Received: 24 February 2020 Revised: 2 July 2020 Accepted: 12 July 2020

DOI: 10.1111/pce.13854

ORIGINAL ARTICLE

A transcriptomic, metabolomic and cellular approach to the

physiological adaptation of tomato fruit to high temperature

Juliana Almeida | Laura Perez-Fons | Paul D. Fraser

Department of Biological Sciences, Royal

Holloway University of London, Egham, UK Abstract
High temperatures can negatively influence plant growth and development. Besides
Correspondence
Paul D. Fraser, Department of Biological yield, the effects of heat stress on fruit quality traits remain poorly characterised. In
Sciences, Royal Holloway University of tomato, insights into how fruits regulate cellular metabolism in response to heat
London, Egham, Surrey TW20 0EX, UK.
Email: p.fraser@rhul.ac.uk 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
Funding information
H2020 Food, Grant/Award Number: H2020 tomato fruits after exposure to transient heat stress have been elucidated at the
programme TomGEM / 679766 transcriptome, cellular and metabolite level. An impact on fruit quality was evident as
nutritional attributes changed in response to heat stress. Fruit carotenogenesis was
affected, predominantly at the stage of phytoene formation, although altered
desaturation/isomerisation arose during the transient exposure to high temperatures.
Plastidial isoprenoid compounds showed subtle alterations in their distribution within
chromoplast sub-compartments. Metabolite profiling suggests limited effects on pri-
mary/intermediary metabolism but lipid remodelling was evident. The heat-induced
molecular signatures included the accumulation of sucrose and triacylglycerols, and a
decrease in the degree of membrane lipid unsaturation, which influenced the volatile
profile. Collectively, these data provide valuable insights into the underlying bio-
chemical and molecular adaptation of fruit to heat stress and will impact on our abil-
ity to develop future climate resilient tomato varieties.

KEYWORDS
carotenoids, fruit quality, fruit ripening, heat stress, isoprenoids, metabolomics, plastoglobuli,
tomato, transcriptomics

1 | I N T RO DU CT I O N systems and to threaten local to global food security (Lesk

et al., 2016; Zhao et al., 2017). Higher temperatures can trigger devel-
Plant growth and development are vulnerable to abiotic stresses such opmental, physiological, cellular stress responses in plants which is
as higher temperatures, leading to detrimental effects on agricultural highly dependent on duration and severity of stress as well as sensi-
yield (Battisti & Naylor, 2009; Bita & Gerats, 2013). Increased global tivity of plant cell type and developmental stage (Larkindale
mean temperatures and extreme climate-related events are now et al., 2007; Ohama et al., 2017). When heat stress is moderate, the
occurring with increased frequency and intensity (Horton et al., 2015; changes occurring to crops may be rapidly reversible, but severe epi-
IPCC, 2013). The consequences arising from the warmer temperatures sodes of elevated temperatures are irreversible and can lead to crop
have already been observed and hold potential to destabilise food failure (Zhang et al., 2010).

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2020 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.

Plant Cell Environ. 2020;1–19. wileyonlinelibrary.com/journal/pce 1

2 ALMEIDA ET AL.

Tomato (Solanum lycopersicum) is among the crops for which yield response to heat, plants induce synthesis of hormones, and other pro-
losses have been well documented when different high-temperature tective molecules including osmoprotectants and antioxidants (Gray &
regimes occur during reproductive phase. Yield (e.g., fruit number and Brady, 2016; Wahid et al., 2007). The antioxidant network, in part, is
weight) is adversely affected by daily mean temperatures above 29
C, based on the action of several molecules including carotenoids,
ranging from a few days—when pollen development or fruit set is tocopherols (vitamin E), ascorbate and phenolic compounds, all with
disturbed—to a whole developmental period (Peet et al., 1998; Press- potential to contribute to fruit nutritional proprieties (Li et al., 2018b).
man et al., 2002; Sato et al., 2000, 2006). Notably, the tomato vegeta- Furthermore, as heat can lead to membrane damage caused by lipid
tive development is less sensitive to episodic temperature increases hyper-fluidity and lipid peroxidation, modifying membrane lipid com-
as structural damages of photosystem II were not detected for tem- position, particularly the acyl moieties of glycerolipids, is another criti-
peratures reaching 38
C (Lu et al., 2017; Spicher et al., 2017). Never- cal aspect of plant thermotolerance (Falcone et al., 2004; Murakami
theless, the influence of heat stress on tomato nutritional composition et al., 2000; Higashi & Saito, 2019) that is linked to fruit quality.
and quality has received less attention. This is surprising considering Changes in 18-carbon (C18) polyunsaturated fatty acids can affect the
tomato is one of the most widely consumed fruits globally enzymatic/nonenzymatic formation of oxylipins derived thereof
(Bergougnoux, 2014), it is grown worldwide and is an important (Feussner & Wasternack, 2002). Lipoxygenase (LOX) pathway pro-
source of vitamins and bioactives in the human diet (Viuda-Martos duces hydroperoxide intermediates for the synthesis of different com-
et al., 2014). pounds, including jasmonic acid and volatiles, the latter an essential
Metabolite composition of tomato fruit is affected by adverse aspect of fruit quality (Tieman et al., 2017). Lipid-derived signaling
environmental conditions (Quinet et al., 2019). While some abiotic molecules as oxidised derivatives constitute important components of
stresses as water deficit lead to an increase in sugars, organic acids, heat stress response, acting, for example, in the control of gene
vitamin C and carotenoids (Albert et al., 2016), high-temperature con- expression related to protective responses (Balogh et al., 2013;
ditions seem to have a diverse impact on fruit quality. Most informa- Farmer & Mueller, 2013; Hou et al., 2016).
tion relies on studies performed with differing post-harvest In this present study, transcriptomic, metabolomic and cellular
conditions. Early studies have demonstrated that the long-known fail- analysis has been applied to tomato fruits at different ripening transi-
ure to achieve normal pigmentation of excised tomatoes ripening at tions, following exposure to transient high-temperature treatment
high temperatures (Tomes, 1963) is associated with changes in ethyl- (40
C day/ 30
C night). These conditions may replicate events of heat
ene production, fruit softening and colour development, a phenotype stress experienced during commercial production. Collectively, the
that could be reversed when fruits were transferred to optimal tem- data provide new insights into the metabolic plasticity of tomato fruit
perature (Lurie et al., 1996; Picton & Grierson, 1988). More recent to heat stress episodes and may contribute generically to the develop-
evaluations corroborated these findings highlighting the heat- ment of climatic resilient crops.
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- 2 | MATERIAL AND METHODS
pounds have also been observed (Hernández et al., 2015; Mulholland
et al., 2003). Vitamin C (ascorbate) and carotenoids (provitamin A) are 2.1 | Plant material, growth conditions and
significantly lower when a heat-stress treatment is imposed during transient high-temperature treatment
the advanced stages of fruit development. However, when the tem-
perature is raised at earlier stages, the lack of effects indicates differ- Tomato plants (Solanum lycopersicum, cv. Ailsa Craig) were grown
ential thermo-sensitivity of fruit developmental stages (Hernández under greenhouse conditions with a 16/8-hr day night photoperiod at
et al., 2015). While these changes in tomato metabolism have been 25
C /19
C, respectively.
reported, a comprehensive evaluation exploring the cellular and For transient heat stress treatment (H), the greenhouse chamber
molecular modifications associated with heat response awaits temperatures were set at 40
C/30
C (day/night) over a 48 hr dura-
elucidation. tion. Photoperiod and lighting were the same as the control plant
Alteration in metabolite composition can be direct consequence chamber, and watering adjusted to keep soil water content near field
of several molecular mechanisms underlying the heat stress capacity. Non-stressed plants were kept at control conditions (C) in a
responses. A typical signature response is a wide-scale transient parallel chamber. Flowers were tagged at anthesis. Treatment H was
reprogramming of gene expression, including the expression of heat imposed on three different groups formed by at least five tomato
shock proteins (HSPs, Kotak et al., 2007; Ohama et al., 2017). The plants (between 12- and 13-week-old), each one used to harvest a
majority of HSPs function as molecular chaperones which act not only specific fruit ripening stage, that was heat-stressed only once.
in protection against stress damage but also in folding, intracellular After H, five biological replicates of fruits, that is, fruits from five dif-
distribution and degradation of proteins (Mishra et al., 2002). Interest- ferent plants, were harvested as follows: mature green (HMG,
ingly, HSPs seem to be important for tomato fruit ripening 37–39 days after anthesis, DAF), breaker (HB, 39–41 DAF) and 3 days
(Fragkostefanakis et al., 2015; Neta-Sharir et al., 2005). To maintain post breaker (HB3, 42–44 DAF). Leaves from heat-stressed plants (HL)
membrane stability and deal with oxidative stress generated in were also collected. Plants dedicated to HB and HB3 treatment could
ALMEIDA ET AL. 3

recover (R) under control conditions, and fruits were harvested at red Components were identified using a mass spectral library built
ripe stage (7 days post breaker, B7); for HB, after 7-days recovery from in-house standards and NIST11 database. Each analytical batch
period (HBR7); for HB3, after 4-days recovery period (HB3R4). Overall was validated with quality control samples.
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 2.4 | Profiling of volatile compounds by GC–MS


liquid N2 upon collection and stored at −80 C until metabolic and
molecular analysis. Frozen fruit samples were ground in liquid N2 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
2.2 | Isoprenoid determination and quantification with deuterated acetophenone-d3 as internal standard (20 ppb). Capped
vials were incubated at 40
C and shaken for 30 min. Volatile compounds
Isoprenoids (carotenoids, tocochromanols and chlorophylls) were were then adsorbed onto a SMPE fibre (Car/DVB/PDSM) for 20 min,
extracted from lyophilised tissue powder (15 mg) as described by followed by desorption into the injection port for 5 min. Chromatographic
Enfissi et al. (2010). Compounds were analysed by reverse-phase separation was conducted in a DB-5MS 30 m × 250 μm × 0.25 μm col-
chromatography using an ultra-performance liquid chromatography umn (J&W Scientific, Folsom, CA), equipped with a 10 m guard column
(UPLC) system (Acquity, Waters) equipped with a Photo Diode Array and using a step-temperature gradient from 40 to 300
C at 5
C/min. The
(PDA) detector (Acquity, Waters). A UPLC BEH-C18 column linear temperature gradient included a 2 min hold-temperature and then
(100 mm × 2.1 mm; 1.7 μm, Acquity, Waters) was used for separation steps at 40, 120, 250
C and 5 min at 300
C. Helium was employed as
as described by Nogueira et al. (2013). Peak identification was the carrier gas and the flow rate was 1 ml/min. The inlet and the mass
achieved by comparison of characteristic UV/Vis spectrum with spectrometer transfer line were heated to 250
C. A 7890B-5977B GC-
authentic standards, reference spectra and retention times (Fraser MS system (Agilent Technologies, Palo Alto, CA) was used in splitless
et al., 2007). Quantification was performed using dose–response cur- mode, and data processing and analysis proceeded using AMDIS (version
ves obtained from authentic standards. 2.73) software.

2.3 | Metabolite profiling by gas chromatography 2.5 | Subchromoplast fractionation

(GC)-MS
Chromoplasts were isolated from fruits (90 g) at B3 to B4 stage, and
Polar extracts were prepared from freeze-dried fruit powder (10 mg), sub-compartments were fractionated using a discontinuous gradient
extracted with 1 ml of solution containing methanol and water acidified of sucrose, according to Nogueira et al. (2013).
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 2.6 | Transmission electron microscopy
extracts, alkaline hydrolysis with KOH was performed with a fruit pow-
der aliquot (10 mg) during 1 hr at 40
C followed by extraction as Pericarp fruit segments were fixed at room temperature in solution
described for isoprenoids. Nonpolar extracts were spiked with deuter- [3% (v/v) glutaraldehyde, 4% (v/v) formaldehyde buffered with 0.1 M
ated myristic acid-d27 as the internal standard. The dried residues were PIPES buffer pH 7.2] and then stored at 4
C for at least 24 hr until
derivatised in methoxyamine hydrochloride (in pyridine) followed by processing. Samples were post-fixed in buffered 1% (w/v) osmium
silylation with N-methyl trimethylsilyl trifluoroacetamide. The GC–MS tetroxide and uranyl acetate, washed, dehydrated in a graded series of
analysis was achieved on Agilent 7890A GC system interfaced with a acetone, and embedded in resin. Ultrathin sections were stained with
5975C mass-selective detector as described in Uluisik et al. (2016). Reynolds lead citrate and imaged on a Tecnai T12 Transmission Elec-
For lipid and fatty acid compositional analysis, extraction was per- tron Microscope (Field Electron and Ion Company).
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, 2.7 | qPCR expression analyses
and water [91:30:7, (v/v/v)]. Regions containing the lipid classes were
identified based on the comparison with authentic standards vis- Total RNA was extracted from frozen leaves and fruit pericarps using
ualised with iodine vapour and scraped from the HTLC plate. Elution the RNeasy kit (Qiagen) according to manufacturer's instructions. RNA
and conversion to fatty acid methyl esters (FAMEs) by acid-catalysed from at least four biological replicates was prepared from each tissue
transmethylation, followed by quantification using GC–MS were per- and ripening stage. RNA quality was assessed by agarose gel electro-
formed as previously described by Nogueira et al. (2013). FAMEs phoresis. Total RNA (1 μg) was treated with DNase and converted into
were quantified using myristic-d27 acid as an internal standard. cDNA using the QuantiTect Reverse Transcription kit (Qiagen),
4 ALMEIDA ET AL.

according to the manufacturer's protocols. Real-time quantitative PCR possessed fruits undergoing specific ripening transitions, from
(qPCR) assays were performed in technical duplicates using RotorGene mature green to breaker (HB) and yellow to light-red transition
SYBR green PCR kit (Qiagen) on Rotor-Gene Q, with approximately (HB3) (Figure 1). For comparison, an additional H treatment at early
10 ng of reverse-transcribed RNA. Primer sequences are listed in to late mature green (HMG) was included. After H, plants were ret-
Table S1. Relative expression was calculated as described by Quadrana urned to control conditions (recovery, R) until fruits ripened
et al. (2013). For reference gene selection, expression stability of five (B7 stage).
known reference genes (CAC, EXP, GAGA, ACT1 and ACT2) (Cheng The levels of plastidial isoprenoids were determined by UPLC-
et al., 2017; Exposito-Rodriguez et al., 2008) was evaluated on control PDA. H negatively influenced the carotenoid levels in tomato fruit,
and heat-stressed samples using GeNorm (Vandesompele et al., 2002). and changes from the heat stress were highly dependent on the fruit
ACT2 and CAC were selected based on lowest expression stability stage used (Table 1). Remarkably, phytoene, the first carotene prod-
values (M) of 0.362 and 0.438, respectively. uct of the pathway, was reduced both in HB (10-fold) and HB3
(4.6-fold) compared to their corresponding non-stressed fruits. The
other carotenes in the pathway, phytofluene and ζ-carotene, when
2.8 | RNA sequencing detected, responded similarly to phytoene when H was applied.
Lycopene, the predominant carotenoid found in red ripe tomato,
Total RNA from three biological replicates samples was isolated using was significantly lower, below detection in HB fruits and reduced by
Trizol RNA Purification kit (Thermo Fisher Scientific). cDNA libraries 50% in HB3 fruits, compared to corresponding non-stressed controls.
were prepared and sequenced by IGA Technology Services facility Interestingly, lycopene levels only partially recovered in HB3R4
(Udine, Italy). Single-end sequence reads (75 nt) at a read depth of 31.3 fruits, to about 60% of the content found in ripe CB7 fruit, despite
million reads on the average per sample (24.1 to 39.9 M reads) were the levels of phytoene and phytofluene precursors being fully
obtained from the NextSeq500 platform (Illumina). Raw reads were restored at this stage (Table 1).
processed using ERNE (Del Fabbro et al., 2013) and Cutadapt The total fruit carotenoid content was consistent with lycopene
(Martin, 2011) software. The reads were mapped onto tomato genome levels at later stages of ripening. While lycopene decreased in HB3 and
(S. lycopersicum, cv. Heinz) reference SL3.0, with gene models ITAG3.10, HB3R4 fruits, total carotenoids remained unchanged both in HB fruits
using STAR (Dobin et al., 2013) applying default parameters. Assembling and in those fruits allowed to recover from heat stress (HBR7). By
and quantification of full-length transcripts were accomplished by contrast, HMG carotenoid levels were mostly similar to the control,
Stringtie (Pertea et al., 2015). The counting was achieved by HTseq- except for a modest increase in β-carotene levels. The same response
count (Anders et al., 2015). Gene ontology (GO) term annotation was was also observed in heat-stressed leaves (HL). Additionally, lutein
performed using Blast2GO Pro (version 5.2.5) (Conesa et al., 2005). levels as well as total carotenoid showed an increase in HL compared
All raw RNA sequencing data are available on NCBI, under the to control conditions.
Bioproject accession number PRJNA603594.

2.9 | Data analyses

Significant differences between the control and heat-stressed condi-

tions were determined by Student's t test or ANOVA followed by a
Dunnett's multiple comparison with the level of significance set to 0.05,
using GraphPad Prism software. For pair-wise differential expression
analysis of transcriptome data, statistical analyses were performed by
DeSeq2 (Love et al., 2014). Differentially expressed genes were deter-
mined using false discovery rate (FDR) ≤ 0.01 (adjusted p-value) and
jfold-changej ≥ 1.5 (or jlog2FCj ≥ 0.58). GO enrichment analysis with
Fisher's Exact Test was conducted using Blast2GO.

3 | RESULTS
F I G U R E 1 Outline of heat stress experiment. Tomato plants were
3.1 | Heat stress at advanced ripening stages kept at 25
C day/20
C night (control, C) or exposed to transient high-
temperature treatment (H) at 40
C/30
C (day/night) for 48 hr. After H,
negatively affects carotenoid accumulation
fruits at the following stages were harvested: mature green (HMG),
breaker (HB) and 3 days post-breaker (HB3). Fruits allowed to recover
Plants grown under control conditions (C) were exposed to 48 hr (R) under normal conditions were harvested at ripe stage (HBR7 and
high-temperature treatment (H). The H was imposed when plants HB3R4) [Colour figure can be viewed at wileyonlinelibrary.com]
 ALMEIDA ET AL.

TABLE 1 Transient changes in isoprenoid profile of tomato fruits and leaves exposed to high-temperature treatment

Fruit Leaf
a a a a a
μg/g DW CMGa HMGa CBa HBa CB3 HB3 CB7 HBR7 HB3R4 CL a HLa
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 t tests or ANOVA/Dunnett's test, * p < .05; ** p < .01). nd, not detected; DW, dry
weight; CB3, nonstressed fruit at 3 days post-breaker; HB3, heat-stressed fruit at 3 days post-breaker. CB7, non-stressed fruit at 7 days post-breaker; HBR7 and HB3R4, fruits HB and HB3, respectively, allowed to
recover under normal conditions and harvested at ripe stage. CL, nonstressed leaf; HL, heat-stressed leaf.
a
CMG, nonstressed fruit at mature green stage; HMG, heat-stressed fruit at mature green stage. CB, nonstressed fruit at breaker stage; HB, heat-stressed fruit at breaker stage.
5
6 ALMEIDA ET AL.

Levels of other lipid-soluble antioxidants were also influenced by The score plot obtained from polar and nonpolar extracts could not
H. α-tocopherol increased 1.2-fold in HMG and HB3 fruits. Interest- discriminate non-stressed and heat-stressed conditions among the
ingly, its levels were still higher even after a recovery period in HBR7 fruit stages evaluated, though a clear separation between early- and
and HB3R4 fruits compared to control conditions (Table 1). late-ripening stages was achieved (Figure. 2a, b). Overall primary
metabolism remained unchanged after H and followed by R
(Table S2).
3.2 | Metabolite profiling of tomato fruit exposed Our high-temperature treatment did not appreciably affect the
to transient heat stress levels of known osmoregulators such as proline and GABA, and only a
few metabolites responded significantly to H mostly in a fruit stage-
Metabolite profiling using GC–MS was carried out on fruits exposed dependent manner (Figure 2c, d). Sucrose was very responsive to
to heat stress and concurrently samples were collected for trans- heat, consistently accumulating in fruits (HMG and HB) at early ripe
criptome analysis. Principal component analysis (PCA) was used to stages (Figure 2c). Some amino acids changed in content, particularly
compare primary metabolism of fruits under C, H and R conditions. threonine was increased in HB and HB3. The tricarboxylic acid (TCA)

F I G U R E 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 t test corrected for multiple comparison using Holm-Sidak's post-test; * Adjusted p < .05,
** p < .01, *** p < .001). Full data set available in Table S2. C22:0, Docosanoic acid; C24-ol, tetracosanol; Ribose/xylose (f), Ribose/xylose in
furanose ring form [Colour figure can be viewed at wileyonlinelibrary.com]
ALMEIDA ET AL. 7

intermediates showed a variable response, with aconitic acid accumu- Analysis of lipid classes revealed a relative increase in the stor-
lating upon H. From non-polar fraction, β-sitosterol levels significantly age lipid triacylglycerol (TAG) under H at both stages B and B3 com-
accumulated in HB and HB3. pared to control (Figure 3a, c). Extraplastidic classes of
After R, primary metabolism of ripe fruits previously heat- phospholipids, phosphatidylcholine (PC), phosphatidylserine (PS) and
stressed at B transition (HBR7) was largely the same than their non- phosphatidylethanolamine (PE) showed variable response to heat.
stressed counterparts, except for high levels of α-tocopherol. This For the fatty acid composition of the lipid classes (Figure 3), the gen-
quantitative tocopherol response to heat was observed both within eral trend was the lower levels of the trienoic fatty acid C18:3
the GC–MS and UPLC-PDA derived datasets confirming the utility of under H, particularly in the plastidial membrane lipids mono-
the approaches (Table S2). galactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol
(DGDG) in comparison to control conditions. This decline was pre-
dominantly mirrored by an increase in the corresponding dienoic
3.3 | Fruit lipid metabolism is highly responsive fatty acid precursor, linoleic acid (C18:2), and other less unsaturated
to heat and saturated acyl moieties (Figure 3b, d). Consistently, a greater
reduction of the C18:3/C18:2 ratio was observed in MGDG and
Lipid remodelling while-decreasing the level of lipid unsaturation is a DGDG (Table S3). In contrast, HB fatty acid composition of the TAG
crucial aspect of plant thermotolerance under suboptimal temperature fraction showed an opposite response with higher levels of the tri-
conditions (Falcone et al., 2004). Nevertheless, the analysis of the enoic acid C18:3 proportion compared to control CB (Figure 3).
total lipid fraction by GC–MS showed no significant differences Overall, tomato fruit response to high-temperature conditions
between C and H in fatty acid composition. To increase the sensitivity includes lipid remodelling, which leads to TAG accumulation, and
and address the potential changes in complex lipid moieties, lipid spe- decreasing the level of membrane lipid unsaturation, particularly tri-
cies were first separated by TLC and then analysed by GC–MS. enoic acid composition.

F I G U R E 3 Lipid profile of tomato fruits after heat stress. Lipids were separated by TLC and quantified by GC–MS. (a, c) Relative abundance
of lipid content (mol % of total) based on detector response calculated from internal standard. The values are shown as mean ± SD of three
measurements from a pool of four biological replicates. (b, d) Heat map representation of changes in fatty acid composition (mol% of total) of
each lipid class under heat. The ratio % in H versus % in C were obtained and showed as log2 fold change. Dark red or dark blue indicates that the
acyl moiety is relatively increased or decreased, respectively, under high-temperature conditions. Grey colour indicates acyl moieties not
detected. MGDG, monogalactosyldiacylglycerol; DGDG, digalactosyldiacylglycerol; PC/PS, phosphatidylcholine/phosphatidylserine; PE,
phosphatidylethanolamine; TAG, triacylglycerol [Colour figure can be viewed at wileyonlinelibrary.com]
8 ALMEIDA ET AL.

The volatile organic compounds (VOC) analysis revealed C18 fatty metabolic pathways but also in compound sequestration (Spicher
acid-derived volatiles were altered in heat-stressed fruits (Table S4). For et al., 2017; Zhang et al., 2010). To ascertain whether high tempera-
the C18:3-derived flavour molecules produced via LOX action, a nota- ture alters the distribution of liposoluble antioxidants into plastidial
ble decrease in C6 volatiles was observed. Interestingly, levels of the sub-compartments, analysis of fractionated chromoplast from heat-
C18:2-decomposition product hexanal were significantly lower only at stressed and non-stressed fruits was carried out (Figure 4).
HB fruits, whilst HB3 exhibited hexanal at the same levels of non- First, the total amounts obtained from the sum of all fractions
stressed fruits. Contrastingly, C5 volatiles as pentanal and 2-pentanone (i.e., plastoglobuli, envelope membranes, stroma, thylakoids) corrobo-
accumulated in HB3 fruits. Carotenoid-derived volatiles detected were rated the lower levels of phytoene, phytofluene and lycopene in HB3
unaffected except for the lycopene derived 6-methyl-5-hepten-2-one; fruits (Table S5). Overall, the proportions of all carotenoids, tocoph-
which was found to be lower in HB3 compared to CB3. These data com- erols and plastochromanol (PC-8), this latter also an antioxidant typi-
plement the lower levels of lycopene found in these samples following cally accumulating at plastoglobuli (Kruk et al., 2014) followed the
H. Finally, the accumulation of different VOCs belonging to terpenoid same distribution pattern immediately after H, suggesting that sub-
parent molecules, such as α-pinene, δ-4-carene, cymene, β-phellandrene compartmentation was not largely altered by heat stress. Yet, the
was detected in at least one ripening stage either immediately after lower phytofluene levels found in HB3 fruits preferentially accumu-
stress and/or after recovering. A similar trend was found for the pheno- lated into plastoglobular fraction (Figure 4b). No changes in the pro-
lic derived molecule o-guaiacol (Table S4). portion of carotenoids and tocopherols arose in the plastoglobular
fraction in the fruits that undergone heat stress at B stage following a
short recovery (HBR4). However, the presence of the fractions termed
3.4 | Chromoplast structure of fruits exposed to membrane II was more pronounced in HBR4, which indicates the per-
high temperatures sistence of thylakoids remnants (Figure 4d).
Analysis of chromoplast ultrastructure by transmission electron
Heat stress-induced changes in carotenoids, tocopherols and neutral microscopy suggested that non-stressed fruit chromoplasts displayed
lipid levels may be associated with perturbations not only in the more mature lycopene crystals (Figure S1) while fruits experiencing

F I G U R E 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 (HB3 and HBR4) by flotation on a discontinuous sucrose gradient. (b, d) Plastidial
isoprenoid profile of fractions. Values represent % of the total amount found in each fraction [Colour figure can be viewed at
wileyonlinelibrary.com]
ALMEIDA ET AL. 9

stress at B stage (HBR4) had smaller lycopene crystals and a notable differentially expressed (DE) transcripts were detected as a result of H
persistence of thylakoid membrane which could reflect delays in fruit imposed (Table S6). In total, 8,141 and 7,006 genes were found DE in
ripening. the comparison CB versus HB and CB3 versus HB3, respectively.
Among the DE, 2067 genes were up-regulated, and 2,368 were
down-regulated under heat, irrespectively of the fruit stage when the
3.5 | Transcriptome analysis stress was imposed. These subsets of commonly heat-stressed regu-
lated genes were used in the gene ontology (GO) enrichment analysis
To address the molecular mechanisms underlying metabolic responses (Figure 5a).
to heat stress, transcriptome analysis of B and B3 fruits under Considering the most specific GO terms, up-regulated genes were
stressed and non-stressed conditions was carried out. Numerous significantly enriched for only a few biological process GOs

F I G U R E 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 (CL)
and heat-stressed (HL, 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 t test, * p < .05, ** p < .01, *** p < .001) between heat and control conditions at corresponding organ/developmental stage are shown
[Colour figure can be viewed at wileyonlinelibrary.com]
10 ALMEIDA ET AL.

(10) associated with general terms as RNA processing, mitotic cell acid biosynthetic process” are closely related to the metabolic repro-
cycle and chromatin remodelling (Figure 5b). When a more relaxed gramming triggered by heat in fruits.
significance threshold (p-value <.01) was applied, GO terms as “mRNA Extreme temperature is known to induce the expression of HEAT
splicing, via spliceosome” (GO:0000398; p-value 2.15 E−03), and SHOCK TRANSCRIPTION FACTORS (Hsfs). In tomato, HsfA1, which is
molecular function “SWI/SNF superfamily-type complex” constitutively expressed and post-translationally regulated, is respon-
(GO:0070603, p-value 4.68 E−04), the latter acting in chromatin sible for the initial heat stress response controlling the HS-induced
remodelling processes (Table S7) were found. DNA de novo methyla- expression of HsfA2 and HsfA3 (Fragkostefanakis et al., 2015; von
tion results in part from the activity of DOMAIN REARRANGED Koskull-Döring et al., 2007). Both HsfA2 and HsfA3 were found up-
METHYLTRANSFERASE (DRM) and chromatin remodelers as DEFEC- regulated under H in RNA-seq dataset compared to control conditions
TIVE IN RNA-DIRECTED DNA METHYLATION1 (DRD1) (Zhang (Table S6). qPCR assays confirmed higher HsfA2 transcript levels not
et al., 2018). As a response to heat, for example, gene encoding only for HB and HB3 but also in heat-stressed leaves (HL), though sig-
tomato homologs of DRM (Solyc10g078190, Solyc05g053260) and nificant differences for HMG were not detected. Importantly, higher
DRD1 (Solyc01g109970) were found up-regulated upon H (Table S6). HsfA2 transcripts were not sustained after R (Figure 5c).
The epigenetic mechanisms also featured when the heat-induced
genes were queried for each comparison separately; “histone modifi-
cation” and “RNA processing” processes were overrepresented among 3.6 | Ripening regulators and targeted pathways
up-regulated HB3 genes (Table S7).
By contrast, genes down-regulated by H were enriched for GOs Given that processes related to fruit ripening were significantly
mainly associated with defence response to biotic stress, hormone enriched among the genes repressed by heat (Figure 5b), we explored
synthesis and signaling pathway, metabolic processes related to lipids, the heat-induced transcriptional regulation associated with ripening
carbohydrate, amino acids, and redox-related compound glutathione as well as downstream targeted pathways such as isoprenoid metabo-
(Figure 5b). GO terms found overrepresented such as “carotenoid bio- lism (Karlova et al., 2014; Li et al., 2019; Quadrana et al., 2013)
synthetic process,” “carbohydrate metabolic process,” “alpha-amino (Figure 6a, Table S8).

F I G U R E 6 Transcriptional regulation of genes involved in fruit ripening and lipid metabolism under heat stress. Bars represent the log2-fold
change based on transcriptome comparison of H versus C at B (dark grey) and at B3 (light grey) for DEG. (a) Ripening-related genes. (b) Lipid-
related genes. Full data set available in Tables S8 and S9. Gene abbreviatures according to Tables S8 and S9
ALMEIDA ET AL. 11

In tomato, climacteric ripening is controlled by several transcrip- 3.7 | Lipid metabolism

tion factors in conjunction with different phytohormones, such as eth-
ylene (Karlova et al., 2014). Genes encoding key ripening-associated Upon heat stress, lipid metabolism-related transcripts were found
transcription factors such as COLORLESS NON-RIPENING (CNR), NON- to be overrepresented among downregulated genes (Figure 5(b)).
RIPENING (NOR), FRUITFULL1 (FUL1), TOMATO AGAMOUS-LIKE1 From a manually curated list derived from tomato loci showing
(TAGL1) and AUXIN RESPONSE FACTOR2A (ARF2A) were strongly homology to Arabidopsis genes associated with acyl-lipid metabo-
suppressed by heat. Interestingly, the expression of RIPENING-INHIBI- lism (Higashi et al., 2015), a subset of genes putatively involved in
TOR (RIN) was not found to be heat-sensitive (Figure 6a). Transcripts the plastidial de novo fatty acid biosynthesis was suppressed
encoding key repressors of photomorphogenesis as PHYTOCHROME under H in both B and B3 stages (Figure 6b, Table S9). First, the
INTERACTING FACTORS (PIFs), which are degraded in the light upon tomato homologs encoding the plastidial pyruvate dehydrogenase
interaction with photoactivated phytochromes (Leivar & complex (PDH-E1 α, E1 β) producing acetyl-CoA precursors that
Monte, 2014), specifically PIF1a and PIF3, as well as DEETIOLATED1 feed fatty acid synthesis; then the subunits of heteromeric acetyl-
(DET1/HP2), CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) CoA carboxylase (α-CT/CAC3, BCCP2), the acyl carrier protein
responded positively to heat at both ripening stages analysed. (ACP), the malonyl-CoA:ACP malonyltransferase (MCMT), the
Ripening-inducible genes related to ethylene biosynthesis and sig- 3-ketoacyl-ACP synthase 3 and 1 (KASIII, KASI) and the reductase
naling, namely 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (KAR), the hydroxyacyl ACP dehydratase (HAD), the enoyl-ACP
and ACC synthase (ACO1, ACS2, ACS4), cell wall metabolism reductase (ENR) were all strongly down-regulated under heat. The
(e.g., polygalacturonase, PG2A), were significantly down-regulated as a same trend was observed for the genes related to the acyl-ACP
result of heat stress exposure (Figure 6a). Moreover, expression of the hydrolysis (FATB) involved in the acyl moieties releasing for the
gene encoding the fruit specific phytoene synthase (PSY1), the first trafficking between plastid and endoplasmic reticulum (ER, Hölzl &
enzyme of carotenoid pathway, was significantly repressed under Dörmann, 2019).
H. This result was also confirmed by qPCR analysis in HB and HB3 For the plastidial galactolipid synthesis, heat-inducible tran-
samples (Figure 5c). Indeed, the expression profile obtained from all scripts included those involved in the remodelling of galactolipids;
samples suggests that PSY1 suppression under heat seemed to be for example, the galactolipid galactosyltransferase (SFR2/GGGT),
linked to later ripening stages when chromoplast differentiates, which converts MGDG to oligogalactolipids and diacylglycerol
because in HMG fruits the PSY1 expression was yet slightly up- (DAG, Higashi & Saito, 2019), and digalactosyl-DAG synthase (DGD).
regulated. In contrast, genes encoding members of the plastid-localized lipid
Besides PSY1, other carotenogenic-related genes were DE phosphatidate phosphatases (LPP), promoting dephosphorylation of
upon heat exposure. Genes encoding 4-diphosphocytidyl-2-C- phosphatidic acid (PA) that yields DAG, were suppressed by heat
methyl-D-erythritol kinase (ISPE), geranylgeranyl pyrophosphate (Figure 6b).
synthase (GGPPS2, GGPPS3), plastidial terminal oxidase (PTOX), In Arabidopsis, fatty acid desaturation, a key aspect of
carotenoid isomerase (CrtISO) were significantly down-regulated. thermotolerance (Falcone et al., 2004), is regulated by genes encoding
Genes associated with vitamin E metabolism showed also lower for the plastid localised fatty acid desaturase (FAB2/SAD, FAD4,
expression (VTE2, VTE3.1, HPPD2) under H. Regarding the plas- FAD5, FAD6, FAD7, FAD8) and the ER-localized fatty acid
toglobuli-related proteins involved in the isoprenoid sequestration, desaturases (FAD2 and FAD3). For the plastid-residing FADs, genes
tomato genes encoding FIBRILLIN (FBN) homologous to those encoding homologs for the 18:0-ACP desaturase (FAB2) and the
described in Arabidopsis thaliana (van Wijk & Kessler, 2017) 16:2/18:2 galactolipid ω3 desaturase (FAD7/FAD8/LeFAD7, Nakamura
responded differently to heat stress. While some highly expressed et al., 2016) were repressed. The latter is in line with the decrease in
members were down-regulated as FBN4, other members with less the plastid membrane trienoic fatty acids (Figure 3). In the present
abundant transcripts in fruits were consistently up-regulated under study, a specific 16:0-MGDG Δ7-desaturase (FAD5) coding gene was
H (Table S8). found to be highly up-regulated after H. In addition, major differences
The sucrose accumulation in heat-stressed fruit may be related to were detected in ER lipid biosynthetic gene transcripts in the HB3
differences in sucrose turnover enzyme abundance (Qin et al., 2016). samples, where the highly expressed gene coding for
Although sucrose accumulated only in HB fruits (Figure 2c), perturba- 18:1-phosphatidylcholine ω6 desaturase (FAD2-1, Lee et al., 2020)
tions in the expression of genes related to sugar metabolism were isoform and 16:2/18:2 galactolipid ω3 desaturase (FAD3, Yu
detected at both B and B3 stages, with notable repression of the tran- et al., 2009) were repressed.
scripts of vascular invertase (VI), cell wall invertase (LIN5) and sucrose For TAG biosynthesis, higher levels of expression were associated
synthase (Susy6) (Figure 6a). Sucrose turnover enzymes coding genes with biosynthetic pathway genes. Different types of enzymes can syn-
were higher repressed in HB than HB3 (e.g., for VI, fold change ratio thesise TAG from DAG, including acyl-CoA dependent enzymes, acyl-
was about four-fold and two-fold in CB vs HB and CB3 vs HB3, respec- CoA:DAG acyltransferases (DGATs), and diacylglycerol acyltransferase
tively), though the transcripts encoding a vacuolar invertase inhibitor (PDAT) which uses PL as acyl donor (Fan et al., 2017). Genes encoding
(VIF) were found slightly up-regulated in HB3, which suggests further DGAT and PDAT were upregulated under heat, following the higher
capping to invertase activity. levels of TAG observed in HB and HB3 (Figure 3). A similar trend was
12 ALMEIDA ET AL.

found for the transcripts encoding proteins associated with TAG detached fruits previously (Lurie et al., 1996). Nevertheless, the lower
hydrolysis, which were predominantly upregulated at both stages carotenoids levels in ripe fruits experiencing heat at B3 transition
analysed (Figure 6b). implies that the length of the recovery period may be critical. Boosting
Fatty acid β-oxidation pathway was overrepresented among the in carotenoid synthesis during fruit ripening is achieved predominantly
genes down-regulated by heat (Figure 5b). In accordance, the genes by the up-regulation of genes encoding key biosynthetic enzymes
encoding enzymes were found actively repressed under heat, such as (Hirschberg, 2001; Enfissi et al., 2017). Thus, the heat-induced tran-
acyl-CoA oxidase (ACX1a, Li et al., 2005) and members of the scriptional misregulation at advanced ripening stages (Figure 6a) may
multifunctional protein (MFP), as well as the peroxisomal isoform of explain the decrease in fruit carotenoid levels. Firstly, both phytoene
long-chain acyl-CoA synthetase (LACS), which activates free fatty formation and subsequent isomerisation are potentially compromised
acids to acyl-CoA thioesters to generate acyl-CoA derivatives. Indeed, as the expression of fruit-specific PSY1 (Figure 5), encoding the major
genes associated with oxylipin biosynthesis were repressed after H, flux-controlling enzyme of carotenogenic pathway (Fraser et al., 2002,
including allene oxide cyclase (AOC3), allene oxide synthase (AOS2) 2007), and CrtISO (Isaacson et al., 2002) were repressed by heat. Sec-
and acyl-hydrolase patatin-like, involved in the production of ondly, efficient carotenoid desaturation conducted by phytoene desa-
jasmonate from polyunsaturated fatty acids. turase (PDS) and ζ-carotene desaturase (ZDS) depends on the redox
For the volatiles production, 13-LOXs and hydroperoxide lyase status of plastoquinone/plastoquinol pool dependent on the activity
(HPL) are the main enzymes catalysing the conversion of C18 polyun- of PTOX (Shahbazi et al., 2007) whose transcripts were found heat-
saturated fatty acids to C5 and C6 volatiles in tomato fruit (Shen sensitive. Finally, methyl-erythritol phosphate (MEP)-derived precur-
et al., 2014). Among the tomato 13-LOXs, LoxC is essential for the sors for carotenogenesis (Almeida et al., 2015; Nogueira et al., 2018)
generation of fruit C5- (1-penten-3-ol, 1-penten-3-one, pentanal, (Z)- might be altered as the expression of ISPE, GGPPS3 and the ripening-
2-penten-1-ol, and 1-pentanol) and C6-flavour volatiles (Shen induced GGPPS2 were suppressed by heat. Together, these perturba-
et al., 2014), while LoxB and LoxA possibly support C5 synthesis tions emphasise the transcriptional heat-sensitivity of the early
(Griffiths et al., 1999). The heat-induced response of genes encoding carotenogenesis in fruits.
tomato 13-LOX varied between HB and HB3, except for LoxB, which Carotenogenic enzymes are also post-translationally regulated by
was downregulated at both ripening stages addressed. Importantly, protease complexes which are highly active under heat (D'Andrea
ripening-inducible LoxC was found only repressed in HB. HPL tran- et al., 2018). The MEP enzyme DXS and carotenogenic PSY are direct
scripts were concordantly repressed under H (Figure 6b, Table S9). substrates of Clp protease complex whose activity, in turn, is
As the levels of β-sitosterol increased after heat stress (Figure 2e), counterbalanced by other chaperones, for example, orange (OR;
the transcripts encoding sterol 22-desaturase (CYP710A11), involved Welsch et al., 2018). Their interaction adjusts DXS and PSY functional
in the conversion of β-sitosterol to stigmasterol, were checked, reveal- forms controlling enzyme level and activity. Interestingly, Clp-
ing a heat-sensitive expression pattern. 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
4 | DISCUSSION post-translational mechanisms curbing the activity of carotenoid-
related enzymes under heat stress. It is worth noting that this
Our study has undertaken an integrative approach to address the response is likely fruit-specific as heat-stressed leaves showed an
metabolic, cellular and molecular changes associated with transient opposite effect on carotenoids; indeed, the increased levels of lutein
heat stress imposed on tomato fruits, elucidating several key features observed in HL were consistent with a previous report using tomato
that impact on fruit quality traits. (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
4.1 | Nutritional attributes were altered by heat which have experienced stress previously irrespective of their devel-
stress opmental stage. The production of tocopherols has been linked to
high-temperature response in tomato leaves supported by transcrip-
Our findings revealed that transient heat stress can alter carotenoid tional regulation (Spicher et al. 2016, 2017). In our study, the lack of
accumulation in tomato fruits, with sensitivity to temperature increas- correlation between fruit tocopherol content (Table 1) and the
ing as ripening advanced. The negative impact of heat on carotenoid expression of genes involved in tocopherol biosynthesis (Figure 6a),
levels was associated with changes in the initial steps in carotene could be associated with the redirection of isoprenoid precursors
formation. from carotenoid formation into tocopherol formation instead
Our study showed that ripe fruits have tremendous plasticity to (Almeida et al., 2015; Fraser et al., 2007). Importantly, carotenoid
restore carotenoid levels following a heat wave, suggesting no perma- and tocopherol heat-responsive genes typically exhibit ripening-


nent damage was achieved. The temperature of 40 C caused moder- associated expression pattern (Quadrana et al., 2013; Sato
ate stress in tomato as reported earlier (Spicher et al., 2017). et al., 2012;) and the changes observed may be due to inhibition of
Reversible effects of heat treatment have been observed on vine fruit ripening.
ALMEIDA ET AL. 13

4.2 | Ripening related processes are misregulated and DRD1 (Gallusci et al., 2016). The up-regulation of tomato homo-
in heat-stressed fruits logs DRM1 and DRD1 (Table S6) might contribute to rearrangements
of epigenome landscape under high-temperature treatment and raises
In tomato, fruit ripening encompasses highly coordinated processes the possibility that plant adaptive responses to heat mediated by epi-
orchestrated by a network of interacting genes and signaling path- genetic mechanisms (Li et al., 2018a; Quadrana et al., 2019; Ohama
ways, which involves differentiation of chloroplasts into chromoplasts et al., 2017) also operate in tomato fruit.
(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 4.3 | Fruit primary metabolism changes in
also in sugar accumulation, tissue softening and volatile production response to heat stress
(Klee & Giovannoni, 2011; Rambla et al., 2015).
Several transcription factors acting as regulators of tomato fruit The absence of signatures commonly shared through all stressed sam-
ripening including CNR, NOR, FUL1, TAGL1 and ARF2a (Bemer ples (Figure 2, Table S2) further supports the idea the thermo-
et al., 2012; Giovannoni, 2007; Hao et al., 2015; Manning et al., 2006; responsive is highly dependent on fruit stage. Among the known
Vrebalov et al., 2009) were transcriptionally suppressed by heat. osmoregulators, higher threonine levels at advanced ripening stages
Besides, repressors of carotenogenesis PIF1a, DET1 and COP1, which are in line with its conserved biomarker for abiotic stress, accumulat-
transduce phytochrome-sensed changes in the environmental light, ing in Arabidopsis leaves under heat stress (Obata & Fernie, 2012).
hence affecting carotenoid biosynthesis and plastid development Notably, citric and malic acids that contribute most to the typical acid-
(Enfissi et al., 2010; Liu et al., 2004; Llorente et al., 2015), were found ity of tomato fruit (Baldwin et al., 2008) remained unaffected.
to be up-regulated under heat conditions. As PIF1a binds to PSY1 pro- Sucrose accumulation in heat-stressed fruit at early ripening stages
moter region and represses PSY1 transcription in tomato fruits (Figure 2) correlated with the sensitivity of sucrose metabolism to high
(Llorente et al., 2015), the higher PIF1a expression may cause temperatures previously reported in tomato male reproductive system
temperature-induced PSY1 repression. and in fruits after pollination (Li et al., 2012; Liu et al., 2016; Sato
Expression profile of the ripening regulators mined from public et al., 2006). Indeed, enhanced LIN and VI activity in tomato has been
database (TomExpress; Zouine et al., 2017) at non-stressed conditions associated with fruit thermotolerance at early developmental stages
supports the view that heat-induced repression of CNR and NOR are (Li et al., 2012; Liu et al., 2016). It is known that, at late-ripening stages,
not likely due to ripening delay (as it cannot be ruled out for FUL1 and sucrose accumulation is limited since invertase activities intensify as ripen-
TAGL1) but rather a bona fide feedback transcriptional regulation of ing progresses, with VI controlling sucrose/hexose ratio (Biais et al., 2014;
fruits in response to higher temperatures. Similarly, for the known Klann et al., 1996; Qin et al., 2016; Yelle et al., 1991). In HB fruits, lower VI
carotenogenic repressors PIF1a, COP1 and DET1 and possibly PIF3, transcripts may explain why sucrose increased. However, control sucrose
all plausibly act as core components of heat-triggered transcriptional levels found at HB3 did not correlate with down-regulation of VI, LIN5 and
reprogramming in tomato fruits. Susy6. In this case, sugar metabolic fluxes at B3 stage may prevent sucrose
Importantly, PIF-dependent signaling is a central pathway for from accumulating under heat. Lack of correlation between invertase
thermoresponsiveness under warmer but non-stressful ambient tem- activity and sucrose/hexose levels has been reported in tomato fruits of
peratures (Rosado et al., 2019; Vu et al., 2019;). Our findings thereby lines with increased LIN activity (Liu et al., 2016).
extend the role of the light signaling components in response to dif- Effects on fruits seem to be minor compared to the vegetative
ferent temperature cues. The prevalence of phytochrome-signaling system or pollen development, where altered carbon metabolism
repressors, together with the suppression of CNR and NOR, may upon exposure to high temperatures can promote yield losses (Ruan
inhibit the ripening program in fruits experiencing heat stress at et al., 2010; Rieu et al., 2017). Moreover, fluctuations in sugar levels
advanced ripening stages, which is in line with the down-regulation of are important, as cell signals, since they can act in crosstalk with hor-
genes involved in ethylene biosynthesis/signalling (ACO1, ACS2, mones (e.g., auxin) and reactive oxygen species (ROS) signaling path-
ACS4, E8), cell wall degradation (PG2a, EXP1) (Li et al., 2019), and also ways during stress responses; sugars can also contribute to stress
with the persistence of thylakoid-like membranes (Figure 4, alleviation by facilitating production of HSP even in reproductive sys-
Figure S1). Moreover, consistent with previous studies in tomato tems, property that correlates to high fruit sensitivity under stress (Liu
(Mishra et al., 2002), fruit heat transcriptional response seems to be et al., 2013). Molecule signaling and protective roles are, therefore,
mediated by Hsfs (Figure 5). Finally, our transcriptome analysis possible to intersect under heat stress in fruits.
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 4.4 | Lipid remodelling triggered by heat stress in
(Figure 5, Table S7). Epigenetic mechanisms as DNA methylation add fruits
another layer of regulation for the tomato ripening program. DNA
methylation relies, in part, on the RNA directed DNA methylation Lipid remodelling was a pronounced feature of heat-stressed fruits,
(RdDM) pathway, dependent on small RNAs and the activity of DRM leading to TAG accumulation, and decreasing the level of membrane
14 ALMEIDA ET AL.

lipid unsaturation (Figure 3, Table S3). Polyunsaturated acyl chains linked to membrane stability. Heat-induced β-sitosterol accumulation
contribute to membrane fluidity and stability and, in response to may contribute to control membrane permeability and membrane pro-
higher temperatures, the degree of unsaturation is decreased to main- tein activity (Guo et al., 2019).
tain optimal fluidity and integrity of membranes (Nishida & TAG accumulation associated with heat stress-induced lipid
Murata, 1996; Murakami et al., 2000; Falcone et al., 2004; Zheng remodelling has been reported in photosynthetic organisms (Légeret
et al., 2011). Moreover, the observed changes in sterols might also be et al. 2016; Mueller et al., 2015; Narayanan et al., 2016). The

F I G U R E 7 Impact of heat stress on tomato fruit. (a) Fruit metabolic processes affected by heat. Compounds targeted on metabolite profiling
are shown in bold. Amino acid (aa) metabolism was omitted for simplicity. Dotted lines indicate multiple metabolic steps; (b) Summary of heat-
induced metabolic changes and associated transcriptional regulation found in B and B3 fruits exposed to high-temperature treatment. Arrows
indicate metabolite changes. Up-regulated and down-regulated genes (or pathways) are indicated in blue and red, respectively. ACP, acyl carrier
protein; CHL, chlorophyll; DAG, diacylglycerol; FA, fatty acid; MVA, mevalonate; MEP, methyl-erythritol phosphate; MGDG,
monogalactosyldiacylglycerol; DGDG, digalactosyldiacylglycerol; PC/PS, phosphatidylcholine/phosphatidylserine; PE, phosphatidylethanolamine;
TAG, triacylglycerol; Suc, sucrose; α-toc, α-tocopherol; TCA, tricarboxylic acid; ox-lipids, oxidised phospholipids; VOC, volatile organic compound
[Colour figure can be viewed at wileyonlinelibrary.com]
ALMEIDA ET AL. 15

substantial heat-induced decrease in C18:3/C18:2 ratio of plastid In conclusion, our findings illustrated the impact of brief exposure
membrane lipids in fruits is similar to previous reports in heat-stressed to high-temperature events on tomato fruit quality and revealed
tomato leaves (Spicher et al., 2016). Besides the prevention of potential molecular mechanisms associated with heat response
physical–chemical damages of membranes, it has been proposed that (Figure 7). Depending on the ripening stage, heat may have under-
selective decline of trienoic acid acyl moieties might confer survival estimated yet significant effects on nutritionally value and other
advantage imposed by cellular oxidative stress associated to excessive quality-related attributes in tomato, with sensitivity to high tempera-
ROS generated under heat (Larkindale et al., 2007; Légeret ture increasing in more advanced ripening stages. Several heat-stress
et al., 2016). As galactolipids are highly enriched in polyunsaturated responsive genes, including fruit ripening regulators, have been identi-
fatty acids, and thus easily prone to lipid peroxidation, photosynthetic fied from transcriptome analysis correlating with the metabolite
organisms may transfer trienoic fatty acids from membrane lipids to changes. Collectively the data acquired provides a significant advance-
TAG sequestered in lipid droplets as a strategy to control the exten- ment to our understanding of fruit metabolic reprogramming associ-
sion of lipid oxidation (Du et al., 2018; Légeret et al., 2016). ated with heat stress. It is now clear that cold storage is not the only
Our transcriptome data provided insights into molecular mecha- stress affecting fruit quality but perturbations in heat will also alter
nisms supporting lipid remodelling (Figure 6b, Table S9). The heat- quality attributes. These data provide an exploitable resource for the
induced decrease in the level of plastid lipid unsaturation coincided development of climate resilient crop varieties.
with the down-regulation of FADs, mainly the FAD7/FAD8 encoding
the plastidial ω3 desaturase. Transcripts of the ER-counterpart desa- ACKNOWLEDG MENTS
turase (FAD3) were only significantly repressed in HB3, following the This work is supported by the H2020 programme No. 679766.
temperature-induced decrease of extraplastidial 18:3-acyl-containing TomGEM; A holistic multi-actor approach towards the design of new
lipids specific to this stage (Figure 3). Lipid remodelling may also be tomato varieties and management practices to improve yield and qual-
supported for the up-regulation of (a) SFR2/GGGT, whose ity in the face of climate change. The authors thank Mr Chris Gerrish
corresponding enzyme contributes to diminish the MGDG/ for the technical assistant with fractionation experiments. IGA Tech-
oligogalactolipids ratio and to release DAG further used for TAG bio- nology Services facility for assistance in the utilisation of RNA-seq
synthesis (Higashi & Saito, 2019; Moellering & Benning, 2011), and data and Dr Genny Enfissi for advice and input in the experimental
(b) DGAT and PDAT, which encode TAG biosynthetic-related approach.
enzymes (Fan et al., 2017). Together, these data suggest that the
higher amounts of 18:3-acyl-containing TAGs upon heat, at B stage, CONFLIC T OF INT ER E ST
may have been derived from C18:3 released from membrane lipids The authors declare no conflict of interest.
than from de novo synthesised fatty acids (Légeret et al., 2016),
therefore reflecting the changes in the proportion of membrane AUTHOR CONTRIBU TIONS
glycerolipid composition (Higashi & Saito, 2019). By contrast, at B3 Juliana Almeida and Paul D. Fraser conceived the original research
stage, 18:3 acyl moieties are likely redirected to lipid oxidation path- plan. Juliana Almeida performed the experimental programme, data
ways (Schilmiller et al., 2007), for example, volatile production. In analysis and statistics. Laura Perez-Fons carried out the volatile analy-
tomato, levels of C18:2 and C18:3 positively correlate to volatile sis and components of the metabolite analysis. Juliana Almeida and
derivatives hexanal and hexenal, respectively (Domínguez Paul D. Fraser wrote the manuscript. All authors contributed to data
et al., 2010; Ties & Barringer, 2012). The lipid remodelling triggered discussion and conclusions. Paul D. Fraser acquired the funding and
by heat under our experimental conditions, affecting plastidial C18:3 accepts to serve as the contact point author for communication. All
availability, may explain the decrease in unsaturated C6 volatiles as authors have read and approved the final manuscript.
hexenal. The increased C18:2 precursors may sustain non-stressed
levels of C18:2-derived hexanal at HB3, as opposed to lower levels OR CID
found in HB, even under a possible constraint in LOX/HPL- Paul D. Fraser https://orcid.org/0000-0002-5953-8900
dependent pathway as suggested by heat-induced repression of HPL
transcripts. C6-volatiles are synthesised via the action of 13-LOX RE FE RE NCE S
and HPL enzymes, whilst C5-volatiles are LOX-dependent though Albert, E., Segura, V., Gricourt, J., Bonnefoi, J., Derivot, L., & Causse, M.
HPL-independent (Shen et al., 2014). C5-volatiles accumulated only (2016). Association mapping reveals the genetic architecture of
tomato response to water deficit: Focus on major fruit quality traits.
in HB3 as, in this case, 18:3-acyl-containing TAGs were unaffected.
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pathway as proposed previously based on tomato HPL-deficient and jasmonate insensitive tomato mutants have altered tocopherol
content and composition. Phytochemistry, 111, 72–83.
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