , DXS2 , and GGDR , which regulate PPP availability. Also, certain genes (i.e., VTE6 , DXS2 , and GGDR ) were co-regulated and shared a similar pattern during maturation in both tissues, suggesting they are developmentally modulated.
... Raspberry fruit metabolic composition and quality traits are influenced both by genetic and environmental factors, as observed in this and previous analyses [25,27,39]. While the present knowledge about the complex genetic architecture of fruit quality attributes has been extensively improved since the development of QTL mapping and GWAS approaches, combined with high throughput metabolomic platforms [40], the impact of the environment on fruit metabolite composition is still poorly understood, its analysis being additionally hampered by current unstable climatic conditions triggered by climate change [41][42][43][44][45]. Furthermore, the metabolic characterization of raspberry genotypes for sensory and nutritional qualities remains essential for fruit breeding strategies. ...
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Promoting the consumption of fruits is a key objective of nutrition policy campaigns due to their associated health benefits. Raspberries are well appreciated for their remarkable flavor and nutritional value attributable to their antioxidant properties. Consequently, one of the objectives of present-day raspberry breeding programs is to improve the fruit’s sensory and nutritive characteristics. However, developing new genotypes with enhanced quality traits is a complex task due to the intricate impacts genetic and environmental factors have on these attributes, and the difficulty to phenotype them. We used a multi-platform metabolomic approach to compare flavor- and nutritional-related metabolite profiles of four raspberry cultivars (‘Glen Ample’, ‘Schönemann’, ‘Tulameen’ and ‘Veten’) grown in different European climates. Although the cultivars appear to be better adapted to high latitudes, for their content in soluble solids and acidity, multivariate statistical analyses allowed us to underscore important genotypic differences based on the profiles of important metabolites. ‘Schönemann’ and ‘Veten’ were characterized by high levels of anthocyanins and ellagitannins, respectively, ‘Tulameen’ by its acidity, and ‘Glen Ample’ for its content of sucrose and β-ionone, two main flavor contributors. Our results confirmed the value of metabolomic-driven approaches, which may foster the development of cultivars with enhanced health properties and flavor.
Background Tomato is one of the most important vegetables as well as a main dietary source of carotenoids. As a diverse kind of phytochemicals and main sources of natural pigments, carotenoids and carotenoid-derived volatile compounds are associated with the sensory, nutritional and flavor quality of tomato products. Scope and approach The current review summarizes the biosynthetic and catabolic pathways of carotenoids, as well as carotenoid biofortification in tomato products throughout the whole agro-food chain, including breeding/selection for suitable tomato varieties, pre-harvest managements, harvesting, and postharvest handlings. Key findings and conclusions: We recommend the integrative and sustainable management of tomato industry along the whole agro-food chain to fight carotenoid deficiency worldwide starting from breeding/selection carotenoids-biofortified tomato cultivars either via classic hybrid, molecular mark assisted breeding, or genetic engineering and genome editing approach, followed by suitable cultivation methods such as deficit irrigation, control of environmental conditions in pre-harvest production period. Meanwhile, harvesting at the proper stage of maturity and appropriate postharvest handlings including chemical regulation, optimum storage conditions, and processing methods during postharvest period can also maximize carotenoid retention. Considering that both fresh and processed tomatoes serve as major dietary sources of carotenoid, effective carotenoid biofortification in tomato products along the whole agro-food chain is essential in providing consumers carotenoid-enriched tomato on the eve of a new era in food fortification, contributing to sustainable tomato industry as well as human nutrition and potential health benefits.
Carotenoid biosynthesis has now been subjected to metabolic engineering for over two decades. The outputs clearly show that carotenoid formation is an integral component of metabolism. Perturbations can affect intermediary metabolism and other isoprenoids. The advances in omic technologies have enabled the quantitative assessment of changes in the transcriptome, proteome and metabolome in response to altered carotenoid biosynthesis. In the present article, the approaches and procedures relating to the capture of the metabolome in response to modulation of the carotenoid biosynthetic pathway are described. These data will contribute to the fundamental understanding of metabolic biology, underpinning future rationale design of New Plant Breeding Techniques (NPBTs) and associated regulatory affairs.
After reaching the stigma, pollen grains germinate and form a pollen tube that transports the sperm cells to the ovule. Due to selection pressure between pollen tubes, pollen grains likely evolved mechanisms to quickly adapt to temperature changes to sustain elongation at the highest possible rate. We investigated these adaptions in tobacco (Nicotiana tabacum) pollen tubes grown in vitro under 22 °C and 37 °C by a multi-omics approach including lipidomic, metabolomic, and transcriptomic analysis. Both glycerophospholipids and galactoglycerolipids increased in saturated acyl chains under heat stress, while triacylglycerols changed less in respect to desaturation but increased in abundance. Free sterol composition was altered, and sterol ester levels decreased. The levels of sterylglycosides and several sphingolipid classes and species were augmented. Most amino acid levels increased during heat stress, including the non-codogenic amino acids γ-amino butyrate and pipecolate. Furthermore, the sugars sedoheptulose and sucrose showed higher levels. Also, the transcriptome underwent pronounced changes with 1,570 of 24,013 genes being differentially upregulated and 813 being downregulated. Transcripts coding for heat shock proteins and many transcriptional regulators were most strongly upregulated but also transcripts that have so far not been linked to heat stress. Transcripts involved in triacylglycerol synthesis increased, while the modulation of acyl chain desaturation seemed not to be transcriptionally controlled, indicating other means of regulation. In conclusion, we show that tobacco pollen tubes are able to rapidly remodel their lipidome under heat stress likely by post-transcriptional and/or post-translational regulation.
The upcoming climate change presents a great challenge for plant growth and development being extremes temperatures among the major environmental limitations to crop productivity. Understanding the repercussions of these extreme temperatures is of high importance to elaborate future strategies to confront crop damages. Tomato plants (Solanum lycopersicum L.) are one of the most cultivated crops and their fruits are consumed worldwide standing out for their organoleptic characteristics and nutritional value. Tomato plants are sensitive to temperatures below 12 °C and above 32 °C. In this study, Micro-Tom cultivar was used to evaluate the effects of extreme temperatures on the plant of tomato and the fruit productivity and quality from the stressed plants, either exposed to cold (4 °C for three nights per week) or heat (32 °C during the day, seven days per week) treatments. Total productivity and the percentage of ripe fruits per plant were evaluated together with foliar stress markers and the contents of photosynthetic pigments and tocochromanols. Fruit quality was also assessed determining lycopene contents, total soluble solids, total acidity and ascorbate contents. High temperatures altered multiple physiological parameters indicating a moderate stress, particularly decreasing fruit yield. As a response to this stress, plants enhanced their antioxidant contents both at leaf and fruit level. Low temperatures did not negatively affect the physiology of plants with similar yields as compared to controls, suggesting chilling acclimation. Both high and low temperatures, but most particularly the former, increased total soluble solids contents indicating that temperature control may be used as a strategy to modulate fruit quality.
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Tomato (Solanum lycopersicum L.) belongs to the Solanaceae family and is the second most important fruit or vegetable crop next to potato (Solanum tuberosum L.). It is cultivated for fresh fruit and processed products. Tomatoes contain many health-promoting compounds including vitamins, carotenoids, and phenolic compounds. In addition to its economic and nutritional importance, tomatoes have become the model for the study of fleshy fruit development. Tomato is a climacteric fruit and dramatic metabolic changes occur during its fruit development. In this review, we provide an overview of our current understanding of tomato fruit metabolism. We begin by detailing the genetic and hormonal control of fruit development and ripening, after which we document the primary metabolism of tomato fruits, with a special focus on sugar, organic acid, and amino acid metabolism. Links between primary and secondary metabolic pathways are further highlighted by the importance of pigments, flavonoids, and volatiles for tomato fruit quality. Finally, as tomato plants are sensitive to several abiotic stresses, we briefly summarize the effects of adverse environmental conditions on tomato fruit metabolism and quality.
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The conversion of oleic acid (C18:1) to linoleic acid (C18:2) in the endoplasmic reticulum is critical to the accumulation of polyunsaturated fatty acids in seeds and other tissues, and this reaction is catalyzed by a Δ-12 desaturase, FATTY ACID DESATURASE2 (FAD2). Here, we report that the tomato (Solanum lycopersicum) genome harbors two genes, SlFAD2-1 and SlFAD2-2, which encode proteins with in vitro Δ-12 desaturase activity. In addition, tomato has seven divergent FAD2 members that lack Δ-12 desaturase activity and differ from canonical FAD2 enzymes at multiple amino acid positions important to enzyme function. Whereas SlFAD2-1 and SlFAD2-2 are downregulated by biotic stress, the majority of divergent FAD2 genes in tomato are upregulated by one or more stresses. In particular, SlFAD2-7 is induced by the potato aphid (Macrosiphum euphorbiae) and has elevated constitutive expression levels in suppressor of prosystemin-mediated responses2 (spr2), a tomato mutant with enhanced aphid resistance and altered fatty acid profiles. Virus induced gene silencing of SlFAD2-7 in spr2 results in significant increases in aphid population growth, indicating that a divergent FAD2 gene contributes to aphid resistance in this genotype. Thus, the FAD2 gene family in tomato is important both to primary fatty acid metabolism and to responses to biotic stress.
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Plant development is highly dependent on the ability to perceive and cope with environmental changes. In this context, PIF proteins are key players in the cellular hub controlling responses to fluctuating light and temperature conditions. Reports in various plant species show that manipulation of PIF4 level affects important agronomical traits. In tomato (Solanum lycopersicum), SlPIF1a and SlPIF3 regulate fruit nutraceutical composition. However, the wider role of this protein family, and the potential of their manipulation for the improvement of other 43 traits, has not been explored. Here we report the effects of constitutive silencing of tomato 44 SlPIF4 on whole-plant physiology and development. Ripening anticipation and higher 45 carotenoid levels observed in SlPIF4-silenced fruits revealed a redundant role of SlPIF4 in the 46 accumulation of nutraceutical compounds. Furthermore, silencing triggered a significant 47 reduction in plant size, flowering, fruit yield, and fruit size. This phenotype was most likely 48 caused by reduced auxin levels and altered carbon partitioning. Impaired thermomorphogenesis 49 and delayed leaf senescence were also observed in silenced plants, highlighting the functional 50 conservation of PIF4 homologs in angiosperms. Overall, this work improves our understanding 51 of the role of PIF proteins – and light signaling – in metabolic and developmental processes that 52 affect yield and composition of fleshy fruits.
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Salinity is one of the most decisive environmental factors threatening the productivity of crop plants. Understanding the mechanisms of plant salt tolerance is critical to be able to maintain or improve crop yield under these adverse environmental conditions. Plant membranes act as biological barriers, protecting the contents of cells and organelles from biotic and abiotic stress, including salt stress. Alterations in membrane lipids in response to salinity have been observed in a number of plant species including both halophytes and glycophytes. Changes in membrane lipids can directly affect the properties of membrane proteins and activity of signaling molecules, adjusting the fluidity and permeability of membranes, and activating signal transduction pathways. In this review, we compile evidence on the salt stress responses of the major membrane lipids from different plant tissues, varieties, and species. The role of membrane lipids as signaling molecules in response to salinity is also discussed. Advances in mass spectrometry (MS)-based techniques have largely expanded our knowledge of salt-induced changes in lipids, however only a handful studies have investigated the underlying mechanisms of membrane lipidome regulation. This review provides a comprehensive overview of the recent works that have been carried out on lipid remodeling of plant membranes under salt treatment. Challenges and future perspectives in understanding the mechanisms of salt-induced changes to lipid metabolisms are proposed.
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Transposable elements (TEs) are mobile parasitic sequences that have been repeatedly coopted during evolution to generate new functions and rewire gene regulatory networks. Yet, the contribution of active TEs to the creation of heritable mutations remains unknown. Using TE accumulation lines in Arabidopsis thaliana we show that once initiated, transposition produces an exponential spread of TE copies, which rapidly leads to high mutation rates. Most insertions occur near or within genes and targets differ between TE families. Furthermore, we uncover an essential role of the histone variant H2A.Z in the preferential integration of Ty1/copia retrotransposons within environmentally responsive genes and away from essential genes. We also show that epigenetic silencing of new Ty1/copia copies can affect their impact on major fitness-related traits, including flowering time. Our findings demonstrate that TEs are potent episodic (epi)mutagens that, thanks to marked chromatin tropisms, limit the mutation load and increase the potential for rapid adaptation.
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Understanding the regulation of fleshy fruit ripening is biologically important, provides insights and opportunities for controlling fruit quality, enhancing nutritional value for animals and humans and improving storage and waste reduction. The ripening regulatory network involves master‐ and downstream transcription factors (TFs) and hormones. Tomato is a model for ripening regulation, which requires ethylene and master TFs including NAC‐NOR and the MADS‐box protein MADS‐RIN. Recent functional characterization showed the classical RIN‐MC gene fusion, previously believed to be a loss‐of‐function mutation, is an active transcription factor with repressor activity. This, and other evidence, has highlighted the possibility that MADS‐RIN itself is not important for ripening initiation but is required for full ripening. In this review, we discuss the diversity of components in the control network, their targets and how they interact to control initiation and progression of ripening. Both hormones and individual TFs affect the status and activity of other network participants which changes overall network signaling and ripening outcomes. MADS‐RIN, NAC‐NOR and ethylene play critical roles in this network but there are unanswered questions about the role of other TFs. Further attention should be paid to relationships between ethylene, MADS‐RIN and NACs in ripening control. This article is protected by copyright. All rights reserved.
Environmental stresses cause membrane damage in terrestrial plants. Studies on the lipids obtained from these plants are required to understand their adaptation to climate change. Recently, a number of plant leaf lipidomic studies converged on the topic of chloroplastic glycerolipid remodeling and triacylglycerol production. In this review, we show that among various abiotic stresses, plant leaves under heat stress specifically increase the levels of galactolipids containing linoleate (18:2) in chloroplasts; phospholipids containing palmitate (16:0), stearate (18:0), and oleate (18:1) in the endoplasmic reticulum and plasma membrane; and triacylglycerol containing α-linolenate (18:3) and hexadecatrienoic acid (16:3) as lipid droplets in the leaves of Arabidopsis thaliana. Recent studies have proposed responsible genes for the lipid remodeling under heat stress, highlighting the importance of the catabolic process of chloroplastic monogalactosyldiacylglycerol. This review comprehensively describes glycerolipid compositional changes in plant leaves under heat stress detected by lipidomic analyses and compares them with those under other abiotic stresses. We will discuss the physiological significance underlying the observed lipid metabolism under heat stress. Detailed knowledge about plant lipid remodeling can aid in the development of solutions to deal with the consequences of climate change, including global warming.
Chloroplasts contain high amounts of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) and low levels of the anionic lipids sulfoquinovosyldiacylglycerol (SQDG), phosphatidylglycerol (PG), and glucuronosyldiacylglycerol (GlcADG). The mostly extraplastidial lipid phosphatidylcholine is found only in the outer envelope. Chloroplasts are the major site for fatty acid synthesis. In Arabidopsis, a certain proportion of glycerolipids is entirely synthesized in the chloroplast (prokaryotic lipids). Fatty acids are also exported to the endoplasmic reticulum and incorporated into lipids that are redistributed to the chloroplast (eukaryotic lipids). MGDG, DGDG, SQDG, and PG establish the thylakoid membranes and are integral constituents of the photosynthetic complexes. Phosphate deprivation induces phospholipid degradation accompanied by the increase in DGDG, SQDG, and GlcADG. During freezing and drought stress, envelope membranes are stabilized by the conversion of MGDG into oligogalactolipids. Senescence and chlorotic stress lead to lipid and chlorophyll degradation and the deposition of acyl and phytyl moieties as fatty acid phytyl esters.
DNA methylation is a conserved epigenetic modification that is important for gene regulation and genome stability. Aberrant patterns of DNA methylation can lead to plant developmental abnormalities. A specific DNA methylation state is an outcome of dynamic regulation by de novo methylation, maintenance of methylation and active demethylation, which are catalysed by various enzymes that are targeted by distinct regulatory pathways. In this Review, we discuss DNA methylation in plants, including methylating and demethylating enzymes and regulatory factors, and the coordination of methylation and demethylation activities by a so-called methylstat mechanism; the functions of DNA methylation in regulating transposon silencing, gene expression and chromosome interactions; the roles of DNA methylation in plant development; and the involvement of DNA methylation in plant responses to biotic and abiotic stress conditions.